Secretome analysis reveals effector candidates associated with broad host range necrotrophy in the fungal plant pathogen Sclerotinia sclerotiorum
© Guyon et al.; licensee BioMed Central Ltd. 2014
Received: 3 February 2014
Accepted: 27 April 2014
Published: 4 May 2014
The white mold fungus Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a remarkably broad host range. The interaction of necrotrophs with their hosts is more complex than initially thought, and still poorly understood.
We combined bioinformatics approaches to determine the repertoire of S. sclerotiorum effector candidates and conducted detailed sequence and expression analyses on selected candidates. We identified 486 S. sclerotiorum secreted protein genes expressed in planta, many of which have no predicted enzymatic activity and may be involved in the interaction between the fungus and its hosts. We focused on those showing (i) protein domains and motifs found in known fungal effectors, (ii) signatures of positive selection, (iii) recent gene duplication, or (iv) being S. sclerotiorum-specific. We identified 78 effector candidates based on these properties. We analyzed the expression pattern of 16 representative effector candidate genes on four host plants and revealed diverse expression patterns.
These results reveal diverse predicted functions and expression patterns in the repertoire of S. sclerotiorum effector candidates. They will facilitate the functional analysis of fungal pathogenicity determinants and should prove useful in the search for plant quantitative disease resistance components active against the white mold.
KeywordsSclerotinia sclerotiorum. Effectors Gene expression Secretome Necrotrophic fungal Pathogen Arabidopsis thaliana
The white mold fungus Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan necrotrophic pathogen infecting over 400 plant species. It is among the most devastating pathogens of soybean, rapeseed and sunflower, causing several hundred million dollar losses annually at the pre- and postharvest stages . S. sclerotiorum host range is remarkably broad, with fruit and vegetable productions also being severely impacted . S. sclerotiorum and its close relative the grey mould fungus Botrytis cinerea are among the few fungal pathogens considered as typical necrotrophs. As such, they derive energy to complete their life cycle mostly from dead plant cells, as opposed to biotrophs that feed on living plant cells.
There is now ample evidence that biotrophic and hemibiotrophic fungi secrete specialized effector proteins manipulating host cell physiology to obtain nutrients, suppress plant defense and ultimately promote infection . Effectors may also trigger plant defense responses, leading to plant resistance, when recognized directly or indirectly by the plant immune system in a gene-for-gene relationship. This results from a co-evolutionary arms race between pathogen effectors, their plant targets, and components of the plant immune system . Necrotrophs have long been considered as less adapted, secreting mostly degrading enzymes and toxins that unspecifically trigger programmed cell death (PCD) and dismantle plant cells.
However, host specific necrotrophs such as Cochliobolus victoriae secrete effector proteins translocated into plant cells that interact with specific corresponding host proteins to facilitate disease progression [5, 6]. This involves the activation of plant PCD instead of its suppression as in the case of infection by biotrophic pathogens. S. sclerotiorum also produces the non-proteic pathogenicity determinant oxalic acid. This molecule induces the synthesis of reactive oxygen species (ROS) and triggers plant PCD late during infection, but has the opposite effect, suppressing ROS burst and PCD induction, at the early stages of infection . The SSITL secreted integrin-like protein of S. sclerotiorum promotes virulence and delays the activation of plant defense responses, supporting the view that S. sclerotiorum secretes effectors to finely manipulate plant physiology . In addition, enzymes secreted by necrotrophs can act as virulence factors independently of their catalytic activity . Effector repertoires vary considerably, notably according to pathogens lifestyle , and it is becoming clear that interactions between necrotrophs and their host plants are considerably more complex and subtle than previously considered. What is the effector candidate repertoire associated with broad host range necrotrophy remains unclear. As a first step towards elucidating the molecular bases for colonization by S. sclerotiorum, its repertoire of effector candidates needs to be determined.
The recent release of genome sequences for a number of plant pathogenic fungi facilitated the search for effector candidates (ECs) at the genome level . Nevertheless, considering that pathogen effector repertoires are typically lineage-specific, the identification of effectors remains challenging . The analysis of S. sclerotiorum genome sequence uncovered sets of genes associated with the manipulation of redox status, including enzymes of OA biosynthesis, the degradation of plant cell wall, and 603 secreted proteins with other functions . Known hallmarks of fungal effectors such as the presence of signal peptides and absence of transmembrane domains, small size and amino-acid composition generally produce lists of hundreds of potential effectors in a given pathogen. Therefore, more sophisticated approaches are required to pinpoint the most relevant ECs for the promotion of infection in S. sclerotiorum secretome.
A limited number of known fungal effector families show conservation at the sequence level or similar predicted functions. This is notably the case for the toxin and cell death elicitor proteins of the Necrosis and ethylene-inducing Like Proteins (NLPs), the cerato-platanin, cyanovirin-N homology (CVNH) and ECP2 families [13–16]. The growing number of characterized fungal effectors suggests conservation at the biochemical function level in the overall effector repertoire of several fungal pathogens. The ability to bind chitin or other cell wall oligosaccharides, masking the presence of the pathogen or dampening damage-induced plant responses, is a feature common to effectors from multiple fungal pathogens [17–20]. Fungal effectors harboring a protease inhibitor activity are also common [21–24]. The biochemical activity of a few other fungal effectors such as M. oryzae Fungalysin metalloprotease AvrPita , U. maydis chorismate mutase cmu1  and peroxidase inhibitor PEP1  may also be part of the arsenal of effector functions in multiple fungal lineages. This hypothesis suggests that thorough annotation of protein domains and prediction of biochemical function of secreted proteins may prove useful to identify novel effectors in S. sclerotiorum.
However, a majority of effectors do not show significant similarity to known sequences in other organisms nor obvious protein domains. Yet other genomic characteristics may help identify EC genes. The rapid evolution of effector genes allows the fungi to overcome selection pressures induced by resistant plant cultivars. A high ratio of non-synonymous over synonymous substitutions (Ka/Ks) in alleles from related strains is a frequently used proxy for inferring fast gene evolution and the action of positive selection . This approach has been used to reveal ECs in several filamentous plant pathogen lineages [29–34]. Positive selection has been detected in B. cinerea genome  suggesting that it may be used to mine S. sclerotiorum genome for ECs. Second, gene duplication is another hallmark of several known fungal effector genes, such as the ToxB host specific toxin of Pyrenophora tritici-repentis [36, 37]. Third, genomic regions with high repeat and transposable element content are enriched in effector genes in several lineages of plant pathogens [37–39] suggesting that genome architecture analysis can assist in the search for EC genes. Finally, effectors can alter host cell function by mimicking plant peptides . These ECs likely elude functional annotation on the basis of primary amino-acid sequence, but may be revealed using three-dimensional structure prediction.
As opposed to Oomycete pathogen genomes in which many effector genes can be identified through conserved sequence motifs [29, 41], the use of conserved sequence motifs, such as the Y/F/WxC motif , has proven limited in revealing fungal effectors across lineages. However, the presence of a signal peptide directing protein secretion and gene expression in planta are relatively universal properties of effectors that can be exploited as first filters to narrow down the list of effector candidates in fungal genomes.
S. sclerotiorum effector proteins would be useful as probes to search for resistance components in plants and to design strategies for inhibiting infections by this devastating but poorly characterized pathogen. In this study, we report a diverse repertoire of S. sclerotiorum effector candidates revealed by an in depth analysis of its predicted secretome. We combined refined secretome annotation, phylogeny, selection and gene duplication analyses, and three-dimensional structure prediction to identify 78 ECs. Among those, we highlight a predicted subtilisin inhibitor, a xylanase, a duplicated gene of unknown function and three toxin mimics as high priority candidates for functional studies. We analyzed in planta expression pattern for 16 EC genes and revealed host-blind and host-regulated ECs.
Definition and annotation of S. sclerotiorumsecretome
Next, we performed three different effector-oriented analyses on the 486 SPEP genes. First, we used Blast2GO, PFAM domain and nuclear localization signal (NLS) searches to annotate 326 SPEPs (Figure 1b). We built a database of known fungal effectors and explored the literature to select 31 S. sclerotiorum ECs among annotated SPEP genes. Second, we defined clusters of orthologous genes (COGs) between S. sclerotiorum and B. cinerea predicted genes using Inparanoid (Figure 1c). A total of 197 SPEP genes grouped in COGs. We aligned S. sclerotiorum and B. cinerea orthologs for these 197 gene pairs and calculated ratios of non synonymous over synonymous substitutions (Ka/Ks) to identify five ECs with signature of positive selection. The 289 SPEP genes with no ortholog in B. cinerea were grouped into clusters based on sequence similarity to identify 29 ECs distributed in 24 families containing genes duplicated in S. sclerotiorum. Finally, we analyzed the taxonomic distribution of SPEP genes across the kingdom Fungi using BlastP searches against a database of 14 complete genomes representative of all major fungal lineages. This identified 70 S. sclerotiorum-specific SPEP genes, most of which had no annotation (Figure 1d). We used protein structure prediction and pattern and fold recognition searches to identify 17 ECs analogous to known protein fold encoded by S. sclerotiorum-specific SPEP genes.
Using effector-oriented analyses, we identified four lists of ECs, containing a total of 78 EC genes (four being common to two lists). We could not predict any enzymatic activity encoded by 33% of the SPEP genes (160), suggesting that S. sclerotiorum effector repertoire encodes diverse functions that are not restricted to plant cell degrading enzymes. Besides, 59.5% of the SPEP genes (289) did not cluster in B. cinerea COGs, revealing a relatively high degree of divergence from this closely related fungal pathogen.
Sclerotiniaeffector candidates showing conserved domains
List of 31 S . sclerotiorum effector candidates selected based on their annotation
SPEPs containing PFAM domains found in fungal effectors
Chitin binding protein
Chitin recognition protein (PF00187.14)
Chitin binding protein
Chitin binding domain (PF03067)
Chitin binding protein
Chitin recognition protein (PF00187.14)
Starch binding domain containing protein
Chitin binding domain (PF03067)
Chitin binding protein
Chitin binding domain (PF03067)
Agglutinin isolectin 3-like
Chitin recognition protein (PF00187.14)
LysM domain protein
Concanavalin A lectin glucanase
Ricin-type beta-trefoil lectin domain (PF00652)
Serine protease inhibitor
Peptidase inhibitor I9 (PF05922)
Serine protease inhibitor
Peptidase inhibitor I9 (PF05922)
Alkaline serine protease alp1
Peptidase inhibitor I9 (PF05922)
SPEPs containing nuclear localization signal (NLS)
PHD-like zinc-binding domain (PF13771); Ring finger domain (PF13639)
Zinc finger CCCH-type domain containing protein
Zinc finger C-x8-C-x5-C-x3-H type (PF00642)
NLS (189–250; 340–349)
NLS (66–120; 143–167; 225–245)
NLS (65–79; 118–138; 223–233)
Ribosomal protein s17
SPEPs showing homology to fungal effectors or fungal effector candidates
Necrosis inducing protein (PF05630)
Homolog to B. cinerea NEP1 (Staats et al., 2007); P. Sojae NIP (Qutob et al., 2002); studied in (Bashi et al., 2011)
Necrosis inducing protein (PF05630)
Homolog to B. cinerea NEP1 (Staats et al., 2007); P. Sojae NIP (Qutob et al., 2002); studied in (Bashi et al., 2011)
AltA-1 allergen analog
Homolog to C. hingginsianum HE651255_CHEC91 Kleemann et al., 2012)
CyanoVirin-N Homology domain (PF08881)
Homolog to C. hingginsianum HE651243_CHEC80 Kleemann et al., 2012)
Deuterolysin metalloprotease (M35) family (PF02102)
Homolog to M. oryzae AvrPita (MGG_15370 - Orbach et al., 2000)
Homolog to B. cinerea BcSpl1 (Frias et al., 2011); H. atroviridis EPL1 (Seidl et al., 2006); M. oryzae MSP1 Jeong et al., 2007); C. hinginsianum HE651160_CHEC5 Kleemann et al., 2012)
A family of subtilisin-inhibitor effector candidates conserved in Ascomycetes
Sclerotiniaeffector candidates showing high Ka/Ks ratios
List of five S . sclerotiorum effector candidates selected based on Ka/Ks > 1 in pairwise comparisons with their B . cinerea orthologs
Ka/Ks vs BC1T
KaKs vs BcT4
Ka/Ks > 1 sites
max Ka/Ks = 0.93
max Ka/Ks = 0.4
Glycoside hydrolase family 11 (PF00457.12)
R36, L43, D74, Q76, S128, N174, S200
P22, K31, T34, A36, S54, A63, S85, S86, G88, S89, Q95, A118, D146, I165, F191, D208, P211, S212, T213, L218, 226I, 230A, 233S, 236A, 237G, 238 T, S246, V253, M258, S259, N260, 261 V, N263, V269, S274, P275, N276, 278Q, H285, A286, A290, H301, S302, P306, S310, N316, K318, S319, S324
Glycoside hydrolase family 15 protein
Glycoside hydrolase family 15 (PF00723.16); Carbohydrate-binding module family 20 (PF00686.14); Carbohydrate-binding module family 25 (PF03423.8)
N53, R56, M81, S91, N128, S313, S361, Q383, Q409, S416, N514, Y526, F537, V549, K586, V589, S605, S617, Q639
Sclerotiniaeffector candidates encoded by recently duplicated genes
The 24 gene clusters containing duplicated S . sclerotiorum SPEP genes
N° Ss - Bc*
S. sclerotiorum SPEPs
Other genes in group
Nearest repeat to SPEPs (distance Kb)
BC1G_01968, SS1G_09509, BC1G_01095, SS1G_00547, BC1G_09386, SS1G_09141, SS1G_05689, BC1G_02407, SS1G_06186, BC1G_12856, SS1G_02784, BC1G_13021
SS1G_04513, SS1G_09104, SS1G_09671, BC1G_12617, BC1G_09286, SS1G_09338, BC1G_00394, SS1G_14236, BC1G_00455
Glycoside hydrolase family 47 protein
BC1G_01945, SS1G_12508, SS1G_12939, BC1G_02687, BC1G_11888, BC1G_10788, SS1G_01984, SS1G_14293
SS1G_08020, BC1G_05350, BC1G_01594, SS1G_11304, BC1G_11407, SS1G_05897
Glycoside hydrolase family 18 protein
BC1G_04114, SS1G_12365, SS1G_03721, SS1G_14379, SS1G_11693, SS1G_10104
BC1G_01964, SS1G_12510, SS1G_00677, SS1G_00773, BC1G_00533
Glycosyl hydrolases family 18 protein
BC1G_10397, SS1G_10773, BC1G_07160, SS1G_13589
SS1G_03681, SS1G_10564, BC1G_10623, BC1G_03527
CFEM domain containing
Xyloglucan-specific endo-betaglucanase A
Cell wall glucanase
BOTY_LTR (13.0) – Gypsy-31_ADe-I ( 35.9)
Bacterial alpha-L-rhamnosidase domain protein
BOTY_LTR (3.9) - BOTY_LTR (11.4)
Tripeptidyl peptidase sed3
BOTY_LTR (1.1) - Mariner-3_AF (5.9)
BOTY_LTR (18.2) - BOTY_LTR (0.6)
LysM domain protein
Alpha-mannosidase family protein
Amidase family protein
Autophagy related lipase
5SrRNA_AN (2.3) - Tad1-14_BG (1.8)
Acetyl xylan esterase
Heterokaryon incompatibility Het-c domain protein
Thioesterase-like domain protein
Cluster051 is remarkable for containing only genes coding for secreted proteins, with only one from B. cinerea and 6 from S. sclerotiorum, such as the SPEP gene SS1G_13371 (Table 3). To get support for the duplication of SS1G_13371 gene ancestor in S. sclerotiorum lineage, we constructed a phylogenetic tree of SS1G_13371 homologs. A total of 58 homologs in 25 fungal species could be retrieved from the JGI database covering 238 complete fungal genomes. We selected the 20 closest homologs to build a parsimony tree based on a 90 amino-acid alignment (Figure 4c). For the 6 S. sclerotiorum genes of cluster 051, the phylogeny revealed clustering based on paralogy rather than orthology, suggesting that this family expanded after the separation of the 25 species analyzed. Gene duplication in this family may have allowed increased accumulation of the corresponding protein, neo-functionalization in some paralogs, or differential regulation.
S. sclerotiorum-specific effector candidates with toxin structural analogs
List of 17 Sclerotinia -specific effector candidates revealed by pattern and fold recognition searches
Selected analog description
S. cerevisiae Exo70 with additional residues to 2.1 Angstrom resolution
Crystal Structure of the two MBT repeats from Sex-Comb on Midleg (SCM) in complex with peptide R-(me)K-S
Predicted nucleotide-binding protein from Vibrio cholerae
Schizosaccharomyces pombe YchF GTPase
Crystal structure of SusD-like carbohydrate binding protein (YP_001298396.1) from
Bacteroides vulgatus ATCC 8482 at 1.70 A resolution
Crystal structure of PAS domain from the mouse EAG1 potassium channel C-terminal portion of human eIF4GI
Pseudomonas mendocina lipase
X-ray structure of the RNA-binding protein SHE2p
Lectin Domain of Lectinolysin complexed with Glycerol
Crystal structure of the conserved herpesvirus fusion regulator complex gH-gL
Penicillin-binding protein 2b (PBP-2b) from Streptococcus pneumoniae (strain 5204)
Saposin-like protein Na-SLP-1
ERAD pathway mediated by the ER-resident protein disulfide reductase ERdj5
Oxidized pea fructose-1,6-bisphosphate phosphatase
C-terminal region of Death Associated Protein 5(DAP5)
Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-angstrom resolution
S. sclerotiorum effector candidate genes show diverse patterns of expression in planta
The set of 16 ECs analyzed presented diverse expression patterns. We observed peaks of expression on N. benthamiana at 6, 24 or 48 hpi for SS1G_08858, SS1G_07749 and SS1G_00849 respectively illustrating the diversity of induction kinetics. On A. thaliana Shahdara accession, peaks of expression occurred at 6, 24 or 48 hpi for SS1G_01593, SS1G_06213 and SS1G_10096 respectively (Figure 7b). At 24 hpi, extensive cell death was visible on leaves of A. thaliana accession Shahdara whereas only limited cell death symptoms were visible on N. benthamiana (Additional file 2: Figure S1), suggesting that the activation of host cell death is not the only determinant of S. sclerotiorum EC induction. We observed a consistent 2- to 4- fold induction between 6 and 24 hpi for SS1G_06213 on all four host plants tested. By contrast, SS1G_08858 was induced >4-fold at 6 hpi on N. benthamiana, at 24 hpi on tomato, and at 48 hpi on A. thaliana (Figure 7c). This result suggests that S. sclerotiorum possess effector genes that are regulated independently of the host being colonized and others that are differentially regulated in a host-dependent manner. Furthermore, SS1G_13371 was induced >2 fold during infection of A. thaliana resistant accession Rubezhnoe, but not during infection of the susceptible accession Shahdara. Conversely, SS1G_00849 was induced >8 fold during infection of the susceptible accession Shahdara, but only ~4 fold during infection of the resistant accession Rubezhnoe (Figure 7d). This data points towards a versatile repertoire of effector candidates the expression of which can be modulated according to the nature of the host plant being colonized.
In their global analysis of S. sclerotiorum genome, Amselem et al.  identified 603 genes encoding non-CAZYme, non-peptidase secreted proteins. These secretome genes did not appear significantly enriched in genes induced in planta. In this study, we combined multiple bioinformatics approaches to identify a total of 745 predicted secreted proteins, among which 486 with experimental evidence for expression in planta (SPEPs). The predicted SPEPs include SsNEP1 and SsITL1 that have proposed to be S. sclerotiorum virulence factors [8, 44]. Since we have chosen to focus the search for effector candidates on these 486 SPEP genes, we have deliberately ignored genes expressed in planta for which experimental evidence is lacking, and enzymes that contribute to the biosynthesis of secondary metabolites as virulence determinants. It is therefore expected that the diversity of S. sclerotiorum virulence factors exceeds that of the candidate effectors presented here. Sequence similarity to known fungal effectors is a powerful method to uncover effector families conserved across species , that allowed us to identify S. sclerotiorum homologs of B. cinerea NEP1  and Spl1 , M. oryzae MGG_15370 and C. hingginsianum CHEC91 and CHEC80. To complement this approach, we used PFAM domain and NLS motif searches to reveal additional effector candidates. We identified putative chitin-binding proteins, putative protease inhibitors, cystein-rich proteins and putative nuclear localized proteins. Effectors with chitin binding activity such as C. fulvum Ecp6, M. oryzae Slp1 and M. graminicola Mg3LysM function in suppressing plant immunity [18, 20, 59]. Similarly, SsITL1 (SS1G_14133) integrin-like secreted protein suppresses plant jasmonic acid and ethylene signaling pathways and enhances susceptibility . These findings suggest that S. sclerotiorum secretes proteins able to suppress plant immunity.
The comparative analysis of Fusarium graminearum secretome and genomes of other ascomycetes revealed a high level of conservation with only 25 F. graminearum specific out of 574 secreted proteins . The taxonomic distribution of S. sclerotiorum SPEP homologues analyzed in this work supports the conservation of more than 50% of SPEP genes across ascomycetes. As proposed by Brown et al. , these core SPEP genes may support S. sclerotiorum epiphytic growth and highlight important distinctions between multiple phases in infection by this fungus . Nevertheless it also revealed 70 SPEP genes (14%) specific to S. sclerotiorum, many of which are unannotated proteins. The systematic prediction of their 3D structure allowed identifying putative structural analogs of some predicted SPEPs and suggests that they may carry unique functions to assist S. sclerotiorum pathogenicity. It will be interesting to take advantage of these predictions to test the biological function of these effector candidates and confront them to experimentally determined structures. Furthermore, in spite of the limited sequence diversity included in the dataset analyzed here, we were able to detect signatures of positive selection in five S. sclerotiorum SPEP genes (2.5% of genes analyzed). Similar frequency (3.2%, 21 out of 642 genes) has been reported in B. cinerea . In the future, an in depth exploration of sequence diversity in S. sclerotiorum should allow to reveal more sites subjected to selection. SPEP genes for which positive selection has been detected encode cell wall degrading enzymes, including SS1G_07749 encoding a putative xylanase. This protein is related to B. cinerea Xyn11 considered as a Pathogen Associated Molecular Pattern (PAMP) . The detection of positive selection in SS1G_07749 is therefore consistent with the hypothesis that PAMPs may be characterized by signatures of positive selection in a background of strong negative selection . It may therefore be hypothesized that a subset of S. sclerotiorum critical secreted enzymes are engaged in an evolutionary arms race with plant pattern recognition receptors, driving opposing forces of natural selection S. sclerotiorum effector genes. Since plant inhibitors are known for many fungal cell wall degrading enzymes, it is also possible that an evolutionary arms race with plant inhibitors drives the evolution of some S. sclerotiorum effector candidates [53, 62, 63]. Remarkably, we also identified 14.4% of species-specific SPEP genes. The extent to which evolutionary constraints imposed by a broad host range contributes to diversification in the effector candidate repertoire of S. sclerotiorum remains to be determined. Detailed functional analysis of effector gene alleles and their plant targets will be needed to address this question.
Although S. sclerotiorum is considered as a typical necrotroph, there is evidence that it colonizes plant tissues through multiple phases involving important transcriptional and physiological reprogramming . Consistent with this model, the phytotoxin oxalic acid dampens plant immune responses at the initial stages of infection and later enhances programmed cell death . In this work, we report the sequential transcriptional activation of S. sclerotiorum candidate effector genes. A >2 fold induction was measured at 6 hpi for several effector candidate genes, whereas no necrotic symptoms are visible at this time, except on A. thaliana Shahdara accession. This suggests that the sequential secretion of effectors is required for the efficient induction of host cell death by S. sclerotiorum, or that some secreted proteins could contribute to S. sclerotiorum virulence independently of host cell death activation. By comparing the expression pattern of selected Blumeria graminis f. sp. hordei genes grown on barley and on A. thaliana, Hacquard et al.  concluded that very divergent hosts do not significantly alter the fungal gene expression program. The expression pattern of some S. sclerotiorum ECs is indeed independent on the host plant being colonized (e.g. SS1G_06213). Nevertheless, other ECs showed differential regulation in a host-dependent manner (e.g. SS1G_08858, SS1G_13371). The transcriptional activation of distinct set of effectors depending on the host being colonized has also been reported for the generalist root endophyte Piriformospora indica in barley and A. thaliana . The growing number of transcriptomic studies on various pathosystems should help determine whether host-dependent modulation of effector gene expression differs according to pathogens lifestyle or host range. We speculate that the white mold fungus benefits from a versatile repertoire of secreted proteins with diverse functions, evolution and expression patterns, to successfully infect a wide range of host plants. A systematic characterization of S. sclerotiorum transcriptome on multiple hosts and the functional analysis of differentially regulated effector genes should prove useful to decipher the molecular determinants of quantitative disease resistance and host range in this fungal pathogen.
In this work, we explored systematically the diversity of candidate virulence genes in the necrotrophic fungal pathogen S. sclerotiorum using in silico structure and evolution analyses. We report the identification of 486 S. sclerotiorum secreted proteins expressed in planta, including 78 ECs. We have analyzed in planta expression for a representative subset of 16 ECs, highlighting diverse predicted functions and expression patterns. This study reveals that besides plant degrading enzymes, S. sclerotiorum genome encodes numerous predicted secreted proteins that may be involved in the interaction between the fungus and its host plants. It will facilitate future investigation on their relevance in the infection process and sheds new light on the underestimated complexity of host colonization by necrotrophic plant pathogens.
Secretome prediction and annotation
We used complete genome and predicted proteomes of Sclerotinia sclerotiorum strain 1980 v.2, Botrytis cinerea strain b05.10 v.1 and strain t4 v.1 described in . The presence of secretion signals was predicted with SignalP v.2 and v.4 [66, 67], transmembrane helices and GPI anchor sequence were predicted with TMHMM  and GPIsom  respectively. For the identification of genes expressed in planta, microarrays data for gene induction fold at 2 days post inoculation on sunflower cotyledons and Expressed Sequence Tags (ESTs) from  were used. ESTs were assigned to the S. sclerotiorum predicted transcript giving the lowest e-value in a BLASTN search. Genes were considered expressed in planta when either (i) showing induction fold ≥1 in during sunflower infection in microarrays data or (ii) being assigned at least one EST in either infection cushion, infected B. napus or infected tomato library. S. sclerotiorum predicted proteins were annotated using Blast2GO , PFAM  and NLStradamus . Predicted proteins shorter than 40 amino-acids were excluded from the analysis. PFAM domains were annotated using HMMER3 searches against the PFAM 26.0 database . We defined non-annotated predicted SPEPs as having no hit to PFAM_A with e-value <0.1. For the identification proteins similar to known fungal effectors, BlastP searches against a local database of 191 effectors with an e-value cutoff of 1e-3.
Definition of ortholog clusters and natural selection analysis
Core ortholog groups (COGs) between S. sclerotiorum 1980 and B. cinerea b05.10 or B. cinerea t4 proteomes were identified using Inparanoid7  with the following parameters: score cutoff 40 bits; sequence overlap cutoff 0.5; group merging cutoff 0.5; scoring matrix BLOSUM62. COGs in which a length difference >10 amino-acids existed between S. sclerotiorum and B. cinerea were discarded. Pairwise ortholog alignments were generated using the needleall program from the EMBOSS package using the following parameters: gapopen 50.0; gapextend 0.2; minscore 100.0; aformat3 MARKX3. Needleall output files were parsed into .axt alignments used as input in Ka/Ks calculator2 . Ka/Ks ratios were calculated for all COG pairs using Yn00 method . The identification of codon sites under positive selection was achieved through Bayesian inference using the Selecton2.2 server  with the “Positive selection enabled (M8, beta + w > =1)” evolutionary model with 8 categories, on alignments of S. sclerotiorum 1980, B. cinerea b05.10 and B. cinerea t4 orthologs.
Protein structure modeling and analysis
Protein structure modeling was performed with the I-TASSER server  and rendered using UCSF Chimera . Site-specific alignment consensus and Ka/Ks ratios were mapped onto protein models using the ‘define attribute’ function in UCSF Chimera. Moving average over a 3 amino-acid window of the percentage consensus in a 99 homologous protein alignment was used to characterize conservation in SS1G_01593 family. Structural analogs were identified using the TM-align program in I-TASSER.
Taxonomic distribution and phylogenetic analyses
Fungal taxonomy trees are based on . The presence of SPEP homologs in 234 fungal species was assessed using BlastP searches against JGI fungi Gene Catalog Proteins  with an e-value cutoff of 1e-5 without low complexity filter. Among retrieved homologs, proteins that had no signal peptide detected with SignalP4 or SignalP2 were discarded. For the global analysis of taxonomic distribution of SPEP genes, the predicted proteomes of Neurospora crassa, Magnaporthe oryzae, Verticilium dahlia, Fusarium oxysporum, Stagonospora nodorum, Pyrenophora tritici-repentis, Alternaria brassicicola, Leptosphaeria maculans, Mycosphaerella graminicola, Aspergillus flavus, Cryptococcus neoformans and Rhizopus oryzae were used in local BlastP searches with e-value cutoff 1. For each SPEP gene, BlastP scores for all hits in a given species were summed up, and SPEP genes were considered as absent if total score is <2. Phylogenetic trees for SS1G_13371 family was generated using the parsimony method with 100 bootstrap replicates with the Extended Majority rule, as implemented in the protpars and consense programs of the Phylip 3.67 package .
Sequence-based clustering and genome distribution of duplicated genes
S. sclerotiorum 1980 and B. cinerea b05.10 proteins were clustered based on sequence similarity by Markov clustering using the orthoMCL function in Biolayout 3D . A self BlastP search on the combined S. sclerotiorum 1980 and B. cinerea b05.10 complete proteomes with e-value cutoff 1e-30 was used as input for orthoMCL. Repeats and transposable elements were identified using RepeatMasker on S. sclerotiorum 1980 supercontigs with the cross_match method at slow speed and “Fungi” as a DNA source. Genomic distances and genome architecture heatmaps were generated according to .
Plant and fungus cultivation, inoculation procedure
Arabidopsis thaliana accession Shahdara and Rubezhnoe-1 were grown in Jiffy pots for four weeks at 22°C with cycles of 9 hours of light per 24 hours. Tomato (Solanum lycopersicum cv. Heinz) were grown for six weeks in pots containing disinfected soil in a greenhouse at 23°C with cycles of ~14 hours of light per 24 hours. Nicotiana benthamiana plants were grown for four weeks at 21°C with cycles of 16 hours of light per 24 hours. S. sclerotiorum strain S55 was first grown for 4 days on PDA plates at 25°C in the dark. Fifty mL of liquid PDB medium were inoculated with 3 agar plugs of PDA cultures and incubated for 4 days at 25°C in the dark, with 150 rpm shaking. Three independent inoculation experiments were performed in which fully grown plant leaves were cut and placed right side up on a wet paper towel in large petri dishes. Mycelium was washed twice in PDB, filtered on Miracloth (Calbiochem, CA), and spread over whole leaf surfaces. Inoculated leaves were incubated for up to 3 days at 25°C with 14 hours of light per 24 hours.
Effector candidate gene expression by quantitative RT-PCR
Plant leaves were harvested immediately and 6, 24 and 48 hours after inoculation, and ground in liquid nitrogen. Total RNA was extracted using a Nucleospin RNA II kit (Machery- Nagel) according to manufacturer’s instructions. RNAs were analyzed and quantified on an Agilent 2100 Bioanalyzer. The first-strand cDNA was synthesized using TRT reverse transcriptase (Roche) according to manufacturer’s instructions. Real-time PCR reactions included 3.5 μL of SYBR green mix (Roche), 1 μL of 5 μM primers (Additional file 3: Table S2) and 200 ng of cDNA. Reactions were performed on a Light Cycler 480 II machine (Roche) under the following conditions: 95°C for 5 minutes; 45 cycles of 95°C for 15 seconds, 65°C for 20 seconds and 72°C for 20 seconds; then 95°C for 10 seconds; 65°C for 15 seconds followed by a progressive in increase in temperature at 0.11°C/second up to 95°C to obtain melt curve. S. sclerotiorum actin (SS1G_08733) and ubiquitin 16 (SS1G_11173) genes were used as controls. The expression of effector gene candidates relative to Ct values of the control genes was determined and analyzed using the LightCycler 480 SW 1.5 software. Fungal cultures were grown in vitro for 3 days and either harvested immediately (Day 0) or inoculated to plants. Values are given as log2 ratio over Day 0 expression. Error bars represent standard deviation calculated from two technical replicates on each of three independent biological experiments.
SR is supported by a Marie Curie CIG grant (“SEPAraTE”, contract 334036) and a starting grant of the European Research Council (“VariWhim”, contract 336808). This work was supported by the French Laboratory of Excellence project "TULIP" (ANR-10-LABX-41; ANR-11-IDEX-0002-02).
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