Genomic Characterization Reveals Insights Into Patulin Biosynthesis and Pathogenicity in Penicillium Species.

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Genomic characterization reveals insights into patulin biosynthesis

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and pathogenicity in Penicillium species

Molecular Plant-Microbe Interactions "First Look" paper • http://dx.doi.org/10.1094/MPMI-12-14-0398-FI • posted 01/27/2015 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.

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Boqiang Li1#, Yuanyuan Zong1#, Zhenglin Du2, Yong Chen1, Zhanquan Zhang1,

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Guozheng Qin1, Wenming Zhao2, and Shiping Tian1*

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Sciences, Beijing, China; 2 Core Genomic Facility, Beijing Institute of Genomics,

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Chinese Academy of Sciences, Beijing, China.

Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of

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*

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Institute of Botany, Chinese Academy of Sciences,

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Xiangshan Nanxincun 20, Haidian District,

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Beijing 100093, China

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Tel: +86-10-6283-6559.

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Fax: +86-10-8259-4675.

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#

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Data accession

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Genome assembly data has been deposited at DDBJ/EMBL/GenBank under the

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accession AYHP00000000 (P. expansum) and JMDK00000000 (P. italicum).

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RNA-sequencing data have been deposited in Sequence Read Archive (SRA) under

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the project no. SRP043647 (accession no. SRX647239, SRX647240, SRX647241,

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SRX647242).

Author for correspondence (e-mail: [email protected])

These authors contributed equally to this work.

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ABSTRACT

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Penicillium species are fungal pathogens that infect crop plants worldwide. P.

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expansum, P. italicum and P. digitatum, major postharvest pathogens of pome and

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citrus, differ in that the former is able to produce the mycotoxin patulin, and has a

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broader host range. The molecular basis of host-specificity of fungal pathogens has

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now become the focus of recent research. The present report provides the whole

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genome sequence of P. expansum (33.52 Mb) and P. italicum (28.99 Mb), and

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identifies differences in genome structure, important pathogenic characters, and

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secondary metabolite (SM) gene clusters in Penicillium species. We identified a total

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of 55 gene clusters potentially related to secondary metabolism, including a cluster of

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15 genes (named PePatA ~ O), that may be involved in patulin biosynthesis in P.

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expansum. Functional studies confirmed that PePatL and PePatK play crucial roles in

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the biosynthesis of patulin, and that patulin production is not related to virulence of P.

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expansum. Collectively, P. expansum contains more pathogenic genes and SM gene

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clusters, in particular an intact patulin cluster, than P. italicum or P. digitatum. These

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findings provide important information relevant to understanding the molecular

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network of patulin biosynthesis and mechanisms of host-specificity in Penicillium

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species.

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INTRODUCTION

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Penicillium is a genus within ascomycetous fungi with great importance in natural

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environment, as well as food and drug production (Cheeseman et al. 2014; Fierro et al.

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1995). Several species, such as P. expansum, P. italicum, and P. digitatum, are well

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known postharvest pathogens that are responsible for losses of up to 10% of harvested

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produce. P. expansum can infect a wide range of fruit and vegetable hosts, except 2

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citrus fruit, and produces the mycotoxin patulin (Andersen et al. 2004). In contrast, P.

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digitatum and P. italicum are restricted to citrus fruit and do not produce patulin.

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Patulin is a tetraketide mycotoxin, that can induce immunological, neurological and

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gastrointestinal diseases leading to serious health problems in humans (Bennett and

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Klich 2003; Pfeiffer et al. 1998). As reviewed by Moake et al (2005), the biosynthetic

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pathway of patulin contains at least ten enzymatic steps. A few genes associated with

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patulin biosynthesis, such as 6-msas encoding a 6-methylsalicylic acid synthase, idh

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encoding an isoepoxydon dehydrogenase, and peab1 encoding an ATP-binding

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cassette transporter, have been cloned from Penicillium species (Wang et al. 1991;

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White et al. 2006). In addition, a fifteen gene patulin cluster was reported in A.

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clavatus (Artigot et al. 2009) and P. expansum (Tannous et al. 2014). Fungal

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phytotoxins have been demonstrated to act as a mediator of pathogenicity or virulence

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in several mycotoxigenic genera that infect many agricultural crops and cause

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significant disease (Möbius and Hertweck 2009; Yoder and Turgeon 1996). Whether

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or not mycotoxin biosynthesis directly contributes to fungal pathogenicity, however,

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remains an open question in many host-pathogen systems. Therefore, exploring the

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molecular basis of pathogenicity and patulin biosynthesis in Penicillium species is

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biologically interesting and may have economic significance.

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Previous studies have indicated that signaling pathways, which connect fungal

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development with mycotoxin biosynthesis, would be scattered throughout the genome

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(Andersen et al. 2013; Fierro et al. 1995; van den Berg et al. 2008). However, the

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complexity of these regulatory networks and their interconnection with other

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regulatory mechanisms remains to be discovered. Comparative genomics is a tool that

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can be used to develop a better understanding of the molecular basis of fungal

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pathogenicity and patulin biosynthesis in Penicillium species. Here, we provide and 3

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report two high-quality draft genomes; a 33.52 Mb P. expansum genome containing

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11,770 predicted protein-coding genes and a 28.99 Mb P. italicum genome containing

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9,369 predicted protein-coding genes. The genome structure of both species was

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comprehensively analyzed and important pathogenic characters and secondary

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metabolite (SM) gene clusters in all three Penicillium species were examined. Notably,

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P. expansum contains more CAZymes and a larger secretome, as well as more SM

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gene clusters, than P. italicum and P. digitatum. We also identified a cluster of 15

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genes potentially related to patulin biosynthetic pathway of P. expansum. Our

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findings lay the foundation for further understanding the molecular basis of

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pathogenicity and patulin biosynthesis in Penicillium species.

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RESULTS

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Genome sequencing, assembly and characterization

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Two Penicillium species, P. expansum T01 and P. italicum B3 were sequenced

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using a whole-genome shotgun-sequencing strategy. The assembled genome of P.

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expansum is 33.52 Mb (Fig. 1; Table 1), and the total length of the eight largest

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scaffolds is 32.9 Mb (approximately 98% of the assembled genome). A high quality

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draft genome (28.99 Mb) of P. italicum B3 was also obtained. The genome of both

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species is slightly larger than the 26 Mb reported for the related species, P. digitatum

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(Marcet-Houben et al. 2012). Repetitive elements comprise 1.78% of the P. expansum

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genome and 1.8% of the P. italicum genome (Table 1). Repetitive elements are

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clustered in genomic regions with a low GC content (Fig. 1C and D), which is similar

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to reports in Colletotrichum graminicola (O'Connell et al. 2012) and Leptosphaeria

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maculans (Rouxel et al. 2011). There is a high co-linearity between the genomes of P.

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expansum and P. italicum (Fig. 1E). A total of 11,770 and 9,369 protein-coding genes 4

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were predicted from the genome sequences of P. expansum and P. italicum,

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respectively. The two Penicillium species also shared a similar profile of Gene

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Ontology (GO) categories, but number of proteins in each functional category is

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greater in P. expansum than in P. italicum (Supplementary Fig. S1).

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Comparative genomics of three Penicillium phytopathogens

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Based on a phylogenetic analysis, P. expansum is more closely related to P.

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italicum than to P. digitatum (Fig. 2A). The three Penicillium species share 6,222 gene

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families (Fig. 2B) using Markov cluster (MCL) analysis (Enright et al. 2002). In

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addition to the core gene families that are present in all three species, P. expansum

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shares more gene families (898) with P. italicum than P. digitatum (224). Notably, 102

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gene families are uniquely shared by P. italicum and P. digitatum. The shared and

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specific gene families in the three Penicillium species were classified by GO analysis

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according to biological processes (Fig. 2C). The majority of the 6,222 core families

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(Circle I) were classified into the functional categories of cellular (2,160 families) and

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metabolic processes (2,554 families). Approximately half of the species-specific gene

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families (Circles V-VII) could not be assigned into a designated GO category (Fig.

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2C). MCL analysis revealed that the three Penicillium species share similar

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multi-member gene families, including a major facilitator superfamily (MFS) of

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transporters, amino acid permeases, and betaine aldehyde dehydrogenases

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(Supplementary Table S1). P. expansum has more members in these gene families

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than P. italicum and P. digitatum.

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Using the CAZymes Analysis Toolkit (CAT) (Park et al., 2010), 640, 460, and 394

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putative CAZymes were identified in P. expansum, P. italicum, and P. digitatum,

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respectively (Fig. 3A; Supplementary Table S2). P. expansum was found to possess a

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greater number of carbohydrate esterases (CEs), glycoside hydrolases (GHs), and 5

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polysaccharide lyases (PLs), and a similar number of glycosyltransferases (GTs),

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relative to P. italicum and P. digitatum (Fig. 3A). The greater number of CEs, GHs

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and PLs in P. expansum may be due to the larger genome size of P. expansum, relative

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to the other two species. CEs, GHs and PLs are associated with the ability to utilize

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the diversity of carbohydrates present in the environment and within host plants. GTs

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are mainly involved in the basal activities of fungal cells (e.g. fungal cell wall

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synthesis, glycogen cycle, trehalose cycle), and so the number and type of GTs tend to

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be conserved in fungi (Lombard et al. 2014; O'Connell et al. 2012). It is well known

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that CAZymes involved in the degradation of plant cell wall are important for the

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pathogenicity of plant pathogens. All three Penicillium species were found to encode

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a smaller number of cellulose degrading enzymes (less than 10 proteins, Fig. 3B),

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compared to hemicellulose and pectin degrading enzymes (Fig. 3C, D and E).

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Pectin is abundant in fruit cell walls, and apple and citrus are the main raw

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materials used for the industrial production of pectin. The three Penicillium species

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have genes encoding proteins in nine of the pectin-targeting CAZyme families,

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including three PL families (PL1, 3, and 4), four GH families (GH28, 78, 95, and 105),

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and two CE families (CE8 and 12). In general, the three Penicillium species have a

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similar number of representatives in each of above pectin-targeting CAZyme families,

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except in the GH78 family. P. expansum has eight genes encoding for GH78 family

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enzymes, which is twice that of P. italicum (3 enzymes) and P. digitatum (4 enzymes)

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(Fig. 3D). Pectin is composed of homogalacturonans (“smooth” regions) and more

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complex rhamnogalacturonans that contain arabinan, galactan and arabinogalactan

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side chains (“hairy” regions). GH78 enzymes exhibit α-L-rhamnosidase activity and

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are involved in the degradation of rhamnogalacturonan backbone of the “hairy”

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region in pectin molecules. The greater number of genes in P. expansum encoding for 6

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GH78 family enzymes may represent an adaption to the structure of apple pectin,

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which has a high proportion of “hairy” regions (Wang et al. 2014). The expression of

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eight GH78 family genes in P. expansum when it was supplied with apple or citrus

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pectin as a carbon source was examined (Fig. 3G). Expression of all eight GH78

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family genes increased with time of culture when either apple or citrus pectin was

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used. Relative expression of four of the genes (PEG03271, PEG05990, PEG10044,

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and PEG11722) was 2 - 6 fold higher after 48 h when P. expansum was cultured on

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apple pectin, rather than citrus pectin, suggesting that those genes may play an

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important role in the degradation of the apple pectin. Besides rhamnogalacturonan

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backbone, the “hairy” region has arabinan and/or arabinogalactan-rich side chains

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substituted onto rhamnose residues (Blackman et al. 2014). Wang et al. (2014)

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reported that the structure of arabinogalactan side chains in apple and citrus pectin are

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different. A total of 17, 13, and 8 CAZymes were identified in P. expansum, P.

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italicum and P. digitatum, respectively, that may function in the degradation of side

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chains in the “hairy” region (Fig. 3E). Since CAZymes in GH43, 51, 53, 54, and 93

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families can also degrade hemicelluloses and other substrates, it is difficult to

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specifically identify the number of GHs involved in the degradation of pectin side

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chains (Amselem et al. 2011; Blackman et al. 2014).

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P. expansum also possesses a larger secretome (927 predicted secreted proteins, 7.7%

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of proteome) than P. italicum (662, 7.1%) and P. digitatum (546, 6.0%) (p < 0.05

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according to Fisher’s exact test). Proteases are abundant in the secretome of all

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species, with P. expansum containing over twice as many secreted proteases as P.

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digitatum (Fig. 3F). Possessing a diversity of secreted proteases is beneficial since it

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allows the fungus to exploit a variety of ambient nutrients and also counteract

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protein-based defense responses of the hosts. As shown in Fig. 3F, more than half of 7

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the identified secreted proteases belong to a super-family of serine proteases. Among

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them, S33 family is lacking in P. italicum and P. digitatum, while P. expansum

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encodes three S33 proteases (Supplementary Table S3). S33 proteases exhibit prolyl

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aminopeptidase activity, which releases N-terminal proline from a peptide. P.

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expansum also has some unique families, such as M6, M12B, M20D, M35, and M77,

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that belong to a super-family of metallo proteases (Supplementary Table S3).

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Identification of gene clusters related to secondary metabolism and patulin

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biosynthesis

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Typically, a SM gene cluster contains one or more “backbone” genes, which

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encode key enzymes of the SM biosynthetic process, such as polyketide synthase

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(PKS), non-ribosomal peptide synthetase (NRPS), polyketide synthase/non-ribosomal

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peptide synthetase hybrid (PKS-NRPS hybrid), dimethylallyl tryptophan synthase

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(DMATS). Using the bioinformatic program SMURF (Khaldi et al. 2010), 71

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predicted SM backbone genes were found to be present in P. expansum

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(Supplementary Table S4), which is distinctly larger than that in P. italicum (38

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backbone genes) and P. digitatum (32 backbone genes) (Fig. 4A). Phylogenetic

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analysis indicated that some of the SM backbone genes present in these three species

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clustered together (Fig. 4B). In addition to possessing an increased number of SM

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backbone genes, P. expansum also contains the largest number of Cytochrome P450

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(CYP450) (Fig. 4C) and transporter (Fig. 4D) genes, both of which play important

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roles in the biosynthesis and transportation of SMs (Del Sorbo et al. 2000; Weng et al.

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2012). Among the transporter genes, P. expansum contains a larger number of MFS

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transporters (527, 54% of total transporters) than P. italicum (386, 42%) and P.

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digitatum (258, 41%) (Fig. 4D). In contrast, a smaller difference in the number of

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ABC transporter genes exists between the three Penicillium species. 8

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In total, 55 SM clusters were identified in P. expansum as compared to 30 SM

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clusters in P. italicum and 24 clusters in P. digitatum (Supplementary Table S5). Some

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SM clusters are shared by all three or by two of the Penicillium species (Fig. 5A and

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B), indicating that the three Penicillium species may produce some similar SMs. Only

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P. expansum, however, possesses the complete patulin gene cluster (Fig. 5B). This 44

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kb cluster consists of 15 genes, named PePatA ~ O. Each of the genes in the patulin

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cluster of P. expansum shares high identity with a corresponding gene in the putative

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patulin cluster of A. clavatus (Table 2), but the order of the genes within the cluster is

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quite different in the two species (Fig. 5C). Among the 15 genes in the patulin cluster

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of P. expansum, one encodes a putative transcription factor (PePatL), three encode

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transporters (PePatA, C and M), nine encode biosynthetic enzymes, and two (PePatF

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and J) have an unknown function (Table 2). PePatF has a SnoaL-like domain, and

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PePatJ has a Cupin domain based on a Pfam analysis. Proteins with those domains

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commonly exhibit enzymatic activity in microorganisms (Dunwell et al. 2000;

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Sultana et al. 2004). PePatK is the backbone gene of the patulin cluster in P.

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expansum. It encodes a 6-methylsalicylic acid synthase (6-MSAS), which belongs to

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the type I PKS group and has 5 different domains (Supplementary Fig. S2). Although

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a few orthologs of patulin genes were also found in P. italicum and P. digitatum, the

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backbone gene (PatK) was absent in both and only three patulin genes (PatC, PatD

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and PatL) were identified in P. italicum (Fig. 5C). PatC and PatD appeared to fuse

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into one predicted gene (Pit_00308) in P. italicum. The loss of the backbone and other

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patulin genes negates the ability to produce patulin in P. italicum and P. digitatum.

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Expression analysis of genes involved in patulin biosynthesis

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The transcriptome of P. expansum cultured in static (patulin permissive) and

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shaking (patulin restrictive) conditions were compared in order to investigate the 9

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expression of genes potentially related to patulin biosynthesis. Approximately 3,034

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genes were differentially expressed (p ≤ 0.001) in both conditions (Fig. 6A), and

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based on GO functional classification, most could be assigned to catalytic activity and

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metabolic process (Supplementary Fig. S3). Among the differentially expressed genes,

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1,642 were up-regulated under static condition, including all 15 genes in the patulin

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cluster, and 1,392 genes were down-regulated (Fig. 6A).

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Differentially expressed genes were enriched in the ko03010 (ribosome) pathway

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based on KEGG analysis (Supplementary Table S6 and S7), and the ortholog of LaeA

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(PEG09421) was significantly up-regulated in the static culture (Supplementary Table

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S8). LaeA is a well-known regulator of secondary metabolism in A. nidulans (Bok

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and Keller, 2004). This protein is thought to activate expression of SM gene clusters

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by chromatin modification mediated by the velvet complex (Bayram et al. 2008;

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Strauss and Reyes-Dominguez, 2011). Differential expression of the SM backbone

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genes, and genes encoding CYP450s, ATP-binding cassette (ABC) transporters, and

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MFS transporters, were also observed (Fig. 6B). Approximately half of the SM

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backbone genes were differentially expressed, including 15 genes encoding PKSs, 16

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genes encoding NRPSs, 4 genes encoding PKS-NPRS hybrids, and 2 genes encoding

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DMATs (Fig. 6B). Among these genes, 23 were up-regulated and 14 down-regulated

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under static condition. In addition, 12 genes encoding ABC transporter were

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up-regulated and 2 were down-regulated. RT-qPCR analysis confirmed that all of the

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genes in the patulin cluster were up-regulated when P. expansum was grown under

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static condition (Fig. 6C), which is conducive to patulin production (Fig. 6D).

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Relationship between patulin production and pathogenicity in P. expansum

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To explore the role of patulin in the pathogenesis of P. expansum, two genes in the

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patulin cluster, PePatK and PePatL, were deleted using a gene replacement strategy 10

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and Agrobacterium-mediated transformation (Fig. 7A). PePatK encodes a putative

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PKS and PePatL encodes a putative transcription factor. These two genes were

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selected as the target genes for the knockout assay since they were predicted to play

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an important role in the biosynthesis of patulin in P. expansum. Three knockout

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mutants were obtained for each target gene identified by PCR (Supplementary Fig. S4)

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and confirmed by Southern blot analysis (Fig. 7B). None of the knockout mutants of

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PePatL and PePatK produced detectable patulin as determined by HPLC analysis

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(Fig. 7D). The knockout mutants of PePatL and PePatK exhibited a slightly smaller

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colony diameter, relative to the growth of the wild-type strain (Fig. 7E). No

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significant difference in sporulation was observed, however, between the knockout

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mutants and the wild-type strain (Fig. 7F). Two pome fruit hosts (apple and pear) and

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one stone fruit host (plum) were used in a pathogenicity assay in order to investigate

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the effect of patulin production on the pathogenicity in P. expansum. Results indicated

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that there was no significant difference in pathogenicity between the knockout

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mutants and the wild-type strain on any of three fruit hosts (Fig. 7G).

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Characterization of PePatL, the putative transcription factor

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A common feature of fungal gene clusters responsible for the production of SMs is

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the presence of a pathway-specific regulatory gene (Yu et al. 1996; Yu and Keller

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2005). PePatL encodes the only putative transcription factor in the patulin cluster. The

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amino-acid sequence of PePatL was compared to several known transcription factors,

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such as GAL4 in Saccharomyces cerevisiae, and AFLR in A. flavus and A. nidulans.

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In this regard, PePatL was found to possess a Zn(II)2Cys6

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binding motif at the N-terminal end of the protein (Fig. 8A). The expression of genes

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in the patulin cluster in both the ∆PePatL mutant and the wild-type strain was

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compared. The results indicated that the examined genes in the patulin cluster were 11

binuclear cluster DNA

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only marginally expressed in the ∆PePatL mutant when it was grown under patulin

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permissive condition (Fig. 8B). Additionally, microscopic analysis of a PePatL-GFP

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fusion protein indicated that PePatL is localized in the nuclei of P. expansum (Fig.

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8C).

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DISCUSSION

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Some fungal plant pathogens cause economic losses not only by decaying the plant

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material but also by producing harmful mycotoxins (Calvo et al. 2002). P. expansum

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is able to infect a great number of fruit hosts and produces patulin in the contaminated

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tissues (Andersen et al. 2004). In contrast, P. italicum and P. digitatum only infect

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citrus fruit but do not produce patulin (Marcet-Houben et al. 2012). The molecular

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basis of host-specificity and patulin biosynthesis of Penicillium species, however, has

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not been elucidated. Bioinformatic analysis on genome sequence of a fungal pathogen

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can be used to assist the discovery of the molecular mechanisms that are responsible

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for pathogenicity and the production of SMs (Fierro et al. 1995; Marcet-Houben et al.

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2012).

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In the present study, the genomes of both P. expansum and P. italicum were

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sequenced (Fig. 1) and a high colinearity was found between the two species (Fig. 1E).

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The number of proteins in various functional categories, however, are greater in P.

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expansum than in P. italicum (Fig. 2, 3, 4 and Supplementary Fig. S1). In particular, P.

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expansum contains more genes encoding CAZymes and secreted proteases than either

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P. italicum or P. digitatum (Fig. 3A-F; Supplementary Table S2 and S3). CAZymes in

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plant pathogens are responsible for the synthesis, degradation, and modification of

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carbohydrates, and thus play an important role in the breakdown of plant cell walls

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and in host-pathogen interactions (Zhao et al. 2014; Zheng et al. 2013). Fungi secrete 12

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enzymes to break down biopolymers from hosts and utilize them as nutrients

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(Marcet-Houben et al. 2012). Secreted proteins from pathogens may also determine

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the progress and success of an infection (Li et al. 2012; Mueller et al. 2008; Qin et al.

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2007). Therefore, the larger number of CAZyme-related genes in P. expansum may be

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partially responsible for the wider range of fruit hosts, relative to P. italicum and P.

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digitatum.

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Several fungal pathogens produce bioactive small molecules, commonly classified

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as SMs, that play a significant role in the development and lifecycle of fungi (Keller

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et al. 2005). During the infection process, fungal pathogens may produce abundant

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levels of SMs in plant hosts (Möbius and Hertweck 2009). SM genes in fungi are

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typically organized in clusters around backbone genes that encode synthases, such as

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PKSs, NRPSs, DMATSs, and PKS-NPRS hybrids (Andersen et al. 2013). In this study,

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P. expansum was found to possess twice as many of these backbone genes than P.

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italicum and P. digitatum (Fig. 4; Supplementary Table S4), and more SM gene

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clusters (Fig. 5; Supplementary Table S5).

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gene clusters present in P. expansum (Fig. 5) may possibly contribute towards its wide

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host range.

The larger number and variety of SM

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In the present study, a cluster of 15 genes putatively responsible for patulin

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biosynthesis in P. expansum was identified (Fig. 5C; Table 2), which has the same

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number of genes as a putative patulin cluster in A. clavatus (Artigot et al. 2009). All

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15 genes in the cluster were up-regulated when P. expansum was cultured under static

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(patulin permissive) condition (Fig. 6A and C), suggesting that all of the genes may be

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potentially related to patulin biosynthesis in P. expansum. A total of 1,642

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up-regulated and 1,392 down-regulated genes were detected by transcriptome analysis

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when P. expansum was cultured under static conditions (Fig. 6A), including the SM 13

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backbone genes, and genes encoding CYP450s, ABC transporters, and MFS

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transporters (Fig. 6B). CYP450s facilitate a wide variety of biochemical reactions in

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microorganisms and are critical for SM production (Kelly and Kelly, 2013). MFS and

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ABC transporters are the two biggest families of fungal transporters and mediate

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transport of intermediates and export toxic end products in the secondary metabolism

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pathway (Del Sorbo et al. 2000). The differential expression of many of these genes in

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P. expansum grown under static vs. shaking conditions indicated that culture condition

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has a significant effect on SM profiles in this fungal pathogen.

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Using an Agrobacterium-mediated transformation method, two genes in the patulin

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cluster, PePatK and PePatL, were deleted. These deletions resulted in a complete

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inhibition of patulin production in the P. expansum knockout mutants (Fig. 7D).

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6-MSAS, encoded by PatK, has been reported to catalyze the first step of patulin

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biosynthesis which involves the conversion of acetyl CoA and malonyl-CoA to

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6-methylsalicylic acid in other Penicillium species (Puel et al. 2010). Our results

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demonstrated that PePatK is also crucial for the biosynthesis of patulin in P. expansum.

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PePatL has a conserved Zn (II)2Cys6 binuclear cluster DNA binding motif (Fig. 8A),

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and is similar to AFLR, a demonstrated transcription factor associated with the

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aflatoxin biosynthetic pathway (Yu et al. 2004). PePatL was observed to be localized

342

in the nuclei of P. expansum (Fig. 8C) and affected the expression of other genes in

343

the patulin cluster (Fig. 8B). These data support the premise that PePatL may act as a

344

specific transcription factor in the patulin biosynthetic pathway. Importantly, a loss of

345

the ability to produce patulin in the ∆PePatK and ∆PePatL mutants, did not impair

346

virulence (Fig. 7G), indicating that patulin does not act as a pathogenicity factor or

347

contribute to the virulence of P. expansum. In fact, most patulin producing fungal

348

species are not plant pathogens. Since patulin can exhibit antibiotic activity against 14

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some bacteria and fungi (Moake et al. 2005), the biological significance of patulin

350

production in P. expansum may be to improve the ability of P. expansum to compete

351

with other microbial organisms in specific environmental niches.

352

In summary, this study provides information on the molecular basis of

353

pathogenicity and patulin biosynthesis in three Penicillium species based on the

354

analysis of the sequenced genome of P. expansum and P. italicum. Of these, the most

355

important ones are that (1) we provide the genome sequence of both P. expansum and

356

P. italicum; (2) we provide an analysis of the composition of each genome and of

357

important pathogenicity-related characteristics in three Penicillium species; (3) we

358

predicted 55 gene clusters potentially related to secondary metabolism and identify a

359

cluster of 15 genes that may be involved in patulin biosynthetic pathway in P.

360

expansum; (4) we determined a crucial role of PePatL and PePatK in the patulin

361

biosynthetic pathway, and demonstrated that patulin production does not play a role in

362

the virulence of P. expansum. We observed that P. expansum contains more

363

pathogenic-related genes and SM gene clusters, in particular an intact patulin cluster,

364

than P. italicum or P. digitatum. The obtained genome sequence data also provide the

365

information that is needed to further explore the molecular basis for other biological

366

characteristics in Penicillium species.

367 368

MATERIALS AND METHODS

369

Fungal strains

370

P. expansum T01 and P. italicum B3 were isolated, from infected apple and sweet

371

orange fruits, respectively, that exhibited typical blue mold symptom. Two strains

372

were purified by single spore isolation, and stored at -80 °C prior to use.

373

Extraction of genomic DNA and total RNA for sequencing 15

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P. expansum T01 and P. italicum B3 were cultured on Potato Dextrose Agar (PDA)

375

plates. After 7 to 10 d, the spores were harvested by flooding with sterile distilled

376

water. The concentration of a spore suspension was adjusted to 1 × 107 spores/ml with

377

a hemocytometer.

378

For genome sequencing, a 1 ml aliquot of a spore suspension was added to 20 ml

379

Czapek Yeast Extract (CY) medium (3 g NaNO3, 1 g K2HPO4·3H2O, 0.5 g KCl, 0.5 g

380

MgSO4·7H2O, 0.01 g FeSO4·7H2O, 30 g sucrose, 5 g yeast extract, 1 l distilled water,

381

pH 5.2) in 50-ml conical flasks and cultured at 25 ºC on a rotary shaker at 200 rpm for

382

3 d. Mycelium was collected by filtering the liquid culture through 4 layers of

383

cheesecloth. The obtained mycelium was then washed thoroughly with cold sterile

384

distilled water. A 2.0 g (wet weight) aliquot of mycelium was used for genomic DNA

385

extraction with a DNeasy Plant Mini Kit (QIAGEN, Valencia, CA, USA).

386

Total RNA was extracted from four different samples of P. expansum and P.

387

italicum, respectively. Sample I: spores collected from PDA plates cultured for 7 d at

388

25 ºC; Sample II: spores incubated in liquid CY medium for 13 h with shaking at 200

389

rpm; sample III: mycelium grown in liquid CY medium for 4 d with shaking at 200

390

rpm; sample IV: mycelium grown in liquid CY medium for 4 d without shaking. Total

391

RNA was isolated from 0.2 g (wet weight) aliquots of each sample using TRIzol.

392

RNA from each sample was pooled equally before sequencing. The RNA library was

393

constructed using an Illumina TruSeq RNA Sample Preparation kit and sequenced on

394

Hiseq2000 with the pair-end mode. RNA-seq data were collected for improvement of

395

gene prediction.

396

Genome sequencing and assembly

397

The genome of P. expansum T01 and P. italicum B3 was sequenced with an

398

Illumina Hiseq2000 using a whole genome shotgun sequencing strategy. Four 16

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sequencing libraries were constructed for P. expansum, with an insert size of 180 bp,

400

300 bp, 3 kb, and 8 kb, respectively. Illumina TruSeq DNA Sample Preparation kits

401

were used for the construction of the 2 pair-end libraries (180 bp and 300 bp). The

402

two large insert-size libraries (3 kb and 8 kb) were constructed using the Roche

403

mate-pair library preparation method. A total number of 21.6 Gb reads sequence was

404

generated by Hiseq2000 using 100 bp pair-end mode, representing approximately

405

527x genome coverage. Clean reads from the two libraries with an insert size of 180

406

and 300 bp were assembled using SOAPdenovo (V1.05) assembler (Li et al. 2008) to

407

construct scaffolds. Then, using the clean reads from the large insert-size libraries of 3

408

and 8 kb, the pre-assembled scaffolds were processed scaffolding again using the

409

stand-alone scaffolding tool, SSPACE (V2.0) (Boetzer et al. 2011). Lastly, the SOAP

410

GapCloser (V1.12) (Li et al. 2009) was used to fill gaps in the obtained scaffolds.

411

Three sequencing libraries were constructed for P. italicum, with an insert size of

412

180bp, 300bp, and 500bp, respectively. A total number of 5.7 Gb reads sequence was

413

generated with 100bp pair-end, representing approximately 193x genome coverage.

414

Gene annotation

415

For gene prediction, a combination of three methods was used. Firstly,

416

homology-based gene prediction was performed using Exonerate (v2.2.0) (Slater and

417

Birney 2005). Then, Augustus (v2.5.5) (Stanke et al. 2004), Glimmer-HMM (v3.0.2)

418

(Majoros et al. 2004) and GeneMark.hmm (v2.3) (Lukashin and Borodovsky 1998)

419

were used to obtain ab initio prediction of gene structure. Lastly, RNA-Seq data were

420

de novo assembled into transcripts by Trinity (Grabherr et al. 2011) to improve

421

predicted gene structure. All of the above data were combined by EVM (Haas et al.

422

2008) to produce a consensus gene set. Then, PASA (Haas et al. 2003) was used to

423

update the EVM gene structures and add UTR annotations. For functional annotation, 17

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protein sequences were blasted against a series of protein databases, including

425

Swiss-Prot, TrEMBL, NCBI nr, and KOG, and the best hit was used to infer their

426

biological function. Gene Ontology annotation was performed using Blast2GO, which

427

assigned homologous sequences aligned by BLAST with Uniprot and NCBI nr

428

database to GO terms.

429

Annotation of specific gene categories

430

The Secondary Metabolite Unknown Region Finder (SMURF) (Khaldi et al. 2010)

431

was used to predict backbone genes and SM gene clusters. The Fungal Cytochrome

432

P450 Database was used to identify Cytochrome P450 enzymes by BLAST (blastp),

433

with an e-value cutoff of 1e-5 and alignment coverage ≥ 80%. Transporters were

434

identified based on BLAST searches (e-value ≤ 1e-10 and > 35 identity of protein

435

sequence) against the Transporter Classification Database and the NCBI protein

436

sequence database. The CAZyme genes were identified using the CAZymes Analysis

437

Toolkit (CAT) (Park et al. 2010). SignalP (v4.1) (Petersen et al. 2011) was used to

438

predict secreted proteins. A MEROPS Batch BLAST analysis (Rawlings et al. 2010)

439

was performed to identify secreted peptidase genes.

440

Transposable element analysis

441

To identify the repetitive sequence in the genome, Repeatmasker was used with the

442

Repbase library (v17.11) and the species option of ‘Fungi’. The repeat sequence was

443

masked with ‘N’.

444

Genome synteny analysis

445

The collinearity between P. expansum and P. italicum genomes was analyzed using

446

SyMAP (v4.0) (Soderlund et al. 2011). The longest 8 scaffolds of P. expansum,

447

representing 98% of the assembled genome, were used in the analysis. The P. italicum

448

genome was aligned to the 8 scaffolds of P. expansum. The P. italicum scaffold 18

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449

sequences were merged together based on the alignment results, and named Pi_1 to

450

Pi_8. The collinearity between the P. expansum and P. italicum genomes was then

451

analyzed using SyMAP.

452

Gene family analysis

453

Gene families were generated using MCL software (v12-068) (Enright et al. 2002),

454

which utilizes a Markov clustering algorithm to cluster proteins into families based on

455

pre-computed sequence similarity information among all genes in all taxa. An

456

all-vs-all BLAST search of each species' protein was performed and the blastp results

457

were filtered using threshold limits of E-values ≤ 1e-5, alignment identity ≥ 30%, and

458

an alignment coverage ≥ 50%. Gene families were generated by MCL software using

459

an inflation value of 2.0.

460

Phylogenetic analysis

461

A whole-genome cladogram displaying the phylogenetic relationships between P.

462

expansum, P. italicum, and P. digitatum was constructed using CVTree with

463

Talaromyces marneffei as an outgroup (O'Connell et al. 2012). The genome sequences

464

of P. digitatum PHI26 and Talaromyces marneffei PM1 were downloaded from NCBI

465

under the accessions of AKCT00000000 (Marcet-Houben et al. 2012) and

466

AGCC00000000 (Woo et al. 2011), respectively. Phylogenetic trees of PKSs，NRPSs，

467

DMATs and Hybrids were constructed using the Maximum-Likelihood approach

468

implemented in PhyML (Guindon et al. 2010).

469

Expression analysis of GH78 family genes induced by apple and citrus pectin

470

For expression analysis of genes in GH78 family, Reverse Transcription –

471

quantitative PCR (RT-qPCR) analysis was performed on an ABI StepOnePlus

472

Real-Time PCR System (Applied Biosystems, CA, USA). A spore suspension of P.

473

expansum was added to 20 ml of pectin-CY medium (CY without sucrose and with 1% 19

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474

pectin from apple or citrus) to obtain a final concentration of 106 spores/ml and

475

cultured at 25 °C with shaking at 180 rpm. Mycelium was harvested using 4 layers of

476

cheesecloth at 24 and 48 h after inoculation. Total RNA was isolated from the

477

mycelium of P. expansum as described above. RT-qPCR was carried out in a total

478

volume of 20 µl, containing 10 µl of 2 × Ultra SYBR Mixture (with high ROX)

479

(CWBIO), 2 µl of the 10× diluted cDNA, 0.4 µl of each primer (10 mM) and 7.2 µl of

480

RNase-free water. The primers used for amplification are listed in Supplementary

481

Table S9. Transcript levels were normalized against the beta-tublin gene (GenBank

482

accession number AF003248.1), and relative expression levels were calculated using

483

the 2−∆∆CT method (Livak and Schmittgen 2001).

484

Patulin production and gene expression analysis under different cultural

485

conditions

486

Spores of P. expansum were inoculated in liquid CY medium as described above,

487

and cultured under shaking (200 rpm) or static conditions at 25 ºC. After 2 and 4 d,

488

the mycelium was harvested, and quickly frozen with liquid nitrogen for RNA

489

extraction. CY medium filtrates were filtered through 0.45 µm filters before being

490

analyzed by HPLC for patulin production. High-performance liquid chromatography

491

system was equipped with an auto sampler (Waters 2707, Waters Corp., MA, USA),

492

binary HPLC pump (Waters 1525, Waters Corp., MA, USA), and an UV/Visible

493

Detector (Waters 2487, Waters Corp., MA, USA). A C18 column (5 µm, 250 × 4.6

494

mm, Intersil ODS-3, GL Sciences, Japan) was used for separation at 30 °C. A mixture

495

of water and acetonitrile (90 : 10, V/V) at a flow rate of 1 ml/min was used as the

496

mobile phase in an isocratic elution mode. Detection of patulin was performed at a

497

wavelength of 276 nm.

498

For transcriptome analysis, total RNA was extracted from the mycelium of cultures. 20

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499

Two independent biological samples from each culture condition were used. The RNA

500

library was constructed using the method described above and sequenced on a

501

Hiseq2500 with the pair-end mode. About 2 Gb reads sequence was generated for

502

each sample. After removing sequencing adapters and trimming consecutive

503

low-quality bases (Quality < 20) from both the 5' and 3' end of the reads, high-quality

504

RNA-seq reads from P. expansum were aligned to the reference genome using

505

Tophat2 (v2.0.11) (Kim et al. 2013) with default parameters. Cufflinks (Trapnell et al.

506

2010) was then used to quantify expression. Abundances are reported as normalized

507

fragments per kb of transcript per million mapped reads (FPKM). Differential

508

expression of a gene is considered to be significant if its expression differs between

509

two samples at a p value ≤ 0.001, as calculated by Cufflinks.

510

For expression analysis of genes in the patulin cluster, total RNA was isolated from

511

the mycelium of P. expansum cultured for 2 and 4 d. RT-qPCR was carried out as

512

described above. The primers used for amplification are listed in Supplementary Table

513

S10.

514

Construction of the replacement vectors and transformation

515

The pLOB 7 and pCAMBIA 1300 vectors were kindly provided by Dr. Jan A.L.

516

van Kan, Wageningen University, The Netherlands and Dr. Zhonghua Ma, Zhejiang

517

University, respectively. Firstly, An EcoRI-HindIII DNA fragment containing the

518

hygromycin phosphotransferase gene hph under control of the oliC promoter and trpC

519

terminator was excised from pLOB 7 and cloned into pCAMBIA 1300 to yield the

520

binary vector, pCHPH. For the construction of gene replacement vectors, two

521

fragments flanking each of the target sequences were amplified and cloned into

522

pCHPH at up- and down stream of hph to yield the vectors, pCHPH-PatK and

523

pCHPH-PatL, respectively. Primers for amplification of the flanking fragments are 21

Page 22 of 138


Boqiang Li MPMI

524

listed in Supplementary Table S11. The gene replacement vector was transformed into

525

the Agrobacterium tumefaciens EHA 105 strain by electroporation. The transformed A.

526

tumefaciens strain was co-cultured with P. expansum spores on a cellophane sheet that

527

was placed on agar plates containing the co-cultivation medium (10 mM

528

K2HPO4·3H2O, 10 mM KH2PO4, 2.5 mM NaCl, 2 mM MgSO4·7H2O, 0.45 mM

529

CaCl2, 9 µM FeSO4·7H2O, 4 mM (NH4)2SO4, 5 mM glucose, 0.5% glycerol, 40 mM

530

MES, 200 µM acetosyringone, and 1.5% agar, pH 5.3). After co-cultivation, the

531

cellophane sheet was transferred to a CYA medium (CY added with 1.5% agar) that

532

was supplemented with 250 µg/ml hygromycin B to select transformants. Stability of

533

the antibiotic-resistant phenotype was tested by repeated transfer of the putative

534

transformants to fresh selection plates for 3 times. The resistant strains were verified

535

by PCR using the primer pair, KL1/H1, for PePatK, and LL1/H1 for PePatL,

536

respectively (Supplementary Table S11). Then, the positive transformants were

537

purified by single-spore isolation, and confirmed by Southern blot analysis to exclude

538

transformants with ectopic integration sites. Genomic DNA for PCR confirmation and

539

Southern blot analysis was isolated following the procedure of Raeder and Broda

540

(1985). For Southern blot analysis, 10 µg of genomic DNA was completely digested

541

with EcoRV. The digests were fractionated by electrophoresis on an 0.8% agarose gel,

542

and transferred to a Hybond-N+ nylon membrane (Mylab, China) using alkaline

543

transfer buffer (0.4 M NaOH and 1 M NaCl). After being washed in neutralization

544

buffer (0.5 M Tris-HCl and 1 M NaCl, pH 7.2) and heated at 80 °C for 1 h, the

545

membrane was hybridized and detected according to the instructions of the Dig

546

Hybridization Detection Kit (Mylab, China). The probes were labeled with DIG by

547

PCR using the primers of KL1/KL2 and LL1/LL2, respectively (Supplementary Table

548

S11). 22



549

Phenotype analysis of gene knockout mutants

550

Analysis of patulin production, colony growth, and sporulation were determined

551

according to the methods of Sanzani et al. (2012) with some modifications. Five µl

552

aliquots of a spore suspension with a concentration of 105 spores/ml were inoculated

553

in the center of 9-cm PDA plates, and incubated at 25 °C in the dark for 10 d. Colony

554

morphology, colony diameter, spore and patulin production were then recorded.

555

Patulin was extracted using acidified, distilled water and determined using the HPLC

556

system described above. For the pathogenicity assay, conidia of mutants ∆PePatK-1

557

and ∆PePatL-3 mutants and the wild-type strain of P. expansum were inoculated in

558

wounded apple (Malus domestica cv. Fuji), pear (Pyrus pyrifolia cv. Huangguan) and

559

plum (Prunus americana) fruit. Four wounds (3 × 3 mm) were made with a sterile

560

nail on the equator of each apple or pear fruit, and two wounds (3 × 3 mm) were made

561

on each plum fruit. A 10 µl spore suspension (1 × 104 spores/ml) was pipetted into

562

each wound. Inoculated fruits were stored at 25 °C for 5 days and lesion diameters

563

were determined daily.

564 565

ACKNOWLEDGMENTS

566

We thank Nancy P. Keller and Manyuan Long for helpful comments on the

567

manuscript. We also thank Dr. J. van Kan and Dr. Z. Ma for supplying vectors. This

568

work was supported by National High Technology Research (863) Program of China

569

(2012AA101607), National Natural Science Foundation of China (31030051) and the

570

Chinese Academy of Sciences (KSCX2-EW-G-6). The funders had no role in study

571

design, data collection and analysis, decision to publish, or preparation of the

572

manuscript.

573

23



574

AUTHOR CONTRIBUTIONS

575

Conceived and designed the experiments: Shiping Tian. Performed the

576

experiments: Yuanyuan Zong, Boqiang Li, Zhanquan Zhang, and Yong Chen.

577

Analyzed the data: Boqiang Li, Zhenglin Du, Guozheng Qin, Wenming Zhao, and

578

Shiping Tian. Wrote the paper: Boqiang Li and Shiping Tian.

579 580

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Moake, M. M., Padilla-Zakour, O. I. and Worobo, R.W. 2005. Comprehensive review of patulin control methods in foods. Compr. Rev. Food Sci. Food Safety 4:8-21. Möbius, N. and Hertweck, C. 2009. Fungal phytotoxins as mediators of virulence. Curr. Opin. Plant Biol. 12:390-398.

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793

Genomics 15:6.

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Y., Sun, Z., Feng, H., Liang, X., Fu, R., Tang, C., Li, Q., Zhang, J., Xie, Z., Deng,

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Q., Li, S., Wang, S., Zhu, J., Wang, L., Liu, H. and Li, P. 2013. The evolution and

797

pathogenic mechanisms of the rice sheath blight pathogen. Nat. Commun.

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799

32

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AUTHOR-RECOMMENDED INTERNET RESOURCES

801

Carbohydrate-Active enZYmes Database: http://www.cazy.org/

802

Fungal Cytochrome P450 Database: http://p450.riceblast.snu.ac.kr

803

Repeatmasker: http://www.repeatmasker.org

804

Transport Classification database (TCDB): http://www.tcdb.org

33

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Table 1. Assembly and genome characteristics of P. expansum T01 and P.

806

italicum B3. Genome features

P. expansum T01

P. italicum B3

Genome size (Mb)

33.52

28.99

Sequencing coverage

527 ×

193 ×

Number of contigs

4294

4873

123.62

161.33

108

822

36

640

Max scaffold length (kb)

9171.49

829.49

N50 scaffold (Kb)

5031.09

205.92

GC content (%)

47.10

47.03

Protein-coding genes

11770

9369

Mean gene length (bp)

1891

1648

Mean CDS length (bp)

1464

1481

3

3

Mean exons length (bp)

486

490

Mean intron length (bp)

80

82

Min protein length (aa)

50

50

Max protein length (aa)

7257

7288

1.78

1.80

N50 contig (Kb) Number of scaffolds (>500bp) Number of scaffolds (>1kb)

Mean exons per gene

Repetitive elements (%) 34


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Table 2. Comparison of genes in the patulin clusters of A. clavatus (Ac) and P. expansum (Pe). ID

Length of ORF (bp)

Gene

Exon

Putative function Ac

Pe

Ac

Pe

Ac

Nucleotide

Protein

identity

identity

(%)

(%)

Pe

PatA

ACLA_093560

PEG03683

Acetate transporter

1007

1041

5

5

63.05

78.09

PatB

ACLA_093570

PEG03684

Carboxylesterase

1872

1927

4

4

63.09

67.2

PatC

ACLA_093580

PEG03685

MFS transporter

1781

1752

3

3

72.18

77.41

PatD

ACLA_093590

PEG03686

Alcohol dehydrogenase

1491

1511

5

7

63.65

73.2

2013

2001

3

3

70.32

79.17

Glucose-methanol-choline PatE

ACLA_093600

PEG03687 oxidoreductase

PatF

ACLA_093610

PEG03688

Hypothetical protein 1

597

600

1

1

71.50

77

PatG

ACLA_093620

PEG03689

Amidohydrolase family protein

1061

1025

2

2

68.33

78.46

PatH

ACLA_093630

PEG03690

m-cresol methyl hydroxylase

1854

1841

5

5

70.80

84.54

35


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Boqiang Li MPMI

m-hydroxybenzyl alcohol PatI

ACLA_093640

PEG03676

1926

1886

5

5

67.38

80.41

hydroxylase PatJ

ACLA_093650

PEG03675

Hypothetical protein 2

1117

1116

3

3

70.38

84.36

PatK

ACLA_093660

PEG03674

6-Methylsalicylic acid synthase

5163

5464

1

2

68.48

77.8

PatL

ACLA_093670

PEG03677

C6 transcription factor

2541

2456

2

2

64.67

66.87

PatM ACLA_093680

PEG03681

ABC transporter

4630

4553

7

7

68.95

76.69

PatN

ACLA_093690

PEG03680

Isoepoxydon dehydrogenase

908

896

3

3

74.40

90.35

PatO

ACLA_093700

PEG03678

Isoamyl alcohol oxidase

1973

1960

5

5

65.47

71.68

808 809 810

36



811 812

FIGURE CAPTIONS

813

Figure 1. Genomes of P. expansum and P. italicum. A, Colony, microscopic

814

morphology of P. expansum, and typical disease symptoms in apple fruit. B, Colony,

815

microscopic morphology of P. italicum, and typical disease symptoms in orange fruit.

816

C and D, Genome organization and gene distribution in P. expansum and P. italicum.

817

The peripheral circle represents the scaffolds. Pe_1 to Pe_8 represents the longest 8

818

scaffolds of P. expansum. The P. italicum genome was aligned to these 8 scaffolds.

819

Based on the results of the alignment, P. italicum scaffold sequences were merged

820

together and designated as Pi_1 to Pi_8. The second circle indicates the GC content

821

with the ratio of GC sites per 50 kb ranged from 0.35 to 0.53. The third circle

822

indicates the gene density with the frequency of sites per 50 kb ranged from 1 to 28.

823

The fourth circle indicates the distribution of repetitive DNA elements with the

824

frequency of sites per 50 kb ranged from 0 to 0.1. E, Genome synteny map between P.

825

expansum and P. italicum.

826 827

Figure 2. Genome comparison between P. expansum (Pe), P. italicum (Pi), and P.

828

digitatum (Pd). A, A whole-genome cladogram showing the phylogenetic

829

relationships between the three Penicillium species was constructed using CVTree

830

with Talaromyces marneffei as an outgroup. Branch lengths corresponding to

831

substitutions per site were calculated using a maximum likelihood approach. B, A

832

Venn diagram showing the number of shared and specific gene families. C, Gene

833

Ontology (GO) categories of gene families according to biological process. Circles I ~

834

VII corresponds to areas I ~ VII in (B).

37

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Boqiang Li MPMI

835

Figure 3. Comparison of genes encoding CAZymes and secreted proteases in P.

836

expansum (Pe), P. italicum (Pi), and P. digitatum (Pd). A, CAZymes identified in

837

the genome of P. expansum, P. italicum, and P. digitatum. PL: Polysaccharide lyase;

838

GT: Glycosyltransferase; GH: Glycoside hydrolase; CE: Carbohydrate esterase; CBM:

839

Carbohydrate-Binding Module; AA: Auxiliary Activity. B, CAZymes involved in

840

cellulose degradation. C, CAZymes involved in hemicellulose degradation. D,

841

CAZymes involved in pectin degradation. E, CAZymes involved in degradation of

842

pectin side chains. F. Secreted proteases. G. RT-qPCR analysis of genes encoding

843

GH78 family enzymes in P. expansum when cultured with either apple or citrus pectin

844

as a carbon source.

845 846

Figure 4. Comparison of genes involved in secondary metabolism in P. expansum

847

(Pe), P. italicum (Pi), and P. digitatum (Pd). A, Secondary metabolite backbone

848

genes. B, Phylogenetic analysis of genes encoding polyketide synthase (PKS),

849

non-ribosomal peptide synthetase (NRPS), polyketide synthase/non-ribosomal peptide

850

synthetase hybrid (PKS-NRPS hybrid), dimethylallyl tryptophan synthase (DMATS)

851

in three Penicillium species.

852

backbone genes of Pd. C, Genes encoding CYP450s. D, Genes encoding transporters.

, backbone genes of Pe;

, backbone genes of Pi;

,

853 854

Figure 5. Comparison of secondary metabolite gene clusters and structure of the

855

patulin gene clusters. A, Venn diagram showing the number of shared secondary

856

metabolite gene clusters in P. expansum (Pe), P. italicum (Pi), and P. digitatum (Pd).

857

B, Clusters shared by three Penicillium species. Red solid bars: clusters shared by Pe,

858

Pi, and Pd; green solid bars: clusters shared by Pe and Pi; yellow solid bars: clusters

859

shared by Pe and Pd; gray solid bars: specific clusters in each Penicillium species; 38

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Boqiang Li MPMI

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blue solid bar: the patulin cluster in Pe; blue hollow bars: the incomplete patulin

861

clusters in Pi and Pd. C, Structure comparison of patulin gene clusters in three

862

Penicillium species and A. clavatus (Ac). Black arrows represent genes not involved

863

in patulin biosynthesis. Patulin clusters in Pe and Ac were artificially divided into four

864

regions (I ~ IV) separated by orange vertical lines.

865 866

Figure 6. Analysis of gene expression in P. expansum cultured under patulin

867

permissive (static) and restrictive (shaking) conditions. A, Scatterplot showing

868

significantly up- and down-regulated genes as revealed by transcriptome analysis. All

869

of the genes in the patulin cluster (dots with red circles) were up-regulated. B,

870

Differential expressions of genes involved in secondary metabolism. Genes included

871

in patulin cluster are marked with red circles. C, RT-qPCR analysis of the expression

872

of patulin cluster genes at 2 and 4 d of static or shaking culture conditions. D, Patulin

873

production at 2 and 4 d of static or shaking culture conditions.

874 875

Fig. 7. Generation of PePatK and PePatL mutants and phenotype analysis. A,

876

Replacement strategy for deletion of PePatK and PePatL. The gene replacement

877

vector was constructed by cloning Flank L and Flank R on each side of the

878

hygromycin resistance cassette of pCAMBIA 1300. Primer pairs KL1/H1 and

879

LL1/H1 were used for flank-spanning PCR to verify the positive transformants. Probe,

880

digoxigenin-labeled Flank L for Southern blot analysis. Restriction sites: A, AscI; E,

881

EcoRV; H, HindIII; K, KpnI; S, SalI, X, XbaI. B, Southern blot analysis of the

882

wild-type strain (WT) and three knockout mutants of each target gene. Genomic DNA

883

was digested with EcoRV and hybridized with a probe (digoxigenin-labeled Flank L).

884

C, Colony morphology of WT and knockout mutants. D, Patulin production in WT 39

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Boqiang Li MPMI

885

and knockout mutants after being cultured for10 d on PDA. ‘ND’ means no patulin

886

production was detected. E and F, Colony diameter and sporulation of WT and

887

knockout mutants incubated on PDA plates at 25 °C for 10 d. G, Lesion diameters

888

after 3, 4, or 5 d in apple, pear, and plum fruit inoculated with either the wild-type

889

strain, ∆PePatK-1, or ∆PePatL-3 knockout mutants and kept at 25 °C. Error bars

890

represent standard deviation of three independent biological replicates. Columns with

891

different letters are significantly different from each other using the least significant

892

difference test (p < 0.05)

893 894

Figure 8. Characterization of PePatL, a putative transcription factor in the

895

patulin gene cluster of P. expansum. A, PePatL possesses a conserved zinc cluster

896

motif present in other demonstrated or putative transcription factors. B, RT-qPCR

897

analysis of the expression of patulin cluster genes in wild-type (WT) and the PePatL

898

knockout mutant of P. expansum cultured under static condition for 4 d. C,

899

Subcellular localization of PePatL. A transformant carrying the empty vector

900

expressing only eGFP was used as a control. PePatL tagged with eGFP is localized in

901

nuclei as determined with the aid of a specific nuclear stain DAPI. Bar = 5 µm.

902

40


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160x109mm (300 x 300 DPI)


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140x122mm (300 x 300 DPI)


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170x180mm (300 x 300 DPI)


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170x101mm (300 x 300 DPI)


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170x77mm (300 x 300 DPI)


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170x153mm (300 x 300 DPI)


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170x196mm (300 x 300 DPI)


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170x78mm (300 x 300 DPI)

Page 49 of 138

Supplementary information Table S1. Top 10 gene families with the most members in P. expansum, P. italicum, and P. digitatum identified using MCL.


Species

P. expansum

P. italicum

P. digitatum

Members 50 37 30 30 28 27 26 26 24 20 33 20 19 18 18 18 16 15 14 14 26 18 17 16 15 14 13 12 11 11

Putative function MFS multidrug transporter MFS drug transporter MFS monosaccharide transporter Amino acid permease Betaine aldehyde dehydrogenase MFS transporter, putative MFS allantoate transporter hypothetical protein hypothetical protein hypothetical protein MFS multidrug transporter Amino acid permease MFS drug transporter MFS monosaccharide transporter MFS transporter hypothetical protein Betaine aldehyde dehydrogenase Ubiquitin carrier protein hypothetical protein hypothetical protein MFS multidrug transporter, putative MFS monosaccharide transporter, putative Betaine aldehyde dehydrogenase, putative Amino acid permease, putative hypothetical protein MAP kinase SakA hypothetical protein Ubiquitin carrier protein MFS transporter, putative hypothetical protein

1

Page 50 of 138

Table S2. CAZymes in P. expansum, P. italicum, and P.digitatum. CAZymes were identified using CAT with the mode of Pfam based annotation. Auxiliary Activities Carbohydrate-Binding Modules Carbohydrate esterases Glycoside hydrolases


Family

Subfamily AA1 AA2 AA3 AA4 AA5 AA6 AA7 AA8 AA9 CBM1 CBM4 CBM14 CBM18 CBM20 CBM21 CBM22 CBM24 CBM32 CBM42 CBM43 CBM46 CBM48 CBM50 CBM63 CBM67 CE1 CE2 CE3 CE4 CE5 CE7 CE8 CE9 CE10 CE12 CE14 CE16 GH1 GH2 GH3 GH5 GH6 GH7 GH10 GH11 GH12 GH13 GH15 GH16 GH17 GH18 GH20 GH25 GH26 GH28 GH30 GH31 GH32 GH33 GH35 GH36 GH37 GH38 GH43

P. expansum

P. italicum

P. digitatum

4 4 28 6 2 2 41 1 5 6 1 1 11 3 1 0 4 2 1 3 1 0 10 1 6 16 2 2 9 4 1 3 2 70 4 1 3 6 10 27 13 1 2 2 1 4 14 3 16 5 16 3 1 1 9 1 6 4 1 4 1 1 1 10

2 3 19 2 1 2 22 1 4 5 0 0 9 1 1 1 4 1 1 3 1 0 7 1 1 13 2 1 6 2 3 3 1 40 3 1 2 3 6 13 8 1 2 1 1 3 12 2 14 4 11 2 0 0 7 1 6 4 0 2 1 1 1 7

2 3 13 4 0 2 15 1 2 4 0 0 4 2 1 0 1 0 1 3 1 1 4 1 2 9 2 1 6 4 1 3 1 27 3 1 1 3 4 14 8 1 2 1 1 2 11 2 15 4 8 2 0 0 8 1 6 3 0 4 1 1 1 3

2

Glycosyltransferases Polysaccharide lyases


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GH47 GH51 GH53 GH54 GH55 GH63 GH64 GH65 GH67 GH71 GH72 GH74 GH75 GH76 GH78 GH79 GH81 GH84 GH92 GH93 GH95 GH105 GH106 GH109 GH114 GH125 GH127 GH128 GH131 GH132 GT1 GT2 GT3 GT4 GT5 GT8 GT15 GT20 GT21 GT22 GT24 GT25 GT28 GT31 GT32 GT33 GT34 GT35 GT39 GT41 GT48 GT50 GT54 GT57 GT58 GT59 GT61 GT62 GT66 GT69 GT71 GT76 GT90 PL1

7 3 1 1 6 2 1 1 1 8 7 1 2 8 8 4 1 1 4 2 1 1 1 14 2 1 2 2 1 2 10 13 1 7 4 6 3 5 2 4 1 2 1 3 12 1 3 1 3 0 1 1 3 2 1 1 1 3 1 2 3 1 8 4

7 2 1 1 5 2 1 1 0 7 6 1 2 8 3 0 1 1 3 2 1 2 0 10 2 1 0 1 1 2 5 13 1 6 4 7 3 5 2 4 1 3 1 2 9 1 3 1 3 0 1 1 1 2 1 1 0 3 1 2 2 1 8 3

6 1 1 1 3 2 1 1 1 6 6 1 1 7 4 1 1 0 4 2 1 1 2 8 2 1 0 1 1 2 3 13 1 7 4 3 3 5 2 4 1 0 1 2 9 1 3 1 3 1 1 1 0 2 1 1 0 3 1 2 2 1 5 3

PL3

1

1

1

PL4

2

1

2

PL20

2

1

0

PL22

1

0

0

3

Page 52 of 138


Table S3. Classification of genes encoding putative secreted proteases in P. expansum, P. italicum, and P. digitatum. Family A represents aspartic peptidases; C represents cysteine peptidases; G represents glutamic peptidases; M represents metallo peptidases; S represents serine peptidases; T represents threonine peptidases. Specific families in P. expansum are highlighted. Gene ID P. expansum PEG00582 PEG02365 PEG04822 PEG07531 PEG07662 PEG10343 PEG10734 PEG09551 PEG00521 PEG06282 PEG09661 PEG03458 PEG06753 PEG00630 PEG07269 PEG10100 PEG06981 PEG03375 PEG02144 PEG06981 PEG06795 PEG06468 PEG09658 PEG03483 PEG09315 PEG01912 PEG02144 PEG04206 PEG00506 PEG00693 PEG00954 PEG02602 PEG02779 PEG03684 PEG03964 PEG04253

Family A1A A1A A1A A1A A1A A1A A1A C13 G1 G1 G1 I9 M6 M12B M14A M20A M20B M20D M28A M28E M28X M35 M35 M43B M77 S8A S8A S8B S9 S9 S9 S9 S9 S9 S9 S9 4

e-value 3.00E-138 2.80E-113 8.30E-96 5.20E-140 1.10E-82 6.10E-73 5.00E-28 2.80E-113 1.90E-78 8.30E-60 7.10E-76 1.90E-08 8.00E-37 1.70E-26 5.20E-51 1.80E-74 1.10E-05 2.20E-57 4.40E-65 5.30E-72 3.00E-49 5.50E-23 1.00E-66 1.70E-32 2.70E-54 8.50E-117 1.00E-05 4.10E-93 1.20E-26 7.50E-32 2.20E-18 1.70E-18 3.30E-31 6.70E-24 3.40E-29 5.10E-47


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PEG04422 PEG05141 PEG05150 PEG05152 PEG06034 PEG06141 PEG06218 PEG06554 PEG06575 PEG06678 PEG06761_06762 PEG07013 PEG07232 PEG07457 PEG08032 PEG08543 PEG09869 PEG09871 PEG11230 PEG11230 PEG11307 PEG00159 PEG03514 PEG04396 PEG04847 PEG04847 PEG05104 PEG05596 PEG05850 PEG08745 PEG09214 PEG10796 PEG01709 PEG01709 PEG02450 PEG03726 PEG04785 PEG04785 PEG01575 PEG05877 PEG09216 PEG02704 PEG06554 PEG11666

S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S10 S10 S10 S10 S10 S10 S10 S10 S10 S10 S10 S12 S12 S12 S12 S12 S12 S15 S28 S28 S33 S33 S33 5

1.20E-23 1.60E-36 2.50E-07 4.40E-16 1.70E-38 5.80E-35 8.80E-48 2.70E-05 3.70E-29 2.20E-24 8.70E-40 1.60E-40 7.90E-32 1.00E-36 3.00E-29 3.10E-43 6.80E-15 5.50E-19 1.10E-30 3.90E-06 3.10E-39 4.60E-88 1.80E-233 3.20E-167 4.90E-45 2.40E-09 4.70E-181 1.80E-93 1.00E-85 5.10E-227 6.70E-68 4.80E-183 4.60E-16 2.30E-11 3.80E-22 8.30E-26 1.50E-10 1.20E-08 1.00E-23 6.70E-210 8.80E-49 3.70E-84 4.70E-07 4.90E-14


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PEG04922 PEG08145 PEG10232 PEG05856 P. italicum Pit_00519 Pit_00642 Pit_00916 Pit_03845 Pit_05636 Pit_04842 Pit_01766 Pit_02086 Pit_06527 Pit_01455 Pit_07649 Pit_06626 Pit_05431 Pit_02721 Pit_02721 Pit_02721 Pit_04191 Pit_05525 Pit_05431 Pit_07261 Pit_06247 Pit_07248 Pit_00968 Pit_01578 Pit_02896 Pit_04334 Pit_05053 Pit_05062 Pit_05063 Pit_05169 Pit_05354 Pit_05457 Pit_06470 Pit_07154 Pit_07630 Pit_07838 Pit_08041 Pit_09321 Pit_00434

S53 S53 S53 T3

2.30E-140 1.30E-68 1.10E-76 4.70E-92

A1A A1A A1A A1A A1A C13 G1 G1 G1 I9 M14A M20A M20B M23B M23B M23B M28A M28A M28E M43B S8A S8B S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S9 S10

2.00E-141 7.50E-82 5.90E-139 2.10E-94 1.80E-108 2.80E-113 3.20E-76 5.00E-83 9.30E-57 6.40E-09 2.40E-55 6.30E-77 1.50E-05 3.30E-06 4.00E-05 1.80E-05 7.70E-50 5.60E-63 1.40E-72 1.70E-32 5.70E-118 5.10E-102 2.60E-33 8.60E-45 5.60E-38 4.30E-28 1.50E-36 1.30E-28 1.30E-16 3.70E-31 5.90E-40 6.90E-40 5.40E-37 3.10E-12 2.20E-132 1.60E-30 8.50E-20 4.60E-31 1.60E-232

6


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Pit_02377 Pit_03376 Pit_03820 Pit_03820 Pit_04359 Pit_06159 Pit_06159 Pit_06436 Pit_08519 Pit_08762 Pit_00279 Pit_03877 Pit_03877 Pit_03072 Pit_04482 Pit_01300 Pit_05898 Pit_08866 Pit_09405 Pit_01915 P. digitatum PDIG_81260 PDIG_10550 PDIG_01760 PDIG_01700 PDIG_01700 PDIG_34290 PDIG_88470 PDIG_42030 PDIG_17180 PDIG_36270 PDIG_04870 PDIG_23080 PDIG_16630 PDIG_23080 PDIG_43170 PDIG_09970 PDIG_54480 PDIG_42710 PDIG_74090 PDIG_34320 PDIG_37590 PDIG_75860 PDIG_63950

S10 S10 S10 S10 S10 S10 S10 S10 S10 S10 S12 S12 S12 S28 S41A S53 S53 S53 S53 T3 A1A A1A A1A A1A A1A A1A C13 G1 G1 G1 M20A M20B M28A M28E M43B S1A S8A S8A S8B S9 S9 S9 S9 7

2.80E-94 8.70E-66 9.40E-45 2.40E-09 4.20E-167 6.60E-83 2.40E-34 1.20E-88 2.30E-231 6.30E-185 1.10E-25 9.70E-12 6.40E-07 3.80E-208 5.90E-05 2.50E-61 2.10E-126 7.90E-143 5.90E-69 1.60E-92 1.50E-139 8.90E-123 5.60E-97 1.00E-05 9.60E-05 2.20E-06 2.80E-113 1.10E-80 1.10E-57 2.60E-69 4.00E-72 7.90E-06 8.80E-51 1.60E-71 1.50E-31 2.20E-26 5.70E-118 5.30E-05 4.00E-89 4.10E-21 2.60E-122 3.80E-24 3.10E-43


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PDIG_38140 PDIG_86120 PDIG_47620 PDIG_86320 PDIG_49610 PDIG_22810 PDIG_29090 PDIG_00340 PDIG_75600 PDIG_62200 PDIG_86640 PDIG_27380 PDIG_67780 PDIG_85970 PDIG_00900

S9 S9 S9 S9 S9 S9 S9 S10 S10 S10 S12 S41A S53 S53 S53

8

6.70E-33 7.90E-06 1.80E-37 5.60E-11 3.10E-39 3.90E-38 2.80E-17 1.90E-184 2.30E-161 1.10E-235 3.60E-28 2.40E-05 6.60E-66 1.30E-123 7.90E-143


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Table S4. List of secondary metabolite backbone genes in P. expansum, P. italicum, and P. digitatum. Backbone genes were identified by the Secondary Metabolite Unknown Region Finder (SMURF). Gene ID P. expansum

Annotated_gene_function

Type

PEG01697 PEG01735 PEG03227 PEG09315 PEG05394 PEG06323 PEG08928 PEG09953 PEG11583 PEG00515 PEG01115 PEG01281 PEG01738 PEG01769 PEG01775 PEG02717 PEG02934 PEG03560 PEG04032 PEG05034

Tryptophan dimethylallyltransferase Dimethylallyl tryptophan synthase, putative Uncharacterized protein Uncharacterized protein Equisetin synthetase, putative Hybrid NRPS/PKS enzyme, putative Hybrid NRPS/PKS enzyme Hybrid NRPS/PKS enzyme, putative Nonribosomal peptide synthetase 14 Nonribosomal peptide synthase Pes1 BcNRPS1, nonribosomal peptide synthetase Nonribosomal peptide synthase, putative Nonribosomal peptide synthase, putative Hybrid NRPS/PKS enzyme, putative HC-toxin synthetase Non-ribosomal peptide synthetase modules and related proteins Uncharacterized protein Nonribosomal peptide synthase Nonribosomal peptide synthase SidE HC-toxin synthetase

DMAT DMAT DMAT DMAT HYBRID HYBRID HYBRID HYBRID HYBRID NRPS NRPS NRPS NRPS NRPS NRPS NRPS NRPS NRPS NRPS NRPS

9


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PEG06412 PEG07110 PEG08931 PEG09317 PEG09713 PEG10459 PEG10745 PEG00300 PEG00384 PEG00818 PEG02534 PEG03094 PEG03565 PEG06465 PEG06502 PEG06847 PEG08009 PEG08344 PEG09077 PEG09299 PEG09300 PEG10085 PEG00306 PEG00688 PEG00806

HC-toxin synthetase Hydroxamate-type ferrichrome siderophore peptide synthetase Aureobasidin A1 biosynthesis complex Non ribosomal peptide synthase Non ribosomal peptide synthase HC-toxin synthetase Predicted protein Predicted protein Non-ribosomal peptide synthetase Predicted protein Nonribosomal peptide synthase, putative Acyl-CoA synthetase Ochratoxin A non-ribosomal peptide synthetase Predicted protein Non-ribosomal peptide synthetase modules and related proteins HC-toxin synthetase Uncharacterized protein Uncharacterized protein NRPS-like enzyme, putative Non-ribosomal peptide synthetase Non-ribosomal peptide synthetase Uncharacterized protein Podospora anserina S mat+ genomic DNA chromosome 2, supercontig 2 Uncharacterized protein Citrinin polyketide synthase

10

NRPS NRPS NRPS NRPS NRPS NRPS NRPS NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like PKS PKS PKS


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Polyketide synthase Polyketide synthase Polyketide synthase, putative (JCVI) Polyketide synthase Catalytic activity: polyketide synthases are multifunctional enzymes Podospora anserina S mat+ genomic DNA chromosome 5, supercontig 8 Conidial pigment polyketide synthase PksP/Alb1 Phenolpthiocerol synthesis polyketide synthase ppsA Polyketide synthase, putative Polyketide synthase PksB Polyketide synthase, putative Polyketide synthase, putative 6-methylsalicylic acid synthase Reducing type I polyketide synthase Polyketide synthase Uncharacterized protein Polyketide synthase, putative Polyketide synthase module KR domain-containing protein Polyketide synthase, putative Polyketide synthase, putative Uncharacterized protein Polyketide synthase, putative Nonribosomal peptide synthetase 7 3-oxoacyl-(Acyl-carrier-protein) synthase

11

PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS-Like PKS-Like


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PEG03599 P. italicum Pit_04265 Pit_06840 Pit_07581 Pit_04044 Pit_02093 Pit_02628 Pit_03045 Pit_05101 Pit_05806 Pit_06839 Pit_07134 Pit_08837 Pit_01462 Pit_02423 Pit_04977 Pit_05015 Pit_05150 Pit_06643 Pit_08791 Pit_09469 Pit_00025 Pit_00973 Pit_01644

3-oxoacyl-[acyl-carrier-protein] synthase

PKS-Like

dimethylallyl tryptophan synthase GliD1 tryptophan dimethylallyltransferase prenyltransferase Pc14g00080 Pc16g04690 Pc22g20400 Pc13g05250 Pc21g10790 putative nonribosomal peptide synthase -like protein nonribosomal peptide synthase GliP2 Pc13g14330 nonribosomal peptide synthase, putative acylglycerophosphoethanolamine acyltransferase, putative putative lovastatin nonaketide synthase protein Pc18g00380 Pc12g09980 Pc12g13170 Pc20g02260 putative nonribosomal siderophore peptide synthase protein NRPS-like enzyme BcPKS5, polyketide synthase Pc13g08690 Pc21g04840

DMAT DMAT DMAT HYBRID NRPS NRPS NRPS NRPS NRPS NRPS NRPS NRPS NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like NRPS-Like PKS PKS PKS

12


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Pit_02885 Pit_03180 Pit_04021 Pit_04272 Pit_04315 Pit_04847 Pit_05376 Pit_05993 Pit_06327 Pit_06758 Pit_06845 Pit_06848 Pit_08360 Pit_00372 Pit_07808 P. digitatum PDIG_21200 PDIG_01630 PDIG_55700 PDIG_78750 PDIG_09880 PDIG_09960 PDIG_16150 PDIG_29980 PDIG_38770

Nonribosomal peptide synthetase 7 polyketide synthase, putative Polyketide synthase, putative reducing type I polyketide synthase 10 polyketide synthase polyketide synthase, putative polyketide synthase, putative reducing type I polyketide synthase Pc21g16000 Pc12g11530 polyketide synthase, putative polyketide synthase, putative Polyketide synthase, putative Pc22g09030 polyketide synthase

PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS-Like PKS-Like

dimethylallyl transferase hybrid PKS-NRPS protein polyketide synthase/peptide synthetase BcPKS5, polyketide synthase TqaB TqaA nonribosomal peptide synthase, putative nonribosomal siderophore peptide synthase Sid2 putative PKS-NRPS protein

DMAT HYBRID HYBRID HYBRID NRPS NRPS NRPS NRPS NRPS

13


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PDIG_39590 PDIG_41980 PDIG_53550 PDIG_55560 PDIG_89680 PDIG_04970 PDIG_53330 PDIG_66040 PDIG_71820 PDIG_15090 PDIG_21220 PDIG_38480 PDIG_46630 PDIG_49740 PDIG_49980 PDIG_53730 PDIG_76300 PDIG_76310 PDIG_83120 PDIG_89200 PDIG_11250 PDIG_36880 PDIG_82960

K01932 nonribosomal peptide synthase Pes1 nonribosomal peptide synthase, putative nonribosomal siderophore peptide synthase, putative nonribosomal peptide synthase, putative no annotation nonribosomal peptide synthase, putative hybrid NRPS/PKS enzyme, putative nonribosomal peptide synthetase TdiA fatty acid synthase S-acetyltransferase K00665 fatty acid synthase, animal type polyketide synthase, putative polyketide synthase, putative putative polyketide synthase polyketide synthase polyketide synthetase PksP polyketide synthase, putative K00680 reducing type I polyketide synthase protein kinase subdomain-containing protein polyketide synthase, putative polyketide synthase beta-ketoacyl synthase (Cem1), putative

14

NRPS NRPS NRPS NRPS NRPS NRPS-Like NRPS-Like NRPS-Like NRPS-Like PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS PKS-Like PKS-Like PKS-Like


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Table S5. List of secondary metabolite gene clusters in P. expansum, P. italicum, and P. digitatum. Gene clusters were identified by the Secondary Metabolite Unknown Region Finder (SMURF). Backbone gene ID Gene ID Annotated_gene_function P. expansum Cluster 1 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072 PEG00072

PEG00084 PEG00083 PEG00082 PEG00081 PEG00080 PEG00079 PEG00078 PEG00077 PEG00076 PEG00075 PEG00074 PEG00073 PEG00072 PEG00071 PEG00070 PEG00069 PEG00068 PEG00067 PEG00066

Short-chain dehydrogenase/reductase 2, putative Cytochrome P450 CYP4/CYP19/CYP26 subfamilies Major facilitator superfamily Uncharacterized protein Uncharacterized protein Pc16g00550 protein (Precursor) Uncharacterized protein Uncharacterized protein Zinc-binding alcohol dehydrogenase, putative Pc16g00440 protein Nonribosomal peptide synthetase 7 Nonribosomal peptide synthetase 7 Nonribosomal peptide synthetase 7 Nonribosomal peptide synthetase 7 Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein

15


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Cluster:2 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306 PEG00306

PEG00314 PEG00313 PEG00312 PEG00311 PEG00310 PEG00309 PEG00308 PEG00307 PEG00306 PEG00305 PEG00304 PEG00303 PEG00302 PEG00301 PEG00300 PEG00299 PEG00298

Cytochrome P450 monooxygenase AAEL014742-PA (Fragment) Cytochrome P450 Putative uncharacterized protein Uncharacterized protein Extracellular guanyl-specific ribonuclease, putative (AFU_orthologue; AFUA_4G01200) Pc12g06530 protein Integral membrane protein Podospora anserina S mat+ genomic DNA chromosome 2, supercontig 2 Podospora anserina S mat+ genomic DNA chromosome 2, supercontig 2 Monocarboxylate permease, putative Uncharacterized protein FAD binding domain protein CFEM domain-containing protein Predicted protein Uncharacterized protein Aspartate aminotransferase (Fragment)

Cluster:3 PEG00384 PEG00384 PEG00384 PEG00384 PEG00384

PEG00393 PEG00392 PEG00391 PEG00390 PEG00389

Function: TRI101 of F. sporotrichioides confers resistance to the trichothecene 4 Predicted protein Uncharacterized protein Uncharacterized protein Uncharacterized protein

16


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Uncharacterized protein Uncharacterized protein UPF0619 GPI-anchored membrane protein Putative uncharacterized protein Pc16g06180 Non-ribosomal peptide synthetase

Cluster:4 PEG00515 PEG00515

PEG00515 PEG00514

Nonribosomal peptide synthase Pes1 MFS multidrug transporter, putative

Cluster:5 PEG00688 PEG00688 PEG00688 PEG00688 PEG00688 PEG00688 PEG00688 PEG00688 PEG00688 PEG00688 PEG00688 PEG00688 PEG00688

PEG00694 PEG00693 PEG00692 PEG00691 PEG00690 PEG00689 PEG00688 PEG00687 PEG00686 PEG00685 PEG00684 PEG00683 PEG00682

MFS lactose permease, putative Carboxylesterase, putative C6 finger domain protein, putative WGS project CABT00000000 data, contig 2.47 Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Endosomal cargo receptor (P24), putative Myosin heavy chain-like protein, putative Similar to sterol 24-c-methyltransferase 1,3-beta-glucanosyltransferase Bgt1 Uncharacterized protein

17


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Cluster:6 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821

PEG00836 PEG00835 PEG00834 PEG00833 PEG00832 PEG00831 PEG00830 PEG00829 PEG00828 PEG00827 PEG00826 PEG00825 PEG00824 PEG00823 PEG00822 PEG00821 PEG00820 PEG00819 PEG00818 PEG00817 PEG00816 PEG00815 PEG00814 PEG00813

Uncharacterized protein Homoaconitase C6 transcription factor, putative Uncharacterized protein Beta-mannosidase, putative Predicted protein Predicted transporter Uncharacterized protein Uncharacterized protein Amino acid transporter Uncharacterized protein Amino acid permease Arylamine N-acetyltransferase 1 Uncharacterized protein Hypothetical oxidoreductase Polyketide synthase Integral membrane protein TmpA Uncharacterized protein Predicted protein Catalytic activity: 2 RH + 2 chloride + H(2)O(2) = 2 RCl + 2 H(2)O Aerobactin siderophore biosynthesis protein iucb Multidrug/pheromone exporter Pyridine nucleotide-disulphide oxidoreductase AMID-like, putative Predicted protein

18


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PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821 PEG00821


Uncharacterized protein Siderophore iron transporter Trichodiene synthase, putative Uncharacterized protein Uncharacterized protein Citrinin biosynthesis transporter CtnC Citrinin polyketide synthase Citrinin biosynthesis oxydoreductase CtnB Citrinin biosynthesis oxygenase CtnA Citrinin biosynthesis transcriptional activator CtnR



FAD-dependent monooxygenase, putative (AFU_orthologue; AFUA_8G02380) 4-hydroxybenzoate polyprenyl transferase, putative (AFU_orthologue; AFUA_6G07240) Predicted protein Cinnamoyl-CoA reductase, putative Polyketide synthase, putative (JCVI) ABC multidrug transporter #N/A Cytochrome P450 Dyp-type peroxidase family protein Similarity to hypothetical protein B7F18.120 - Neurospora crassa Similarity to hypothetical protein B7F18.110 - Neurospora crassa Uncharacterized protein BcNRPS1, nonribosomal peptide synthetase

19


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O-methyltransferase, putative Similar to cytochrome P450 Methyltransferase SirN-like, putative Uncharacterized protein Glutathione S-transferase Similar to An14g02830 Predicted protein Similarity to hypothetical protein encoded by An14g02830 - Aspergillus niger Oxidoreductase Polyketide synthase

Cluster:8 PEG01152 PEG01152 PEG01152 PEG01152 PEG01152 PEG01152 PEG01152

PEG01152 PEG01151 PEG01150 PEG01149 PEG01148 PEG01147 PEG01146

Polyketide synthase Predicted protein Pathway: trichothecene synthesis 2-(R)-hydroxypropyl-CoM dehydrogenase MFS transporter Cysteine transporter Predicted transporter

Cluster:9 PEG01281 PEG01281 PEG01281 PEG01281

PEG01281 PEG01280 PEG01279 PEG01278

Nonribosomal peptide synthase, putative Major facilitator superfamily ABC multidrug transporter, putative C6 finger domain protein, putative

20


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Maltase MalT MFS maltose permease, putative Uncharacterized protein Killer toxin sensitivity protein (Iki1), putative Protein transport protein sec22 Uncharacterized protein Uncharacterized protein

Cluster:10 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697

PEG01708 PEG01707 PEG01706 PEG01705 PEG01704 PEG01703 PEG01702 PEG01701 PEG01700 PEG01699 PEG01698 PEG01697 PEG01696 PEG01695 PEG01694 PEG01693

Aryl-alcohol dehydrogenase Adenosine deaminase family protein Uracil permease, putative Adenosine deaminase Adenosine deaminase, putative #N/A Oxidoreductase L-2,4-diaminobutyrate decarboxylase Cytochrome P450 Catalase Methyltransferase Tryptophan dimethylallyltransferase Long-chain fatty-acid-CoA ligase Uncharacterized protein Catalytic activity: polyketide synthases are multifunctional enzymes Phytanoyl-CoA dioxygenase family protein

21


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PEG01697 PEG01697 PEG01697 PEG01697 PEG01697 PEG01697


O-acetyltransferase, putative Allantoate permease, putative Alpha-ketoglutarate-dependent taurine dioxygenase Fungal specific transcription factor domain protein Dihydrodipicolinate synthase, putative MFS nicotinic acid transporter

Cluster:11 PEG01738 PEG01738 PEG01738 PEG01738 PEG01738 PEG01738 PEG01738 PEG01738 PEG01738 PEG01738 PEG01738

PEG01738 PEG01737 PEG01736 PEG01735 PEG01734 PEG01733 PEG01732 PEG01731 PEG01730 PEG01729 PEG01728

Nonribosomal peptide synthase, putative Cytochrome P450 oxidoreductase, putative FAD binding domain-containing protein Dimethylallyl tryptophan synthase, putative WD-repeat protein, putative Uncharacterized protein Histone acetylase complex subunit, putative MRNA splicing protein Yju2 20S cyclosome subunit (Cut9/Cdc16), putative Endoplasmic reticulum-resident kdel protein P450 family sporulation-specific N-formyltyrosine oxidase Dit2, putative


PEG01786 PEG01785 PEG01784 PEG01783

MFS transporter, SHS family, lactate transporter Uncharacterized protein Uncharacterized protein Uncharacterized protein

22


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PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775 PEG01775

PEG01782 PEG01781 PEG01780 PEG01779 PEG01778 PEG01777 PEG01776 PEG01775 PEG01774 PEG01773 PEG01772 PEG01771 PEG01770 PEG01769 PEG01768 PEG01767 PEG01766 PEG01765

Hydantoin racemase C6 finger domain protein, putative Predicted protein Uncharacterized protein TPR domain protein PQ loop repeat protein Uncharacterized protein HC-toxin synthetase Uncharacterized protein Putative uncharacterized protein Predicted protein Uncharacterized protein Uncharacterized protein Hybrid NRPS/PKS enzyme, putative Podospora anserina S mat+ genomic DNA chromosome 5, supercontig 8 #N/A Catalytic activity: Acyl-[acyl-carrier protein] + NADP(+) trans-2 Tetracycline resistance protein TetB/drug resistance transporter



Conidial pigment polyketide synthase PksP/Alb1 Conidial pigment biosynthesis oxidase Arb2/brown2 #N/A Uncharacterized protein Glutamate carboxypeptidase, putative

23


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Predicted protein Uncharacterized protein Similar to An03g00300 Exopolyphosphatase, putative Oxidoreductase, short-chain dehydrogenase/reductase family Short-chain dehydrogenase



C6 transcription factor, putative Permease of the major facilitator superfamily Glycosyl hydrolases family 32 superfamily Amino acid transporter, putative Cytochrome c mitochondrial import factor (Cyc2), putative Phosphoserine aminotransferase HET-s/LopB domain protein Uncharacterized protein Uncharacterized protein Uncharacterized protein MFS multidrug transporter, putative MFS multidrug transporter, putative Uncharacterized protein Predicted protein Phenolpthiocerol synthesis polyketide synthase ppsA Uncharacterized protein Beta-glucosidase, putative

24


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PEG02331 PEG02331 PEG02331 PEG02331 PEG02331 PEG02331 PEG02331 PEG02331 PEG02331


UDP-glucosyl transferase family protein Uncharacterized protein Mitochondrial GTPase (Mss1), putative Cytokinesis protein SepA/Bni1 Uncharacterized protein Formate dehydrogenase Uncharacterized protein Leucyl-tRNA synthetase Uncharacterized protein

Cluster:15 PEG02534 PEG02534 PEG02534 PEG02534 PEG02534 PEG02534 PEG02534 PEG02534 PEG02534 PEG02534 PEG02534 PEG02534 PEG02534 PEG02534

PEG02544 PEG02543 PEG02542 PEG02541 PEG02540 PEG02539 PEG02538 PEG02537 PEG02536 PEG02535 PEG02534 PEG02533 PEG02532 PEG02531

Sugar transporter, putative Acetyltransferase Aspartic protease Golgi (Precursor) UDP-glucuronate 5-epimerase, putative Uncharacterized protein Predicted protein Homeobox transcription factor (RfeB), putative Uncharacterized protein Small nuclear ribonucleoprotein SmD3, putative Nonribosomal peptide synthase, putative Uncharacterized protein Similar to An07g05870 MFS transporter

25


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NADPH:quinone reductase and related Zn-dependent oxidoreductases S-adenosylmethionine synthetase 4-coumarate:CoA ligase, putative Uncharacterized protein MFS transporter, putative Uncharacterized protein Uncharacterized protein

Cluster:16 PEG02620 PEG02620 PEG02620 PEG02620 PEG02620 PEG02620 PEG02620 PEG02620 PEG02620 PEG02620 PEG02620 PEG02620 PEG02620 PEG02620 PEG02620

PEG02620 PEG02619 PEG02618 PEG02617 PEG02616 PEG02615 PEG02614 PEG02613 PEG02612 PEG02611 PEG02610 PEG02609 PEG02608 PEG02607 PEG02606

Polyketide synthase, putative Uncharacterized protein Uncharacterized protein NAD-dependent epimerase/dehydratase, putative Hydroxysteroid dehydrogenase, putative Scytalone dehydratase, putative Uncharacterized protein F-box domain containing protein Multidrug/pheromone exporter Similarity to hypothetical protein encoded by An18g00300 - Aspergillus niger Remark: catalyzes the cyclization of squalene into hopene Catalytic activity: RH + Reduced flavoprotein + O2 = ROH + Oxidized flavoprotein (Precursor) Pc16g12210 protein Short-chain dehydrogenases/reductase, putative Uncharacterized protein

26


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Non-ribosomal peptide synthetase modules and related proteins Uncharacterized protein Ankyrin repeat protein Branched-chain amino acid aminotransferase/4-amino-4-deoxychorismate lyase Cytochrome P450 CYP3/CYP5/CYP6/CYP9 subfamilies Fatty acid synthase alpha subunit, putative ABC multidrug transporter, putative



Inorganic phosphate transporter Monooxygenase, putative Predicted protein Uncharacterized protein Predicted protein Uncharacterized protein 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase Lipase/esterase, putative Uncharacterized protein Ste50p MFS sugar transporter, putative Sorbitol/xylitol dehydrogenase, putative C6 transcription factor, putative Uncharacterized protein Polyketide synthase PksB

27


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Uncharacterized protein MFS transporter, putative Fungal specific transcription factor domain protein Uncharacterized protein Uncharacterized protein Chitin deacetylase, putative Uncharacterized protein Uncharacterized protein OAH class protein Permease of the major facilitator superfamily C6 transcription factor Uncharacterized protein Alkaline ceramidase family protein Predicted protein Ceramide synthase membrane component (LAG1), putative Sugar transporter, putative



Acyl-CoA synthetase Uncharacterized protein Uncharacterized protein Protein copper regulated Folylpolyglutamate synthase Uncharacterized protein Predicted protein

28


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PEG03094 PEG03094

PEG03087 PEG03086

#N/A GCN5-related N-acetyltransferase


PEG03228 PEG03227

MFS multidrug transporter Uncharacterized protein



NACHT and WD40 domain protein Predicted protein Aryl-alcohol dehydrogenase, putative Integral membrane protein Ochratoxin A non-ribosomal peptide synthetase GMC oxidoreductase, putative Uncharacterized protein 3-oxoacyl-(Acyl-carrier-protein) synthase MFS multidrug transporter, putative Nonribosomal peptide synthase MFS monocarboxylate transporter 4-hydroxy-2-oxovalerate aldolase, putative Zn(II)2Cys6 transcription factor Function: S. cerevisiae PPR1 regulates expression of pyrimidine biosynthetis genes Allantoate permease

Cluster:22

29


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Predicted protein Uncharacterized protein Uncharacterized protein Siderochrome-iron transporter MirC Rho GTPase ModA, putative Uncharacterized protein 3-oxoacyl-[acyl-carrier-protein] synthase SAGA complex component mRNA-capping enzyme subunit alpha Histone acetyltransferase esa1 Uncharacterized protein Mitochondrial F1F0 ATP synthase subunit F (Atp17), putative Uncharacterized protein HEAT repeat protein DNA-directed RNA polymerase subunit Sister chromatid cohesion protein Mis4, putative NADH-ubiquinone oxidoreductase 64 kDa subunit, putative

Cluster:23 PEG03640 PEG03640 PEG03640 PEG03640 PEG03640 PEG03640


MFS multidrug transporter, putative Transcription activator of gluconeogenesis acuK #N/A Dienelactone hydrolase Uncharacterized protein Nitroreductase family protein

30


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Citrinin biosynthesis oxydoreductase CtnB Polyketide synthase, putative Salicylate hydroxylase, putative Predicted protein Zinc-binding oxidoreductase, putative Polyketide synthase, putative Fungal specific transcription factor, putative



Cytochrome P450 oxidoreductase, putative Amidohydrolase family protein Uncharacterized protein Glucose-methanol-choline (Gmc) oxidoreductase Alcohol dehydrogenase MFS transporter Carboxylesterase family protein Acetate transporter #N/A Isoepoxydon dehydrogenase (Fragment) #N/A C6 transporter Cytochrome P450 oxidoreductase Cupin RmlC-type 6-methylsalicylic acid synthase

31


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Reducing type I polyketide synthase Uncharacterized protein WGS project CABT00000000 data, contig 2.1 FAD binding domain protein Cytochrome p450 Acyl transferase Phosphatidyl synthase NADP(+)-dependent glycerol dehydrogenase Uncharacterized protein Uncharacterized protein Uncharacterized protein Glutathione transferase, putative Uncharacterized protein Alcohol dehydrogenase GroES domain protein

Cluster:26 PEG04032 PEG04032 PEG04032 PEG04032 PEG04032 PEG04032 PEG04032 PEG04032

PEG04041 PEG04040 PEG04039 PEG04038 PEG04037 PEG04036 PEG04035 PEG04034

MFS multidrug transporter, putative MFS alpha-glucoside transporter 3-phytase A Similar to An14g04210 Predicted protein Similarity to the aminopeptidase (Precursor) C6 transcription factor, putative C6 transcription factor, putative

32


Page 81 of 138

PEG04032 PEG04032 PEG04032 PEG04032 PEG04032 PEG04032 PEG04032 PEG04032 PEG04032 PEG04032 PEG04032 PEG04032


Uncharacterized protein Nonribosomal peptide synthase SidE Mitochondrial enoyl reductase, putative MFS monocarboxylate transporter, putative Low-density lipoprotein receptor YWTD repeat Antibiotic biosynthesis monooxygenase family protein 3-hydroxyacyl-CoA dehyrogenase Alpha methylacyl-CoA racemase Function: facB of A. nidulans is required for acetate induction of acetamidase Cytochrome P450, putative Cation diffusion facilitator family transporter Uncharacterized protein



HC-toxin synthetase ABC multidrug transporter SitT Acetyltransferase SidF Enoyl-CoA hydratase/isomerase family protein Uncharacterized protein


PEG05400 PEG05399 PEG05398 PEG05397

Fungal specific transcription factor domain protein Uncharacterized protein MFS transporter, putative O-methyltransferase, putative

33


Page 82 of 138



Zinc-binding dehydrogenase family oxidoreductase, putative Uncharacterized protein Equisetin synthetase, putative Predicted protein Signal peptidase I Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein MFS transporter, putative Uncharacterized protein Major facilitator superfamily transporter



Quinone oxidoreductase, putative Uncharacterized protein Arginase family protein Similarity to hypothetical protein SPAC11D3.06 - Schizosaccharomyces pombe MATE efflux family protein subfamily, putative Predicted protein Prenyl cysteine carboxyl methyltransferase, putative Polyketide synthase

Cluster:30

34


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Oxidoreductase, short-chain dehydrogenase/reductase family Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Histidine acid phosphatase Acetyltransferase Predicted protein Similar to alternative sulfate transporter Predicted protein Putative uncharacterized protein Pc21g14080 Chitosanase, putative MFS monosaccharide transporter, putative Endo-1,4-beta-mannosidase Amino acid permease, putative Proline permease PrnB Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein

35


Page 84 of 138



MFS monocarboxylate transporter, putative Amino acid permease (Can1), putative Acyl-CoA dehydrogenase family protein Cytochrome P450 monooxygenase Uncharacterized protein Uncharacterized protein Uncharacterized protein Hybrid NRPS/PKS enzyme, putative MFS transporter, putative Putative uncharacterized protein Formate/nitrite transporter family protein, putative Taurine catabolism dioxygenase TauD Predicted protein Aldehyde dehydrogenase (AldH12), putative Methionine aminopeptidase MFS transporter



Uncharacterized protein Uncharacterized protein Monocarboxylate permease #N/A Uncharacterized protein ABC multidrug transporter, putative

36


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#N/A Thioredoxin reductase Uncharacterized protein Uncharacterized protein Uncharacterized protein #N/A Polyketide synthase, putative

MFS aflatoxin efflux pump, putative C6 zinc finger domain protein LD-carboxypeptidase ATP-dependent RNA helicase (Hrh1), putative Uncharacterized protein Uncharacterized protein Sterol glucosyltransferase Similar to An12g06240 Glycosyl hydrolase, putative Predicted protein Cellobiose dehydrogenase, putative HC-toxin synthetase 2-dehydropantoate 2-reductase Uncharacterized protein Uncharacterized protein Dehydrogenases with different specificities

37


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PEG06412 PEG06412

PEG06407 PEG06406

Uncharacterized protein Dehydrogenase with different specificitie



Predicted protein Predicted protein Uncharacterized protein Uncharacterized protein Ankyrin repeat protein



Uncharacterized protein MFS multidrug transporter Predicted protein Transcriptional regulator GARP complex subunit Vps53, putative GARP complex subunit Vps53, putative COPII-coated vesicle membrane protein Erv46, putative Thiol methyltransferase, putative Molybdenum cofactor sulfurase protein Uncharacterized protein Putative uncharacterized protein Pc22g07630 Podospora anserina S mat+ genomic DNA chromosome 2, supercontig 2 Predicted protein O-methyltransferase, putative

38


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PEG06502 PEG06502 PEG06502 PEG06502

PEG06503 PEG06502 PEG06501 PEG06500

LolO-1 Non-ribosomal peptide synthetase modules and related proteins LolE-1 LolC



Peroxisomal ABC transporter (PXA1), putative General stress response phosphoprotein phosphatase Psr1/2, putative Uncharacterized protein Uncharacterized protein MFS transporter, putative Putative uncharacterized protein Protein kinase domain-containing protein 16 kDa allergen Uncharacterized protein Predicted protein Predicted protein Similar to transcription factor Cmr1 Short-chain dehydrogenase, putative Polyketide synthase module

Cluster:37 PEG06654_06655 PEG06654_06655 PEG06654_06655

PEG06667 PEG06666 PEG06665

NADP(+)-dependent dehydrogenase Predicted protein #N/A

39


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PEG06654_06655 PEG06654_06655 PEG06654_06655 PEG06654_06655 PEG06654_06655 PEG06654_06655 PEG06654_06655 PEG06654_06655 PEG06654_06655 PEG06654_06655


Siderophore biosynthesis protein, putative Proline utilization protein PrnX-like, putative Pyrroline-5-carboxylate reductase Uncharacterized protein Uncharacterized protein Uncharacterized protein 37S ribosomal protein Mrp10, mitochondrial Cytochrome protein Carbonyl reductase



Oxidoreductase, 2OG-Fe(II) oxygenase family, putative Pantothenate transporter, putative #N/A Pc13g07590 protein Uncharacterized protein Restriction of telomere capping protein 5 Fatty acid synthase subunit alpha, putative Fatty acid synthase subunit alpha, putative HC-toxin synthetase Uncharacterized protein ABC transporter, putative ABC transporter, putative Uncharacterized protein

#N/A

40


Page 89 of 138



Uncharacterized protein Putative uncharacterized protein Glycylpeptide N-tetradecanoyltransferase Folylpolyglutamate synthase Aldehyde dehydrogenase, putative Ethanolamine utilization protein (EutQ) Amino acid transporter FAD binding domain protein Cytochrome P450 oxidoreductase



Similar to amino acid permease Haloacid dehalogenase, type II Nuclear migration protein, putative Cytochrome b5, putative MRNA splicing factor RNA helicase (Cdc28), putative Protein kinase, putative Hydroxamate-type ferrichrome siderophore peptide synthetase



MFS allantoate transporter, putative Uncharacterized protein Putative uncharacterized protein U3 small nucleolar ribonucleoprotein subunit (Imp3), putative Uncharacterized protein

41


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PEG07181

PEG07181

Polyketide synthase, putative

Cluster:41 PEG07447 PEG07447 PEG07447 PEG07447 PEG07447 PEG07447 PEG07447 PEG07447 PEG07447 PEG07447 PEG07447 PEG07447


Hexose transporter Triacylglycerol lipase Alpha/beta-hydrolase Peroxidase Similar to transcription factor Cys6 C6 transcription factor, putative Uncharacterized protein NADH-cytochrome b-5 reductase Arylsulfotransferase Uncharacterized protein Hexokinase-1 Polyketide synthase, putative

Cluster:42 PEG08009 PEG08009 PEG08009

PEG08011 PEG08010 PEG08009

Uncharacterized protein Uncharacterized protein Uncharacterized protein

Cluster:43 PEG08344 PEG08344 PEG08344

PEG08346 PEG08345 PEG08344

C6 transcription factor (Mut3), putative High-affinity hexose transporter, putative Uncharacterized protein

42


Page 91 of 138



Aureobasidin A1 biosynthesis complex Uncharacterized protein Alcohol dehydrogenase Hybrid NRPS/PKS enzyme Uncharacterized protein Similarity: the predicted ORF is rich in proline Cytochrome P450 monooxygenase CYP52G11

Cluster:45 PEG09077 PEG09077 PEG09077 PEG09077 PEG09077 PEG09077 PEG09077 PEG09077 PEG09077 PEG09077


L-fucose permease, putative Endoplasmic reticulum calcium ATPase, putative 14-3-3 family protein Pc14g01720 protein Uncharacterized protein Enolase Proteasome regulatory particle subunit Rpt4, putative Mannosyl-oligosaccharide alpha-1,2-mannosidase Peptidase, putative NRPS-like enzyme, putative


PEG09311 PEG09310

Uncharacterized protein Uncharacterized protein

43


Page 92 of 138



Uncharacterized protein #N/A Similar to ribonuclease p/mrp subunit Similar to tetratricopeptide repeat domain protein Uncharacterized protein Chromosome segregation protein BIR1, putative Uncharacterized protein Uncharacterized protein Bli-3 protein, putative Non-ribosomal peptide synthetase Non-ribosomal peptide synthetase Catalytic activity: FAD binding oxidoreductase O-methyltransferase Transcription factor spt3, putative Diphthine synthase, putative MFS transporter



Histidinol-phosphate aminotransferase Uncharacterized protein Uncharacterized protein Uncharacterized protein Non ribosomal peptide synthase Aminotransferase GliI

44


Page 93 of 138



Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein



Predicted protein C6 transcription factor, putative Chromatin structure remodeling complex protein RSC3 Aminotransferase Uncharacterized protein C6 transcription factor Predicted protein MFS transporter, DHA2 family, methylenomycin A resistance protein Predicted protein GliK Glutathione S-transferase O-methyltransferase Cytochrome P450 monooxigenase Non ribosomal peptide synthase Dipeptidase CRAL/TRIO domain protein MFS transporter, putative

45


Page 94 of 138



Uncharacterized protein Uncharacterized protein Uncharacterized protein NRPS-like enzyme, putative NRPS-like enzyme, putative Uncharacterized protein Multidrug resistance protein 4



Predicted protein Polyketide synthase Uncharacterized protein Aminohydrolase, putative Allantoin permease, putative Asp glu racemase Cytochrome P450 Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Cytochrome P450 oxidoreductase GliF Putative uncharacterized protein Uncharacterized protein Isoamyl alcohol oxidase Uncharacterized protein

46


Page 95 of 138

PEG09953 PEG09953

PEG09953 PEG09952

Hybrid NRPS/PKS enzyme, putative Flavin-containing amine oxidasedehydrogenase, putative



MFS transporter, putative MFS transporter, putative Sensor histidine kinase/response regulator, putative Protein fluG Aldehyde dehydrogenase Pc20g02280 protein Uncharacterized protein Uncharacterized protein

Cluster:51 PEG10459 PEG10459 PEG10459 PEG10459 PEG10459 PEG10459 PEG10459 PEG10459 PEG10459 PEG10459 PEG10459

PEG10465 PEG10464 PEG10463 PEG10462 PEG10461 PEG10460 PEG10459 PEG10458 PEG10457 PEG10456 PEG10455

Integral membrane protein Cytochrome P450 oxidoreductase, putative TRI7-like toxin biosynthesis protein, putative Uncharacterized protein Cytochrome P450 monooxygenase Uncharacterized protein HC-toxin synthetase Uncharacterized protein Predicted protein Cell wall protein, putative Serine/arginine repetitive matrix protein 1

47


Page 96 of 138



Checkpoint protein kinase, putative Ribosome biogenesis protein nsa2 Aconitate hydratase, putative Uncharacterized protein conserved in bacteria Uncharacterized protein C6 transcription factor (OTam), putative



Alcohol dehydrogenase, putative Uncharacterized protein Uncharacterized protein MFS transporter, putative RING finger protein Uncharacterized protein Uncharacterized protein Peroxisomal membrane protein (PmpP24), putative Ribosome biogenesis protein, putative Uncharacterized protein Uncharacterized protein ABC multidrug transporter, putative Predicted protein #N/A Uncharacterized protein Pc20g12730 protein Pectinesterase family protein

48


Page 97 of 138



Urea amidolyase, putative C6 transcription factor Lactam utilization protein LamB Uncharacterized protein C4-dicarboxylate transporter/malic acid transport protein, putative Aspartyl protease, putative AAT family amino acid transporter #N/A Putative uncharacterized protein #N/A Aflatoxin biosynthesis ketoreductase nor-1 ToxD-like zinc binding oxidoreductase, putative Zn(II)2Cys6 transcription factor

Cluster:53 PEG10948 PEG10948 PEG10948 PEG10948 PEG10948 PEG10948 PEG10948 PEG10948 PEG10948 PEG10948


Predicted protein Pc12g02650 protein Poly(ADP)-ribose polymerase PARP, putative Predicted protein #N/A Ribokinase Predicted protein Predicted transporter C6 transcription factor, putative Uncharacterized protein

49


Page 98 of 138

PEG10948 PEG10948 PEG10948 PEG10948

PEG10950 PEG10949 PEG10948 PEG10947

Putative uncharacterized protein Uncharacterized protein Uncharacterized protein MFS transporter, putative

Cluster:54 PEG11284 PEG11284 PEG11284 PEG11284 PEG11284 PEG11284 PEG11284 PEG11284 PEG11284


Serine/threonine-protein kinase bud32 Serine/threonine-protein kinase bud32 Gamma-cysteine synthetase regulatory subunit, putative Uncharacterized protein Polyketide synthase, putative Uncharacterized protein Beta-ketoacyl synthase domain-containing protein Monooxygenase FAD-binding protein Major facilitator superfamily


Major Facilitator Superfamily protein Alpha/beta hydrolase, putative Integral membrane protein, putative Uncharacterized protein Nonribosomal peptide synthetase 14

Pit_00030

putative o-methyltransferase protein

Cluster:55 PEG11583 PEG11583 PEG11583 PEG11583 PEG11583 P. italicum Cluster:1 Pit_00025

50


Page 99 of 138

Pit_00025 Pit_00025 Pit_00025 Pit_00025 Pit_00025


putative bifunctional p-450:nadph-p450 reductase protein enniatin synthetase Zinc-binding dehydrogenase family oxidoreductase, putative conserved hypothetical protein BcPKS5, polyketide synthase

Cluster:2 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372 Pit_00372

Pit_00384 Pit_00383 Pit_00382 Pit_00381 Pit_00380 Pit_00379 Pit_00378 Pit_00377 Pit_00376 Pit_00375 Pit_00374 Pit_00373 Pit_00372 Pit_00371 Pit_00370 Pit_00369 Pit_00368 Pit_00367

Uridine kinase CTLH domain-containing protein NADH-ubiquinone oxidoreductase 64 kDa subunit, putative Sister chromatid cohesion protein Mis4, putative DNA-directed RNA polymerase I 13.1 kDa polypeptide HEAT repeat protein NADH-ubiquinone oxidoreductase subunit mitochondrial F1F0 ATP synthase subunit F (Atp17), putative hypothetical protein PDIG_82930 Histone acetyltransferase esa1 mRNA-capping enzyme subunit alpha Putative 3-oxoacyl-[acyl-carrier-protein] synthase, mitochondrial Putative 3-oxoacyl-[acyl-carrier-protein] synthase, mitochondrial agmatinase, putative Rho GTPase ModA, putative Siderochrome-iron transporter MirC similar to An02g14170 conserved hypothetical protein

51


Page 100 of 138

Pit_00372

Pit_00366

cytochrome P450

Cluster:3 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973 Pit_00973

Pit_00986 Pit_00985 Pit_00984 Pit_00983 Pit_00982 Pit_00981 Pit_00980 Pit_00979 Pit_00978 Pit_00977 Pit_00976 Pit_00974 Pit_00975 Pit_00973 Pit_00972 Pit_00971 Pit_00970 Pit_00969 Pit_00968 Pit_00967

DNA mismatch repair protein Msh5, putative Heat shock protein/chaperonin HSP78, putative Elongation factor 1-beta hypothetical protein PDIG_14970 ketoreductase, putative period circadian protein, putative Ubiquitin carrier protein Cell wall biogenesis protein phosphatase Ssd1, putative RNA export mediator Gle1, putative 1,3-beta-glucanosyltransferase Bgt1 Myosin heavy chain-like protein, putative Endosomal cargo receptor (P24), putative similar to EF-hand calcium-binding domain protein 6-methylsalicylic acid synthase RNase H domain containing protein alcohol oxidase C6 finger domain protein, putative protein kinase carboxylesterase, putative MFS lactose permease, putative

Cluster:4

52


Page 101 of 138

Pit_01462 Pit_01462 Pit_01462

Pit_01464 Pit_01463 Pit_01462

estradiol 17 beta-dehydrogenase, putative short chain type dehydrogenase, putative acylglycerophosphoethanolamine acyltransferase, putative

Cluster:5 Pit_02093 Pit_02093 Pit_02093 Pit_02093 Pit_02093 Pit_02093 Pit_02093 Pit_02093 Pit_02093 Pit_02093 Pit_02093

Pit_02101 Pit_02100 Pit_02099 Pit_02098 Pit_02097 Pit_02096 Pit_02095 Pit_02094 Pit_02093 Pit_02092 Pit_02091

RNase L inhibitor of the ABC superfamily, putative Formin binding protein (FNB3), putative conserved hypothetical protein conserved hypothetical protein Phosphoglycerate kinase 26 proteasome complex subunit Sem1, putative mitochondrial outer membrane protein Iml2 MFS multidrug transporter, putative Nonribosomal peptide synthase Pes1 ABC multidrug transporter, putative MFS transporter

Cluster:6 Pit_02423 Pit_02423 Pit_02423 Pit_02423 Pit_02423 Pit_02423 Pit_02423

Pit_02426 Pit_02425 Pit_02424 Pit_02423 Pit_02422 Pit_02421 Pit_02420

MFS multidrug transporter, putative enoyl reductase putative lovastatin nonaketide synthase protein putative lovastatin nonaketide synthase protein Zn2/Cys6 DNA-binding protein Pc21g06130 translation initiation factor eIF-2B alpha subunit

53


Page 102 of 138



putative kinetochore protein nuf2 conserved hypothetical protein AP-3 adaptor complex subunit beta, putative Amidohydrolase Serine/threonine-protein phosphatase

Cluster:7 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628 Pit_02628

Pit_02640 Pit_02639 Pit_02638 Pit_02637 Pit_02636 Pit_02635 Pit_02634 Pit_02632 Pit_02633 Pit_02631 Pit_02630 Pit_02629 Pit_02628 Pit_02627 Pit_02626 Pit_02625 Pit_02624

MFS transporter, putative MFS transporter, putative iron sulfur assembly protein, putative Tor Tor Ketoreductase conserved hypothetical protein 40S ribosomal protein S17, putative DUF408 domain protein SH3 domain protein Pc22g20420 Siderophore biosynthesis lipase/esterase, putative HC-toxin synthetase ABC multidrug transporter SitT Acetyltransferase SidF Enoyl-CoA hydratase/isomerase family protein Pc22g20360

54


Page 103 of 138

Cluster:8 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885 Pit_02885

Pit_02897 Pit_02896 Pit_02895 Pit_02894 Pit_02893 Pit_02892 Pit_02891 Pit_02890 Pit_02889 Pit_02888 Pit_02887 Pit_02885 Pit_02886 Pit_02884 Pit_02883 Pit_02882

MFS peptide transporter, putative Extracellular lipase, putative glucoamylase precursor efflux pump antibiotic resistance protein hypothetical protein PDIP_42520 MFS transporter Zinc-binding alcohol dehydrogenase, putative serine/threonine-protein kinase HT1, putative C6 zinc finger domain protein cytochrome P450 amidohydrolase 2 Nonribosomal peptide synthetase 7 UbiA-like prenyltransferase, putative cytochrome P450 hypothetical protein PDIG_49760 short-chain dehydrogenase/reductase family protein

Cluster:9 Pit_03045 Pit_03045 Pit_03045 Pit_03045 Pit_03045 Pit_03045

Pit_03045 Pit_03044 Pit_03043 Pit_03042 Pit_03041 Pit_03040

Hydroxamate-type ferrichrome siderophore peptide synthetase Protein kinase, putative MRNA splicing factor RNA helicase (Cdc28), putative Cytochrome b5, putative Nuclear migration protein, putative Haloacid dehalogenase, type II

55


Page 104 of 138

Pit_03045

Pit_03039

probable general amino acid permease

Cluster:10 Pit_03180 Pit_03180 Pit_03180

Pit_03181 Pit_03180 Pit_03179

cytochrome P450 polyketide synthase, putative cytochrome P450, putative



unnamed protein product Rieske 2Fe-2S family protein, putative C6 transcription factor, putative leucoanthocyanidin dioxygenase, putative choline transport protein Sensor histidine kinase/response regulator, putative Pc22g00740 unnamed protein product conserved hypothetical protein hypothetical protein DUF1212 domain membrane protein DUF341 family oxidoreductase, putative Polyketide synthase, putative BcBOA13 protein conserved hypothetical protein MFS gliotoxin efflux transporter thioredoxin reductase GliT-like, putative

56


Page 105 of 138

Pit_04021 Pit_04021 Pit_04021

Pit_04016 Pit_04015 Pit_04014

ABC multidrug transporter, putative MFS transporter monocarboxylate permease



methionine aminopeptidase 2 Pc14g00020 Pc14g00030 conserved hypothetical protein conserved hypothetical protein Hybrid NRPS/PKS enzyme, putative putative zinc-binding dehydrogenase family protein



Zn2/Cys6 DNA-binding protein aminoadipic semialdehyde synthase, putative reducing type I polyketide synthase 10 short chain dehydrogenase conserved hypothetical protein conserved hypothetical protein cytochrome P450 2D18 NAD(P)-binding protein FAD-binding domain-containing protein dimethylallyl tryptophan synthase GliD1 short-chain dehydrogenase

57


Page 106 of 138



FAD binding domain protein Transcription initiation factor TFIID subunit 13 Pentatricopeptide repeat protein cell cycle control protein cwf18 Pc16g02550 unnamed protein product hypothetical protein PDIG_42580 50S ribosomal protein L17 conserved hypothetical protein Pc16g02600 40S ribosomal protein S23

Cluster:14 Pit_04315 Pit_04315 Pit_04315 Pit_04315 Pit_04315 Pit_04315 Pit_04315 Pit_04315 Pit_04315 Pit_04315 Pit_04315 Pit_04315

Pit_04320 Pit_04319 Pit_04318 Pit_04317 Pit_04316 Pit_04315 Pit_04314 Pit_04313 Pit_04311 Pit_04312 Pit_04310 Pit_04308

2,3-diketo-5-methylthio-1-phosphopentane phosphatase, putative ABC fatty acid transporter Cell wall biogenesis protein Mhp1, putative conserved hypothetical protein kinetochore protein fta4, putative polyketide synthase prenyl cysteine carboxyl methyltransferase, putative 60S ribosomal protein L5 hypothetical protein hypothetical protein conserved hypothetical protein

58


Page 107 of 138

Pit_04315 Pit_04315

Pit_04307 Pit_04306

nitrate transporter CrnA nitrite reductase NiiA


Pit_04848 Pit_04847 Pit_04312

short chain dehydrogenase/reductase family oxidoreductase, putative polyketide synthase, putative hypothetical protein

Cluster:16 Pit_04977 Pit_04977 Pit_04977 Pit_04977 Pit_04977


maltose permease MAL61 C6 and C2H2 transcription factor RegA-like C6 transcription factor (Mut3), putative hexose carrier protein hypothetical protein PDIP_75340

Cluster:17 Pit_05101 Pit_05101 Pit_05101 Pit_05101 Pit_05101 Pit_05101 Pit_05101 Pit_05101 Pit_05101

Pit_05110 Pit_05109 Pit_05108 Pit_05107 Pit_05106 Pit_05105 Pit_05104 Pit_05103 Pit_05102

oxidoreductase, short-chain dehydrogenase/reductase family Disrupter of telomere silencing protein Dot5, putative Mitochondrial protein sorting (Msf1), putative conserved hypothetical protein Phosphoethanolamine Glutaredoxin Grx5, putative chitinase putative short-chain dehydrogenase reductase family protein hypothetical protein ACLA_082690

59


Page 108 of 138



nonribosomal peptide synthase Arg5,6p ankyrin repeat protein branched-chain-amino-acid aminotransferase TOXF cytochrome P450 3-oxoacyl-[acyl-carrier-protein] synthase ABC multidrug transporter, putative


Pit_05152 Pit_05151 Pit_05150

O-methyltransferase hypothetical protein PDIP_78140 NRPS-like enzyme, putative



GNAT family N-acetyltransferase, putative oxidoreductase, short chain dehydrogenase/reductase family superfamily F-box domain protein conserved hypothetical protein Translation initiation factor 2 alpha subunit, putative Sulfate transporter, putative Cation transporting ATPase, putative conserved hypothetical protein polyketide synthase, putative aldo-keto reductase Gamma-cysteine synthetase regulatory subunit, putative

60


Page 109 of 138



Serine/threonine-protein kinase bud32 DNA-directed RNA polymerase DNA-directed RNA polymerase Mitotic spindle checkpoint protein (Mad2), putative DUF218 domain protein C-x8-C-x5-C-x3-H type zinc finger protein Golgi transport protein Sly1, putative


Pit_05808 Pit_05807 Pit_05806

cytochrome P450 monooxygenase O-methyltransferase, putative putative nonribosomal peptide synthase -like protein



toxin biosynthesis putative n-terminal domain protein reducing type I polyketide synthase glycosyltransferase family 8 protein cre-bre-3 protein Flavin-containing amine oxidasedehydrogenase, putative Salicylate hydroxylase, putative hypothetical protein PDIP_10370 DNA helicase, putative DNA repair protein Ntg1, putative ubiquitin conjugating enzyme Pex4-Penicillium chrysogenum

61


Page 110 of 138

Pit_05993 Pit_05993 Pit_05993

Pit_05984 Pit_05982 Pit_05981

hypothetical protein PDIG_05650

Cluster:22 Pit_06643 Pit_06643 Pit_06643 Pit_06643 Pit_06643 Pit_06643 Pit_06643 Pit_06643 Pit_06643 Pit_06643

Pit_06643 Pit_06642 Pit_06641 Pit_06640 Pit_06639 Pit_06638 Pit_06636 Pit_06635 Pit_06634 Pit_06633

NRPS-like protein conserved hypothetical protein Putative succinate-semialdehyde dehydrogenase succinate-semialdehyde dehydrogenase, putative Putative succinate-semialdehyde dehydrogenase Sensor histidine kinase/response regulator, putative

Cluster:23 Pit_06758 Pit_06758

Pit_06759 Pit_06758

MFS transporter Fum1p

Cluster:24 Pit_06845 Pit_06845 Pit_06845 Pit_06845

Pit_06847 Pit_06846 Pit_06845 Pit_06844

salicylate hydroxylase, putative c6 zinc finger domain containing protein polyketide synthase, putative MFS multidrug resistance transporter

1,3-beta-glucanosyltransferase Gel1

Chain A, Solution Structure Of The Class I Hydrophobin Dewa Chain A, Solution Structure Of The Class I Hydrophobin Dewa MFS transporter, putative

62


Page 111 of 138



cytochrome P450 monooxigenase GliC2



cytochrome P450 oxidoreductase putative aspartyl protease C4-dicarboxylate transporter/malic acid transport protein, putative pectinesterase family protein nonribosomal peptide synthase, putative ABC multidrug transporter, putative Pc22g14460 putative taurine catabolism dioxygenase protein Ribosome biogenesis protein, putative cytochrome P450 monooxigenase GliC2 Peroxisomal membrane protein (PmpP24), putative conserved hypothetical protein similar to An16g07980 RING finger protein MFS transporter, putative hypothetical protein PDIG_00100 toxin biosynthesis ketoreductase

tryptophan dimethylallyltransferase nonribosomal peptide synthase GliP2 AMP-dependent synthetase/ligase conserved hypothetical protein

63


Page 112 of 138

Pit_07134

Pit_07122

conserved hypothetical protein



fungal specific transcription factor domain protein related to multidrug resistant protein C6 zinc finger domain protein TPA: RTA1 domain protein, putative (AFU_orthologue; AFUA_7G01970) prenyltransferase leucoanthocyanidin dioxygenase, putative Cytochrome P450 oxidoreductase, putative

Cluster:27 Pit_07808 Pit_07808 Pit_07808 Pit_07808 Pit_07808 Pit_07808 Pit_07808 Pit_07808 Pit_07808 Pit_07808 Pit_07808 Pit_07808 Pit_07808

Pit_07817 Pit_07816 Pit_07815 Pit_07814 Pit_07813 Pit_07812 Pit_07811 Pit_07810 Pit_07809 Pit_07808 Pit_07807 Pit_07806 Pit_07805

C6 finger domain protein LCCL domain-containing protein unnamed protein product cytochrome P450 52A13 unnamed protein product MFS transporter, putative oxidoreductase, short-chain dehydrogenase/reductase family conserved hypothetical protein conserved hypothetical protein polyketide synthase polyketide synthase RNase H domain containing protein protein kinase

64


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Pit_07808 Pit_07808 Pit_07808

Pit_07804 Pit_07803 Pit_07802

Pc22g23690 Putative methylmalonate-semialdehyde dehydrogenase Hypercellular protein HypA

Cluster:28 Pit_08360 Pit_08360 Pit_08360 Pit_08360 Pit_08360


MFS allantoate transporter, putative hypothetical protein PDIP_85080 U3 small nucleolar ribonucleoprotein protein imp3 conserved hypothetical protein Polyketide synthase, putative

Cluster:29 Pit_08837 Pit_08837 Pit_08837 Pit_08837 Pit_08837 Pit_08837 Pit_08837 Pit_08837 Pit_08837 Pit_08837 Pit_08837 Pit_08837

Pit_08837 Pit_08836 Pit_08835 Pit_08834 Pit_08833 Pit_08832 Pit_08831 Pit_08830 Pit_08829 Pit_08828 Pit_08827 Pit_08826

nonribosomal peptide synthase, putative TqaJ quinone oxidoreductase conserved hypothetical protein F-box domain protein Indoleamine 2,3-dioxygenase family protein conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein Cytochrome c oxidase assembly protein, putative unnamed protein product conserved hypothetical protein

65


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Cluster:30 Pit_09469 Pit_09469 Pit_09469 Pit_09469 Pit_09469 Pit_09469 Pit_09469 Pit_09469 Pit_09469 P. digitatum Cluster:1 PDIG_01630 PDIG_01630 PDIG_01630 PDIG_01630 PDIG_01630 PDIG_01630

Pit_09477 Pit_09476 Pit_09475 Pit_09474 Pit_09473 Pit_09472 Pit_09471 Pit_09470 Pit_09469

conserved hypothetical protein stomatin-like protein 2 hypothetical protein hypothetical protein PDIP_40210 Protein hir1 GTP-binding protein YchF PUA RNA binding domain protein, putative Pc16g06190 NRPS-like enzyme

PDIG_01670 PDIG_01660 PDIG_01650 PDIG_01640 PDIG_01630 PDIG_01620

MFS transporter, putative Cytochrome P450 monooxygenase, putative Alpha/beta hydrolase, putative Dipeptidyl aminopeptidase/acylaminoacyl peptidase Polyketide synthase Transcription factor ACEII

Cluster:2 PDIG_04970 PDIG_04970 PDIG_04970 PDIG_04970 PDIG_04970

PDIG_04970 PDIG_04960 PDIG_04950 PDIG_04940 PDIG_04930

hypothetical protein hypothetical protein Putative succinate-semialdehyde dehydrogenase Sensor histidine kinase/response regulator, putative MFS transporter, putative

66


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PDIG_04970 PDIG_04970

PDIG_04920 hypothetical protein PDIG_04910 MFS transporter, putative

Cluster:3 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960 PDIG_09960

PDIG_09980 PDIG_09970 PDIG_09960 PDIG_09950 PDIG_09940 PDIG_09930 PDIG_09920 PDIG_09910 PDIG_09900 PDIG_09890 PDIG_09880 PDIG_09870 PDIG_09860 PDIG_09850 PDIG_09840 PDIG_09830 PDIG_09820

Cluster:4 PDIG_11250 PDIG_11250

PDIG_11250 Mycocerosic acid synthase PDIG_11240 Polyketide synthase, putative

hypothetical protein Trypsin-like serine protease, putative HC-toxin synthetase FAD dependent oxidoreductase, putative hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein Nonribosomal peptide synthase, putative Transferase family protein hypothetical protein Luciferase-like monooxygenase, putative hypothetical protein Alpha-L-arabinofuranosidase MFS sugar transporter, putative

67


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hypothetical protein Hexokinase-1 hypothetical protein hypothetical protein C6 transcription factor, putative

Cluster:5 PDIG_15090 PDIG_15090 PDIG_15090 PDIG_15090 PDIG_15090


MFS lactose permease, putative hypothetical protein hypothetical protein hypothetical protein hypothetical protein

Cluster:6 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150

PDIG_16260 PDIG_16250 PDIG_16240 PDIG_16230 PDIG_16220 PDIG_16210 PDIG_16200 PDIG_16190 PDIG_16180 PDIG_16170 PDIG_16160

Glutathione S-transferase kappa 1 hypothetical protein Oxidoreductase, 2OG-Fe(II) oxygenase family, putative hypothetical protein hypothetical protein hypothetical protein Restriction of telomere capping protein 5 Fatty acid synthase subunit alpha, putative Branched-chain amino acid aminotransferase Fatty acid synthase subunit beta, putative Benzoate 4-monooxygenase cytochrome P450

68


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PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150 PDIG_16150


HC-toxin synthetase hypothetical protein ABC transporter, putative hypothetical protein hypothetical protein Glycylpeptide N-tetradecanoyltransferase Tetrahydrofolylpolyglutamate synthase (Met7), putative Phosphoinositide phosphatase (Sac1), putative RAB GTPase Ypt5, putative Formation of crista junctions protein 1

Cluster:7 PDIG_21220 PDIG_21220 PDIG_21220 PDIG_21220 PDIG_21220 PDIG_21220 PDIG_21220 PDIG_21220 PDIG_21220 PDIG_21220 PDIG_21220 PDIG_21220 PDIG_21220

PDIG_21330 PDIG_21320 PDIG_21310 PDIG_21300 PDIG_21290 PDIG_21280 PDIG_21270 PDIG_21260 PDIG_21250 PDIG_21240 PDIG_21230 PDIG_21220 PDIG_21210

40S ribosomal protein S0 30S ribosomal protein S15 hypothetical protein DEAD/DEAH box helicase (Sbp4), putative Phosphatidate cytidylyltransferase, putative Rad2-like endonuclease, putative Actin-related protein RO7, putative hypothetical protein Cytochrome P450, putative hypothetical protein Short-chain oxidoreductase, putative hypothetical protein hypothetical protein

69


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PDIG_21220 PDIG_21220

PDIG_21200 hypothetical protein PDIG_21190 Benzoate 4-monooxygenase cytochrome P450, putative

Cluster:8 PDIG_29980 PDIG_29980 PDIG_29980 PDIG_29980 PDIG_29980 PDIG_29980 PDIG_29980 PDIG_29980 PDIG_29980 PDIG_29980 PDIG_29980 PDIG_29980 PDIG_29980

PDIG_29990 PDIG_29980 PDIG_29970 PDIG_29960 PDIG_29950 PDIG_29940 PDIG_29930 PDIG_29920 PDIG_29910 PDIG_29900 PDIG_29890 PDIG_29880 PDIG_29870

ABC multidrug transporter SitT HC-toxin synthetase Siderophore biosynthesis lipase/esterase, putative hypothetical protein hypothetical protein hypothetical protein 40S ribosomal protein S17, putative hypothetical protein Ketoreductase Tor hypothetical protein Iron-sulfur cluster assembly accessory protein Isa2, putative MFS transporter, putative

Cluster:9 PDIG_38480 PDIG_38480 PDIG_38480 PDIG_38480 PDIG_38480 PDIG_38480


C6 transcription factor, putative hypothetical protein hypothetical protein Malic enzyme hypothetical protein hypothetical protein

70


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Polyketide synthase, putative hypothetical protein U3 small nucleolar ribonucleoprotein subunit (Imp3), putative hypothetical protein MFS allantoate transporter, putative

Cluster:10 PDIG_38770 PDIG_38770 PDIG_38770 PDIG_38770 PDIG_38770 PDIG_38770 PDIG_38770 PDIG_38770

PDIG_38800 PDIG_38790 PDIG_38780 PDIG_38770 PDIG_38760 PDIG_38750 PDIG_38740 PDIG_38730

hypothetical protein hypothetical protein C6 transcription factor, putative hypothetical protein hypothetical protein hypothetical protein Alcohol dehydrogenase, putative hypothetical protein



C6 transcription factor (AmyR), putative 3-demethylubiquinone-9 3-methyltransferase hypothetical protein hypothetical protein hypothetical protein CaaX farnesyltransferase beta subunit Ram1 hypothetical protein hypothetical protein

71


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PDIG_39590 PDIG_39590

PDIG_39600 hypothetical protein PDIG_39590 hypothetical protein

Cluster:12 PDIG_41980 PDIG_41980 PDIG_41980 PDIG_41980 PDIG_41980 PDIG_41980 PDIG_41980 PDIG_41980 PDIG_41980

PDIG_41980 PDIG_41970 PDIG_41960 PDIG_41950 PDIG_41940 PDIG_41930 PDIG_41920 PDIG_41910 PDIG_41900

Nonribosomal peptide synthase Pes1 MFS multidrug transporter, putative hypothetical protein hypothetical protein Phosphoglycerate kinase hypothetical protein hypothetical protein Formin binding protein (FNB3), putative RNase L inhibitor of the ABC superfamily, putative

Cluster:13 PDIG_46630 PDIG_46630 PDIG_46630 PDIG_46630 PDIG_46630 PDIG_46630 PDIG_46630 PDIG_46630 PDIG_46630 PDIG_46630


hypothetical protein Putative intracellular invertase Amino acid transporter, putative Cytochrome c mitochondrial import factor (Cyc2), putative Phosphoserine aminotransferase MFS multidrug transporter, putative hypothetical protein Phenolpthiocerol synthesis polyketide synthase ppsA hypothetical protein Beta-glucosidase, putative

72


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UDP-glucosyl transferase family protein hypothetical protein Mitochondrial GTPase (Mss1), putative Cytokinesis protein SepA/Bni1 hypothetical protein Formate dehydrogenase Leucyl-tRNA synthetase hypothetical protein

Cluster:14 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740

PDIG_49830 PDIG_49820 PDIG_49810 PDIG_49800 PDIG_49790 PDIG_49780 PDIG_49770 PDIG_49760 PDIG_49750 PDIG_49740 PDIG_49730 PDIG_49720 PDIG_49710 PDIG_49700 PDIG_49690

Epd1 hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein Nonribosomal peptide synthetase 7 UbiA-like prenyltransferase, putative hypothetical protein hypothetical protein hypothetical protein hypothetical protein

73


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PDIG_49740 PDIG_49740 PDIG_49740 PDIG_49740

PDIG_49680 PDIG_49670 PDIG_49660 PDIG_49650

Zinc-binding alcohol dehydrogenase, putative hypothetical protein hypothetical protein hypothetical protein



hypothetical protein Cytochrome P450 oxidoreductase, putative hypothetical protein hypothetical protein Histone acetylase complex subunit, putative MRNA splicing protein Yju2 20S cyclosome subunit (Cut9/Cdc16), putative P450 family sporulation-specific N-formyltyrosine oxidase Dit2, putative

Cluster:16 PDIG_53550 PDIG_53550 PDIG_53550 PDIG_53550 PDIG_53550 PDIG_53550 PDIG_53550

PDIG_53550 PDIG_53540 PDIG_53530 PDIG_53520 PDIG_53510 PDIG_53500 PDIG_53490

HC-toxin synthetase hypothetical protein hypothetical protein Class III chitinase, putative Plasma membrane H(+)ATPase, putative Glutamyl-tRNA synthetase hypothetical protein

Cluster:17

74


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2-dehydropantoate 2-reductase family protein, putative hypothetical protein hypothetical protein Glutamine dependent NAD+ synthetase, putative NADH pyrophosphatase, putative hypothetical protein hypothetical protein Flavin dependent monooxygenase, putative hypothetical protein Conidial pigment polyketide synthase PksP/Alb1

Cluster:18 PDIG_55560 PDIG_55560 PDIG_55560 PDIG_55560 PDIG_55560 PDIG_55560


Hydroxamate-type ferrichrome siderophore peptide synthetase L-ornithine N5 monooxygenase Palmitoyltransferase akr1 hypothetical protein hypothetical protein hypothetical protein


PDIG_55700 Hybrid NRPS/PKS enzyme, putative PDIG_55690 hypothetical protein

Cluster:20 PDIG_66040

PDIG_66040 hypothetical protein

75


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PDIG_66040 PDIG_66040

PDIG_66030 High-affinity hexose transporter, putative PDIG_66020 C6 transcription factor (Mut3), putative



hypothetical protein Aldehyde dehydrogenase family protein, putative hypothetical protein hypothetical protein Sterigmatocystin 8-O-methyltransferase hypothetical protein hypothetical protein hypothetical protein

Cluster:22 PDIG_76310 PDIG_76310 PDIG_76310 PDIG_76310 PDIG_76310 PDIG_76310 PDIG_76310 PDIG_76310 PDIG_76310 PDIG_76310 PDIG_76310

PDIG_76340 PDIG_76330 PDIG_76320 PDIG_76310 PDIG_76300 PDIG_76290 PDIG_76280 PDIG_76270 PDIG_76260 PDIG_76250 PDIG_76240

hypothetical protein hypothetical protein hypothetical protein hypothetical protein Polyketide synthase, putative hypothetical protein hypothetical protein Vacuolar basic amino acid transporter 3 Cytochrome P450, putative hypothetical protein Zinc-binding oxidoreductase, putative

76


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Zinc-binding oxidoreductase CipB hypothetical protein Cell wall protein PhiA hypothetical protein Aspartate aminotransferase Dihydrodipicolinate synthetase family protein MFS multidrug transporter, putative

Cluster:23 PDIG_78750 PDIG_78750 PDIG_78750 PDIG_78750 PDIG_78750 PDIG_78750 PDIG_78750 PDIG_78750 PDIG_78750 PDIG_78750 PDIG_78750 PDIG_78750

PDIG_78750 PDIG_78740 PDIG_78730 PDIG_78720 PDIG_78710 PDIG_78700 PDIG_78690 PDIG_78680 PDIG_78670 PDIG_78660 PDIG_78650 PDIG_78640

Equisetin synthetase, putative hypothetical protein hypothetical protein hypothetical protein Zinc-binding dehydrogenase family oxidoreductase, putative hypothetical protein Acetolactate synthase O-methyltransferase, putative hypothetical protein C6 transcription factor, putative hypothetical protein hypothetical protein


PDIG_83130 hypothetical protein PDIG_83120 hypothetical protein

77


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PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960 PDIG_82960

PDIG_83110 PDIG_83100 PDIG_83090 PDIG_83080 PDIG_83070 PDIG_83060 PDIG_83050 PDIG_83040 PDIG_83030 PDIG_83020 PDIG_83010 PDIG_83000 PDIG_82990 PDIG_82980 PDIG_82970 PDIG_82960 PDIG_82950 PDIG_82940 PDIG_82930 PDIG_82920 PDIG_82910 PDIG_82900 PDIG_82890 PDIG_82880 PDIG_82870

hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein Oxidoreductase, short-chain dehydrogenase/reductase family hypothetical protein hypothetical protein hypothetical protein hypothetical protein Siderochrome-iron transporter MirC Rho GTPase ModA, putative hypothetical protein Putative 3-oxoacyl-[acyl-carrier-protein] synthase, mitochondrial mRNA-capping enzyme subunit alpha Histone acetyltransferase esa1 hypothetical protein hypothetical protein HEAT repeat protein DNA-directed RNA polymerase subunit Sister chromatid cohesion protein Mis4, putative hypothetical protein NADH-ubiquinone oxidoreductase 64 kDa sub

78


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Table S6. Differentially expressed genes enriched in the top 20 KEGG pathways ranked by q-value.

* †

Pathway ID

Name

M/N*

m/n†

p-value

q-value

ko03010 ko00030 ko00710 ko00680 ko04620 ko00230 ko00624 ko04623 ko00100 ko04621 ko00900 ko00982 ko00010 ko00195 ko01051 ko00140 ko04622 ko00350 ko00051 ko05162

Ribosome Pentose phosphate pathway Carbon fixation in photosynthetic organisms Methane metabolism Toll-like receptor signaling pathway Purine metabolism Polycyclic aromatic hydrocarbon degradation Cytosolic DNA-sensing pathway Steroid biosynthesis NOD-like receptor signaling pathway Terpenoid backbone biosynthesis Drug metabolism - cytochrome P450 Glycolysis / Gluconeogenesis Photosynthesis Biosynthesis of ansamycins Steroid hormone biosynthesis RIG-I-like receptor signaling pathway Tyrosine metabolism Fructose and mannose metabolism Measles

148/4213 39/4213 31/4213 74/4213 9/4213 142/4213 188/4213 15/4213 35/4213 9/4213 23/4213 120/4213 135/4213 2/4213 2/4213 34/4213 13/4213 201/4213 138/4213 20/4213

91/1177 22/1177 18/1177 33/1177 7/1177 54/1177 68/1177 9/1177 16/1177 6/1177 10/1177 43/1177 46/1177 2/1177 2/1177 14/1177 7/1177 65/1177 47/1177 9/1177

4.22E-18 1.64E-04 4.00E-04 0.0014662 0.0027065 0.0051199 0.0073123 0.0091835 0.0182237 0.0175748 0.0797376 0.0341521 0.0664852 0.0780017 0.0780017 0.0659244 0.0432947 0.0906584 0.0645935 0.0769107

1.07E-15 0.0207369 0.0337466 0.0927402 0.1369512 0.2158894 0.2642866 0.290428 0.4610586 0.4940483 0.7759086 0.7854988 0.8009878 0.8054867 0.8054867 0.8339433 0.8425813 0.8495026 0.8601137 0.8648176

M indicates the number of genes assigned into a certain KEGG pathway, N indicates the total number of genes assigned into all KEGG pathway. m indicates the number of specific genes assigned into a certain KEGG pathway, n indicates the total number of specific genes assigned into all KEGG pathway.

79


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Table S7. Differentially expressed genes involved in ribosome pathway. FPKM FPKM log2 Gene ID p-value q-value (Shaking) (Static) (Shaking/Static)

Regulation Annotation


515.677 184.191 595.651 1056.3 604.628 5.75647 1272.18 834.035 429.773 558.746 889.322 294.856 162.536 15.1022

981.369 465.837 1331.86 2011.1 1562.79 188.386 2528.96 3298.08 980.865 1093.43 1758.27 682.08 455.964 75.7981

0.92833 1.33863 1.1609 0.928973 1.37 5.03237 0.991243 1.98344 1.19048 0.968595 0.983378 1.20993 1.48816 2.3274

5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05

0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195

up up up up up up up up up up up up up up

PEG07263

348.46

608.731

0.804811

0.0001

0.000374

up


2614.32 122.576 95.7062 346.665 557.278 802.075

6526.53 252.994 295.273 885.52 1460.32 1911.54

1.31988 1.04542 1.62536 1.35298 1.38982 1.25293

5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05

0.000195 0.000195 0.000195 0.000195 0.000195 0.000195

up up up up up up

80

60S ribosomal protein L10 60S ribosomal protein P0 Ribosomal L18ae protein family Ribosomal protein L15 40S ribosomal protein S8 Integral membrane protein Pth11-like 60S ribosomal protein L40 60S ribosomal protein L38, putative Uncharacterized protein 60S ribosomal protein L13 60S ribosomal protein L7 60S ribosomal protein L18 Uncharacterized protein U3 small nucleolar ribonucleoprotein subunit Whole genome shotgun sequence assembly, scaffold_162, strain Mel28 60S ribosomal protein L29, putative 40S ribosomal protein S7e 60S ribosomal protein L22, putative 40S ribosomal protein 40S ribosomal protein S19 Alkaline serine protease


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308.04 253.906 56.7226 455.903 1993.16 804.062 484.272 696.685 65.245

854.836 634.669 253.051 910.644 5803.15 1950.61 1419.16 1614.38 387.414

1.47253 1.32171 2.15744 0.998161 1.54178 1.27855 1.55115 1.2124 2.56994

5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05

0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195

up up up up up up up up up

PEG09351

697.086

1761.66

1.33752

5.00E-05

0.000195

up


209.435 1138.04 504.269 496.182 768.45 501.173 349.055 3046.31

611.358 2980.75 1242.84 1254.87 1842.44 1293.93 1010.65 11670.2

1.54551 1.38912 1.30138 1.3386 1.26159 1.36838 1.53376 1.9377

5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05

0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195

up up up up up up up up

PEG03042

1005.02

2371.47

1.23856

5.00E-05

0.000195

up

PEG03796 PEG04250 PEG04291 PEG04341

601.65 262.771 240.675 1125.49

1340.26 831.886 649.737 2574.94

1.15551 1.66258 1.43277 1.19399

5.00E-05 5.00E-05 5.00E-05 5.00E-05

0.000195 0.000195 0.000195 0.000195

up up up up

81

Ribosomal protein L34 protein, putative Inosine-5'-monophosphate dehydrogenase Uncharacterized protein 40S ribosomal protein S11 40S ribosomal protein S26E Ribosomal protein L16a 60S ribosomal protein L27 60S acidic ribosomal protein P1 Integral membrane protein Pth11-like, NAD-dependent D-isomer specific 2-hydroxyacid dehydrogenase 40S ribosomal protein S22 40S ribosomal protein S13 60S ribosomal protein L4 60S ribosomal protein L8 60S ribosomal protein L30-2 60S ribosomal protein L28 Ribosomal protein L26 60S ribosomal protein L37a Oxidoreductase family, NAD-binding Rossmann fold protein 40S ribosomal protein S6 40S ribosomal protein S10b Cytosolic large ribosomal subunit protein L7A Nitrilase family protein (Nit3),


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919.82 789.192 662.39 756.834 714.558 621.63 939.349 460.165 354.245 585.772 1.41298 10.8923 906.518 491.887 626.292 664.353 101.02 673.165 714.876 475.513 180.308 334.72 539.951 700.167 18.6357

2239.96 2188.31 1349.45 2436.09 1638.02 1495.2 2126.62 1244.16 966.173 1401.49 16.9313 29.4909 2136.45 1181.55 1945.65 1529.59 298.77 1903.98 1992.31 1335.93 362.04 875.454 1419.12 2021.68 46.6133

1.28405 1.47137 1.02661 1.68652 1.19683 1.26621 1.17883 1.43495 1.44753 1.25855 3.58287 1.43695 1.23681 1.26428 1.63534 1.20312 1.56439 1.49999 1.47868 1.49029 1.00569 1.38708 1.39409 1.52978 1.32267

0.0003 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 0.0001 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05

0.001027 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000374 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195

82

up up up up up up up up up up up up up up up up up up up up up up up up up

Ribosomal protein S13p/S18e 60S ribosomal protein L23 40S ribosomal protein S1 60S ribosomal protein L36 60S acidic ribosomal phosphoprotein P2 40S ribosomal protein S15 40S ribosomal protein S17 40S ribosomal protein S11 40S ribosomal protein Rps16 60S ribosomal protein L24a Integral membrane protein Uncharacterized protein Ribosomal protein S28e 40S ribosomal protein S24 Uncharacterized protein 40S ribosomal protein S5 Predicted protein 40S ribosomal protein S9 60S ribosomal protein L21 40S ribosomal protein S4 60S ribosomal protein L37 60S ribosomal protein L35Ae 40S ribosomal protein S10a Uncharacterized protein Integral membrane protein


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PEG11247 PEG11661 PEG11709 PEG01151 PEG01260 PEG01323 PEG01437 PEG00670 PEG00790 PEG00565 PEG00601 PEG00178 PEG00233 PEG00348 PEG02008 PEG03566 PEG03865 PEG05106 PEG05496 PEG11667 PEG01498 PEG00169

380.297 659.151 829.848 1.41604 16.5418 476.578 725.738 563.767 580.912 0.28506 697.703 778.942 443.616 0.910984 16.0651 169.653 31.3999 13.3171 287.703 12.4866 215.119 4542.2

973.008 1288.12 2107.83 65.2849 56.2958 1062.78 1653.73 1531.55 1690.64 2.31231 1636.72 2060.98 1140.52 13.6957 1.44373 15.4052 2.31255 5.28582 149.493 3.61034 2.15029 946.05

1.35533 0.966588 1.34484 5.52681 1.76691 1.15706 1.1882 1.44182 1.54118 3.02 1.23012 1.40374 1.36231 3.91015 -3.47605 -3.4611 -3.7632 -1.33308 -0.944497 -1.79017 -6.64447 -2.2634

5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 5.00E-05 0.0008 5.00E-05 5.00E-05 5.00E-05 0.00065 5.00E-05 5.00E-05 5.00E-05 0.00025 5.00E-05 5.00E-05 5.00E-05 5.00E-05

0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.000195 0.002489 0.000195 0.000195 0.000195 0.002061 0.000195 0.000195 0.000195 0.000868 0.000195 0.000195 0.000195 0.000195

83

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40S ribosomal protein S0 60S ribosomal protein L5 Ribosomal protein Predicted protein Cell cycle control protein Cdc123 Uncharacterized protein Transcriptional corepressor Cyc8 Uncharacterized protein 60S ribosomal protein L35 Uncharacterized protein Ubiquitin 60S ribosomal protein L9 40S ribosomal protein S23 Zinc finger protein Integral membrane protein Integral membrane protein Uncharacterized protein Predicted protein Integral membrane protein Uncharacterized protein Uncharacterized protein Uncharacterized protein

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Table S8. Expressions of genes encoding components of velvet complex in P. expansum.


Gene VeA VelB LaeA

Gene ID

FPKM* (Shaking)

FPKM (Static)

p-value

Regulation

PEG11106 PEG05220 PEG09421

48.9627 17.0981 28.2504

45.2133 16.3137 56.3033

>0.001 >0.001 5.00E-05

/ / up

* †

FPKM: Fragments per kilobase of transcript per million fragments mapped. /: No significant difference was found.

84

†

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Table S9. Primers used for RT-qPCR in the expression assay of GH78 family genes. Gene ID

Primer sequences (5’ - 3’)

F: ATCGCGACCGACAAAATCAG R: CGGCAATATGCTTCCCATTC F: ATGCACCGTCCTCGAGAATT PEG03271 R: GGTCAGAGCTGCTGGAGTTGT F: TCGCATCTCCTGGACCTTCT PEG05990 R: GGCGCACGGAAATCTCATAC F: TCGTACGGTGCATTCAAGGA PEG08945 R: ACCGAACATAATTGCCATGCT F: CGTGTGAATGGAACGCAAGT PEG10044 R: AGGCTCAGGTTTCCGCATAA F: GCCGAAGGTCATCGATATCC PEG10046 R: CACGAGATCCGAGGGCAAT F: CCACAACCAAATACCCCCTAA PEG10982 R: CGTCCACATTTGCTGGTACAC F: CCGATGCCAAGGTTGTTCTG PEG11722 R: ATCTCGTTTTGCACCATCCA F: CTCCAGCTCGAGCGTATGAAC Beta-tubulin R: GGCTCCAAATCGACGAGAAC PEG00739

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Table S10. Primers used for RT-qPCR in the expression analysis of genes in the patulin cluster. Gene Molecular Plant-Microbe Interactions "First Look" paper • http://dx.doi.org/10.1094/MPMI-12-14-0398-FI • posted 01/27/2015 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.

PePatA PePatB PePatC PePatD PePatE PePatF PePatG PePatH PePatI PePatJ PePatK PePatL PePatM PePatN PePatO Beta-tubulin

Primer sequences (5’ - 3’) F: AAAGGCCGGTGCATTGATC R: TTGGAGGCTTTGGTGAGCAT F: GCCAGGCTATGCGATTGAGT R: GCTGGAACCCTGTCCATTGT F: TCCACCTGCGAATATCCCTTA R: CATCGCCAGTGCCATTTTC F: ATGAGATTCGTCTGCGCAAAG R: CTACCCAAGCGGGATGAGATT F: CATTCTCATCGGGCCTGAGT R: TCGAAGCTCTTCCGGACATG F: GCGAGTGAATTCGGCCAAT R: GTCCGACCCAAAGGATGAAG F: CGGCCGTCTTGAAGGAAAT R: CTTGCCGTAGCGGGTGAATA F: CATTTATCGGCGGTGTTCTGA R: GATCAACGCTTGCACGATAGC F: GCAAACTCATTCCGCAAGGA R: TGGTTCTTGCCATCGATCAC F: CGCCAGACATACCGCCATA R: TTTGGTCGATCGGGACTGTT F: GACGCTGGGCTACTGGATTG R: TCGTGCGTGAGGCCAGTAT F: GCAGGAGATCCGTTTCAGACA R: CCACTGACCGACGGTTACAAC F: ACCCACAGCTGCACATGGA R: AGCGAGAAGAGGCGGAAGA F: CGTTCGATGTCGCTAGCAAA R: GGCGATAATCACGTCAATTCG F: TCGCCTCCTGGTGTGTATCTT R: AAGCGTGCCCAGTCATTCAG F: CTCCAGCTCGAGCGTATGAAC R: GGCTCCAAATCGACGAGAAC

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Talbe S11. Primers used for gene replacement and transformants confirmation. Fragment Primer Sequences (5’ - 3’) KL1 TCGCCTTCTACATACCCCTC PatK-Flank L KL2 CTGTTGTGCTTTGGCATTGG KR1 CTAGCCGCCTAGACAACGAA PatK-Flank R KR2 TGCTGAAGAAGTCCTCTGTGG LL1 TGTCGGCCTTTCCGGGACGA PatL-Flank L LL2 GGATCGATTGTGATGTGATG LR1 TCTCAACGTTGCAGGAGTCG PatL- Flank R LR2 GGTACTGGGATGCGTTGTCC H1 TTCCTTTGCCCTCGGACGAG

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Figure S1. Gene Ontology classification of genes in P. expansum and P. italicum. Outside cycle represents P. expansum, and inside cycle represents P. italicum. A,


Functional categories according to biological process. B, Functional categories according to cellular component. C, Functional categories according to molecular function.

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Figure S2. Modulation of PePatK in the patulin cluster of P. expansum. Domain definitions: ACP, acyl carrier protein; AT, acyltransferase; DH, dehydratase; KR,


ketoreductase domain; KS, β-ketoacyl synthase.

Figure S3. Functional classification of differentially expressed genes using transcriptome analysis under static and shaking conditions in P. expansum.

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Figure S4. Confirmation of positive transformants using flank-spanning PCR. A, a 2.9 kb fragment was amplified with primer pair KL1/H1 from the PePatK


transformants deriving from homologous integration. B, a 2.8 kb fragment was amplified with primer pair LL1/H1 from the PePatL transformants deriving from homologous integration. The primers used for amplification are listed in Supplementary Table S11.

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