Comparative genomic and transcriptional analyses of the carbohydrate-active enzymes and secretomes of phytopathogenic fungi reveal their significant roles during infection and development

Lyu, Xueliang; Shen, Cuicui; Fu, Yanping; Xie, Jiatao; Jiang, Daohong; Li, Guoqing; Cheng, Jiasen

doi:10.1038/srep15565

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Published: 04 November 2015

Comparative genomic and transcriptional analyses of the carbohydrate-active enzymes and secretomes of phytopathogenic fungi reveal their significant roles during infection and development

Xueliang Lyu^1,2,
Cuicui Shen^1,2,
Yanping Fu²,
Jiatao Xie²,
Daohong Jiang^1,2,
Guoqing Li^1,2 &
…
Jiasen Cheng^1,2

Scientific Reports volume 5, Article number: 15565 (2015) Cite this article

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Abstract

Our comparative genomic analysis showed that the numbers of plant cell wall (PCW)- and fungal cell wall (FCW)-degradation-associated carbohydrate-active enzymes (CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi. However, our transcriptional analyses of CAZyme-encoding genes in Melampsora larici-populina, Puccinia graminis and Sclerotinia sclerotiorum showed that many genes encoding PCW- and FCW-degradation-associated CAZymes were significantly up-regulated during the infection of both necrotrophic fungi and biotrophic fungi, indicating an existence of a universal mechanism underlying PCW degradation and FCW reorganization or modification, which are both intimately involved in necrotrophic and biotrophic fungal infection. Furthermore, our results showed that the FCW reorganization or modification was also related to the fungal development. Additionally, our transcriptional analysis of the secretome of S. sclerotiorum showed that many secreted protein-encoding genes were dramatically induced during infection. Among them, a small, cysteine-rich protein SsCVNH was experimentally confirmed to be essential for the virulence and sclerotial development, indicating that the small secreted proteins might also play crucial roles as potential effectors in host-non-specific necrotrophic fungi.

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Introduction

Many fungal carbohydrate-active enzymes (CAZymes) and secreted proteins directly participate in the interactions between phytopathogenic fungi and their hosts. Among the secretome, CAZymes are a large group, but not all the members of this group are secreted proteins. CAZymes are responsible for the breakdown, biosynthesis or modification of glycoconjugates and oligo- and polysaccharides by degrading, modifying, or creating glycosidic bonds. They participate in many important biological processes, including cell wall synthesis, signaling and energy production¹. Currently, there are four main classes of CAZymes: glycosyltransferases (GTs), glycoside hydrolases (GHs), polysaccharide lyases (PLs) and carbohydrate esterases (CEs)². The latter three classes of CAZymes are often known as cell wall degrading enzymes (CWDEs) because they play central roles in plant cell wall (PCW) decomposition by fungi and bacteria³. There are many proteins with carbohydrate-binding modules (CBMs) that can potentiate CAZyme activities by targeting and promoting a prolonged interaction with the substrate, although they have no catalytic activity per se except some CAZymes with CBMs². Generally speaking, PCW consists of carbohydrate (including cellulose, hemicellulose, pectin and lignin), structural proteins and aromatic compounds^4,5, while fungal cell wall (FCW) is primarily composed of chitin, glucans, mannans and glycoproteins⁶. Owing to the distinct components of PCW and FCW, fungi have developed two categories of CAZymes: PCW- and FCW-degrading CAZymes, which have different enzyme activities and substrates, to degrade or modify PCW and FCW respectively. Because breakthrough of PCW is an essential step for the colonization of host tissues for all phytopathogens, CWDEs, such as pectinases and xylanases, have been identified as important pathogenicity or virulence factors^7,8. A large number of CAZyme-encoding genes in the genomes of phytopathogenic fungi are associated with PCW degradation. It is well known that PCW-degrading CAZymes play crucial roles during fungal infection. There are a large number of genes encoding FCW-degrading enzymes in phytopathogenic fungal genomes, however, the expression patterns and the exact roles of these FCW-degrading CAZymes during fungal infection and development are unknown.

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic, phytopathogenic fungus with a remarkably broad host range and worldwide distribution. At least 408 described species of plants from 278 genera in 75 families, including many economically important crops such as rapeseed (Brassica spp.), sunflowers, soybeans and peanuts, are susceptible to this pathogen⁹. White mold caused by this pathogen often leads to a momentous loss of crop production. Importantly, for many important crops such as rapeseed, there are almost no resistant cultivars that can be used for production. Thus, Sclerotinia disease is difficult to control and has raised considerable concern. There are several key steps in the life cycle of S. sclerotiorum, including vegetative growth, infection, sclerotial development, sclerotial myceliogenic germination, sclerotial carpogenic germination and apothecium formation (stipe). These steps are all important for the lifecycle of S. sclerotiorum. For example, the sclerotium produced by S. sclerotiorum is a pigmented, hard, asexual resting structure that is capable of surviving for years in soil^10,11,12. Sclerotia germinate myceliogenically to produce saprophytic or infectious mycelia or carpogenically to form apothecia. Airborne ascospores derived from apothecia are the principal source of primary inocula and the most important means of S. sclerotiorum dispersal^13,14,15.

The secretomes play crucial roles during plant-pathogen interactions. Notably, most effectors of fungi and oomycetes are small secreted proteins (especially small, secreted, cysteine-rich proteins)^{16,17,18,19,20}, with the exception of some non-proteinaceous toxins and secondary metabolites. Many effectors have been well documented in host-specific necrotrophic fungi, hemibiotrophic fungi, biotrophic fungi and oomycetes^{16,17,18,19,20}. However, most of the secreted proteins in host-non-specific, typically necrotrophic fungi with a remarkably broad host range, such as S. sclerotiorum, are poorly understood. Secretome analysis revealed large numbers of predicted effector candidates in the genome of S. sclerotiorum²¹ and recent studies demonstrated that the non-CWDE secreted proteins played crucial roles during infection^22,23. However, the large-scale transcriptional responses of genes encoding the secretome during the infection, vegetative and reproductive developmental stages of S. sclerotiorum and the exact biological functions of most of these effector candidates are unknown.

In this study, we compared the numbers of the CAZyme-encoding genes in the genomes of different phytopathogenic fungi and investigated the global transcriptional responses of the CAZyme-encoding genes and secretome-encoding genes during the infection and development of phytopathogenic fungi. To confirm our hypotheses, 16 genomes of phytopathogenic fungi (including biotrophs, hemibiotrophs and necrotrophs) and three expression profiles [including two reported microarray data of rust fungi and one digital gene expression profiling (DGE) data of S. sclerotiorum] were chosen for comparative analysis. Additionally, as an example, one small, cysteine-rich, secreted protein (SsCVNH) was chosen for functional studies. Our results increase the understanding of the roles of CAZymes and the secretomes in the infection and development of phytopathogenic fungi and facilitate the discovery of novel effector candidates in host-non-specific necrotrophic fungi.

Results

Comparative genomic analysis of the CAZymes in phytopathogenic fungi

Considering the representativeness and accessibility of available genome data, nine genomes of hemibiotrophic and necrotrophic fungi (Magnaporthe oryzae, Fusarium graminearum, F. oxysporum, F. verticillioides, Phaeosphaeria nodorum, Pyrenophora tritici-repentis, Verticillium dahliae, Botrytis cinerea and S. sclerotiorum), seven genomes of biotrophic fungi (Blumeria graminis, Ustilago maydis, Cladosporium fulvum, Melampsora larici-populina, Puccinia graminis, P. triticina and P. striiformis) and two yeast genomes (Saccharomyces cerevisiae and Schizosaccharomyces pombe) were chosen for the comparative analysis of CAZymes. Our results showed that the numbers of genes encoding PCW- and FCW-degradation-associated CAZymes and their respective related CBMs in necrotrophic and hemibiotrophic fungi were both significantly larger than that in most of biotrophic fungi except for C. fulvum (Fig. 1, Table S1), indicating both PCW- and FCW-degradation-associated CAZymes may play crucial roles during the infection and (or) development of necrotrophic and hemibiotrophic fungi.

DGE of S. sclerotiorum

To explore the expression patterns of CAZyme-encoding genes during the infection and development of necrotrophic fungi, we used DGE based on deep sequencing technology to illuminate the transcriptional responses of CAZyme-encoding genes during the infection stage and different developmental stages (including vegetative growth, sclerotial development, sclerotial myceliogenic germination, sclerotial carpogenic germination and apothecium formation) of the necrotrophic fungus S. sclerotiorum, which is used as an example. Sequencing quality evaluation and sequencing saturation analyses guaranteed the data quality for further DGE analysis (Figure S1 and S2). Differentially expressed genes were identified during the various developmental stages compared with the vegetative growth stage (Figure S3). To validate the DGE data, twelve genes were selected randomly for quantitative reverse transcription PCR (qRT-PCR) analysis. The data were presented as fold changes in gene expression normalized to the β-tubulin gene^24,25,26. Pearson’s correlation coefficient (R value) was used to measure the consistency of the qRT-PCR and DGE data. The R values ranged from 0.88 to 1.00, indicating the qRT-PCR results were strongly correlated with the DGE data (Figure S4). These results showed that the expression patterns of the twelve genes in the DGE and qRT-PCR data were accordant and our DGE data was reliable for further transcriptional analysis.

The expression patterns of CAZyme-encoding genes in S. sclerotiorum

Among the 501 identified CAZyme-encoding genes in S. sclerotiorum, the expression of 315 was significantly regulated during different developmental stages, compared with vegetative growth stage (Table S2a). Our result showed that the geometric mean of the expression values of the PCW- and FCW-degrading CAZyme-encoding genes and their respective related CBMs showed dramatic induction during the infection and various developmental stages of S. sclerotiorum (Fig. 2a), indicating both of the PCW- and FCW-degrading CAZymes were activated during these biological processes. As expected, gene expression cluster analysis showed that a large number of genes encoding PCW-degradation-associated CAZymes and related CBMs were significantly up-regulated during infection (Fig. 2b). For example, 73 (39.2%) out of a total of 186 genes encoding PCW-degradation-associated CAZymes and related CBMs, were significantly up-regulated during infection (Fig. 2d, Table S3). This result confirmed the important roles of plant CWDEs during infection. Additionally, a large number of genes encoding FCW-degradation-associated CAZymes and related CBMs were also dramatically induced during infection (Fig. 2c). For example, 19 (18.8%) from a total of 101 genes encoding FCW-degradation-associated CAZymes and related CBMs, were significantly up-regulated during infection (Fig. 2e, Table S3). Microscopic observation showed that the invasive hyphal tips were clearly different from the vegetative hyphal tips: the latter were thinner and their morphology was normal, whereas the former were thicker and their morphology was blistered, swollen and malformed (Fig. 3). This result further suggested FCW reorganization or modification was intimately involved in the infection processes of necrotrophic fungi. During the vegetative and reproductive developmental stages, there are 36 (19.4%), 37 (19.9%), 32 (17.2%) and 31 (16.7%) out of a total of 186 genes encoding PCW-degrading enzymes and related CBMs which were significantly up-regulated during sclerotial development, sclerotial myceliogenic germination, sclerotial carpogenic germination and apothecium formation, respectively, compared with the vegetative growth stage (Fig. 2d, Table S3), indicating the expression of these genes encoding PCW-degrading enzymes and related CBMs was induced not by specific plant factors but by the developmental regulation of S. sclerotiorum or by tough environmental factors which are unfavorable for vegetative growth (such as nutrient starvation). Notably, a large number of genes encoding FCW-degrading CAZymes and related CBMs were also significantly induced during the vegetative and reproductive developmental stages of S. sclerotiorum. For example, there are 28 (27.7%), 21 (20.8%), 18 (17.8%) and 19 (18.8%) from a total of 101 genes encoding FCW-degrading enzymes and related CBMs that were significantly up-regulated during sclerotial development, sclerotial myceliogenic germination, sclerotial carpogenic germination and apothecium formation, respectively, compared with the vegetative growth stage (Fig. 2e, Table S3). These results indicated that the FCW reorganization or rearrangement also played important roles in the vegetative and reproductive development of S. sclerotiorum and the FCW was in a state of dynamic change during various developmental stages. Taken together, the CAZyme-encoding genes exhibited diverse expression patterns during the infection and multiple developmental stages of S. sclerotiorum, compared with the vegetative stage, indicating that the CAZymes might play versatile roles in these developmental processes.

Transcriptional analyses of CAZyme-encoding genes in M. larici-populina and P. graminis

To detect the expression patterns of CAZyme-encoding genes during the infection of phytopathogenic biotrophic fungi, we examined the documented gene expression profiles (microarray data) during the infection of M. larici-populina²⁷ and P. graminis²⁸ urediniospores, which are used as examples. The results showed that the expression of a large number of genes encoding CAZymes and related CBMs in M. larici-populina and P. graminis was significantly regulated during infection. In details, 75 (49.7%) out of a total of 151 genes encoding PCW-degradation-associated CAZymes and related CBMs were significantly induced during at least one infection stage of M. larici-populina urediniospores on poplar leaves, compared with the stage of dried-urediniospores in vitro (Table S4a), the proportion varied from 17.9% to 36.4% during different infection stages (Fig. 4e, Table S4a). There are 39 (40.2%) and 37 (38.1%) out of a total of 97 genes encoding PCW-degradation-associated CAZymes and related CBMs that were significantly induced during the infection of P. graminis urediniospores on wheat and barley, respectively, compared with the stage of urediniospores in vitro (Fig. 4g, Table S4b). Surprisingly, many genes encoding FCW-degrading CAZymes and related CBMs in M. larici-populina and P. graminis were also significantly regulated during infection. In details, there are 26 (36.1%) from a total of 72 genes encoding FCW-degradation-associated CAZymes and related CBMs which were significantly induced during at least one infection stage of M. larici-populina urediniospores on poplar leaves, compared with the stage of dried-urediniospores in vitro (Table S4a), the proportion varied from 13.9% to 23.6% during different infection stages (Fig. 4f). There are 22 (36.1%) and 22 (36.1%) from a total of 61 genes encoding FCW-degradation-associated CAZymes and related CBMs that were significantly induced during the infection of P. graminis urediniospores on wheat and barley, respectively, compared with the stage of urediniospores in vitro (Fig. 4h, Table S4b). These results indicated that the PCW degradation and FCW reorganization or modification might also be intimately involved in the infection processes of biotrophic fungi, although the numbers of genes encoding PCW and FCW-degradation associated CAZymes in biotrophic fungi were smaller than those in necrotrophic fungi.

Transcriptional analysis of the secretome of S. sclerotiorum

We compared the total numbers of secreted proteins, small secreted proteins and cysteine-rich, small secreted proteins from the 18 fungi described above, respectively. The result showed P. striiformis, M. larici-populina, F. verticillioides, M. oryzae, P. graminis, F. oxysporum and P. triticina had more secreted proteins, especially small secreted proteins, compared with the other species (Figure S5). To decipher the biological functions of the S. sclerotiorum secretome, the gene expression patterns of the secretome were clustered based on our DGE data. The result showed that many genes encoding secreted proteins were significantly regulated during the infection, sclerotial development, sclerotial myceliogenic germination, sclerotial carpogenic germination and apothecium formation stages, compared with the vegetative growth stage (Figure S6, Table S2b), indicating their diversified roles during these biological processes. Gene functional enrichment analysis was performed on the significantly up-regulated genes encoding secreted proteins during different developmental stages. Many Funcat functional categories and gene ontology (GO) items were enriched during various developmental stages, especially during the sclerotial development and infection stages (Figure S7, Table S5), indicating their important and diverse roles in these biological processes. Interestingly, compared with the vegetative growth stage, the “cell wall” associated functional modules (such as “cell wall organization”, “fungal-type cell wall” and “cell wall modification”) were significantly over-represented during different developmental stages (Figure S7, Table S5), which is in accord with our hypothesis that the FCW is in a state of dynamic change during various developmental stages. Additionally, the results showed the PCW- and FCW-degradation associated functional modules (such as “extracellular polysaccharide degradation”, “extracellular lignin degradation”, “cellulose binding”, “chitin binding”, “hydrolase activity”, “cellulase activity”, “cutinase activity”, “polygalacturonase activity”, “beta-galactosidase activity” and “mannan endo-1,6-alpha-mannosidase activity”) were also over-represented during different developmental stages (Figure S7, Table S5), intimating the significant roles of CAZymes in corresponding biological processes.

SsCVNH is a predicted cysteine-rich, small secreted protein with a CVNH domain

To experimentally validate the important roles of small secreted proteins during the infection, vegetative and reproductive developmental stages of S. sclerotiorum, a predicted secreted protein-encoding gene (SS1G_02904) was selected as an example for further study. We chose this gene because of the following reasons: (i) In planta expression analysis can help to unearth candidate effector genes¹⁸ and SS1G_02904 had the highest expression fold change during the infection in our DGE data; (ii) Many fungal effectors are cysteine-rich, small secreted proteins^17,29 and SS1G_02904 consists of 153 aa and contains 10 cysteine residues that account for more than 6.5% of the total amino acids; (iii) Some fungal effectors show conserved protein domains and SS1G_02904 was identified as an effector candidate that shares 35% identity with a Colletotrichum hingginsianum effector candidate^21,30. SS1G_02904 is a predicted secreted protein with a signal peptide (1–20 aa) in its N-terminus and a CVNH domain (pfam08881, E-value = 2.65e-06) in its C-terminus (Fig. 5a); hence, it is designated as SsCVNH. The CVNH domain corresponds to a carbohydrate-binding module^31,32. The predicted three-dimensional structure of SsCVNH is highly similar to the structure of cyanovirin-N³³ (Fig. 5b). Although the CVNH domain was found to be widely distributed in cyanobacteria, filamentous ascomycetes and the fern Ceratopteris richardii³⁴, protein sequence similarity search (blastp) result showed that SsCVNH had homologs only in Sclerotinia and Botryotinia, using an E-value lower than 1e-6 as the threshold (Fig. 5c). These results indicated that the three-dimensional structure of the CVNH domain was more evolutionarily conserved than the protein sequence.

The expression patterns of SsCVNH

DGE data showed that SsCVNH was not expressed during the vegetative growth stage and was significantly induced during the stages of sclerotial development, sclerotial myceliogenic germination, sclerotial carpogenic germination, apothecium formation and infection (Fig. 6a). QRT-PCR analysis further showed that the expression of SsCVNH was significantly up-regulated during the initial stage of sclerotial development at 3 days post-inoculation (dpi) and peaked during the mature stage of sclerotial development at 7 dpi. The expression change of this gene reached more than 600-fold during sclerotial development (Fig. 6b). When pure, actively growing hyphal fragments of S. sclerotiorum without any culture medium were inoculated onto Arabidopsis thaliana (Col-0) leaves, the transcripts of SsCVNH rapidly increased by more than 15-fold during the early infection stage at 3 hours post-inoculation (hpi) and maintained at high expression levels during the later infection stages (Fig. 6c). These results were coincident with the DGE data, suggesting that SsCVNH may be involved in the infection, vegetative and reproductive development of S. sclerotiorum.

SsCVNH is secreted from the hyphal tips

Bioinformatics analysis revealed that SsCVNH might be a secreted protein due to the presence of a signal peptide in its N-terminus. An immunofluorescence technique was used to confirm this hypothesis. FLAG-tagged SsCVNH strains under the control of the P_EF − 1α promoter were engineered. Western blot analysis showed FLAG-tagged SsCVNH could be detected in the hyphae of these engineered strains (Fig. 7a). The mycelia of the engineered SsCVNH-FLAG strains were inoculated on the onion bulb lower epidermis prior to live cell imaging of the infected onion epidermal cells to examine the subcellular localization of SsCVNH during infection. To eliminate the possible interference of the autofluorescence of plant or fungal tissues, two different secondary antibodies conjugated with rhodamine red-X (RRX) or fluorescein isothiocyanate (FITC) were used independently to exhibit the specificity of fluorescence signal. The result showed fluorescence could be observed in the hyphal tips of the SsCVNH-FLAG engineered strains. No fluorescence was observed in the wild-type strain. Similar results were obtained with different antibodies tagged with FITC or RRX (Fig. 7b), indicating that SsCVNH was indeed a secreted protein that was secreted from the hyphal tips during the infection of S. sclerotiorum.

SsCVNH-silenced transformants showed significantly reduced virulence and abnormal sclerotial development

Due to the presence of the multi-nucleated cells in S. sclerotiorum, RNAi technology was used to characterize the biological functions of SsCVNH. QRT-PCR was used to examine the transcript accumulation in SsCVNH-silenced transformants. The results showed that the sclerotial development of the transformants (SsCVNH-89, SsCVNH-84 and SsCVNH-40) with dramatically reduced SsCVNH expression was completely blocked on PDA medium at 20 °C. However, the wild-type strain and the transformant SsCVNH-46 with slightly reduced SsCVNH expression showed normal sclerotial development (Fig. 8a,d). The growth rate of the silenced transformants was also significantly reduced, although there was no obvious difference between the morphology of the hyphal tips of the silenced transformants and the wild-type strain (Fig. 8c,f). The virulence of the SsCVNH-silenced transformants was significantly reduced and only small lesions developed on the detached leaves of Brassica napus at 2 dpi. Furthermore, the decrease in virulence was positively correlated with the silencing efficiency of different transformants (Fig. 8b,e). Similar result was observed when the SsCVNH-silenced transformants were inoculated on detached tomato leaves (Figure S8), indicating that the virulence reduction was not host species-specific. These results indicated that SsCVNH played crucial roles in the virulence, sclerotial development and growth rate of S. sclerotiorum. In conclusion, our results indicated that small secreted proteins in typically necrotrophic fungi might function as potential effectors similar to those in hemibiotrophs and biotrophs.

Discussion

To date, whether the phytopathogenic fungi use FCW-degrading enzymes for the degradation or modification of their own cell walls, the walls of antagonistic fungi, or for plant polysaccharides during infection is unknown³⁵. Our results indicated that the expression levels of many genes encoding FCW-degrading enzymes were significantly regulated during the infection of phytopathogenic fungi. Meanwhile, our results indicated different kinds of FCW-degrading enzymes participated in this biological process and their proportions varied during different infection stages, indicating the FCW reorganization or modification process is sophisticatedly and stringently regulated. The present results support the hypothesis that these FCW-degrading enzymes may play significant roles in shaping their own cell walls because the FCW reorganization or remodeling could be clearly observed during infection. The FCW reorganization or rearrangement itself may be essential for phytopathogenic fungal infection. This hypothesis may also be adapted to the development of phytopathogenic fungi because the FCW components are obviously different in various tissues during different developmental stages. Our results indicated that the FCW was in a state of dynamic change during the infection and development of phytopathogenic fungi, which is consistent with the previous studies on FCW dynamics^6,36. Additionally, our results indicated that the PCW degradation and FCW reorganization are intimately involved in both necrotrophic and biotrophic fungal infection. But the total numbers of genes encoding PCW- and FCW-degrading CAZymes and associated CBMs in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi. Furthermore, the proportions of differentially expressed CAZyme-encoding genes are also obviously different during infection, indicating the capacity of PCW degradation and FCW reorganization is different in necrotrophic, hemibiotrophic and biotrophic fungi. This is consistent with the lifestyles of different types of fungi, because the nutrient acquisition of necrotrophic and hemibiotrophic fungi is based on host cell killing at the last stage of infection³⁷, while biotrophic fungi absorb nutrients from living cells³⁸.

Our result showed a total of 235 secreted protein-encoding genes were significantly up-regulated during infection (Table S2b). These strongly induced genes are likely to represent “key offensive or repressive forces” or “effector candidates” in S. sclerotiorum that target the plant defense system during the early stages of infection. Notably, many proteinaceous fungal effectors are cysteine-rich, small secreted proteins^16,17,29. In this study, SsCVNH was used as an example to elucidate the significant roles of these types of effector candidates during the infection, vegetative and reproductive development of S. sclerotiorum. In many reports, most effectors or small secreted proteins seemed to have little effect on pathogen development. Interestingly, this may not be the case in S. sclerotiorum because, to date, at least two small secreted proteins (SSITL and Ss-Caf1) have been reported to play important roles in the sclerotial development of S. sclerotiorum^22,23. Similarly, our results demonstrated that SsCVNH also played significant roles in sclerotial development, not only in infection. Additionally, in our large-scale gene function studies of the S. sclerotiorum secretome, SsCVNH was not a unique example to show that the secreted proteins have diverse biological functions during multiple developmental stages of S. sclerotiorum (data not shown here). These results suggest a scenario in which different groups of secreted proteins synergistically function during various developmental stages.

Our result showed that SsCVNH was a secreted protein with a CVNH domain. Cyanovirin-N (CVN) has previously been identified only in the aqueous extracts from the cyanobacterium Nostoc ellipsosporum³⁹. Based on our PSI-BLAST search in the NCBI non-redundant protein sequence database, CVNHs are found in a restricted range of eukaryotic organisms as diverse as the fern Ceratopteris richardii, the filamentous ascomycetes (e.g., Tuber borchii, Neurospora, Magnaporthe, Sclerotinia, Botrytis, Aspergillus, Fusarium, Colletotrichum and Trichoderma) and the basidiomycetes (e.g., Rhizoctonia solani) but are lacking in other lineages (data not shown). This patchy organism distribution suggests the acquisition of the CVNH domain by some organisms after the separation of the main evolutionary lineages and the occurrence of lateral gene transfer events. CVNH-containing organisms have strikingly different lifestyles, ranging from symbiotic, saprotrophic to pathogenic. Meanwhile, in these organisms, there are both sclerotium-producing fungi and non-sclerotium-producing fungi. These phenomena suggest that the CVNHs are not sufficient for infection and sclerotial development but are necessary in some cases. CVNH-related proteins in ferns and cyanobacteria are found from the secretome. However, owing to data limitations, previous studies reported that no secretion signals were identified in any of the fungal CVNHs in filamentous ascomycetes and only three types of domain architectures of CVNHs have been reported^31,34. Due to the increasing availability of genomic sequences, we find there are more than three types of domain architectures of CVNHs, including SsCVNH with a signal peptide in the N-terminus before the CVNH domain, thereby indicating that these CVNHs may also be secreted proteins in filamentous ascomycetes. Our result further confirmed this hypothesis and supplemented the knowledge concerning the domain architectures of CVNHs.

Materials and Methods

Data collection and bioinformatics analyses

The predicted proteomes of M. oryzae, F. graminearum, F. oxysporum, F. verticillioides, P. nodorum, P. tritici-repentis, V. dahliae, B. cinerea, S. sclerotiorum, U. maydis, P. graminis, P. triticina, P. striiformis, S. cerevisiae and S. pombe were downloaded from the Broad Institute (http://www.broadinstitute.org/science/projects/projects). The predicted proteomes of C. fulvum and M. larici-populina were obtained from the DOE Joint Genome Institute (JGI) site⁴⁰ and the predicted proteome of B. graminis was downloaded from BluGen (http://www.blugen.org/)⁴¹. The microarray data of M. larici-populina and P. graminis used in this study were downloaded from Gene Expression Omnibus (GEO) at NCBI (http://www.ncbi.nlm.nih.gov/geo/) and their accession numbers are GSE21624 and GSE25020, respectively^27,28. Protein structure modeling was performed using the Phyre2 server⁴² (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=inde7x) and rendered using UCSF Chimera⁴³. COBALT⁴⁴ (http://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeAd) was used to generate the amino acid alignment, which was viewed and edited with “Jalview” software⁴⁵. The CAZymes were identified by the Hmmscan program in the HMMER 3.0 package⁴⁶ using the family-specific HMM profiles of CAZymes from the dbCAN database⁴⁷. The sub-classification of CAZymes (PCW-degrading enzymes, FCW-degrading enzymes and their respective related CBMs) was performed according to Amselem et al.³⁵. The SignalP⁴⁸ was used to identify secreted proteins. In this study, we defined cysteine-rich, small, secreted proteins as the secreted proteins consisting of at least 76 aa and that are less than 300 aa in length and contain at least 4% cysteine residues. Gene expression patterns were clustered using “cluster”⁴⁹ and the hierarchical clustering algorithm was used based on the average-linkage method⁵⁰. The dendrogram and colored image were produced by “javaTreeview”⁵¹. The functional enrichment analyses were performed according to FungiFun2⁵².

Bacterial and fungal strains, plant and culture conditions

The virulent S. sclerotiorum wild-type strain Ep-1PNA367 was used and routinely cultured on potato dextrose agar (PDA, Difco, Detroit, MI, USA) at a neutral pH at 20 °C. S. sclerotiorum transformants were cultured on PDA amended with 80 μg/ml hygromycin B (Calbiochem, San Diego, CA, USA). Escherichia coli strain DH5α was used to propagate all of the plasmids and A. tumefaciens strain EHA105 was used for the transformation of fungi. Seedlings of A. thaliana (ecotype Columbia-0) were grown in the greenhouse at 20 ± 2 °C for one month under a 12 h light/dark cycle with 70% relative humidity.

Sample preparation, RNA extraction, cDNA production and DGE analysis

For DGE analysis, the Ep-1PNA367 strain was grown or treated under different conditions and samples were collected for RNA extraction during the following stages: (i) Vegetative stage: fresh mycelia of the Ep-1PNA367 strain were placed on a cellophane membrane overlaid onto PDA medium at 20 °C and the mycelia were collected at 12, 24, 36, 48 and 60 h; (ii) Sclerotial formation stage: the colonies growing on the cellophane membrane overlaid onto PDA medium were further incubated under the same conditions and then the cultures were collected at 84, 96, 108, 120 and 132 h; (iii) Early stages of infection: fresh hyphal fragments of the Ep-1PNA367 strain were overlaid onto sterilized cheese cloth, which was overlaid onto the leaves of the A. thaliana ecotype Columbia-0, followed by inoculation at 20 °C with 100% relative humidity and then, the cheese cloth with hyphae was rolled up from the leaves at 9 h and 12 h; (iv) Sclerotial myceliogenic germination stage: sclerotia were surface sterilized with sodium hypochlorite and were placed onto a PDA plate at 20 °C to induce myceliogenic germination; the sclerotia were harvested when approximately 50% had germinated; (v) Sclerotial carpogenic germination stage: sclerotia were dried at room temperature and pretreated in a freezer (4–6 °C) for up to one month; then, the sclerotia were surface sterilized and placed onto wet sterilized sands in a plate at 15 °C to induce carpogenic germination; the sclerotia were harvested when approximately 50% germinated (stipes having only emerged from the sclerotia); and (vi) Early apothecial formation stage: sclerotia were allowed to grow in the same incubator and the stipes were cut and collected immediately before apothecium formation for RNA extraction. Finally, different time-point samples from the vegetative stage, sclerotial formation stage and infection stage were pooled together in equal quantities for RNA extraction. For qRT-PCR analysis, the samples used for validation of the DGE data were prepared independently under the same conditions described above. To evaluate the expression levels of SsCVNH in different transformants, the transformants and the wild-type strain were cultured on PDA at 20 °C for 7 dpi before total RNA extraction. This time-point was selected for qRT-PCR analysis because the expression of SsCVNH peaks at 7 dpi. For both DGE and qRT-PCR analysis, total RNA extraction was conducted according to Zhu et al.²³. Briefly, total RNA was isolated with TriZOL reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocols. The total RNA was incubated for 30 min at 37 °C with 10 units of DNase I (Roche Applied Science, Shanghai, China) to remove residual genomic DNA. The first cDNA strand was synthesized from 1.0 μg of total RNA by Moloney murine leukemia virus reverse transcriptase (Promega, Madison, USA) using oligo(dT)₁₈ primers. For DGE analysis, tag-based transcriptome sequencing methods were used to perform the DGE (Solexa/Illumina, Shenzhen, China) analysis. The DGE raw sequences were transformed into clean tags and subsequently mapped to the S. sclerotiorum transcript database from the Broad Institute (http://www.broadinstitute.org/annotation/genome/sclerotinia_sclerotiorum/MultiDownloads.html). All of the clean tags were mapped to the reference sequences with no more than 1 nt mismatch allowed. Clean tags mapped to reference sequences from multiple genes were filtered. The remaining clean tags were designated as unambiguous clean tags. The number of unambiguous clean tags for each gene was calculated prior to normalization to the number of transcripts per million clean tags (TPM)⁵³. The complete expression datasets are available at the GEO database (NCBI) as series GSE65301.

Identification of differentially expressed genes

For DGE experiment, rigorous algorithms were developed to identify differentially expressed genes (or significantly regulated genes) between the two cDNA libraries according to Audic et al.⁵⁴. In this study, the P-value and false discovery rate (FDR) were manipulated to determine differentially expressed genes⁵⁵. An FDR ≤ 0.001 and an absolute value of log₂Ratio ≥ 1 (more than 2.0 fold change) were used as thresholds to determine significant differences in gene expression. For microarray experiment, a Student t test with FDR multiple testing correction was applied to the data using the INVEX web⁵⁶. Transcripts with an adjusted P value < 0.05 and an absolute value of log₂Ratio ≥ 1 (more than 2.0 fold change) in transcript level were considered as significantly differentially expressed.

Quantification of gene expression by qRT-PCR

QRT-PCR was used to validate the DGE data and evaluate the expression levels of SsCVNH in different transformants. The mRNA transcripts were measured using a SYBR Green I real-time RT-PCR assay in a CFX96™ real-time PCR detection systems (Applied Biosystems). The thermal cycling conditions were 95 °C for 2 min for predegeneration, 40 cycles of 95 °C for 10 s for denaturation, 58 °C for 20 s for annealing and 72 °C for 20 s for extension. All of the reactions were run in triplicate by monitoring the dissociation curve to control the dimers. The S. sclerotiorum β-tubulin gene was used as a normalizer²⁵. After amplification, data acquisition and analysis were performed using the Bio-Rad CFX Manager^TM Software (version 2.0). The 2^−ΔΔCT method was chosen as the calculation method⁵⁷. The difference in the cycle threshold (CT) values of the genes and the housekeeping gene β-tubulin (called ΔC_T) was calculated as follows: ΔΔCT = (ΔC_T of genes at each time point)-(ΔC_T of the initial control). See supplementary Table S6 for the primers for qRT-PCR.

Western blot analysis and the subcellular localization of SsCVNH

To generate the SsCVNH-FLAG fusion constructs (Figure S9a), the promoter P_EF−1α was PCR amplified using the primers P_EF−1α − 1 F/R and subsequently digested with Xho I and Sac I. The PCR products of SsCVNH were amplified with the primers FLAG-SsCVNH F/R and subsequently digested with Sac I and Sma I. These two fragments were sequentially ligated into the pCH vector through intermediate constructs. The primers are shown in Supplementary Table S6. The SsCVNH-FLAG construct was transformed into the wild-type strain using the Agrobacterium-mediated transformation method as previously described⁵⁸. Briefly, the A. tumefaciens cells with the SsCVNH-FLAG constructs were diluted in minimal medium⁵⁹ amended with 50 μg/ml kanamycin and incubated overnight at 28 °C. Then the A. tumefaciens cells were diluted in induction medium⁵⁹ and incubated for 6 h at 28 °C with gentle shaking, before they were co-cultivated with fresh S. sclerotiorum mycelial plugs on a cellophane membrane placed on co-induction medium (induction medium with agar) at 20 °C for 2 days. The cellophane membrane was then transferred to the selective medium [PDA amended with 80 μg/ml hygromycin B and 200 μg/ml cefotaxime sodium (DingGuo, Beijing, China)] after the mycelial plugs were removed. Colonies that were regenerated through the selective medium after incubation at 20 °C for 3–6 days were transferred to the selective medium for subcultivation. Total proteins were extracted from the transformants by cell lysis buffer for western blotting and IP (Beyotime, Wuhan, Hubei, China). A total of 5 μl of ANTI-FLAG M2 monoclonal antibody (Sigma, St Louis, MO, USA) was added to 1 ml of the protein extracts and incubated at room temperature for 2 hours. Next, protein A + G agarose (Beyotime, Wuhan, Hubei, China) was added to the protein extracts and incubated at room temperature for 1 hour prior to collection by centrifugation. The extracts were washed five times with cell lysis buffer and then protein loading buffer was added for the following western blot analysis. Tissues from onion epidermal cells inoculated with SsCVNH-FLAG-engineered strains were sampled at 36 hpi. The lower epidermis was peeled and cut into 1-cm pieces near the inoculation site and fixed with 4% paraformaldehyde for 10 min. Subsequently, the sample was washed 3 times with 1 × phosphate-buffered saline (PBS, pH 7.4) (5 min each wash), incubated in 0.2% Triton X-100 for 10 min and washed 3 additional times with 1 × PBS (5 min each wash). After removing the Triton X-100, the sections were blocked for 30 min in 1% (w/v) BSA and washed 3 times with 1 × PBS (5 min each wash). The sections were subsequently incubated with primary anti-FLAG-tag mouse monoclonal antibody for 2 h at 37 °C, rinsed 3 times with 1 × PBS (5 min each rinse) and incubated in secondary rhodamine red-X-conjugated (RRX) or fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG for 2 h at 37 °C. Subsequently, the onion tissues from the inoculated lower epidermis were rinsed, viewed and photographed using a confocal laser scanning microscope (OLYMPUS® microscope FV1000). The 570 nm and 492 nm absorption laser lines with corresponding appropriate specific emission filter sets were used when imaging RRX and FITC, respectively.

Construction of RNAi vectors and the transformation of S. sclerotiorum

Two strategies described by Nguyen⁶⁰ and Yu⁵⁸ were adopted to construct the S. sclerotiorum RNAi vectors. Briefly, a 336 bp fragment from SsCVNH was amplified with the primers RNAi-SsCVNH F/R from the S. sclerotiorum cDNA library and (i) directly ligated into the pCXDPH vector digested by Xcm I (New England Biolabs, Beverly, MA, USA) to produce the pRNAi-1 vector (Figure S9b); and (ii) digested with two pairs of suitable enzymes, followed by ligation of both of the digested fragments into the pCIT vector in the inverse orientation. Finally, the fusion fragment of PtrpC-SsCVNH-intron-SsCVNH-TtrpC was digested with Sac І and Xho І and subsequently ligated into the pCH vector⁵⁸ to produce the pRNAi-2 vector (Figure S9c). The primers used to generate these RNAi vectors are shown in Supplementary Table S6. The Agrobacterium-mediated transformation method used to transform S. sclerotiorum is described above.

Characterization of the SsCVNH-silenced transformants

To evaluate virulence, at least nine individual, fully expanded leaves of Brassica napus and tomato were detached and inoculated with a single 0.5-cm diameter fresh mycelium-colonized PDA plug of each silenced transformant and the wild-type strain for moisture culture at 20 °C. Disease severity was measured using the average lesion diameter at 48 hpi. To assay growth rates, the virulent wild-type strain and the silenced transformants were cultivated on PDA at 20 °C for 3 days. Mycelial agar discs were collected from the active colony edge and inoculated in the center of the PDA Petri dish at 20 °C prior to examination of hyphal growth. After growth on PDA at 20 °C for 48 h, the tip hyphal morphology of the virulent wild-type strain and the silenced transformants was observed under a light microscope. Each experiment was performed at least three times.

Additional Information

How to cite this article: Lyu, X. et al. Comparative genomic and transcriptional analyses of the carbohydrate-active enzymes and secretomes of phytopathogenic fungi reveal their significant roles during infection and development. Sci. Rep. 5, 15565; doi: 10.1038/srep15565 (2015).

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Acknowledgements

This research was supported by the Special Fund for Agro-scientific Research in the Public Interest (grant number 201303025), the National Basic Research Program (2012CB114000), Nature Science Foundation of Hubei Province (2015CFB294) and the earmarked fund for China Agriculture Research System (CARS-13).

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State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, Hubei Province, China
Xueliang Lyu, Cuicui Shen, Daohong Jiang, Guoqing Li & Jiasen Cheng
The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei Province, China
Xueliang Lyu, Cuicui Shen, Yanping Fu, Jiatao Xie, Daohong Jiang, Guoqing Li & Jiasen Cheng

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Xueliang Lyu
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Cuicui Shen
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J.C. and D.J. designed research; X.L. and C.S. performed research; X.L., Y.P., J.X., D.J., G.L. and J.C. analyzed data; and X.L. and J.C. wrote the paper. All authors read and approved the final manuscript.

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Lyu, X., Shen, C., Fu, Y. et al. Comparative genomic and transcriptional analyses of the carbohydrate-active enzymes and secretomes of phytopathogenic fungi reveal their significant roles during infection and development. Sci Rep 5, 15565 (2015). https://doi.org/10.1038/srep15565

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Received: 26 June 2015
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DOI: https://doi.org/10.1038/srep15565

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