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Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536 Kai Dou a , Zhiying Wang a,∗ , Rongshu Zhang b , Na Wang a , Haijuan Fan a , Guiping Diao a , Zhihua Liu a,∗ a b

School of Forestry, Northeast Forestry University, 26 Hexing Road, 150040 Harbin, China The College of Landscape, Northeast Forestry University, 26 Hexing Road, 150040 Harbin, China

a r t i c l e

i n f o

Article history: Received 12 September 2013 Received in revised form 26 March 2014 Accepted 3 April 2014 Available online xxx Keywords: Trichoderma asperellum Aspartic protease ASP55 Biocontrol Prokaryotic expression

a b s t r a c t Proteases secreted by fungi belonging to the genus Trichoderma play important roles in biocontrol. In this study, the coding sequence and promoter region of the novel aspartic protease gene Asp55 were cloned from strain Trichoderma asperellum ACCC30536. Many cis-elements involved in phytopathogenic and environmental stress responses were identified in the Asp55 promoter region and may be recognized by MYB or WRKY transcription factors. The expression pattern of Asp55 under eight culture conditions was investigated by RT-qPCR. The expression level of Asp55 was up-regulated by poplar stem powder, Alternaria alternata cell wall fragments and A. alternata fermentation liquid, while it was down-regulated by carbon and nitrogen source starvation, and by powdered poplar leaves and roots. Additionally, the expression patterns of 15 genes encoding MYB transcription factors (Myb1 to Myb15) were also analyzed by RT-qPCR. Myb2 showed the most similar expression pattern with Asp55. The cDNA of Asp55 was expressed in Escherichia coli BL21, and recombinant ASP55 (rASP55) was purified. The purified rASP55 was evaluated for enzymatic activity and showed inhibitory effect on phytopathogenic A. alternata. © 2014 Elsevier GmbH. All rights reserved.

1. Introduction The genus Trichoderma is well known for its biological control ability and has been subjected to numerous investigations, especially those relating to multiple biocontrol mechanisms. As biocontrol agents, Trichoderma spp. play roles not only by parasitizing plant-pathogenic fungi but also promoting plant growth and further stimulating plant defense responses. The extracellular proteases secreted by Trichoderma spp. can contribute to all of these mechanisms. The proteases of Trichoderma spp. and their biocontrol roles have been reported previously (Viterbo et al. 2004; Yang et al. 2013). Firstly, proteases produced by Trichoderma spp. play a role in mycoparasitism. For example, a serine protease Prb1 specifically secreted by Trichoderma harzianum under simulated mycoparasitic conditions was purified and characterized (Geremia et al. 1993), and transformants of T. harzianum that over-expressed the Prb1 protease exhibited improved biocontrol activity against Rhizoctonia solani (Flores et al. 1997). Transcriptome-wide gene expression

∗ Corresponding authors. Tel.: +86 0451 82191512. E-mail addresses: [email protected] (Z. Wang), [email protected] (Z. Liu).

analyses of T. harzianum CECT 2413 and Trichoderma atroviride IMI206040 revealed that many genes encoding proteases had upregulated expression levels in the presence of the fungal prey Botrytis cinerea and R. solani (Suárez et al. 2007; Seidl et al. 2009). One possible role of proteases in mycoparasitism is in the degradation of the phytopathogen’s cell walls. Protease Pra1 from T. harzianum has an affinity for fungal cell walls (Elad et al. 2001). An 18.8 kDa protease from T. harzianum 1051 may have the capacity to hydrolyze the cell walls of the fungal phytopathogen Crinipellis perniciosa (De Marco and Felix 2002). Another putative role of proteases is that they break down hydrolytic enzymes secreted by phytopathogens. Proteases from T. harzianum T39 and T. harzianum NCIM1185 can deactivate endo- and exo-polygalacturonase produced by the phytopathogen B. cinerea (Kapat et al. 1998; Elad and Kapat 1999). Secondly, proteases also play an important role in the interactions of Trichoderma spp. with plants. When the roots of cucumber seedlings growing in an aseptic hydroponics medium were colonized by Trichoderma asperellum T-203, two aspartic proteases, PapA and PapB, were identified by SDS–PAGE and peptide sequence analyses (Viterbo et al. 2004). This indicates the beneficial effects of the proteases to cucumber because the Trichoderma strain must colonize the plant’s roots to stimulate plant growth and defense responses (Benítez et al. 2004).

http://dx.doi.org/10.1016/j.micres.2014.04.006 0944-5013/© 2014 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Dou K, et al. Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536. Microbiol Res (2014), http://dx.doi.org/10.1016/j.micres.2014.04.006

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Among the Trichoderma spp., T. asperellum is an excellent biocontrol agent. It can parasitize phytopathogens or inhibit their growth and development. Additionally, it can enhance plant growth and immunity. T. asperellum was an efficient antagonist against Fusarium solani, R. solani and Sclerotinia sclerotiorum (Qualhato et al. 2013). T. asperellum PR11 can enhance the growth of cacao and its resistance to Phytophthora megakarya (Tchameni et al. 2011). Due to its effective biocontrol ability, a T. asperellum-based bioformulation was also developed to control R. solani (Kakvan et al. 2013) and Thielaviopsis paradoxa (Wijesinghe et al. 2011). However, no reports on the mechanisms behind the interactions of T. asperellum with woody plants or woody plant pathogens were uncovered. And the roles of the aspartic proteases of T. asperellum in these interactions are still unknown. In this study, a novel protease gene Asp55 from T. asperellum ACCC30536 was cloned and analyzed. The regulation pattern of Asp55 in the presence of Populus davidiana × Populus bolleana, Alternaria alternata or under different culture conditions was investigated by RT-qPCR. The expression patterns of 15 genes encoding MYB transcription factors (Myb1 to Myb15) were also analyzed under the same culture conditions by RT-qPCR. Additionally, the heterologous expression of Asp55 in Escherichia coli BL21 was conducted and the properties of the recombinase were characterized. 2. Materials and methods 2.1. Strains, plasmids and plant materials T. asperellum ACCC30536 and the phytopathogenic fungi, A. alternata (poplar leaf wither), were used in this study. T. asperellum was cultured on Potato Dextrose Agar (PDA) slant culture-medium for 7 days then stored at 4 ◦ C. A. alternata was inoculated in 200 ml of 1/2 Potato Dextrose (PD) medium and cultured for 10 days at 28 ◦ C and 200 rpm. Mycelium of A. alternata was used to prepare cell wall fragments as described by Morissette et al. (2006). Fermentation liquid was filtered using 0.2 ␮m filters (Pall Corporation, MI, USA) to remove spores. Both cell wall fragments and filtered fermentation liquid were stored at −20 ◦ C. The aseptic P. davidiana × P. bolleana Loucne (Shanxin poplar) seedlings were cultured in liquid woody plant medium (WPM) (Faisal et al. 2012) at 25 ◦ C. Stems, roots and leaves were ground into powder and then stored at −20 ◦ C. E. coli BL21 and vector pGEX-4T-2 (GE Healthcare UK Ltd., Buckinghamshire HP7 9NA, England) were employed for the prokaryotic expression experiment. E. coli BL21 was cultured in Luria-Bertani (LB) medium and stored at −80 ◦ C. 2.2. Gene cloning and sequence analyses Two primers, forward asp1, 5 -CAGCAAAGCTCGCGTTGCATCATG-3 , and reverse asp2, 5 -GAACGGCTCTCTATGCAATATTG-3 , were designed to amplify the Asp55 DNA and cDNA sequences. Primers used for amplifying promoter region of Asp55 were

as follows: forward pr1 5 -ATCATGATTATTCATCAATTGT-3 and reverse pr2 5 -TCTCGTCTGAAATATATTGGCT-3 . Once sequenced, Asp55 DNA and Asp55 cDNA sequences were deposited in GenBank (KF029762 and KF976813, respectively). The protein family of ASP55 was analyzed using the Pfam program (http://pfam. sanger.ac.uk/). A multiple sequence alignment was conducted using the ClustalX program (http://www.ebi.ac.uk/Tools/ clustalw2/). The phylogenetic tree was constructed using the neighbor-joining method in the MEGA 5.10 program. The sequence of ASP55 was used as the general BLASTP query in T. asperellum CBS 433.97, T. harzianum CBS 226.95, T. atroviride ATCC74058 and Trichoderma virens Gv29-8 genomes within the website JGI (http://genome.jgi.doe.gov/Trias1/Trias1.home.html). Each of the obtained sequences was aligned with ASP55 using the NCBI Align two option (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

2.3. Differential expression levels of Asp55 and Myb1 to Myb15 from T. asperellum The expression levels of Asp55 and Myb1 to Myb15 were investigated using the following eight conditions: minimal media (MM) containing 15 g/l NaH2 PO4 , 5 g/l (NH4 )2 SO4 , 600 mg/l CaCl2 ·2H2 O, 600 mg/l MgSO4 ·7H2 O, 5 mg/l FeSO4 , 2 mg/l CoCl2 , 1.6 mg/l MnSO4 , 1.4 mg/l ZnSO4 and 5 g/l glucose; MM without the glucose carbon source (MM-C); MM without nitrogen ammonium sulfate (MMN); MM containing 1% (w/v) cell wall fragments of the fungal plant pathogen A. alternata (MM + CW); MM containing 5% (v/v) fermentation liquid of A. alternata (MM + ferm); MM containing 1% root powder of P. dividiana × P. bolleana (MM + root); MM containing 1% poplar stem powder MM + stem); and MM containing 1% poplar leaf powder (MM + leaf). Conidia (1 × 106 ) of T. asperellum ACCC30536 were inoculated in 1/4 PD medium at 25 ◦ C for 48 h under continuous shaking at 180 rpm. After the conidia germinated, the mycelia were transferred into MM and incubated at 25 ◦ C for 2 h. The mycelia were then harvested, rinsed with sterile distilled water and transferred into the following separate inducing conditions: MM, MM-C, MMN, MM + CW, MM + ferm, MM + root, MM + stem and MM + leaf. Then, mycelia were harvested after being induced for 0, 2, 4, 8, 12, 24, 48 and 72 h, and stored at −80 ◦ C for RNA extractions. Total RNAs were extracted from the mycelia using TRIzol Reagent (Invitrogen, California, USA) and then subjected to RTqPCR. The RT-qPCR was analyzed using the IQ5 real-time PCR detection system (Bio-Rad Laboratories Co., Ltd., Shanghai, China). The expression level of Asp55 was calculated from the threshold cycle according to the 2−CT (Livak and Schmittgen 2001). RNA samples in which the Asp55 expression level was highest in upregulation and lowest in down-regulation were used in RT-qPCR profiling of the transcription factors Myb1 to Myb15. The RT-qPCR primers are shown in Table 1 and Supplemental Table 1.

Table 1 Primers for RT-qPCR. Gene

Primers

Sequences (5 –3 )

Tm/◦ C

Size of product/bp

Asp55

Asp55-L Asp55-R MYB2-L MYB2-R ␣tu-L ␣tu-R ␤tu-L ␤tu-R Act-L Act-R

TAATGCCAACGCCGCCACTT GCCGCACACCACCATCTTCA TTCTTCCCAGGACGCAATTCAGG ATCGCAGCATCCCATTGGTGTAA CACATGGTTGACTGGTGCCCTA CTCGCCCTCTTCCATACCCTCT CAAACCGCCCTGTGCTCCAT TCGGCTGAGGCATCCTGGTAT AGGCAACCTTCTCGCCAACG TCGCTTCTCGACAATGCCAACT

59.0 59.0 59.3 59.1 58.6 59.0 59.0 58.9 59.0 58.9

254

MYB2(gw1.11.1172.1) ˛-tublin ˇ-tublin actin

259 240 245 256

Please cite this article in press as: Dou K, et al. Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536. Microbiol Res (2014), http://dx.doi.org/10.1016/j.micres.2014.04.006

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Fig. 1. The promoter sequence of aspartic protease gene Asp55 from T. asperellum ACCC30536.

2.4. Vector construction and E. coli transformation Primers containing restriction enzyme sites were designed to amplify the Asp55 cDNA sequence (KF976813). The forward primer asp3, 5 -ATCGGAATTCCCGCCGATGCAATGTTCCCACGG-3 , contains an EcoRI site. The reverse primer asp4, 5 CGATCTCGAGTTGCAATATTGACAACCAAAGCC-3 , contains a XhoI site. The recombinant vector pGEX-Asp55 was constructed and transformed into E. coli TOP10 competent cells (YPH-Bio Co., Ltd., Beijing, China). A positive clone was identified by colony PCR and plasmid restriction analysis. The positive recombinant plasmid pGEX-Asp55 was then transformed into E. coli BL21 to obtain the expression strain BL21-Asp55.

2.5. SDS-PAGE analysis and detection of recombinant aspartic protease rASP55 activity E. coli transformant BL21-Asp55 and control transformant BL21pGEX were induced following the procedures described in the Glutathione S-transferase (GST) Gene Fusion System Handbook (GE Healthcare UK Ltd, Buckinghamshire HP7 9NA, England). Isopropyl ˇ-d-1-thiogalactopyranoside (IPTG) was then added to LB culture medium at a final concentration of 1.0 mM. The E. coli cells were harvested after growing for 1, 2, 3, 4, and 5 h at 30 ◦ C. After adding 1× loading buffer, boiled for 5 min and centrifuged for 10 min at 8000 rpm, the supernatants were loaded into a slab gel of 10% SDSPAGE.

Please cite this article in press as: Dou K, et al. Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536. Microbiol Res (2014), http://dx.doi.org/10.1016/j.micres.2014.04.006

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Fig. 2. The protein family of ASP55 from T. asperellum ACCC30536 and multiple sequences alignment of 21 aspartic proteases. (a) Protein family of ASP55; (b) multiple sequences alignment; AGU16978: T. asperellum ACCC30536; EHK42884: T. atroviride IMI 206040; EHK22591: T. virens Gv29-8; ABK64120: T. harzianum; EGR49837: T. reesei QM6a; EGX96125: Cordyceps militaris CM01; EJP68415: Beauveria bassiana ARSEF 2860; EFY93202: Metarhizium acridum CQMa 102; EFZ01182: M. anisopliae ARSEF 23; EGU78232: Fusarium oxysporum Fo5176; XP 389018: F. graminearum PH-1; CCF37190: Colletotrichum higginsianum; EFQ26606: Glomerella graminicola M1.001; ACF20292: T.asperellum T4; AAT09023: T. asperellum T203; AAU11329: T. asperellum T203; CAL25572: T. harzianum CECT 2413; CAL30188: T. harzianum CECT 2413; CAL25579: T. harzianum CECT 2413; CAI91181: T. harzianum CECT 2413; Triat1:129404: T. atroviride IMI206040.

The transformant BL21-Asp55 was induced with 1.0 mM IPTG (final concentration) at 30 ◦ C. The induced cells were harvested after culturing for 30 min and then processed using the E. coli Protein Extraction Solution (HaiGene, Harbin, China) following the manufacturer’s instructions. The recombinant protein ASP55 (rASP55) was purified as described in the Glutathione S-transferase (GST) Gene Fusion System Handbook (GE Healthcare UK Ltd, Buckinghamshire HP7 9NA, England) and then used for enzymatic activity assays as described by Yang et al. (2013) and Lowry et al. (1951). Transformant BL21-pGEX was used as the control. 2.6. Antagonistic activity assay of purified rASP55 against A. alternata A 5 mm diameter disk of A. alternata mycelia was placed at the center of PDA medium that contained 10% (v/v) purified rASP55. As a control, purified rASP55 was heat-denatured and then added to PDA medium at a concentration of 10% (v/v). After cultivating for 5 days, the antagonistic activity was evaluated as described by Liu and Yang (2007). Five replicates were performed.

was also cloned. Many cis-elements known to be involved in phytopathogenic and environmental stress responses were identified in the Asp55 promoter (Fig. 1). The MYB transcription factor was predicted to bind to the cis-elements of the Asp55 promoter. 3.2. Multiple sequence alignment and phylogenetic tree The Pfam protein family prediction indicated that ASP55 belongs to the Asp family (PF00026) (Fig. 2a). A multiple sequence alignment showed that the catalytic motif “DTGS” is highly conserved in the 21 aspartic proteases (Fig. 2b). The phylogenetic tree showed that ASP55 was most closely related to the aspartic proteases from T. atroviride (EHK42884), T. virens (EHK22591), T. harzianum (ABK64120) and Trichoderma reesei (EGR49837), as shown in Branch 1 (Fig. 3). The aspartic proteases TaAsp (ACF20292) from T. asperellum strain T4 (Yang et al. 2013), and PapA (AAT09023) and PapB (AAU11329) from T. asperellum strain T203 (Viterbo et al. 2004) of Branch 3 (Fig. 3) were distantly related to ASP55. 3.3. Similar sequences to Asp55 from four different Trichoderma spp. genomes

3. Results 3.1. Cloning and sequence analysis of aspartic protease ASP55 The DNA sequence of Asp55 is 1920 bp in length. The cDNA sequence of Asp55 is 1602 bp, encoding 533 amino acids with a calculated molecular mass of 55.4 kDa and a predicted isoelectric point of 4.39. SignalP prediction showed that the ASP55 sequence is cleaved by a signal peptidase between positions 21 and 22 (AFA||AP). The 1500 bp length sequence in the promoter region

To locate genes similar to Asp55 from Trichoderma species used in biocontrol and determine the differences among them, we screened the genomes of four Trichoderma spp. that were reported to be biocontrol agents using the BlastP program within the JGI Genome Portal website. The Pfam protein family prediction indicated that they belonged to the Asp family (PF00026). We listed only three aspartic proteases in each Trichoderma genome (Table 2). The level of similarity between ASP55 and the 12 aspartic proteases ranged from 33 to 99%. The 12 aspartic proteases were acid proteins

Please cite this article in press as: Dou K, et al. Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536. Microbiol Res (2014), http://dx.doi.org/10.1016/j.micres.2014.04.006

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AGU16978 T. asperellum ACCC30536 (in this study)

98

EHK42884 T. atroviride IMI206040

100

EHK22591 T. virens Gv29-8

79 56

5

ABK64120 T. harzianum T88 88

EGR49837 T. reesei QM6a EGX96125 Cordyceps militaris CM01

60

Branch 1 (Motif “DSGT”)

EJP68415 Beauveria bassiana ARSEF 2860

100

EFY93202 Metarhizium acridum CQMa 102

71 100

EFZ01182 M. anisopliae ARSEF 23 EGU78232 Fusarium oxysporum Fo5176

100

XP 389018 Fusarium graminearum PH-1

100

CCF37190 Colletotrichum higginsianum EFQ26606 C. graminicola M1.001

100

CAL25572 T. harzianum CECT 2413

(P1324) (Suarez et al., 2007)

CAL30188 T. harzianum CECT 2413

64

Branch 2 (Motif “D-S/T-GS”)

(P7959) (Suarez et al., 2007)

ACF20292 T. asperellum T4 84

100

56 100

(TaAsp) (Yang et al., 2013) CAI91181 T. harzianum CECT 2413 (P6281) (Samolski et al., 2009) AAT09023 T. asperellum T203 (PapA) (Viterbo et al., 2004) Triat1:129404 T. atroviride IMI206040 (Seidl et al., 2009) AAU11329 T. asperellum T203 (PapB) (Viterbo et al., 2004) 100 CAL25579 T. harzianum CECT 2413 (P9438) (Suarez et al., 2007)

Branch 3 (Motif “DTGT”)

0.1 Fig. 3. Phylogenetic tree analysis of 21 aspartic protease. Phylogenetic tree was constructed using neighbor-joining method in MEGA 5.10 program. Bootstrap method was used for the test of phylogeny. The number of bootstrap replications was 1000.

by pI analysis. Only Asp1-Tas, Asp1-Tha, Asp1-Tat and Asp1-Tvi were predicted to have two introns, the others having only one intron (Table 2). The 13 aspartic proteases were divided into three groups according to the phylogenetic tree, with ASP55, ASP1-Tha, ASP1-Tat and ASP1-Tvi being classified into group 1 (Fig. 4). 3.4. Expression patterns of Asp55 and Myb1 to Myb15 in response to different culture conditions RT-qPCR was conducted to analyze the expression of Asp55 in T. asperellum ACCC30536 after being cultured in eight different conditions. The results indicated that the Asp55 gene was mainly

up-regulated in MM, MM + stem, MM + CW and MM + ferm, with the expression level peaking at 48, 24, 48, and 24 h, respectively. The peak expression level was 3.9-, 3.5-, 5.9- and 5.5-fold higher, respectively, than that of the control (Fig. 5). The Asp55 gene was down-regulated in MM-N, MM-C, MM + root and MM + leaf, with the lowest expression level occurring at 8, 12, 2, and 2 h, respectively. The expression level was 7.7, 15, 8.8 and 9.5%, respectively, lower than that of the control (Fig. 5). Furthermore, transcription factor gene Myb2 showed a similar regulatory modulation with Asp55 in all of the eight conditions, and Myb1, Myb4, Myb10, Myb12 showed similar regulation patterns in seven of the conditions (Table 3 and Supplemental Fig. 1). Interestingly, all of the 15

Table 2 The properties of protenase ASP from different Trichoderma spp. genomes. Similar sequences

Scaffold location

Similarity with ASP55/%

AA

pI

MM/kDa

Signal cleavage site

Introns

CD

Asp1-Tas Asp2-Tas Asp3-Tas Asp1-Tha Asp2-Tha Asp3-Tha Asp1-Tat Asp2-Tat Asp3-Tat Asp1-Tvi Asp2-Tvi Asp3-Tvi

scaffold 2:1067028−1068947(−) scaffold 5:839242−842214(−) scaffold 11:358958−360973(+) scaffold 1:1729021−1730911 (+) scaffold 3:2018752-2021117 (+) scaffold 7:59945-61383(+) contig 26:1764897−1766803 (−) contig 27:4021559−4023407 (+) contig 22:1544617−1545722 (−) scaffold 6:1584643−1586740 (−) scaffold 2:665631−667436 (+) scaffold 79:455631−456712 (+)

99 53 48 86 50 54 95 53 33 86 50 50

531 733 613 530 730 466 531 549 351 529 546 336

4.39 5.00 5.15 4.49 4.83 4.59 4.40 4.90 4.41 4.42 4.69 4.68

55.26 76.08 64.06 55.54 75.76 49.73 55.06 58.27 36.67 55.11 58.04 35.67

21 and 22: AEA-AD 17 and 18: AQALE 17 and 18: AQARP 18 and 19: AEAVV 17 and 18: AQALD — 21 and 22: AEAAD 17 and 18: AQALE — — 17 and 18: AQALD —

2 1 1 2 1 1 2 1 1 2 1 1

60–390 60–375 160–480 60–390 60–375 15–330 60–400 60–385 10–320 60–390 60–380 20–330

Asp1-Tas, Asp1 gene of T. asperellum CBS 433.97; Asp1-Tha, Asp1 gene of T. harzianum CBS 226.95; Asp1-Tat, Asp1 gene of T. atroviride ATCC74058; Asp1-Tvi, Asp1 gene of T. virens Gv29-8; AA, number of amino acids; MM, molecular mass; (+), plus strand; (−), minus strand; introns, number of introns; CD, putative conserved domain.

Please cite this article in press as: Dou K, et al. Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536. Microbiol Res (2014), http://dx.doi.org/10.1016/j.micres.2014.04.006

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100 ASP1-Tas

Myb genes (Myb1 to Myb15) were up-regulated at 24 h when cultured in MM + ferm and down-regulated at 2 h when cultured in MM + root (Table 3).

100

ASP55 ASP1-Tat

100

Group 1

ASP1-Tvi 92

3.5. SDS-PAGE analyses and enzymatic properties

ASP2-Tat

100

Compared with the control transformant BL21-pGEX, rASP55 showed one clearly visible protein band with a molecular mass of approximately 81.4 kDa in an SDS-PAGE gel (Fig. 6). This result indicated that the ASP55 protein had been successfully synthesized in the E. coli cells. Additionally, we measured the activity of rASP55. The highest activity was 9.52 U/ml at pH 5.5 and 30 ◦ C.

ASP1-Tha

ASP2-Tas

99

ASP3-Tha

99

100

ASP2-Tha ASP3-Tvi ASP3-Tas

100

3.6. Inhibition of purified rASP55 to A. alternata

0.1

Folds

Compared with those of the control, the mycelia of A. alternata expanded slowly in the PDA medium containing 10% (v/v) purified rASP55 (Fig. 7). The inhibition rate was 15.1%. However, the

a

Fig. 4. Phylogenetic tree of aspartic proteases from 4 biocontrol Trichoderma spp. genomes. Phylogenetic tree was constructed using neighbor-joining method in MEGA 5.10 program. Bootstrap method was used for the test of phylogeny. The number of bootstrap replications was 1000.

b

1.2 1 0.8 0.6 0.4 0.2

0

2

4

8

12

24

48

72

0

0

2

4

Induced time [h]

Folds

72

4

0.6

3

0.4

2 1 0

2

4

8

12

24

48

72

0

0

2

4

Induced time [h]

8

12

24

48

72

24

48

72

24

48

72

Induced time [h]

e

7

f

1.2

6

1

5

Folds

48

5

0.2

0.8

4

0.6

3

0.4

2

0.2

1 0

2

4

8

12

24

48

72

0

0

2

4

Induced time [h]

Folds

24

6

1

5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

12

d

7

0.8

0

8

Induced time [h]

c

1.2

0

Group 3

ASP3-Tat

100

5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

Group 2

ASP2-Tvi

8

12

Induced time [h]

g

h

1.2 1 0.8 0.6 0.4 0.2

0

2

4

8

12

Induced time [h]

24

48

72

0

0

2

4

8

12

Induced time [h]

Fig. 5. Differential expression of Asp55 from T. asperellum ACCC30536 in response to different treatment. X-axis: time points; Y-axis: folds = expression level of treatment/expression level at 0 h; (a) cultured in MM; (b) cultured in MM-C (depleting carbon glucose); (c) cultured in MM-N (depleting nitrogen ammonium sulfate); (d) induced by MM + CW (adding 1% Alternaria alternata cell wall); (e) induced by MM + ferm (adding 5% A. alternata fermentation liquid); (f–h) induced by MM + root, MM + stem and MM + leaf (adding 1% powder roots, stems and leaves of Populus dividiana × P. bolleana), respectively. All the experiments were performed three times.

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Table 3 Differential expression of transcription factor genes Myb1-Myb15 from T. asperellum ACCC30536.

Gene

con

MM-48h

MM-C-12h

MM-N-48h

MM+CW-48h

MM+Ferm-24h

MM+Root-2h

MM+Stem-24h

MM+Leaf-2h

Asp55

1

Myb1

1

3.89↑

0.15↓

0.33↓

5.95↑

5.53↑

0.09↓

3.48↑

0.09↓

5.65↑

1.46↑

Myb2

1

0.79↓

2.28↑

3.02↑

0.24↓

11.07↑

0.14↓

1.79↑

0.81↓

0.54↓

30.34↑

48.22↑

0.16↓

158.40↑

Myb3

0.07↓

1

5.26↑

5.59↑

1.88↑

9.51↑

16.17↑

0.07↓

5.98↑

0.53↓

Myb4

1

0.79↓

0.27↓

0.73↓

5.11↑

9.99↑

0.21↓

2.23↑

0.40↓

Myb5

1

2.87↑

0.75↓

1.18↑

2.20↑

2.19↑

0.17↓

0.99

0.54↓

Myb6

1

0.70↓

0.78↓

0.66↓

2.37↑

4.38↑

0.55↓

3.53↑

1.78↑

Myb7

1

1.99↑

3.72↑

2.64↑

0.85↓

5.47↑

0.35↓

2.75↑

0.95↓

Myb8

1

0.65↓

0.84↓

1.37↑

2.91↑

2.47↑

0.15↓

2.37↑

0.23↓

Myb9

1

0.83↓

0.53↓

0.84↓

0.62↓

1.32↑

0.61↓

0.67↓

1.05↑

Myb10

1

2.21↑

0.76↓

1.01↑

2.24↑

2.82↑

0.71↓

1.46↑

0.76↓

Myb11

1

6.49↑

23.40↑

1.12↑

112.00↑

1493.96↑

0.24↓

479.48↑

1.00

Myb12

1

4.06↑

0.37↓

0.83↓

2.51↑

1.15↑

0.52↓

0.56↓

0.83↓

Myb13

1

5.69↑

1.73↑

1.37↑

4.42↑

3.85↑

0.76↓

1.60↑

0.92↓

Myb14

1

2.34↑

1.38↑

0.72↓

0.37↓

1.46↑

0.51↓

0.85↓

1.93↑

Myb15

1

1.43↑

0.84↓

0.93↓

1.84↑

2.79↑

0.78↓

0.97

1.02↑

↑: Up-regulation; ↓: down-regulation; con: control ratio; numbers mean “expression level of treatment/expression level at 0 h”; numbers in frame mean “having the same trend with Asp55 in a same condition”; MM-48 h: induced by MM for 48 h; MM-C-12 h: induced by MM-C for 12 h; MM-N-48 h: induced by MM-N for 48 h; MM + CW-48 h: induced by MM + CW for 48 h; MM + ferm-24 h: induced by MM + ferm for 24 h; MM + root-2 h: induced by MM + root for 2 h; MM+stem-24 h: induced by MM + stem for 24 h; MM + leaf-2 h: induced by MM + leaf for 2 h.

sporulation quantity of A. alternata could not be inhibited by purified rASP55. 4. Discussion Aspartic proteases widely exist and show diversity in different Trichoderma species. From the results of genome searches, there were 18 different aspartic proteases in T. atroviride ATCC74058, 19 in T. harzianum CBS226.95, 19 in T. asperellum CBS433.97 and 17 in T. virens Gv29-8. The possible reason is that they act as synthetic in Trichoderma when it interacts with a plant or phytopathogen. Presently, the majority of these remain unstudied and only a few have been identified. In this study, we compared the novel ASP55 with some identified aspartic proteases (Viterbo et al. 2004; Liu and Yang 2007; Suárez et al. 2007; Martinez et al. 2008; Samolski et al. 2009; Seidl et al. 2009; Kubicek et al. 2011; Yang

et al. 2013). From the results of a multiple alignment (Fig. 2b), they all corresponded with Szecsi’s description (1992), which stated that aspartic proteases contained the characteristic catalytic motifs “F/I/L-D-T-G-S” and “D-T/S-G-S/T” in the region of the two catalytic aspartic residues. In the 21 aspartic proteases compared, the first catalytic motif “DTGS” was highly conserved and the second catalytic motif “D-T/S-G-S/T” was presented in three forms, “DSGT”, “DTGT”, or “D-S/T-GS” (Fig. 2b). The differences in the second catalytic motif were related to their evolutionary relationship, “DSGT”, “D-S/T-GS” and “DTGT” occurred in the proteases of Branch 1, Branch 2 and Branch 3, respectively (Fig. 3). Through comparison, the second catalytic motif could be used as a characteristic to identify different aspartic proteases in Trichoderma spp. and ASP55 was identified to be a novel aspartic protease in T. asperellum. The transcription level of aspartic proteases in Trichoderma spp. was related to the nutritional content of the culture medium.

Fig. 6. SDS-page analysis of recombinant protein ASP55 from BL21-Asp55. (a) Unpurified protein; (b) purified protein; 1, 3, 5: the supernatant of transformant BL21-Asp55 induced for 2, 3, 4 h; 2, 4, 6: E. coli cells of transformant BL21-Asp55 induced for 2, 3, 4 h; 7: the supernatant of control transformant BL21-pGEX induced for 4 h; 8: the E. coli cells of control transformant BL21-pGEX induced for 4 h; 9, 11: protein marker; 10: purified ASP55.

Please cite this article in press as: Dou K, et al. Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536. Microbiol Res (2014), http://dx.doi.org/10.1016/j.micres.2014.04.006

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Fig. 7. Inhibition of purified rASP55 to A. alternata. alternata was cultured on PDA plates for five days. (a) PDA medium was supplemented with 10% (v/v) rASP55; (b) PDA medium was supplemented with 10% (v/v) heat-denatured rASP55.

Northern analysis showed no detectable aspartic protease gene SA76 signal in T. harzianum cultured with glucose (2%) and ammonium (5 g/L) as the carbon and nitrogen sources, respectively, while under carbon or nitrogen starvation the signal could be observed but was weak (Liu and Yang 2007). In this study, the expression level of Asp55 became up-regulated after 24 h in MM conditions (Fig. 5a). Glucose and ammonium sulfate as the carbon and nitrogen sources, respectively, were essential for the expression of Asp55, which was markedly down-regulated under MM-C and MM-N conditions at 2 h and remained depressed until 72 h (Fig. 5b and c). From the result of previous studies, aspartic proteases could be induced by phytopathogens and plants. Atanasova et al. (2013) investigated the genome-wide transcriptional response of Trichoderma during an interaction with R. solani. The result was that aspartic proteases were consistently up-regulated in T. atroviride and T. virens during the interaction process (Atanasova et al. 2013). In other studies, aspartic proteases P1324, P7959 and P9438 of T. harzianum CECT 2413 were induced by B. cinerea cell walls (Suárez et al. 2007) and Triat1:129404 of T. atroviride was up-regulated during a confrontation of T. atroviride with R. solani (Seidl et al. 2009). Compared with other aspartic proteases, Asp55 was also upregulated in the interaction of T. asprerellum with phytopathogenic A. alternata (Fig. 5d and e). Thus, ASP55 may play a role in the interaction of T. asperellum with A. alternata. The antagonistic activity assay further verified the role of ASP55. In the Trichoderma–plant interaction, the expression of Asp55 was down-regulated when induced by a 1% root and leaf powder of P. dividiana × P. bolleana (Fig. 5f and g). However, Viterbo et al. (2004) thought that the two aspartic proteases PapA and PapB of T. asparellum T-203 were induced in response to plant root attachments. Samolski et al. (2009) revealed that an aspartic protease P6281 of T. harzianum CECT 2413 was significantly up-regulated in the interaction with tomato plants. Compared with the former investigations, our result was different. One possible reason is that the plant root used in this study is not bioactive, and thus cannot induce the expression of Asp55. Another reason may be due to the differences in plant materials. Previous research used herbaceous plants, but our experiment used the woody plant Populus. Interestingly, although the root powder had no bioactivity, the 1% stem powder can induce the up-regulation of Asp55 in our study (Fig. 5h). This may be due to the induction of some bioactive molecules in stems, which are different from those in the roots and leaves. Studies on transcription factors in Trichoderma spp. has mainly been carried out in T. reesei and focused on transcription factors of cellulose degrading-related genes, such as studies of a

regulator of cellulose and xylanase genes (ACEII, zinc binuclear cluster) (Aro et al. 2001) and a beta-glucosidase regulator (BglR, Zn(II)2Cys6 binuclear cluster) (Nitta et al. 2012). However, no transcription factors of aspartic proteases in Trichoderma species were identified. Since approximately seven MYB recognition sites were identified in the promoter region of Asp55 (Fig. 1), MYB may be a transcription factor of Asp55. RT-qPCR analysis was conducted to determine if there was a correlation between Asp55 and Myb at the transcriptional level. This was a preliminary screening for transcription factors and further experiments by yeast one-hybrid or other methods are being performed. In a previous study, a 55 kDa aspartic protease SA76 from T. harzianum was successfully expressed in Saccharomyces cerevisiae, and recombinant SA76 showed direct activity against phytopathogens. The inhibition rate of mycelial growth was 38% for Fusarium oxysporum, 21% for R. solani, 19% for Valsa sordida Nit, 9% for Phytophthora sojae and 7% for S. sclerotiorum (Liu and Yang 2007). Another aspartic protease, TAASP from T. asperellum was successfully expressed in Pichia pastoris and also showed inhibition against the mycelial growth of phytopathogens (Yang et al. 2013). The inhibition rate was 8.0% for F. oxysporum, 10.9% for A. alternata, 16.6% for Cytospora chrysosperma, 22.8% for S. sclerotiorum and 18.2% for R. solani. In our study, purified rASP55 can inhibit the mycelial growth of the phytopathogen A. alternata. The aspartic protease ASP55 from Trichoderma has biocontrol functions and the recombinant aspartic protease could antagonize phytopathogens by inhibiting mycelial growth. 5. Conclusion In this study, a novel aspartic protease gene Asp55 was cloned from T. asperellum ACCC30536, which has biocontrol applications. The transcription of Asp55 could be induced by cell wall and fermentation liquid of phytopathogenic A. alternata. Asp55 was also successfully expressed in E. coli BL21. The purified rASP55 showed protease activity and inhibited the mycelial growth of A. alternata. This work would help us learn more about the role of aspartic proteases in biocontrol and provide a practical reference for applications of the genus Trichoderma. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (NSFC: 31170601), Chinese National

Please cite this article in press as: Dou K, et al. Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536. Microbiol Res (2014), http://dx.doi.org/10.1016/j.micres.2014.04.006

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Scientific Research Special Project in Forestry Public Industry (201104069), Heilongjiang Province Post Doctorate Science Foundation (LBH-Q10156) and Science and Technology Innovation Talents Foundation of Harbin City of China (2011RFQXN051). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.micres.2014.04.006. References Aro N, Saloheimo A, Ilmén M, Penttilä M. ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei. J Biol Chem 2001;276(26):24309–14. Atanasova L, Le Crom S, Gruber S, Coulpier F, Seidl-Seiboth V, Kubicek CP, Druzhinina IS. Comparative transcriptomics reveals different strategies of Trichoderma mycoparasitism. BMC Genomics 2013;14(1):1–15. Benítez T, Rincón AM, Limón MC, Codón AC. Biocontrol mechanisms of Trichoderma strains. Int Microbiol 2004;7:249–60. De Marco JL, Felix CR. Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches’ broom disease. BMC Biochem 2002;3(1):3. Elad Y, Kapat A. The role of Trichoderma harzianum protease in the biocontrol of Botrytis cinerea. Eur J Plant Pathol 1999;105(2):177–89. Elad Y, Freeman S, Monte E. Biocontrol agents: mode of action and interaction with other means of control. IOBC/WPRS Bull 2001;24(3). Faisal M, Alatar AA, Ahmad N, Anis M, Hegazy AK. An efficient and reproducible method for in vitro clonal multiplication of Rauvolfia tetraphylla L. and evaluation of genetic stability using DNA-based markers. Appl Biochem Biotechnol 2012;168(7):1739–52. Flores A, Chet I, Herrera-Estrella A. Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinase-encoding gene prb1. Curr Genet 1997;31(1):30–7. Geremia RA, Goldman GH, Jacobs D, Ardrtes W, Vila SB, Van Montagu M, Herrera-Estrella A. Molecular characterization of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum. Mol Microbiol 1993;8(3):603–13. Kakvan N, Heydari A, Zamanizadeh HR, Rezaee S, Naraghi L. Development of new bioformulations using Trichoderma and Talaromyces fungal antagonists for biological control of sugar beet damping-off disease. Crop Prot 2013;53(0):80–4. Kapat A, Zimand G, Elad Y. Effect of two isolates of Trichoderma harzianum on the activity of hydrolytic enzymes produced by Botrytis cinerea. Physiol Mol Plant Pathol 1998;52(2):127–37. Kubicek C, Herrera-Estrella A, Seidl-Seiboth V, Martinez D, Druzhinina I, Thon M, Zeilinger S, Casas-Flores S, Horwitz B, Mukherjee P, Mukherjee M, Kredics L, Alcaraz L, Aerts A, Antal Z, Atanasova L, Cervantes-Badillo M, Challacombe J, Chertkov O, McCluskey K, Coulpier F, Deshpande N, von Dohren H, Ebbole D, Esquivel-Naranjo E, Fekete E, Flipphi M, Glaser F, Gomez-Rodriguez E, Gruber S, Han C, Henrissat B, Hermosa R, Hernandez-Onate M, Karaffa L, Kosti I, Le Crom S, Lindquist E, Lucas S, Lubeck M, Lubeck P, Margeot A, Metz B, Misra M, Nevalainen H, Omann M, Packer N, Perrone G, Uresti-Rivera E, Salamov A,

9

Schmoll M, Seiboth B, Shapiro H, Sukno S, Tamayo-Ramos JA, Tisch D, Wiest A, Wilkinson H, Zhang M, Coutinho P, Kenerley C, Monte E, Baker S, Grigoriev I. Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma. Genome Biol 2011;12(4):R40. Liu Y, Yang Q. Cloning and heterologous expression of aspartic protease SA76 related to biocontrol in Trichoderma harzianum. FEMS Microbiol Lett 2007;277(2):173–81. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001;25(4):402–8. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1):265–75. Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE, Chapman J, Chertkov O, Coutinho PM, Cullen D, Danchin EG, Grigoriev IV, Harris P, Jackson M, Kubicek CP, Han CS, HoI. Larrondo LF, de Leon AL, Magnuson JK, Merino S, Misra M, Nelson B, Putnam N, Robbertse B, Salamov AA, Schmoll M, Terry A, Thayer N, Westerholm-Parvinen A, Schoch CL, Yao J, Barabote R, Nelson MA, Detter C, Bruce D, Kuske CR, Xie G, Richardson P, Rokhsar DS, Lucas SM, Rubin EM, DunnColeman N, Ward M, Brettin TS. Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat Biotechnol 2008;26(5):553–60. Morissette J, Laurin N, Brown JP. Sequestosome 1: mutation frequencies, haplotypes, and phenotypes in familial Paget’s disease of bone. J Bone Miner Res 2006;21(S2):P38–44. Nitta M, Furukawa T, Shida Y, Mori K, Kuhara S, Morikawa Y, Ogasawara W. A new Zn(II)2Cys6-type transcription factor BglR regulates ␤-glucosidase expression in Trichoderma reesei. Fungal Genet Biol 2012;49(5):388–97. Qualhato T, Lopes F, Steindorff A, Brandão R, Jesuino R, Ulhoa C. Mycoparasitism studies of Trichoderma species against three phytopathogenic fungi: evaluation of antagonism and hydrolytic enzyme production. Biotechnol Lett 2013;35(9):1461–8. Samolski I, de Luis A, Vizcaíno J, Monte E, Suárez MB. Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray. BMC Microbiol 2009;9(1):217. Seidl V, Song L, Lindquist E, Gruber S, Koptchinskiy A, Zeilinger S, Schmoll M, Martínez P, Sun J, Grigoriev I, Herrera-Estrella A, Baker SE, Kubicek CP. Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of a fungal prey. BMC Genomics 2009;10(1):567. Suárez MB, Vizcaíno JA, Llobell A, Monte E. Characterization of genes encoding novel peptidases in the biocontrol fungus Trichoderma harzianum CECT 2413 using the TrichoEST functional genomics approach. Curr Genet 2007;51(5):331–42. Szecsi PB. The aspartic proteases. Scand J Clin Lab Invest Suppl 1992;210:5–22. Tchameni SN, Ngonkeu MEL, Begoude BAD, Wakam Nana L, Fokom R, Owona AD, Mbarga JB, Tchana T, Tondje PR, Etoa FX, Kuaté J. Effect of Trichoderma asperellum and arbuscular mycorrhizal fungi on cacao growth and resistance against black pod disease. Crop Prot 2011;30(10):1321–7. Viterbo A, Harel M, Chet I. Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots. FEMS Microbiol Lett 2004;238(1):151–8. Wijesinghe CJ, Wijeratnam RSW, Samarasekara JKRR, Wijesundera RLC. Development of a formulation of Trichoderma asperellum to control black rot disease on pineapple caused by (Thielaviopsis paradoxa). Crop Prot 2011;30(3): 300–6. Yang X, Cong H, Song J, Zhang J. Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris. World J Microb Biotechnol 2013;29(11):2087–94.

Please cite this article in press as: Dou K, et al. Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536. Microbiol Res (2014), http://dx.doi.org/10.1016/j.micres.2014.04.006