Plant and Cell Physiology Advance Access published May 5, 2015
The Eucalyptus-Leptocybe invasa Interaction
The Transcriptome And Terpene Profile Of Eucalyptus grandis Reveals Mechanisms Of Defence Against The Insect Pest, Leptocybe invasa
Corresponding author: Dr S. Naidoo Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research
Tel: +27 12 420 4974 Email: [email protected]
Subject area: Environmental and stress responses
Number of figures: 2 (2X colour) Number of tables: 2 (1X black and white, 1X colour) Number of supporting materials: 8 Number of figures: 4 (3X black and white, 1X colour) Number of tables: 4 (3X black and white, 1X colour)
© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: [email protected]
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Institute (GRI), University of Pretoria, Private bag x20, Pretoria, 0028, South Africa
The Eucalyptus-Leptocybe invasa Interaction
The Transcriptome and Terpenoid Profile Of Eucalyptus grandis Reveals Mechanisms Of Defence Against The Insect Pest, Leptocybe invasa
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Caryn N Oates , Carsten Külheim , Alexander A Myburg , Bernard Slippers and Sanushka Naidoo
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Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research
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Research School of Biology, Australian National University, 116 Daley Rd, Canberra, 0200, ACT,
Australia
Abbreviations
ANOVA: analysis of variance BGI: Beijing Genomics Institute BLAST: Basic Local Alignment Search Tool cmk: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase-encoding gene dxr: deoxyxylulose-5-phosphate reductase-encoding gene dxs: deoxyxylulose-5-phosphate synthase-encoding gene EgARF: Eucalyptus grandis ADP ribosylation factor-encoding gene EgARP: Eucalyptus grandis auxin responsive protein-encoding gene EgCAD: Eucalyptus grandis cinnamyl-alcohol dehydrogenase-encoding gene EgDIR: Eucalyptus grandis disease resistance-responsive dirigent-like protein-encoding gene EgEFE: Eucalyptus grandis ethylene-forming enzyme-encoding gene EgFBA: Eucalyptus grandis fructose bisphosphate aldolase-encoding gene EgOMT: Eucalyptus grandis O-methyltransferase-encoding gene
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Institute (GRI), University of Pretoria, Private bag x20, Pretoria, 0028, South Africa
Egr: Eucalyptus grandis clone FPKM: fragments per kilobase of transcript per million fragments mapped GC/MS: gas chromatography coupled to mass spectrometry GC: Eucalyptus grandis x Eucalyptus camaldulensis hybrid GO: gene ontology gpps: geranylgeranyl pyrophosphate synthase-encoding gene hdr: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase-encoding gene hds: 4-hydroxy-3-methylbut-2-enyl diphosphate synthase-encoding gene
hmgs: hydroxymethylglutaryl-CoA synthase-encoding gene ippi: isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase-encoding gene JA: jasmonic acid mcs: 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase-encoding gene MEP pathway: methylerythritol phosphate pathway MIQE: Minimum Information for Publication of Real-Time PCR Experiments MVA pathway: mevalonate pathway RNA-Seq: RNA sequencing RT-qPCR: real time quantitative polymerase chain reaction TPS: terpene synthase-encoding gene
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hmgr: hydroxymethylglutaryl-CoA reductase-encoding gene
Abstract
Plants have evolved complex defences that allow them to protect themselves against pests and pathogens. However, there is relatively little information regarding the Eucalyptus defensome. Leptocybe invasa is one of the most damaging pests in global Eucalyptus forestry and essentially nothing is known regarding the molecular mechanisms governing the interaction between the pest and host. The aim of the study was to investigate changes in the transcriptional landscape and terpene profile of a resistant and susceptible Eucalyptus genotype in an effort to improve our understanding of this interaction. We used
validated using RT-qPCR. Terpene profiles were investigated using gas chromatography coupled to mass spectometry on uninfested and oviposited leaves. We found 698 and 1115 significantly differentially expressed genes from the resistant and susceptible interactions, respectively. Gene Ontology enrichment and Mapman analyses identified putative defence mechanisms including cell wall reinforcement, protease inhibitors, cell cycle suppression and regulatory hormone signalling pathways. There were significant differences in the mono- and sesquiterpene profiles between genotypes and between control and infested material. A model of the interaction between Eucalyptus and L. invasa was proposed from the transcriptomic and chemical data.
Keywords
Defence response, Eucalyptus, Leptocybe invasa, RNA-Seq, terpene profile
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RNA-Seq to investigate transcriptional changes following L. invasa oviposition. Expression levels were
Introduction
Plants have evolved a complex, multi-layered system of defences to protect themselves against insect pests. These include physical or chemical barriers with toxic, repellant or anti-nutritive properties (Mithöfer and Boland, 2012). These defences may be further divided into constitutive or inducible types. Constitutive defences form the plant’s first line of defence and provide generalised protection against most potential attackers (Fürstenberg-Hägg et al., 2013). The inducible defences comprise a multifaceted, broad-spectrum system that is sequentially activated following invader recognition (Jones
pathways through a sophisticated network that ensures the most appropriate response is elicited (Tena et al., 2011). Inducible defences include the oxidative burst, synthesis of secondary metabolites and defence-associated proteins, barrier reinforcement and indirect defences (Fürstenberg-Hägg et al., 2013). Suppression of the plant’s primary metabolism and cell cycle are additional responses that have been described as responses to various pests (Rawat et al., 2012). The efficacy of the inducible defence response is dependent on the speed and magnitude of the induction resulting in either plant resistance or susceptibility to the attacker (Jones and Dangl, 2006).
Terpenoids are a common and diverse group of plant secondary metabolites that play important roles in all aspects of their life cycles (Mithöfer and Boland, 2012, Padovan et al., 2013). The role of terpenoids in plant defence is well-established and alterations in terpene profiles following pest or pathogen attack have been widely reported. These compounds are also important signalling molecules and are involved in plant priming (Troncoso et al., 2012), attracting natural enemies of the pest (Bruinsma et al., 2009) and repelling enemies of the plant (Martín et al., 2014). A defining characteristic of the focal genus of this study, Eucalyptus, are the high foliar concentrations of terpenes that it is able to produce along with the largest number of terpene synthase-encoding genes reported in plants to date (Padovan et al., 2013, Myburg et al., 2014). Our understanding of the role played by terpenes in plant-galling insect interactions
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and Dangl, 2006, Pieterse et al., 2012). The recognition signal is transferred to downstream defence
is limited and studies addressing this knowledge gap will illuminate the role of these compounds in plant defence.
Gall-inducing insects include some of the most devastating pests in agriculture and forestry, for example Phylloxera aphids on grapes (Nabity et al., 2013) and Mayetiola destructor (Hessian fly) on wheat (Stuart et al., 2012). Galls are abnormal plant tissue structures that are induced and maintained by the insect and provide both nourishment and protection (Inbar et al., 2010). Gall development occurs through a process of tissue redifferentiation where a change in the identity of a host plant cell results in the formation of a
assume control of its host’s cellular machinery is such that the physiology, morphology, anatomy, development and chemistry are altered in favour of the pest (Inbar et al., 2010, Oliveira and Isaias, 2010, Compson et al., 2011). Furthermore, the insect is capable of avoiding or actively suppressing the host’s immune system, thus reducing its exposure to toxic chemicals and preventing the release of volatile compounds that may trigger indirect defences (Tooker and De Moraes, 2008). The mechanism of gall development remains poorly understood. Work in model systems such as the interaction between the Hessian fly and wheat are starting to unravel this process (Stuart et al., 2012). For example, a study of the Hessian fly salivary gland detected numerous secreted salivary gland protein transcripts that allegedly encode effectors and may provide the means for the insect to manipulate its host (Chen et al., 2004).
A relatively recently described galling insect, Leptocybe invasa Fisher and La Salle (Hymenoptera: Eulophidae), has emerged as one of the most damaging pests of global Eucalyptus forestry resulting in the complete failure of some industrially important clones (Nyeko et al., 2009, Nyeko et al., 2010, DittrichSchröder et al., 2012). Eucalyptus species constitute some of the most widely grown and economically valuable plantation trees in the world (Grattapaglia et al., 2012). This pest was first described in Israel in 2000 following the extensive damage it caused in the region and has since spread to Africa, the Mediterranean Basin, South East Asia, Europe and South America (Mendel et al., 2004, Kim et al., 2009, Nyeko et al., 2009, Thu et al., 2009, Kumari et al., 2010). Leptocybe invasa is an Australian gall-forming
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new, gall-specific cell type with specialized functions (Oliviera and Isaias, 2010). The ability of a galler to
wasp that preferentially oviposits on immature leaf and stem tissue (Mendel et al., 2004). The larvae are endophytic herbivores that cause the development of coalescing galls on young leaves, petioles and twigs of susceptible trees. An infestation can have devastating effects on a plantation as it may result in stunted growth, die-back and death of infested trees (Nyeko et al., 2009, Thu et al., 2009, Kumari et al., 2010).
Biological control is currently considered to be the key tool in controlling this pest (Wingfield et al., 2008, Kim et al., 2009, Kulkarni et al., 2010). A number of parasitoid wasp species have been introduced as
Quadrastichus mendeli and Selitrichodes kryceri in Israel (Kim et al., 2009). Furthermore, varying degrees of tolerance and resistance are observed among species and genotypes of Eucalyptus indicating the potential for using resistant planting stock in affected areas (Nyeko et al., 2009, Dittrich-Schröder et al., 2012). Defence mechanisms providing resistance or tolerance to L. invasa are essentially unknown and, therefore, restrict the design of biotechnological strategies that can be used against the pest.
This study aims to describe the responses that govern the interaction between L. invasa and Eucalyptus by comparing the transcriptional landscape and terpene profile changes that take place during a resistant and susceptible interaction. RNA-sequencing (RNA-Seq) is a next-generation sequencing technique that allows rapid and accurate profiling of total RNA (Mortazavi et al., 2008). We show that RNA-Seq of Eucalyptus mRNA provides a robust means for investigating induced transcriptional responses in Leptocybe-challenged plants. This study identified genes that showed differential expression profiles in response to L. invasa oviposition. Categorisation and enrichment analysis of these genes identified putative defence mechanisms that are employed by Eucalyptus in response to the gall wasp. Analysis of the mono- and sesquiterpene profiles showed distinct differences between the genotypes before and after oviposition. This information was used to propose a model of the defence response of the host. Improving the current knowledge of the induced transcriptional responses between E. grandis and the invasive gall wasp will lead to improved and integrated control strategies by illuminating key defence mechanisms that
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biological control agents of L. invasa including Selitrichodes neseri in South Africa (Kelly et al., 2012), and
may be manipulated to improve resistance. This information, in combination with biological control and silvicultural practices will help minimise the losses caused by L. invasa.
Results
Infestation of Eucalyptus
Infested and uninfested leaves were collected from an E. grandis clone (Egr, Mondi, RSA) and an E.
challenged groups showed L. invasa oviposition, particularly along the midribs (Figure S1). To confirm the resistant and susceptible identity of the Egr and GC clones, ramets were observed for gall development. The susceptible clone showed extensive gall development, whereas the resistant genotype showed signs of oviposition but no subsequent galling. Total RNA obtained from the infested midrib had high RNA quality scores following Experion analyses. This material was considered suitable for RNA-Seq analysis.
RNA-Sequencing Data Analysis
RNA-Seq of the resistant and susceptible samples yielded 23-29 million and 38-46 million paired-end reads, respectively (Table S2). The resistant and susceptible samples were collected concurrently but sequenced in different batches, which accounts for the differences in read number. Good quality scores were obtained for all samples (data not shown). The reads were mapped to the E. grandis v1.0 genome available at www.phytozome.net. Cufflinks identified 28,703-29,850 and 30,541-31,251 genes in Egr and GC, respectively, that showed expression values (fragments per kilobase of transcript per million fragments mapped, FPKM) greater than 0 and were considered as expressed (Table S2). Cuffdiff identified 698 Egr and 1115 GC significantly differentially expressed genes relative to their respective controls. The resistant interaction showed 504 up-regulated and 194 down-regulated genes; the susceptible interaction showed 665 up-regulated and 450 down-regulated genes (Figure S2). The two
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grandis x E. camaldulensis hybrid clone (GC, Mondi) after seven days exposure to L. invasa. All of the
interactions show 440 common differentially expressed genes that possess a 0.90 correlation coefficient. Gene expression was validated by RT-qPCR analysis and showed a correlation coefficient of 0.91 (Figure S3). The subsequent analyses required genes annotated with an Arabidopsis thaliana ortholog. The resistant interaction included 406 annotated genes and the susceptible interaction included 645 annotated genes (Figure S2). These datasets are available as Table S3.
Gene Ontology Categorisations
genes was investigated to identify putative biological pathways involved in the interactions. Figure 1 summarises the enriched terminal nodes for each of the interactions. The terminal nodes describe more specific associated activity than their parent terms and thus provide a greater level of detail regarding the putative defence mechanisms. Genes from the resistant and susceptible interactions were divided into 32 and 38 enriched GO terms, respectively, with 19 common between them (Figure 1). In general, the gene ontologies that were enriched in the resistant Egr outline a range of defence mechanisms that may be involved in resistance to L. invasa. For example, “terpenoid biosynthetic process” and “flavonoid biosynthetic process” imply that the synthesis of secondary metabolites is involved. Observation of the terpenoid-related gene ontologies in the up-regulated, resistant dataset prompted the investigation into the Eucalyptus terpene metabolism-related gene expression profiles and levels of mono- and sesquiterpenes in the two genotypes. The susceptible interaction, as expected, also included a number of gene ontologies that appear to be unsuccessful defence mechanisms. However, this interaction 220 contained more terms that were related to primary metabolic functions than did the resistant interaction.
Terpenoid Metabolism-Related Gene Expression
The expression profiles of terpenoid metabolism-related genes were compared between the resistant and susceptible genotypes and any significant differences between them are shown (Table 1). Many of these
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The gene ontology (GO) biological processes functional categorization of the differentially expressed
genes are involved in the monoterpene biosynthesis through the methylerythritol phosphate (MEP) pathway including deoxyxylulose-5-phosphate synthase (dxs), deoxyxylulose-5-phosphate reductase (dxr),
4-diphosphocytidyl-2-C-methyl-D-erythritol
kinase
(cmk),
2-C-methyl-D-erythritol-2,4-
cyclodiphosphate synthase (mcs), 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (hds) and 4hydroxy-3-methylbut-2-enyl diphosphate reductase (hdr) (McGarvey and Croteau, 1995, Külheim et al., 2011). Additionally, two of these genes are involved in sesquiterpene biosynthesis through the mevalonate
(MVA)
pathway
namely
hydroxymethylglutaryl-CoA
synthase
(hmgs)
and
hydroxymethylglutaryl-CoA reductase (hmgr) (Külheim et al., 2011). Downstream of the MEP and MVA are
isopentenyl
pyrophosphate:dimethylallyl
pyrophosphate
isomerase
(ippi)
and
geranylgeranyl pyrophosphate synthase (gpps) that show significant differential expression (Külheim et al., 2011). The majority of genes that have been implicated in the up-regulation of terpenes were induced in both resistant and susceptible samples (Webb et al., 2013). There are six terpene synthase genes, encoding enzymes that catalyse the final step in the biosynthetic pathway, that showed significant differential expression in this study, four of them in the resistant interaction and four in the susceptible interaction (Table 1). Five of the terpene synthase genes belong to the TPSa subfamily that synthesises sesquiterpenes and one (Eucgr.K00881) belongs to the TPSb subfamily that synthesises monoterpenes. This gene was significantly up-regulated in Egr and down-regulated in GC, although this was not significant. These expression patterns indicate that the pathway is likely activated in both interactions and may lead to alterations in the terpene profiles of the two genotypes.
Terpenoid Chemical Profiling
The resistant genotype contained higher concentrations of monoterpenes, Egr had 16.05±2.61 mg/g dry weight and 12.56±1.35 mg/g dry weight in GC. Following L. invasa oviposition, Egr monoterpene levels increased to 16.60±2.56 mg/g dry weight, while the GC levels increased to 12.61±1.28 mg/g dry weight. Total sesquiterpene content in uninfested Egr was approximately four times higher than in GC (0.26±0.05 mg/g dry weight and 0.06±0.01 mg/g dry weight). There was a small increase in total sesquiterpene
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pathways
concentration in challenged Egr (0.32±0.06 mg/g dry weight), whereas there was a three-fold increase in sesquiterpenes in GC (0.17±0.03 mg/g dry weight). These increases in mono- and sesquiterpene concentrations seem to mirror the up-regulation of the terpene metabolism-related gene expression profiles that were observed. The two-way ANOVA analysis showed that there were distinct differences in the mono- and sesquiterpenes in both genotypes (Table 2, Figure S4). There is also an induced response of specific terpenoids to L. invasa infestation. A Student’s T-test was performed for each terpene measured to determine where the differences were located (Figure S4). It is clear from these results that there are specific terpene profiles in each genotype. For most monoterpenes, the compounds were
Interestingly, four of the six sesquiterpenes were present in either Egr or GC, but not both. The remaining two cases, viridiflorene and α-gurjuene, showed varying concentrations in one genotype compared to the other.
Mapman Gene Categorisation
The annotated genes were further analysed with Mapman to visualise the genes on maps of cellular processes. These categorisations provided an additional line of evidence for the putative responses involved in the Eucalyptus-L. invasa interaction. The results from Mapman generally supported the GO results with some additional categories being highlighted such as signalling and transcriptional regulators (Figure 2). This analysis was performed to help refine the proposed model of Eucalyptus mechanisms employed during the defence response.
Discussion
The purpose of this study was to investigate transcriptional and chemical changes in resistant and susceptible Eucalyptus genotypes induced by L. invasa oviposition. This comparison provided insight and generated numerous hypotheses into the putative host cellular processes that govern the relationship.
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identified in both interactions, but at different levels, both between genotypes and after infestation.
These were differences in transcripts related to phytohormones, cell wall modifications, cell cycle and secondary metabolites and differences in the terpenoid profiles, which we suggest contribute to resistance in Egr.
Phytohormones
Phytohormones play an important role in the regulation of defences. A complex network governs the relationship and communication between the different hormones which provides a powerful regulatory
six hormone metabolic pathways showed differential expression in this study (Figure 2). These include abscisic acid, auxins, brassinosteroids, ethylene, jasmonic acid (JA) and salicylic acid. These hormones have been associated with plant defence and, interestingly, with gall development. Two of these, JA and auxin, showed different expression profiles between Egr and GC.
The role of up-regulated JA metabolic genes in Egr is consistent with this hormone’s well-described defence function in regulating resistance against phytophagous insects (Wasternack and Hause, 2013). These genes are also up-regulated in GC where their role is less clear. Tooker and Helms (2014) proposed that galling insects actively manipulate phytohormones such that the success or failure of gall development could be dependent on the manner of hormonal interactions.
We observed an up-regulation of auxin metabolism-related genes in both Egr and GC. Auxins have been shown to be involved in insect gall development across interactions involving different species (Tooker et al., 2011, Bartlett and Connor, 2014, Bedetti et al., 2014). Auxins may contribute to the production of gallspecific tissues in the susceptible interaction. In the resistant interaction, manipulation by L. invasa may cause the observed changes in gene expression; however, there is currently no evidence to support this.
Defence Mechanisms
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potential for fine-tuning responses to abiotic and biotic stresses (Pieterse et al., 2012). Genes related to
Downstream of hormone signalling pathways, the inducible defences result in activation or suppression of a combination of responses. One of these that demonstrated differences in the expression profiles of genes between the resistant and susceptible interactions was cell wall metabolism (Figures 1, 2). Cell wall reinforcement through lignin or suberin deposition has been suggested as a defence mechanism against galling insects by preventing the establishment of a feeding site (Liu et al., 2007). Alternatively, galls are composed of heavily lignified tissue that creates a protective microenvironment for the insect (Oliviera and Isaias, 2010). This result is an example of a pathway that is likely employed in both resistant
A second defence mechanism that displayed interesting differences between Egr and GC was the expression profiles of eleven protease inhibitors (Figure 2, Table S4). All of these genes showed similar fold changes in the two genotypes. However, five showed significant differences in the uninfested absolute FPKM values and eight showed significant differences in the absolute infested FPKM values, six of which were higher in Egr than GC. The higher levels of protease inhibitor transcripts may allow for an increased rate of protein synthesis in the resistant interaction and thus a more effective defence response. Protease inhibitors act by blocking insect gut proteases thereby reducing the ability of the insect to digest plant material (Bode et al. 2013). The up-regulation of protease-encoding genes and genes encoding other defence-related proteins, such as lectins and the pathogenesis-related proteins, are commonly reported plant responses to herbivorous insects (Wu et al., 2008, Hartl et al., 2010, Sinha et al., 2011, Bode et al., 2013). The synthesis of protease inhibitors is likely an important defence mechanism in the Eucalyptus-L. invasa interaction.
Suppression of the Cell Cycle
The mechanism of gall development, although poorly understood, requires tissue redifferentiation. Oliviera and Isaias (2010) defined tissue redifferentiation as the process whereby novel cell types with
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and susceptible interactions but with different functions in each.
specialised functions are formed following a change in native cell identity. A study of transcriptional changes in susceptible rice following infestation by the gall midge, Orseolia oryzae, identified upregulation of genes involved in the cell cycle and was hypothesised to lead to vegetative tissue production (Rawat et al., 2012). In our study, GO and Mapman analyses identified a number of down-regulated genes from both interactions related to DNA metabolism and the cell cycle (Figures 1, 2). It is possible that the plant attempts to reduce the availability of manipulable targets for L. invasa gall development although extensive experimentation is required to substantiate this hypothesis.
Six terpene synthase-encoding genes, out of 113 in the E. grandis genome (Myburg et al. 2014) showed significant differential expression in this study (Table 1, Figure 2). Terpene synthases are typically multiproduct enzymes capable of producing a range of terpenes (Padovan et al., 2013). Therefore, these genes may provide the means of altering the concentrations of a number of terpenoids as seen in this study. While most terpenes that have different foliar concentration are monoterpenes, only one monoterpene synthase is differentially expressed, the others are classified as sesquiterpene synthases. Both post-transcriptional regulation and non-significant differential expression that lead to significant differences in enzyme abundance could cause this discrepancy.
Elevated expression levels of either individual genes in the MEP pathway or the up-regulation of the entire pathway are often associated with increases in monoterpene concentrations (Webb et al., 2013). We show that six of the seven genes involved in the MEP pathway are up-regulated upon L. invasa oviposition. However, this is not completely mirrored by increases in monoterpene concentrations. It is possible that the time point for the increase in monoterpenes was too early or that most newly synthesised monoterpenes were released from the leaves into the atmosphere. Alternatively, Tooker et al. (2008) demonstrated that the Hessian fly was capable of selectively down-regulating the production of certain volatiles in susceptible wheat biotypes.
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Terpenoid Profiles
Recently, Eucalyptus species have been shown to release large amounts of terpenes into the atmosphere where they can act as signaling molecules. These signals can be used to signal other plants or act as insect repellants or attractants. We therefore expected to observe qualitative changes in the terpene profiles with some terpenes that repel insects to be reduced in GC, while others that may attract predators to be increased in Egr. Candidates for terpenes that attract insects are linalool, limonene, αand
β-phellandrene,
terpinolene,
p-cymene,
β-ocimene,
β-caryophyllene,
α-farnesene
and
aromadendrene (Paré and Tumlinson, 1999, Gershenzon and Dudareva, 2007, Kant et al., 2009).
would expect to be down regulated (Isman 2000, Kant et al., 2009). These systems are however highly complex and a single terpene could be responsible for the attraction of a predatory wasp.
Indirect defences allow the plant to interact with the natural enemies of the pest. The distinct, induced changes in the terpenoid profiles observed between the two genotypes in this study (Table 2, Figure S4) could allow Eucalyptus to provide information to parasitoids of L. invasa about the location of their prey in the form of altered olfactory cues. For example, P. rapae and P. xylostella feeding on Brassica oleracea resulted in JA-induced volatile release that attracted parasitoids of the herbivores (Bruinsma et al., 2009). In Egr it may attract parasitoids, whereas, L. invasa may actively be distorting the message in GC. As a means to test this hypothesis, head-space analysis could be performed in the two genotypes to measure terpenes that are released into the atmosphere. This will determine if specific compounds are released to attract predatory wasps.
Future Work
While there is relatively little information regarding the Eucalyptus defensome, this study provides valuable insight into the mechanisms potentially contributing to defence in E. grandis. Some of these hypotheses need to be tested in E. grandis however, functional genetic studies are, as yet, not routinely
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Terpenes that repel insects are for example 1,8-cineole, α-terpineol and bicyclogermacrene, which we
performed in Eucalyptus. An integration of microscopic, transcriptomic, proteomic and metabolomic changes over time will provide a more in-depth understanding of the immunological network at play in the E. grandis-L. invasa model system. This will illuminate biotechnological targets to be exploited in the future as well as improve the knowledge base on immune functioning of long-lived woody plants and trees in general.
Materials and Methods
Two-year-old ramets of Egr and GC were coppiced and subsequently grown in a field cage insectarium enclosed by a mesh that excluded L. invasa. After four months, ramets of each clone were divided into two groups comprising three replicates of six plants. The control group remained in the insectarium while the test group was exposed to a natural infestation by L. invasa in an unwalled nursery for seven days.
RNA extraction and sequencing
Infested and uninfested leaves were collected from the test and control groups and frozen in liquid nitrogen. Midribs were then excised and total RNA was extracted using the protocol described by Naidoo et al. (2013). Samples were treated using Qiagen RNase-free DNase I enzyme (Qiagen Inc, Valencia, California, USA) and purified using the Qiagen RNeasy Mini Kit following manufacturer’s instructions. The concentration and quality of the RNA samples was tested using the Bio-Rad Experion analyser (Bio-Rad, Hercules, California, USA). Total RNA was submitted to the Beijing Genomics Institute (BGI) for RNA-Seq analysis (50 bp paired-end reads, 20 million reads per Egr sample, 30 million reads per GC sample).
Mapping and analysis of reads against the Eucalyptus grandis v1.0 genome assembly
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Leptocybe invasa infestation trial
RNA-Seq data was analysed using the Galaxy workspace (Giardine et al., 2005, Blankenberg et al., 2010, Goecks et al., 2010). FASTQC v0.52 was used to verify RNA-Seq data quality. Reads were mapped to the E. grandis v1.0 genome assembly using Bowtie (Langmead et al., 2009) and Tophat2 v2.0.9 (Trapnell et al., 2010). Mapped reads were assembled into transcripts and FPKM values were calculated with Cufflinks v2.1.1 (Trapnell et al., 2010). Recommended, default settings were used in both cases. Cufflinks was set to use a maximum intron length of 20,000 bp, a minimum isoform fraction of 0.05, pre mRNA fraction of 0.05 and perform quartile normalisation and bias correction for each sample. Significant differential expression between uninfested and infested samples of each genotype as well as
infested versus GC infested) was calculated with Cuffdiff v2.1.1 (Trapnell et al., 2010) and the following parameters, a minimum alignment count of 1000, a false discovery rate of 0.05 and perform quartile normalisation and bias correction.
Gene ontology enrichment analysis
Differentially expressed genes in each genotype were assigned an Arabidopsis thaliana (TAIR 10) annotation based on a reciprocal BLAST search (www.eucgenie.org) where possible. Annotated genes were analysed for Gene Ontology (GO) term over-representation using the Cytoscape v3.0.2 (Shannon et al., 2003) plugin, BiNGO v2.44 (Maere et al., 2005). BiNGO was set to use a hypergeometric test and a Benjamini and Hochberg false discovery rate of 0.05. Mapman v3.6.0RC1 (Thimm et al., 2004) was used to display the data on maps of biological processes to visualise which genes are involved in various described defences.
Terpenoid Chemical Profiling
Leaves (6-8) from three infested and uninfested biological replicates comprising six ramets of each clone were harvested for chemical profiling. Leaves showing L. invasa oviposition were harvested at random
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between the uninfested samples and infested samples (i.e. Egr uninfested versus GC uninfested and Egr
from ramets and included various developmental stages. Ethanol extracts of leaf tissue were separated by gas chromatography and detected by mass spectroscopy on an Agilent 6890 GC/MS using an Alltech AT-35 (35% phenyl, 65% dimethylpolyoxylane) column (Alltech, Wilmington, DE) and Helium as the carrier gas. The column was 60 m long with an internal diameter of 0.25 mm and with a stationary phase film thickness of 0.25 µm. The temperature program was as follows: 100°C for 5 min, ramping to 200°C at -1
-1
20°Cmin followed by a ramp to 250°C at 5°Cmin , and held at 250°C for 4 min. The total elution time was 25 minutes. An FID and an Agilent 5973 Mass Spectrometer dual setup through an SGE MS/FID splitter detected the separated components. Peaks were identified by comparison of mass spectra to
Deerfield, IL) and major peaks were verified by reference to authentic standards. The area under each peak was measured manually with the help of MSD Chemstation Data Analysis (Agilent Technologies) and converted to a relative concentration by comparison to the internal standard (dodecane). Separate samples were collected to measure the fresh- to dry weight ratio and terpene concentrations were calculated relative to dry weight. We performed two-way analysis of variance (ANOVA) in R version 3.0.2 of the concentrations of each chemical to determine genotypic (Egr versus GC) and oviposition (treatment) effects (p-value
The Eucalyptus-Leptocybe invasa Interaction
The Transcriptome And Terpene Profile Of Eucalyptus grandis Reveals Mechanisms Of Defence Against The Insect Pest, Leptocybe invasa
Corresponding author: Dr S. Naidoo Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research
Tel: +27 12 420 4974 Email: [email protected]
Subject area: Environmental and stress responses
Number of figures: 2 (2X colour) Number of tables: 2 (1X black and white, 1X colour) Number of supporting materials: 8 Number of figures: 4 (3X black and white, 1X colour) Number of tables: 4 (3X black and white, 1X colour)
© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: [email protected]
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Institute (GRI), University of Pretoria, Private bag x20, Pretoria, 0028, South Africa
The Eucalyptus-Leptocybe invasa Interaction
The Transcriptome and Terpenoid Profile Of Eucalyptus grandis Reveals Mechanisms Of Defence Against The Insect Pest, Leptocybe invasa
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1
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Caryn N Oates , Carsten Külheim , Alexander A Myburg , Bernard Slippers and Sanushka Naidoo
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Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research
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Research School of Biology, Australian National University, 116 Daley Rd, Canberra, 0200, ACT,
Australia
Abbreviations
ANOVA: analysis of variance BGI: Beijing Genomics Institute BLAST: Basic Local Alignment Search Tool cmk: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase-encoding gene dxr: deoxyxylulose-5-phosphate reductase-encoding gene dxs: deoxyxylulose-5-phosphate synthase-encoding gene EgARF: Eucalyptus grandis ADP ribosylation factor-encoding gene EgARP: Eucalyptus grandis auxin responsive protein-encoding gene EgCAD: Eucalyptus grandis cinnamyl-alcohol dehydrogenase-encoding gene EgDIR: Eucalyptus grandis disease resistance-responsive dirigent-like protein-encoding gene EgEFE: Eucalyptus grandis ethylene-forming enzyme-encoding gene EgFBA: Eucalyptus grandis fructose bisphosphate aldolase-encoding gene EgOMT: Eucalyptus grandis O-methyltransferase-encoding gene
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Institute (GRI), University of Pretoria, Private bag x20, Pretoria, 0028, South Africa
Egr: Eucalyptus grandis clone FPKM: fragments per kilobase of transcript per million fragments mapped GC/MS: gas chromatography coupled to mass spectrometry GC: Eucalyptus grandis x Eucalyptus camaldulensis hybrid GO: gene ontology gpps: geranylgeranyl pyrophosphate synthase-encoding gene hdr: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase-encoding gene hds: 4-hydroxy-3-methylbut-2-enyl diphosphate synthase-encoding gene
hmgs: hydroxymethylglutaryl-CoA synthase-encoding gene ippi: isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase-encoding gene JA: jasmonic acid mcs: 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase-encoding gene MEP pathway: methylerythritol phosphate pathway MIQE: Minimum Information for Publication of Real-Time PCR Experiments MVA pathway: mevalonate pathway RNA-Seq: RNA sequencing RT-qPCR: real time quantitative polymerase chain reaction TPS: terpene synthase-encoding gene
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hmgr: hydroxymethylglutaryl-CoA reductase-encoding gene
Abstract
Plants have evolved complex defences that allow them to protect themselves against pests and pathogens. However, there is relatively little information regarding the Eucalyptus defensome. Leptocybe invasa is one of the most damaging pests in global Eucalyptus forestry and essentially nothing is known regarding the molecular mechanisms governing the interaction between the pest and host. The aim of the study was to investigate changes in the transcriptional landscape and terpene profile of a resistant and susceptible Eucalyptus genotype in an effort to improve our understanding of this interaction. We used
validated using RT-qPCR. Terpene profiles were investigated using gas chromatography coupled to mass spectometry on uninfested and oviposited leaves. We found 698 and 1115 significantly differentially expressed genes from the resistant and susceptible interactions, respectively. Gene Ontology enrichment and Mapman analyses identified putative defence mechanisms including cell wall reinforcement, protease inhibitors, cell cycle suppression and regulatory hormone signalling pathways. There were significant differences in the mono- and sesquiterpene profiles between genotypes and between control and infested material. A model of the interaction between Eucalyptus and L. invasa was proposed from the transcriptomic and chemical data.
Keywords
Defence response, Eucalyptus, Leptocybe invasa, RNA-Seq, terpene profile
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RNA-Seq to investigate transcriptional changes following L. invasa oviposition. Expression levels were
Introduction
Plants have evolved a complex, multi-layered system of defences to protect themselves against insect pests. These include physical or chemical barriers with toxic, repellant or anti-nutritive properties (Mithöfer and Boland, 2012). These defences may be further divided into constitutive or inducible types. Constitutive defences form the plant’s first line of defence and provide generalised protection against most potential attackers (Fürstenberg-Hägg et al., 2013). The inducible defences comprise a multifaceted, broad-spectrum system that is sequentially activated following invader recognition (Jones
pathways through a sophisticated network that ensures the most appropriate response is elicited (Tena et al., 2011). Inducible defences include the oxidative burst, synthesis of secondary metabolites and defence-associated proteins, barrier reinforcement and indirect defences (Fürstenberg-Hägg et al., 2013). Suppression of the plant’s primary metabolism and cell cycle are additional responses that have been described as responses to various pests (Rawat et al., 2012). The efficacy of the inducible defence response is dependent on the speed and magnitude of the induction resulting in either plant resistance or susceptibility to the attacker (Jones and Dangl, 2006).
Terpenoids are a common and diverse group of plant secondary metabolites that play important roles in all aspects of their life cycles (Mithöfer and Boland, 2012, Padovan et al., 2013). The role of terpenoids in plant defence is well-established and alterations in terpene profiles following pest or pathogen attack have been widely reported. These compounds are also important signalling molecules and are involved in plant priming (Troncoso et al., 2012), attracting natural enemies of the pest (Bruinsma et al., 2009) and repelling enemies of the plant (Martín et al., 2014). A defining characteristic of the focal genus of this study, Eucalyptus, are the high foliar concentrations of terpenes that it is able to produce along with the largest number of terpene synthase-encoding genes reported in plants to date (Padovan et al., 2013, Myburg et al., 2014). Our understanding of the role played by terpenes in plant-galling insect interactions
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and Dangl, 2006, Pieterse et al., 2012). The recognition signal is transferred to downstream defence
is limited and studies addressing this knowledge gap will illuminate the role of these compounds in plant defence.
Gall-inducing insects include some of the most devastating pests in agriculture and forestry, for example Phylloxera aphids on grapes (Nabity et al., 2013) and Mayetiola destructor (Hessian fly) on wheat (Stuart et al., 2012). Galls are abnormal plant tissue structures that are induced and maintained by the insect and provide both nourishment and protection (Inbar et al., 2010). Gall development occurs through a process of tissue redifferentiation where a change in the identity of a host plant cell results in the formation of a
assume control of its host’s cellular machinery is such that the physiology, morphology, anatomy, development and chemistry are altered in favour of the pest (Inbar et al., 2010, Oliveira and Isaias, 2010, Compson et al., 2011). Furthermore, the insect is capable of avoiding or actively suppressing the host’s immune system, thus reducing its exposure to toxic chemicals and preventing the release of volatile compounds that may trigger indirect defences (Tooker and De Moraes, 2008). The mechanism of gall development remains poorly understood. Work in model systems such as the interaction between the Hessian fly and wheat are starting to unravel this process (Stuart et al., 2012). For example, a study of the Hessian fly salivary gland detected numerous secreted salivary gland protein transcripts that allegedly encode effectors and may provide the means for the insect to manipulate its host (Chen et al., 2004).
A relatively recently described galling insect, Leptocybe invasa Fisher and La Salle (Hymenoptera: Eulophidae), has emerged as one of the most damaging pests of global Eucalyptus forestry resulting in the complete failure of some industrially important clones (Nyeko et al., 2009, Nyeko et al., 2010, DittrichSchröder et al., 2012). Eucalyptus species constitute some of the most widely grown and economically valuable plantation trees in the world (Grattapaglia et al., 2012). This pest was first described in Israel in 2000 following the extensive damage it caused in the region and has since spread to Africa, the Mediterranean Basin, South East Asia, Europe and South America (Mendel et al., 2004, Kim et al., 2009, Nyeko et al., 2009, Thu et al., 2009, Kumari et al., 2010). Leptocybe invasa is an Australian gall-forming
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new, gall-specific cell type with specialized functions (Oliviera and Isaias, 2010). The ability of a galler to
wasp that preferentially oviposits on immature leaf and stem tissue (Mendel et al., 2004). The larvae are endophytic herbivores that cause the development of coalescing galls on young leaves, petioles and twigs of susceptible trees. An infestation can have devastating effects on a plantation as it may result in stunted growth, die-back and death of infested trees (Nyeko et al., 2009, Thu et al., 2009, Kumari et al., 2010).
Biological control is currently considered to be the key tool in controlling this pest (Wingfield et al., 2008, Kim et al., 2009, Kulkarni et al., 2010). A number of parasitoid wasp species have been introduced as
Quadrastichus mendeli and Selitrichodes kryceri in Israel (Kim et al., 2009). Furthermore, varying degrees of tolerance and resistance are observed among species and genotypes of Eucalyptus indicating the potential for using resistant planting stock in affected areas (Nyeko et al., 2009, Dittrich-Schröder et al., 2012). Defence mechanisms providing resistance or tolerance to L. invasa are essentially unknown and, therefore, restrict the design of biotechnological strategies that can be used against the pest.
This study aims to describe the responses that govern the interaction between L. invasa and Eucalyptus by comparing the transcriptional landscape and terpene profile changes that take place during a resistant and susceptible interaction. RNA-sequencing (RNA-Seq) is a next-generation sequencing technique that allows rapid and accurate profiling of total RNA (Mortazavi et al., 2008). We show that RNA-Seq of Eucalyptus mRNA provides a robust means for investigating induced transcriptional responses in Leptocybe-challenged plants. This study identified genes that showed differential expression profiles in response to L. invasa oviposition. Categorisation and enrichment analysis of these genes identified putative defence mechanisms that are employed by Eucalyptus in response to the gall wasp. Analysis of the mono- and sesquiterpene profiles showed distinct differences between the genotypes before and after oviposition. This information was used to propose a model of the defence response of the host. Improving the current knowledge of the induced transcriptional responses between E. grandis and the invasive gall wasp will lead to improved and integrated control strategies by illuminating key defence mechanisms that
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biological control agents of L. invasa including Selitrichodes neseri in South Africa (Kelly et al., 2012), and
may be manipulated to improve resistance. This information, in combination with biological control and silvicultural practices will help minimise the losses caused by L. invasa.
Results
Infestation of Eucalyptus
Infested and uninfested leaves were collected from an E. grandis clone (Egr, Mondi, RSA) and an E.
challenged groups showed L. invasa oviposition, particularly along the midribs (Figure S1). To confirm the resistant and susceptible identity of the Egr and GC clones, ramets were observed for gall development. The susceptible clone showed extensive gall development, whereas the resistant genotype showed signs of oviposition but no subsequent galling. Total RNA obtained from the infested midrib had high RNA quality scores following Experion analyses. This material was considered suitable for RNA-Seq analysis.
RNA-Sequencing Data Analysis
RNA-Seq of the resistant and susceptible samples yielded 23-29 million and 38-46 million paired-end reads, respectively (Table S2). The resistant and susceptible samples were collected concurrently but sequenced in different batches, which accounts for the differences in read number. Good quality scores were obtained for all samples (data not shown). The reads were mapped to the E. grandis v1.0 genome available at www.phytozome.net. Cufflinks identified 28,703-29,850 and 30,541-31,251 genes in Egr and GC, respectively, that showed expression values (fragments per kilobase of transcript per million fragments mapped, FPKM) greater than 0 and were considered as expressed (Table S2). Cuffdiff identified 698 Egr and 1115 GC significantly differentially expressed genes relative to their respective controls. The resistant interaction showed 504 up-regulated and 194 down-regulated genes; the susceptible interaction showed 665 up-regulated and 450 down-regulated genes (Figure S2). The two
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grandis x E. camaldulensis hybrid clone (GC, Mondi) after seven days exposure to L. invasa. All of the
interactions show 440 common differentially expressed genes that possess a 0.90 correlation coefficient. Gene expression was validated by RT-qPCR analysis and showed a correlation coefficient of 0.91 (Figure S3). The subsequent analyses required genes annotated with an Arabidopsis thaliana ortholog. The resistant interaction included 406 annotated genes and the susceptible interaction included 645 annotated genes (Figure S2). These datasets are available as Table S3.
Gene Ontology Categorisations
genes was investigated to identify putative biological pathways involved in the interactions. Figure 1 summarises the enriched terminal nodes for each of the interactions. The terminal nodes describe more specific associated activity than their parent terms and thus provide a greater level of detail regarding the putative defence mechanisms. Genes from the resistant and susceptible interactions were divided into 32 and 38 enriched GO terms, respectively, with 19 common between them (Figure 1). In general, the gene ontologies that were enriched in the resistant Egr outline a range of defence mechanisms that may be involved in resistance to L. invasa. For example, “terpenoid biosynthetic process” and “flavonoid biosynthetic process” imply that the synthesis of secondary metabolites is involved. Observation of the terpenoid-related gene ontologies in the up-regulated, resistant dataset prompted the investigation into the Eucalyptus terpene metabolism-related gene expression profiles and levels of mono- and sesquiterpenes in the two genotypes. The susceptible interaction, as expected, also included a number of gene ontologies that appear to be unsuccessful defence mechanisms. However, this interaction 220 contained more terms that were related to primary metabolic functions than did the resistant interaction.
Terpenoid Metabolism-Related Gene Expression
The expression profiles of terpenoid metabolism-related genes were compared between the resistant and susceptible genotypes and any significant differences between them are shown (Table 1). Many of these
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The gene ontology (GO) biological processes functional categorization of the differentially expressed
genes are involved in the monoterpene biosynthesis through the methylerythritol phosphate (MEP) pathway including deoxyxylulose-5-phosphate synthase (dxs), deoxyxylulose-5-phosphate reductase (dxr),
4-diphosphocytidyl-2-C-methyl-D-erythritol
kinase
(cmk),
2-C-methyl-D-erythritol-2,4-
cyclodiphosphate synthase (mcs), 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (hds) and 4hydroxy-3-methylbut-2-enyl diphosphate reductase (hdr) (McGarvey and Croteau, 1995, Külheim et al., 2011). Additionally, two of these genes are involved in sesquiterpene biosynthesis through the mevalonate
(MVA)
pathway
namely
hydroxymethylglutaryl-CoA
synthase
(hmgs)
and
hydroxymethylglutaryl-CoA reductase (hmgr) (Külheim et al., 2011). Downstream of the MEP and MVA are
isopentenyl
pyrophosphate:dimethylallyl
pyrophosphate
isomerase
(ippi)
and
geranylgeranyl pyrophosphate synthase (gpps) that show significant differential expression (Külheim et al., 2011). The majority of genes that have been implicated in the up-regulation of terpenes were induced in both resistant and susceptible samples (Webb et al., 2013). There are six terpene synthase genes, encoding enzymes that catalyse the final step in the biosynthetic pathway, that showed significant differential expression in this study, four of them in the resistant interaction and four in the susceptible interaction (Table 1). Five of the terpene synthase genes belong to the TPSa subfamily that synthesises sesquiterpenes and one (Eucgr.K00881) belongs to the TPSb subfamily that synthesises monoterpenes. This gene was significantly up-regulated in Egr and down-regulated in GC, although this was not significant. These expression patterns indicate that the pathway is likely activated in both interactions and may lead to alterations in the terpene profiles of the two genotypes.
Terpenoid Chemical Profiling
The resistant genotype contained higher concentrations of monoterpenes, Egr had 16.05±2.61 mg/g dry weight and 12.56±1.35 mg/g dry weight in GC. Following L. invasa oviposition, Egr monoterpene levels increased to 16.60±2.56 mg/g dry weight, while the GC levels increased to 12.61±1.28 mg/g dry weight. Total sesquiterpene content in uninfested Egr was approximately four times higher than in GC (0.26±0.05 mg/g dry weight and 0.06±0.01 mg/g dry weight). There was a small increase in total sesquiterpene
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pathways
concentration in challenged Egr (0.32±0.06 mg/g dry weight), whereas there was a three-fold increase in sesquiterpenes in GC (0.17±0.03 mg/g dry weight). These increases in mono- and sesquiterpene concentrations seem to mirror the up-regulation of the terpene metabolism-related gene expression profiles that were observed. The two-way ANOVA analysis showed that there were distinct differences in the mono- and sesquiterpenes in both genotypes (Table 2, Figure S4). There is also an induced response of specific terpenoids to L. invasa infestation. A Student’s T-test was performed for each terpene measured to determine where the differences were located (Figure S4). It is clear from these results that there are specific terpene profiles in each genotype. For most monoterpenes, the compounds were
Interestingly, four of the six sesquiterpenes were present in either Egr or GC, but not both. The remaining two cases, viridiflorene and α-gurjuene, showed varying concentrations in one genotype compared to the other.
Mapman Gene Categorisation
The annotated genes were further analysed with Mapman to visualise the genes on maps of cellular processes. These categorisations provided an additional line of evidence for the putative responses involved in the Eucalyptus-L. invasa interaction. The results from Mapman generally supported the GO results with some additional categories being highlighted such as signalling and transcriptional regulators (Figure 2). This analysis was performed to help refine the proposed model of Eucalyptus mechanisms employed during the defence response.
Discussion
The purpose of this study was to investigate transcriptional and chemical changes in resistant and susceptible Eucalyptus genotypes induced by L. invasa oviposition. This comparison provided insight and generated numerous hypotheses into the putative host cellular processes that govern the relationship.
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identified in both interactions, but at different levels, both between genotypes and after infestation.
These were differences in transcripts related to phytohormones, cell wall modifications, cell cycle and secondary metabolites and differences in the terpenoid profiles, which we suggest contribute to resistance in Egr.
Phytohormones
Phytohormones play an important role in the regulation of defences. A complex network governs the relationship and communication between the different hormones which provides a powerful regulatory
six hormone metabolic pathways showed differential expression in this study (Figure 2). These include abscisic acid, auxins, brassinosteroids, ethylene, jasmonic acid (JA) and salicylic acid. These hormones have been associated with plant defence and, interestingly, with gall development. Two of these, JA and auxin, showed different expression profiles between Egr and GC.
The role of up-regulated JA metabolic genes in Egr is consistent with this hormone’s well-described defence function in regulating resistance against phytophagous insects (Wasternack and Hause, 2013). These genes are also up-regulated in GC where their role is less clear. Tooker and Helms (2014) proposed that galling insects actively manipulate phytohormones such that the success or failure of gall development could be dependent on the manner of hormonal interactions.
We observed an up-regulation of auxin metabolism-related genes in both Egr and GC. Auxins have been shown to be involved in insect gall development across interactions involving different species (Tooker et al., 2011, Bartlett and Connor, 2014, Bedetti et al., 2014). Auxins may contribute to the production of gallspecific tissues in the susceptible interaction. In the resistant interaction, manipulation by L. invasa may cause the observed changes in gene expression; however, there is currently no evidence to support this.
Defence Mechanisms
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potential for fine-tuning responses to abiotic and biotic stresses (Pieterse et al., 2012). Genes related to
Downstream of hormone signalling pathways, the inducible defences result in activation or suppression of a combination of responses. One of these that demonstrated differences in the expression profiles of genes between the resistant and susceptible interactions was cell wall metabolism (Figures 1, 2). Cell wall reinforcement through lignin or suberin deposition has been suggested as a defence mechanism against galling insects by preventing the establishment of a feeding site (Liu et al., 2007). Alternatively, galls are composed of heavily lignified tissue that creates a protective microenvironment for the insect (Oliviera and Isaias, 2010). This result is an example of a pathway that is likely employed in both resistant
A second defence mechanism that displayed interesting differences between Egr and GC was the expression profiles of eleven protease inhibitors (Figure 2, Table S4). All of these genes showed similar fold changes in the two genotypes. However, five showed significant differences in the uninfested absolute FPKM values and eight showed significant differences in the absolute infested FPKM values, six of which were higher in Egr than GC. The higher levels of protease inhibitor transcripts may allow for an increased rate of protein synthesis in the resistant interaction and thus a more effective defence response. Protease inhibitors act by blocking insect gut proteases thereby reducing the ability of the insect to digest plant material (Bode et al. 2013). The up-regulation of protease-encoding genes and genes encoding other defence-related proteins, such as lectins and the pathogenesis-related proteins, are commonly reported plant responses to herbivorous insects (Wu et al., 2008, Hartl et al., 2010, Sinha et al., 2011, Bode et al., 2013). The synthesis of protease inhibitors is likely an important defence mechanism in the Eucalyptus-L. invasa interaction.
Suppression of the Cell Cycle
The mechanism of gall development, although poorly understood, requires tissue redifferentiation. Oliviera and Isaias (2010) defined tissue redifferentiation as the process whereby novel cell types with
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and susceptible interactions but with different functions in each.
specialised functions are formed following a change in native cell identity. A study of transcriptional changes in susceptible rice following infestation by the gall midge, Orseolia oryzae, identified upregulation of genes involved in the cell cycle and was hypothesised to lead to vegetative tissue production (Rawat et al., 2012). In our study, GO and Mapman analyses identified a number of down-regulated genes from both interactions related to DNA metabolism and the cell cycle (Figures 1, 2). It is possible that the plant attempts to reduce the availability of manipulable targets for L. invasa gall development although extensive experimentation is required to substantiate this hypothesis.
Six terpene synthase-encoding genes, out of 113 in the E. grandis genome (Myburg et al. 2014) showed significant differential expression in this study (Table 1, Figure 2). Terpene synthases are typically multiproduct enzymes capable of producing a range of terpenes (Padovan et al., 2013). Therefore, these genes may provide the means of altering the concentrations of a number of terpenoids as seen in this study. While most terpenes that have different foliar concentration are monoterpenes, only one monoterpene synthase is differentially expressed, the others are classified as sesquiterpene synthases. Both post-transcriptional regulation and non-significant differential expression that lead to significant differences in enzyme abundance could cause this discrepancy.
Elevated expression levels of either individual genes in the MEP pathway or the up-regulation of the entire pathway are often associated with increases in monoterpene concentrations (Webb et al., 2013). We show that six of the seven genes involved in the MEP pathway are up-regulated upon L. invasa oviposition. However, this is not completely mirrored by increases in monoterpene concentrations. It is possible that the time point for the increase in monoterpenes was too early or that most newly synthesised monoterpenes were released from the leaves into the atmosphere. Alternatively, Tooker et al. (2008) demonstrated that the Hessian fly was capable of selectively down-regulating the production of certain volatiles in susceptible wheat biotypes.
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Terpenoid Profiles
Recently, Eucalyptus species have been shown to release large amounts of terpenes into the atmosphere where they can act as signaling molecules. These signals can be used to signal other plants or act as insect repellants or attractants. We therefore expected to observe qualitative changes in the terpene profiles with some terpenes that repel insects to be reduced in GC, while others that may attract predators to be increased in Egr. Candidates for terpenes that attract insects are linalool, limonene, αand
β-phellandrene,
terpinolene,
p-cymene,
β-ocimene,
β-caryophyllene,
α-farnesene
and
aromadendrene (Paré and Tumlinson, 1999, Gershenzon and Dudareva, 2007, Kant et al., 2009).
would expect to be down regulated (Isman 2000, Kant et al., 2009). These systems are however highly complex and a single terpene could be responsible for the attraction of a predatory wasp.
Indirect defences allow the plant to interact with the natural enemies of the pest. The distinct, induced changes in the terpenoid profiles observed between the two genotypes in this study (Table 2, Figure S4) could allow Eucalyptus to provide information to parasitoids of L. invasa about the location of their prey in the form of altered olfactory cues. For example, P. rapae and P. xylostella feeding on Brassica oleracea resulted in JA-induced volatile release that attracted parasitoids of the herbivores (Bruinsma et al., 2009). In Egr it may attract parasitoids, whereas, L. invasa may actively be distorting the message in GC. As a means to test this hypothesis, head-space analysis could be performed in the two genotypes to measure terpenes that are released into the atmosphere. This will determine if specific compounds are released to attract predatory wasps.
Future Work
While there is relatively little information regarding the Eucalyptus defensome, this study provides valuable insight into the mechanisms potentially contributing to defence in E. grandis. Some of these hypotheses need to be tested in E. grandis however, functional genetic studies are, as yet, not routinely
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Terpenes that repel insects are for example 1,8-cineole, α-terpineol and bicyclogermacrene, which we
performed in Eucalyptus. An integration of microscopic, transcriptomic, proteomic and metabolomic changes over time will provide a more in-depth understanding of the immunological network at play in the E. grandis-L. invasa model system. This will illuminate biotechnological targets to be exploited in the future as well as improve the knowledge base on immune functioning of long-lived woody plants and trees in general.
Materials and Methods
Two-year-old ramets of Egr and GC were coppiced and subsequently grown in a field cage insectarium enclosed by a mesh that excluded L. invasa. After four months, ramets of each clone were divided into two groups comprising three replicates of six plants. The control group remained in the insectarium while the test group was exposed to a natural infestation by L. invasa in an unwalled nursery for seven days.
RNA extraction and sequencing
Infested and uninfested leaves were collected from the test and control groups and frozen in liquid nitrogen. Midribs were then excised and total RNA was extracted using the protocol described by Naidoo et al. (2013). Samples were treated using Qiagen RNase-free DNase I enzyme (Qiagen Inc, Valencia, California, USA) and purified using the Qiagen RNeasy Mini Kit following manufacturer’s instructions. The concentration and quality of the RNA samples was tested using the Bio-Rad Experion analyser (Bio-Rad, Hercules, California, USA). Total RNA was submitted to the Beijing Genomics Institute (BGI) for RNA-Seq analysis (50 bp paired-end reads, 20 million reads per Egr sample, 30 million reads per GC sample).
Mapping and analysis of reads against the Eucalyptus grandis v1.0 genome assembly
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Leptocybe invasa infestation trial
RNA-Seq data was analysed using the Galaxy workspace (Giardine et al., 2005, Blankenberg et al., 2010, Goecks et al., 2010). FASTQC v0.52 was used to verify RNA-Seq data quality. Reads were mapped to the E. grandis v1.0 genome assembly using Bowtie (Langmead et al., 2009) and Tophat2 v2.0.9 (Trapnell et al., 2010). Mapped reads were assembled into transcripts and FPKM values were calculated with Cufflinks v2.1.1 (Trapnell et al., 2010). Recommended, default settings were used in both cases. Cufflinks was set to use a maximum intron length of 20,000 bp, a minimum isoform fraction of 0.05, pre mRNA fraction of 0.05 and perform quartile normalisation and bias correction for each sample. Significant differential expression between uninfested and infested samples of each genotype as well as
infested versus GC infested) was calculated with Cuffdiff v2.1.1 (Trapnell et al., 2010) and the following parameters, a minimum alignment count of 1000, a false discovery rate of 0.05 and perform quartile normalisation and bias correction.
Gene ontology enrichment analysis
Differentially expressed genes in each genotype were assigned an Arabidopsis thaliana (TAIR 10) annotation based on a reciprocal BLAST search (www.eucgenie.org) where possible. Annotated genes were analysed for Gene Ontology (GO) term over-representation using the Cytoscape v3.0.2 (Shannon et al., 2003) plugin, BiNGO v2.44 (Maere et al., 2005). BiNGO was set to use a hypergeometric test and a Benjamini and Hochberg false discovery rate of 0.05. Mapman v3.6.0RC1 (Thimm et al., 2004) was used to display the data on maps of biological processes to visualise which genes are involved in various described defences.
Terpenoid Chemical Profiling
Leaves (6-8) from three infested and uninfested biological replicates comprising six ramets of each clone were harvested for chemical profiling. Leaves showing L. invasa oviposition were harvested at random
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between the uninfested samples and infested samples (i.e. Egr uninfested versus GC uninfested and Egr
from ramets and included various developmental stages. Ethanol extracts of leaf tissue were separated by gas chromatography and detected by mass spectroscopy on an Agilent 6890 GC/MS using an Alltech AT-35 (35% phenyl, 65% dimethylpolyoxylane) column (Alltech, Wilmington, DE) and Helium as the carrier gas. The column was 60 m long with an internal diameter of 0.25 mm and with a stationary phase film thickness of 0.25 µm. The temperature program was as follows: 100°C for 5 min, ramping to 200°C at -1
-1
20°Cmin followed by a ramp to 250°C at 5°Cmin , and held at 250°C for 4 min. The total elution time was 25 minutes. An FID and an Agilent 5973 Mass Spectrometer dual setup through an SGE MS/FID splitter detected the separated components. Peaks were identified by comparison of mass spectra to
Deerfield, IL) and major peaks were verified by reference to authentic standards. The area under each peak was measured manually with the help of MSD Chemstation Data Analysis (Agilent Technologies) and converted to a relative concentration by comparison to the internal standard (dodecane). Separate samples were collected to measure the fresh- to dry weight ratio and terpene concentrations were calculated relative to dry weight. We performed two-way analysis of variance (ANOVA) in R version 3.0.2 of the concentrations of each chemical to determine genotypic (Egr versus GC) and oviposition (treatment) effects (p-value
