BACKGROUND
colletotrichum truncatum  andrus w.d.
moore causes anthracnose disease of many leguminous species, including lentil , soybean  merr.
), fababean , and pea   <cit> .
this fungal pathogen employs a bi-phasic hemibiotrophic infection strategy to colonize lentil plants.
the anthracnose infection is initiated by the attachment of conidia to aerial parts of the host plant.
conidia germinate immediately after adhesion and differentiate to form infection structures called appressoria devoted to mechanical penetration.
after the appressorium has been formed, a thin infection or penetration peg emerging from the base of the appressorium pierces the host cuticle and cell wall, and grows in between the plant cell wall and plasma membrane.
it differentiates into large, bulbous invasive primary hyphae that are biotrophic in nature.
these hyphae are likely to interact with the host plasma membrane but pull away after plasmolysis , indicating either initial or weak host-pathogen association.
this situation differs from that of haustoria  and the biotrophic invasive hyphae produced by obligate biotrophic fungi, such as powdery mildews and rusts, and other hemibiotrophs like magnaporthe oryzae, the causal agent of rice blast, where the host plasma membrane remains adhered to fungal structures upon plasmolysis  <cit> .
the primary hyphae of c. truncatum are entirely confined to the first infected epidermal cell throughout the biotrophic phase  <cit> .
thereafter, the fungus switches to the necrotrophic phase that is associated with the production of thin secondary hyphae that ramify intra- and inter-cellularly, killing and macerating host tissues by hydrolytic enzymes ahead of infection.
therefore, the biotrophy-necrotrophy switch  is critical in anthracnose development.
during the transition, c. truncatum probably secretes a range of proteins to establish a compatible interaction with its host, including some that may exclusively be involved in switching the pathogen to the necrotrophic phase.
in silico analyses of whole genome sequences have predicted that fungal and oomycete phytopathogens possess large repertoires of secretory proteins, which constitute  <dig> to 10% of predicted proteomes of m. oryzae  <cit> , phytophthora sojae, p. ramorum  <cit>  and ustilago maydis  <cit> .
these secretory proteins play diverse roles in virulence and pathogenicity, and interactions with host cells.
secreted hydrolytic enzymes like cell wall degrading enzymes contribute to penetration of the plant cuticle and cell wall, and to tissue maceration during the onset of the necrotrophic stage  <cit> .
other secretory proteins, such as chitin deacetylase and proteins that bind pathogen cell wall components like chitin binding protein avr <dig> of cladosporium fulvum, modify the pathogen cell wall and are critical in evading the host defense surveillance system  <cit> .
secreted effector proteins alter biochemical, physiological, and morphological processes in host plant cells, thereby facilitating infection  or triggering defense responses   <cit> .
some of these proteins are active in the apoplast where they may interfere with host plant defense processes, e.g.
by inhibiting plant proteases and lytic enzymes.
other fungal effector proteins enter into host cells, though the mechanism of entry of effectors is still a mystery.
however, the role of rxlr  and deer motifs  in host cell targeting of oomycete effectors has been established  <cit> .
recently, kale and colleagues  <cit>  have shown that fungal effectors contain functional variants of rxlr motifs, and that the oomycete and fungal rxlr and deer motifs bind to host cell surface phosphatidylinositol-3-phosphate.
this binding may facilitate the uptaking of effectors through endocytosis.
one of the functions of fungal and oomycete effectors  is to disrupt the host signal transduction pathways that mediate defense responses.
until now, only one effector, cgdn <dig> has been identified from colletotrichum.
this effector averts hr cell death during the biotrophic phase of infection and is essential for c. gloeosporioides pathogenicity on stylosanthes guianensis  <cit> .
studies conducted to date have investigated the secretome at different stages of the infection process, including penetration , biotrophic and necrotrophic stages.
kleeman et al.
<cit>  identified  <dig> putative soluble secretory proteins from a cdna library prepared from mature appressoria of c. higginsianum grown in vitro.
sixteen candidate effector proteins were discovered from venturia inaequalis, the causal agent of apple scab, on the basis of their small size , presence of cysteine residues and of a putative sp  <cit> .
using a yeast signal sequence trap screen, krijger et al.
<cit>  identified secreted proteins from c. graminicola mycelia grown in vitro on corn cell walls and leaf extract.
recently, takahara and colleagues  <cit>  developed a fluorescence-activated cell sorting method to purify the intracellular biotrophic hyphae from c. higginsianum from homogenates of infected arabidopsis leaves and constructed a biotrophy-specific cdna library.
mosquera et al.
<cit>  identified four biotrophy associated secreted proteins from the invasive hyphae of m. oryzae by interaction transcriptome analysis.
so far, few reports have addressed the critical switch point of the hemibiotrophic infection process.
only one gene encoding a switch regulator, clta <dig>  from c. lindemuthianum, has been identified.
mutants harboring disrupted clta <dig> were unable to switch to the necrotrophic phase.
this transcriptional factor is indispensable for pathogenicity on common bean  <cit> .
the objective of the current study was to identify proteins putatively secreted during the in planta biotrophy-necrotrophy switch of c. truncatum.
to catalogue such proteins, we have generated  <dig> ests from a directional cdna library constructed from susceptible canadian lentil cultivar 'eston' inoculated with c. truncatum isolate ct- <dig>  by biocomputational analyses of these ests, we identified eleven c. truncatum candidate effector genes.
in planta functional analysis of three effectors was carried out using pvx-based agroinfiltration in tobacco leaves.
RESULTS
generation of c. truncatum ests
excised leaflets of canadian lentil cultivar 'eston' inoculated with isolate ct- <dig> of c. truncatum were collected and examined microscopically to ensure that the pathogen was at the biotrophic to necrotrophic transition stage where secondary hyphae had become visible .
this corresponded to 48- <dig> hours after inoculation  and we referred to this period of infection as the biotrophy-necrotrophy switch.
total rna was extracted, and poly a+ mrna was isolated and used for construction of a directional cdna library.
using 5'-end single pass sequencing,  <dig> ests were generated with an average read length of more than  <dig> nucleotides.
blastx analysis with a cutoff e value ≤ 10- <dig> identified 57% as plant sequences, 39% as fungal sequences and the remainder  as unassignable.
the high abundance of fungal transcripts was further verified by semiquantitative rt-pcr using the fungal 60s ribosomal gene in an infection time-course study that included control mixtures .
an accumulation of around 40% of fungal 60s ribosomal transcripts, which, in turn, reflect the fungal rna content, was predicted during the biotrophy-necrotrophy switch.
the abundance of plant origin transcripts had declined with fungal proliferation in infected host tissues as shown by rt-pcr analysis of l. culinaris 60s ribosomal gene in the same infection time-course used for assessing the fungal biomass .
the fungal transcriptome contains sequences encoding putative secretory proteins
fungal sequences  were analyzed for features indicative of secreted proteins.
because of directional cloning, we could analyze the coding sequences from the 5'-end.
orf finder, and signalp and ipsort algorithms were exploited to deduce orfs and sps within the orfs.
one hundred sixty-two predicted orfs  were predicted to encode proteins with n-terminal sps.
all  <dig> ests were deposited in the ncbi genebank est database .
using contigexpress software , these orfs could be assembled into  <dig> contigs and  <dig> singletons, resulting in a total of  <dig> unigenes.
clone ids belonging to various contigs are listed in the additional file  <dig>  the average g+c content of these unigenes was around 59%.
we refer to the deduced proteins encoded by these unigenes as putative secretory proteins.
we also employed blastx and orf finder algorithms to investigate whether the first methionine within the amino acid translation represented the true n-terminal methionine to confirm the orf of selected unigenes.
the orfs were then queried against the ncbi non-redundant protein database using blastp algorithm.
fungal effectors are most likely to be small, soluble, extracellular secreted proteins that do not become cross-linked into the fungal cell wall  <cit> .
therefore, predicted orfs from these unigenes were screened for the size of the encoded polypeptide chain and the presence of transmembrane domains, cysteine residues, transmembrane domain and glycosylation sites, including glycosyl-phosphatidylinositol  modification.
however, with the steady increase in the number of fungal phytopathogen genomes being sequenced, the likelihood that orthologs found in other species is increasing.
hence, some candidate effectors were identified based on orthologs in other phytopathogens.
comparison of the protein sequences encoded by the unigenes to the currently annotated databases and their sequence analyses revealed four groups.
the most highly represented group comprised hydrolytic enzymes, which included  <dig> unigenes , followed by  <dig> ceaps ,  <dig> candidate effector proteins  and  <dig> proteins  classified as "other proteins".
based on these analyses, a total of  <dig> unigenes were predicted to encode either proteins with transmembrane domain or gpi addition signal.
among them, six were grouped with hydrolases.
the remaining  <dig> unigenes encoded putatively soluble secretory proteins, including hydrolases.
a list of the clone ids, the top hit for each sequence and the corresponding blast score are compiled in table  <dig>  an e value cutoff ≤ 10- <dig> was used to annotate these unigenes.
four sequences had no match at ≤ 10- <dig> but contained conserved signatures.
therefore, they were classified according to sequence characteristics belonging to corresponding groups and listed in table  <dig>
* unigenes with e value > 10- <dig> are classified according to their conserved signatures as described in the text.
hydrolytic enzymes
sixty-three unique sequences showed significant similarity to members of the enzyme classes   <dig>  and  <dig> .
most of them had been annotated as cell wall degrading enzymes .
ten unigenes encoded enzymes acting on ester bonds ; most of them were reported as pectinolytic enzymes .
these included esterases, pectinesterase, extracellular lipase, phosphoserine phosphatase and cutinase.
four depolymerase pectinolytic enzymes  were also retrieved from this library.
unigenes ct21- <dig> and ct21- <dig> were similar to exopolygalacturonase and endo-polygalacturonase, respectively.
four discrete unigenes contig  <dig>  ct21- <dig>  ct21- <dig> and ct21- <dig> encoded pectate lyase  and contig  <dig> showed homology to pectin lyase .
thirty unique sequences, including ct21- <dig> and ct21- <dig> mentioned above represented the ec  <dig> subclass  <dig>  glycosidases .
glycosidases hydrolyze the bonds between carbohydrates and release sugars from plant cell walls to provide the fungus with carbon sources and support host penetration and colonization  <cit> .
these included xyloglucan-specific endo-β- <dig>  4-glucanase, cell wall glucanosyltransferase, cellulase , glycosyl hydrolase family proteins, β-glucosidase, arabinosidase, β-glucanase, mannan endo- <dig> -β-mannosidase and α-galactosidase.
sixteen unigenes belonged to the ec  <dig> subclass  <dig>  of enzymes acting on peptide bonds .
correlation between peptidase activity and virulence for several phytopathogenic fungi has already been established.
cell envelope-associated proteins
forty-three unigenes carrying sp, transmembrane helices or gpi anchor addition signal, including  <dig> unigenes homologous to functionally uncharacterized genes from fungi, were identified as ceaps.
among them, six unigenes were classified under the hydrolytic enzymes category.
in almost all cases except the unigene ct21- <dig>  the best match for these unigenes was found within the ascomycota.
interestingly, most of the identified genes have not been previously reported in the genus colletotrichum.
c-terminal gpi anchor sites were predicted in the translated amino acid sequences of  <dig> unigenes.
interestingly, we identified a member of the gpi transamidase complex  that is implicated in en bloc synthesis of gpi proteins.
three unigenes contig  <dig>  contig  <dig> and ct21- <dig> encoded gpi anchored serine-threonine rich proteins.
two unigenes, contig  <dig> and ct21- <dig>  were predicted to possess gpi anchor addition signals and cysteine rich fungal extracellular membrane  domains .
unigene ct21- <dig> was identified as a hypothetical protein that possesses a gpi addition signal.
contigs  <dig> and  <dig> were matched to a putative gpi-anchored protein and gpi-anchored serine rich protein, respectively.
clones ct21- <dig> and ct21- <dig> were matched to the gpi anchored cell wall organization protein ecm <dig> from pyrenophora tritici-repentis and glycolipid anchored surface protein gas <dig> from c. graminicola, respectively.
the predicted products of the three discrete unigenes ct21- <dig>  ct21- <dig> and ct21- <dig> resembled the biotrophy associated secreted glycoprotein colletotrichum intracellular hypha  <dig>  from c. lindemuthianum, the causal agent of common bean anthracnose  <cit> .
the orf of ct21- <dig> encoded a protein of  <dig> amino acids containing  <dig> lysin motif  domains.
these lysms recognize and bind n-acetyl d-glucosamine  <cit> , and may play a role protecting fungal chitin from plant chitinases.
the predicted orfs from three discrete clones, contig  <dig>  ct21- <dig> and ct21- <dig> encoded a protein that was similar to clock controlled protein  <dig> from neurospora crassa, induced by light and during conidial development  <cit> .
contig  <dig> encoded a major facilitator superfamily  of sugar transporter that contained six transmembrane domains.
mfs transporters are involved in the symport, antiport and uniport of various substrates, such as sugars, krebs cycle intermediates, phosphate esters, oligosaccharides and antibiotics  <cit> .
contig  <dig> was similar to glucose-regulated protein  <dig>  in that it is not an extracellular protein.
grp <dig> is a subgroup of heat shock protein  <dig> and located in the er lumen where it assists in the post-translational import and folding of proteins  <cit>  as well as the reversible binding of misfolded and underglycosylated proteins  <cit> .
clone ct21- <dig> was matched to a toxb precursor of p. tritici-repentis.
toxb is a host specific chlorosis toxin and was identified as a potential pathogenicity factor in p. tritici-repentis, the causal agent of tan spot of wheat  <cit> .
several enzymes were found to possess transmembrane domains and hence were classified as ceaps.
the cu/zn superoxide dismutase  is a key enzyme in the dismutation of superoxide radicals resulting from cellular oxidative metabolism into hydrogen peroxide  <cit> .
clone ct21- <dig> matched an enzyme called peptidyl-prolyl cis-trans isomerase b  .
ppiase possesses a chaperone activity to fold proteins into active configuration by catalyzing cis/trans isomerization on proline-peptide bonds  <cit> .
an enzyme chitin deacetylase  involved in modifying fungal cell wall was also identified.
two unigenes, ct21- <dig> and ct21- <dig>  encoded metalloreductase and ascorbate peroxidase oxidoreductases, respectively.
the metalloreductase contained a transmembrane domain, hence was classified as ceap, whereas the ascorbate peroxidase lacked a transmembrane domain, and was therefore included under the category of other proteins.
candidate effector proteins
it is assumed that fungal effectors are likely to be small , cysteine-rich and soluble extracellular proteins that do not become cross linked into fungal cell walls  <cit> .
with the exponential rise in the number of fungal phytopathogen genomes being sequenced, the likelihood of finding effector orthologs in other species is increasing.
for example, c. fulvum lysm effector ecp <dig> has orthologs from  <dig> filamentous fungal species, including  <dig> plant pathogens  <cit> .
we identified  <dig> secreted proteins  that met characteristic criteria, including  <dig> proteins homologous to functionally uncharacterized proteins from fungal species.
the predicted proteins encoded by these unigenes shared the characteristics of being small , lacking a transmembrane domain, and being relatively rich in cysteine .
the functionally annotated homologs to two additional clones  also shared these features.
these candidate effector proteins were predicted to possess cysteine disulfide bridges that may aid protein stability in the extracellular milieu and inside the host cell to protect them from degradation  <cit> .
putative disulfide bond spacing was conserved between predicted orfs and similar proteins available in the public domain.
among the functionally annotated proteins, two discrete unigenes  were similar to c. fulvum lysm effector protein ecp <dig> induced during the colonization of tomato  <cit> .
however, unlike ecp <dig>  contig <dig> and ct21- <dig> contained  <dig> and  <dig> lysm, respectively.
contig  <dig> exhibited similarity to an eliciting plant response-like protein that contained a cp domain.
cp proteins from ceratocystis fimbriata are surface proteins that induce necrosis and elicit phytoalexin synthesis in plants  <cit> .
a unigene  was matched to a small secreted protein  from nectria haematococca with  <dig> cysteine residues  in the mature protein.
contig  <dig> was similar to a hr-inducing protein from ceratocystis ulmi.
homologs to  <dig> unigenes were hypothetical proteins.
other proteins
the predicted peptides of a group of  <dig> unigenes showed similarities to diverse proteins from fungal genome sequences.
the encoded peptide of clone ct21- <dig> was similar to a chloroperoxidase related protein.
chloroperoxidase catalyzes the insertion of chlorine, bromine and iodine atoms into organic acceptor molecules.
est clone ct21- <dig> was aligned with glutaminase, an enzyme catalyzing the hydrolysis of glutamine to glutamic acid.
glutaminases play a role in the acquisition of nitrogen from less preferred sources  <cit> .
the oxidoreductase enzyme l-ascorbate peroxidase catalyzes the chemical reaction l-ascorbate + h2o <dig> ↔dehydroascorbate +  <dig> h2o.
the remaining  <dig> unigenes could not be classified by homology search into known or putative proteins .
however, sequence analyses of predicted peptides revealed that most of these unigenes were rich in glycine or leucine.
ct21- <dig> was identified as a nudix/mutt family protein nudix hydrolase .
nudix hydrolases hydrolyze substrates of general structure nucleoside diphosphate linked to another moiety, x to yield nucleotide monophosphate and p-x  <cit> .
time-course expression analysis of genes encoding putative effector proteins and ceaps
using semiquantitative rt-pcr, we examined transcript levels of genes encoding candidate effectors and ceaps  identified from the est library in different cell types formed in vitro, viz.
ungerminated conidia, mycelia and mycelia grown in minimal media supplemented with lentil leaflet cell walls .
expression was also analyzed in planta at various stages of plant infection, namely appressorial penetration , primary hyphae  and secondary hyphae  .
time-course expression profiles were obtained for  <dig> candidate effector encoding genes .
when considering the in planta infection process, two kinds of expression patterns could be distinguished.
est clones contig  <dig>   <dig>   <dig>  ct21- <dig>  and ct21- <dig> were expressed at both in planta biotrophic- and necrotrophic-developmental stages.
expression at lower level was also detected in the appressorial penetration stage for two clones, contigs  <dig> and  <dig>  three clones, contigs  <dig>   <dig>  and ct21- <dig> were expressed constitutively in all in vitro cell types.
clones, contig  <dig> and ct21- <dig> identified as homologs of lysm effector ecp <dig>  <cit> , were expressed in mycelia, whereas they were undetectable in conidia and cell wall treated mycelia, suggesting the possible involvement of these effectors genes in vegetative growth.
transcripts from four genes  were exclusively accumulated during the necrotrophic phase of the infection process.
among them, two genes  also showed expression in the cell wall treated mycelia, however, at a lower level than during the in planta necrotrophic phase, whereas contig  <dig> appeared to be expressed in mycelia and cell wall treated mycelia.
clone ct21- <dig> showed constitutive expression in all cell types tested.
another subclass of ceaps tested for expression was cih <dig> protein encoding unigenes .
all three were constitutively expressed in all cell types tested, indicating their role in vegetative development of the pathogen.
in addition, their transcripts were also accumulated at both biotrophic- and necrotrophic- in planta developmental stages, suggesting that cih <dig> glycoprotein is a basal component of the cell wall of c. truncatum as described previously  <cit> .
functional analysis of selected candidate effectors by pvx agroinfiltration assay
to investigate if candidate effectors have elicitor activity, we expressed  <dig> candidate effector proteins  and m. oryzae cp protein by mean of agroinfiltration in tobacco leaves.
est analysis of contig  <dig> revealed a cdna of 784-bp with an orf of 417-bp encoding a 139-aa preprotein.
a sp of 15-aa with a cleavage site in between glycine- <dig> and serine- <dig> was predicted in the preprotein, and  <dig> cysteine residues were found in the mature protein .
using disulfind server, three cystine disulfide bridges were predicted in the mature protein at a confidence level of >  <dig> .
this protein showed no homology to any functionally characterized protein.
the cdna of contig  <dig> was 775-bp in size with an orf of 414-bp encoding a 138-aa preprotein.
a sp of 21-aa with a cleavage site between valine- <dig> and serine- <dig>  and a cp domain spanned from 23-aa to 138-aa were predicted at the n- and c-termini, respectively.
the cp domain contains  <dig> cysteine residues, which are known to form two disulphide bonds  <cit> .
proteins belonging to the cp family share common characteristics, such as a small size, an n-terminal sp for secretion and a conserved cysteine residue pattern  <cit> .
protein encoded by unigene contig  <dig> possessed all features of a cp protein, hence we termed it ctcp <dig>  sequence alignment of ctcp <dig> with homologues from other fungal species revealed that ctcp <dig> showed significant similarity with fungal proteins of cp family like m. oryzae sm <dig> , hypocrea atroviridis eliciting plant response like protein  <dig> , hypocrea virens snodprot <dig>  and gibberella pulicaris snodprot-fs  .
based on sequence features, ctcp <dig> belongs to epl <dig> cluster of cp proteins  <cit> .
recently, a m. oryzae cp protein mgsm <dig> was shown to induce hr and confer broad-spectrum disease resistance in arabidopsis  <cit> .
est analysis of contig  <dig> revealed a cdna of 686-bp with an orf of 426-bp encoding a 142-aa preprotein.
a sp of 17-aa with a cleavage site between alanine- <dig> and isolecine- <dig> was predicted at the n-terminus of the preprotein, and  <dig> cysteine residues were predicted in the mature protein .
this protein showed significant homology to the hr inducing protein of ceratocystis ulmi and contained 4-cysteine residues.
the orfs of three candidate effectors from c. truncatum and mgsm <dig> from m. oryzae with and without sp were cloned into pvx vector pgr <dig>  and transformed into agrobacterium tumefaciens strain gv <dig>  we used the empty vector pgr <dig> as negative control, which upon transient expression showed no micro- and/or macroscopic phenotype.
we also incorporated two r/avr protein pairs as positive controls that have been shown to interact and trigger cell death in tobacco: pto/avrpto  <cit>  and cf9/avr <dig>  <cit> .
agroinfiltration of recombinant a. tumefaciens strains carrying pgr106-mgsm11- <dig> in tobacco leaves resulted in macroscopic cell death  <dig> days after infiltration, whereas the rest of the strains were unable to cause any macroscopic phenotype .
transient co-expression of the genes that encode avr/r protein pairs  elicited a spotted faint glazing on the tobacco leaf surface  <dig> h post infiltration, which progressed to tissue collapse and became confluent  <dig> days post infiltration.
to investigate whether ctcp <dig> protein causes any microscopic cell death, we collected leaf tissues from the edges of pgr <dig>  pgr106-ctcp11- <dig>  pgr106-ctcp122- <dig>  pgr106-mgsm11- <dig> and pgr106-mgsm122- <dig> infiltration zones for polyphenolic compound- associated autofluorescence under ultraviolet  light.
the pgr <dig>  pgr106-ctcp11- <dig>  pgr106-ctcp122- <dig> and pgr106-mgsm122- <dig> edges had no autofluorescence under uv light, whereas pgr106-mgsm <dig> edges showed strong autofluorescence under uv light .
discussion
the morphological and nutritional transition of a hemibiotrophic pathogen from biotrophy to necrotrophy plays a critical role in disease development.
during this switch, fungal phytopathogens may secrete large repertoires of secretory proteins to manipulate host innate immunity in concert with a destructive arsenal of cwdes that manifest the disease phenotype.
our strategy in this study was to catalogue genes encoding secretory proteins from c. truncatum by constructing a directional cdna library representing the biotrophy-necrotrophy switch and then to exploit biocomputational tools to scan resulting fungal ests for proteins containing canonical n-terminal sps.
to date, cdna library- and est-based studies have been conducted to identify secretory protein encoding genes from haustoria of puccinia striiformis f.sp.
tritici  <cit> , c. higginsianum appressoria  <cit> , in planta infection stages of venturia inaequalis  <cit>  and mycelia of the corn pathogen c. graminicola grown in vitro under different secretion inducing conditions  <cit> .
there has been no previous est-based analysis of the biotrophy-necrotrophy switch for any fungal pathogen.
our library contained  <dig> fungal unigenes encoding secretory proteins.
functional classification of the secretory proteins based on homology searches and sequence analyses revealed the presence of four potential groups: hydrolytic enzymes, ceaps, candidate effector proteins and other proteins.
hemibiotrophs orchestrate a physiological switch from asymptomatic infection to massive cell death and tissue dissolution, presumably resulting from the coordinated secretion of cell-death elicitors and hydrolytic enzymes  <cit> .
with  <dig> of  <dig> unigenes belonging to ec classes  <dig> and  <dig>  hydrolases and lyases are a major group of enzymes identified at the biotrophy-necrotrophy switch.
in general, fungal pathogens secrete a battery of cwdes capable of hydrolyzing host cell wall components  to accommodate a necrotrophic life style.
these cwdes include carbohydrate esterases , glycosyl hydrolases, polysaccharide lyases  and peptidases, which the fungal pathogens utilize to degrade the host plant cell wall for nutrition  <cit> .
the glycosidases like galactosidase and arabinosidase may liberate sugar moieties that can be used as a nutritional source for c. truncatum during the necrotrophic growth.
glycosidases may also have a role in decreasing the steric hinderance for other cwdes by removing protruding side chains from the backbone of polymers and providing increased access for endo forms of cwdes  <cit> .
pectinases weaken the cell wall and expose cell wall components to other enzymes, such as cellulases and hemicellulases.
genome sequencing has revealed that the hemibiotrophic phytopathogen m. oryzae is predicted to possess  <dig> glycosyl hydrolases,  <dig> polysaccharide lyases, and  <dig> carbohydrate esterases  <cit> .
our library retrieved  <dig> glycosyl hydrolases,  <dig> polysaccharide lyases, and  <dig> carbohydrate esterases.
another class of identified hydrolases was peptidases that may have a function in the extracellular processing of fungal secreted proteins  <cit> , degradation of proteinaceous components involved in the plant response against pathogens  <cit>  or degradation of host tissue  <cit> .
esterases, glycosyl hydrolases, lyases, and peptidases could also weaken host cell walls, allowing for an easier access to nutrients.
the interaction between a pathogen and its host is to a large extent orchestrated by the proteins that are secreted or localized to or at the cell membrane or cell wall .
the ceaps are secreted through the endoplasmic reticulum  pathway and tethered either to the fungal cell membrane or wall.
some ceaps lack a transmembrane domain but possess c-terminal gpi anchor addition signal.
gpi anchoring is an important mechanism to tether extracellular proteins to the plasma membrane or cell wall  <cit> .
the carboxyl termini of these proteins have a sequence motif that is recognized by a protein complex located in the er, known as the gpi transamidase .
the gpi transamidase complex cleaves the protein at a position within this motif, termed the omega site, and transfers the gpi anchor en bloc to the newly generated c terminus of the protein.
a genome-wide inventory of secretory proteins of p. sojae and p. ramorum revealed putative secretomes of  <dig>  and  <dig>  proteins, respectively  <cit> , of which a subset of approximately  <dig> proteins contain putative gpi anchor addition signals.
the majority of elicitins  from phytophthora possess a gpi anchor  <cit> .
components of the cell envelope of pathogens can be rapidly acclimatized by a changing environment.
the ability to respond to a changing milieu is critical for pathogens to effectively evade, tolerate and/or suppress host immune responses.
therefore, the structure and composition of the cell envelope is dynamic and changes during different stages of in planta growth.
we observed an abrogation or a reduction in transcript levels of genes encoding ceaps during the biotrophic phase and an abrupt induction in the necrotrophic phase .
the switch from voluminous biotrophic hyphae to thin necrotrophic hyphae involves an increased surface area to volume ratio, therefore may require elevated transcription of some of these cell surface protein encoding unigenes.
this ratio and thinner cell walls of the secondary hyphae would be advantageous for both efficient nutrient uptake and secretion of wall degrading enzymes, and possibly toxins, during the necrotrophic growth of hemibiotrophic fungal pathogens  <cit> .
the predicted products of three discrete unigenes ct21- <dig>  ct21- <dig> and ct21- <dig> resembled the biotrophy associated protein cih <dig> from c. lindemuthianum  <cit> .
the cih <dig> was shown to be secreted into the cell wall of invasive biotrophic hyphae and the plant-pathogen interface.
in the present study, we found that the c. truncatum cih <dig> homolog was expressed constitutively in all in vitro cell types tested and both during the biotrophic- and necrotrophic- phases.
this result was contradictory to studies with c. higginsianum where the cih <dig> protein was specifically expressed in the biotrophic phase  <cit> .
it suggests either the transcription level of cih <dig> does not reflect the protein level, or c. truncatum may employ a different approach to host invasion that is unlike that of other species of colletotrichum.
based on the in planta transcript profiling of nine genes encoding putative effector proteins, we identified two types of expression patterns: biotrophic/necrotrophic-specific and necrotrophic-specific.
transient expression of three candidate effectors in tobacco leaves revealed that none of them was able to elicit an hr response.
surprisingly, our data on ctcp <dig> were in contrast of that recently published on m. oryzae cp protein, mgsm <dig>  mgsm <dig>  upon transient expression, stimulated hr and conferred broad-spectrum resistance to arabidopsis against botrytis cinerea and pseudomonas syringae pv.
tomato  <cit> .
the predicted products of two discrete unigenes  showed homology to extracellular protein  <dig>  from c. fulvum.
lysms are n-acetyl d-glucosamine  binding modules that have been found in mammalian and plant pathogenic fungi as well as in saprophytes  <cit> .
therefore, they may also have a role in counter defense by protecting fungal cell walls against plant chitinases.
bolton et al.
<cit>  demonstrated that ecp <dig> was induced by the plant pathogenic fungus c. fulvum during the initial infection of tomato and acted as a virulence factor.
during infection, lysm domain-containing protein ecp <dig> sequesters chitin oligosaccharides that are released from the cell walls of invading hyphae to avert the elicitation of host immunity.
this sequestering may represent a common strategy of host immune suppression by fungal pathogens, because lysm effectors are widely conserved in the fungal kingdom  <cit> .
we identified several ests that may encode novel effectors.
using bioinformatics tools, we identified  <dig> soluble secreted proteins of unknown function .
three of them  were not expressed in the biotrophic phase while induced during the necrotrophic phase.
their expression was also detected either in some or all of the in vitro cell types.
such genes may be important for establishing fungal necrotrophy.
one  was constitutively expressed during all in planta developmental stages except appressorial penetration and in all in vitro cell types , suggesting its general role in both vegetative development and pathogenesis.
CONCLUSIONS
we catalogued proteins putatively secreted by c. truncatum and several candidate effectors had been mined from the est library.
it was observed that the transcript levels of candidate effector genes were either induced during the necrotrophic growth of the pathogen compared with that during the biotrophic growth or elevated during both phases.
through in planta expression of candidate effectors, we demonstrated the functional role of one effector.
in summary, our study suggests that suppression of host defense during the biotrophic phase and the subsequent unmasking of pamps or elicitors during the necrotrophic phase may be involved in pathogenesis of c. truncatum.

 colletotrichum truncatum andrus w.d. moore causes anthracnose disease of many leguminous species, including lentil, soybean merr.), fababean, and pea. this fungal pathogen employs a bi-phasic hemibiotrophic infection strategy to colonize lentil plants. the anthracnose infection is initiated by the attachment of conidia to aerial parts of the host plant. conidia germinate immediately after adhesion and differentiate to form infection structures called appressoria devoted to mechanical penetration. after the appressorium has been formed, a thin infection or penetration peg emerging from the base of the appressorium pierces the host cuticle and cell wall, and grows in between the plant cell wall and plasma membrane. it differentiates into large, bulbous invasive primary hyphae that are biotrophic in nature. these hyphae are likely to interact with the host plasma membrane but pull away after plasmolysis, indicating either initial or weak host-pathogen association. this situation differs from that of haustoria and the biotrophic invasive hyphae produced by obligate biotrophic fungi, such as powdery mildews and rusts, and other hemibiotrophs like magnaporthe oryzae, the causal agent of rice blast, where the host plasma membrane remains adhered to fungal structures upon plasmolysis. the primary hyphae of c. truncatum are entirely confined to the first infected epidermal cell throughout the biotrophic phase. thereafter, the fungus switches to the necrotrophic phase that is associated with the production of thin secondary hyphae that ramify intra- and inter-cellularly, killing and macerating host tissues by hydrolytic enzymes ahead of infection. therefore, the biotrophy-necrotrophy switch is critical in anthracnose development. during the transition, c. truncatum probably secretes a range of proteins to establish a compatible interaction with its host, including some that may exclusively be involved in switching the pathogen to the necrotrophic phase. in silico analyses of whole genome sequences have predicted that fungal and oomycete phytopathogens possess large repertoires of secretory proteins, which constitute to 10% of predicted proteomes of m. oryzae, phytophthora sojae, p. ramorum and ustilago maydis. these secretory proteins play diverse roles in virulence and pathogenicity, and interactions with host cells. secreted hydrolytic enzymes like cell wall degrading enzymes contribute to penetration of the plant cuticle and cell wall, and to tissue maceration during the onset of the necrotrophic stage. other secretory proteins, such as chitin deacetylase and proteins that bind pathogen cell wall components like chitin binding protein avr of cladosporium fulvum, modify the pathogen cell wall and are critical in evading the host defense surveillance system. secreted effector proteins alter biochemical, physiological, and morphological processes in host plant cells, thereby facilitating infection or triggering defense responses. some of these proteins are active in the apoplast where they may interfere with host plant defense processes, e.g. by inhibiting plant proteases and lytic enzymes. other fungal effector proteins enter into host cells, though the mechanism of entry of effectors is still a mystery. however, the role of rxlr and deer motifs in host cell targeting of oomycete effectors has been established. recently, kale and colleagues have shown that fungal effectors contain functional variants of rxlr motifs, and that the oomycete and fungal rxlr and deer motifs bind to host cell surface phosphatidylinositol-3-phosphate. this binding may facilitate the uptaking of effectors through endocytosis. one of the functions of fungal and oomycete effectors is to disrupt the host signal transduction pathways that mediate defense responses. until now, only one effector, cgdn has been identified from colletotrichum. this effector averts hr cell death during the biotrophic phase of infection and is essential for c. gloeosporioides pathogenicity on stylosanthes guianensis. studies conducted to date have investigated the secretome at different stages of the infection process, including penetration, biotrophic and necrotrophic stages. kleeman et al. identified putative soluble secretory proteins from a cdna library prepared from mature appressoria of c. higginsianum grown in vitro. sixteen candidate effector proteins were discovered from venturia inaequalis, the causal agent of apple scab, on the basis of their small size, presence of cysteine residues and of a putative sp. using a yeast signal sequence trap screen, krijger et al. identified secreted proteins from c. graminicola mycelia grown in vitro on corn cell walls and leaf extract. recently, takahara and colleagues developed a fluorescence-activated cell sorting method to purify the intracellular biotrophic hyphae from c. higginsianum from homogenates of infected arabidopsis leaves and constructed a biotrophy-specific cdna library. mosquera et al. identified four biotrophy associated secreted proteins from the invasive hyphae of m. oryzae by interaction transcriptome analysis. so far, few reports have addressed the critical switch point of the hemibiotrophic infection process. only one gene encoding a switch regulator, clta from c. lindemuthianum, has been identified. mutants harboring disrupted clta were unable to switch to the necrotrophic phase. this transcriptional factor is indispensable for pathogenicity on common bean. the objective of the current study was to identify proteins putatively secreted during the in planta biotrophy-necrotrophy switch of c. truncatum. to catalogue such proteins, we have generated ests from a directional cdna library constructed from susceptible canadian lentil cultivar'eston' inoculated with c. truncatum isolate ct- by biocomputational analyses of these ests, we identified eleven c. truncatum candidate effector genes. in planta functional analysis of three effectors was carried out using pvx-based agroinfiltration in tobacco leaves.  generation of c. truncatum ests excised leaflets of canadian lentil cultivar'eston' inoculated with isolate ct- of c. truncatum were collected and examined microscopically to ensure that the pathogen was at the biotrophic to necrotrophic transition stage where secondary hyphae had become visible. this corresponded to 48- hours after inoculation and we referred to this period of infection as the biotrophy-necrotrophy switch. total rna was extracted, and poly a+ mrna was isolated and used for construction of a directional cdna library. using 5'-end single pass sequencing, ests were generated with an average read length of more than nucleotides. blastx analysis with a cutoff e value ≤ 10- identified 57% as plant sequences, 39% as fungal sequences and the remainder as unassignable. the high abundance of fungal transcripts was further verified by semiquantitative rt-pcr using the fungal 60s ribosomal gene in an infection time-course study that included control mixtures. an accumulation of around 40% of fungal 60s ribosomal transcripts, which, in turn, reflect the fungal rna content, was predicted during the biotrophy-necrotrophy switch. the abundance of plant origin transcripts had declined with fungal proliferation in infected host tissues as shown by rt-pcr analysis of l. culinaris 60s ribosomal gene in the same infection time-course used for assessing the fungal biomass. the fungal transcriptome contains sequences encoding putative secretory proteins fungal sequences were analyzed for features indicative of secreted proteins. because of directional cloning, we could analyze the coding sequences from the 5'-end. orf finder, and signalp and ipsort algorithms were exploited to deduce orfs and sps within the orfs. one hundred sixty-two predicted orfs were predicted to encode proteins with n-terminal sps. all ests were deposited in the ncbi genebank est database. using contigexpress software, these orfs could be assembled into contigs and singletons, resulting in a total of unigenes. clone ids belonging to various contigs are listed in the additional file the average g+c content of these unigenes was around 59%. we refer to the deduced proteins encoded by these unigenes as putative secretory proteins. we also employed blastx and orf finder algorithms to investigate whether the first methionine within the amino acid translation represented the true n-terminal methionine to confirm the orf of selected unigenes. the orfs were then queried against the ncbi non-redundant protein database using blastp algorithm. fungal effectors are most likely to be small, soluble, extracellular secreted proteins that do not become cross-linked into the fungal cell wall. therefore, predicted orfs from these unigenes were screened for the size of the encoded polypeptide chain and the presence of transmembrane domains, cysteine residues, transmembrane domain and glycosylation sites, including glycosyl-phosphatidylinositol modification. however, with the steady increase in the number of fungal phytopathogen genomes being sequenced, the likelihood that orthologs found in other species is increasing. hence, some candidate effectors were identified based on orthologs in other phytopathogens. comparison of the protein sequences encoded by the unigenes to the currently annotated databases and their sequence analyses revealed four groups. the most highly represented group comprised hydrolytic enzymes, which included unigenes, followed by ceaps, candidate effector proteins and proteins classified as "other proteins". based on these analyses, a total of unigenes were predicted to encode either proteins with transmembrane domain or gpi addition signal. among them, six were grouped with hydrolases. the remaining unigenes encoded putatively soluble secretory proteins, including hydrolases. a list of the clone ids, the top hit for each sequence and the corresponding blast score are compiled in table an e value cutoff ≤ 10- was used to annotate these unigenes. four sequences had no match at ≤ 10- but contained conserved signatures. therefore, they were classified according to sequence characteristics belonging to corresponding groups and listed in table * unigenes with e value > 10- are classified according to their conserved signatures as described in the text. hydrolytic enzymes sixty-three unique sequences showed significant similarity to members of the enzyme classes and. most of them had been annotated as cell wall degrading enzymes. ten unigenes encoded enzymes acting on ester bonds; most of them were reported as pectinolytic enzymes. these included esterases, pectinesterase, extracellular lipase, phosphoserine phosphatase and cutinase. four depolymerase pectinolytic enzymes were also retrieved from this library. unigenes ct21- and ct21- were similar to exopolygalacturonase and endo-polygalacturonase, respectively. four discrete unigenes contig ct21- ct21- and ct21- encoded pectate lyase and contig showed homology to pectin lyase. thirty unique sequences, including ct21- and ct21- mentioned above represented the ec subclass glycosidases. glycosidases hydrolyze the bonds between carbohydrates and release sugars from plant cell walls to provide the fungus with carbon sources and support host penetration and colonization. these included xyloglucan-specific endo-β- 4-glucanase, cell wall glucanosyltransferase, cellulase, glycosyl hydrolase family proteins, β-glucosidase, arabinosidase, β-glucanase, mannan endo- -β-mannosidase and α-galactosidase. sixteen unigenes belonged to the ec subclass of enzymes acting on peptide bonds. correlation between peptidase activity and virulence for several phytopathogenic fungi has already been established. cell envelope-associated proteins forty-three unigenes carrying sp, transmembrane helices or gpi anchor addition signal, including unigenes homologous to functionally uncharacterized genes from fungi, were identified as ceaps. among them, six unigenes were classified under the hydrolytic enzymes category. in almost all cases except the unigene ct21- the best match for these unigenes was found within the ascomycota. interestingly, most of the identified genes have not been previously reported in the genus colletotrichum. c-terminal gpi anchor sites were predicted in the translated amino acid sequences of unigenes. interestingly, we identified a member of the gpi transamidase complex that is implicated in en bloc synthesis of gpi proteins. three unigenes contig contig and ct21- encoded gpi anchored serine-threonine rich proteins. two unigenes, contig and ct21- were predicted to possess gpi anchor addition signals and cysteine rich fungal extracellular membrane domains. unigene ct21- was identified as a hypothetical protein that possesses a gpi addition signal. contigs and were matched to a putative gpi-anchored protein and gpi-anchored serine rich protein, respectively. clones ct21- and ct21- were matched to the gpi anchored cell wall organization protein ecm from pyrenophora tritici-repentis and glycolipid anchored surface protein gas from c. graminicola, respectively. the predicted products of the three discrete unigenes ct21- ct21- and ct21- resembled the biotrophy associated secreted glycoprotein colletotrichum intracellular hypha from c. lindemuthianum, the causal agent of common bean anthracnose. the orf of ct21- encoded a protein of amino acids containing lysin motif domains. these lysms recognize and bind n-acetyl d-glucosamine, and may play a role protecting fungal chitin from plant chitinases. the predicted orfs from three discrete clones, contig ct21- and ct21- encoded a protein that was similar to clock controlled protein from neurospora crassa, induced by light and during conidial development. contig encoded a major facilitator superfamily of sugar transporter that contained six transmembrane domains. mfs transporters are involved in the symport, antiport and uniport of various substrates, such as sugars, krebs cycle intermediates, phosphate esters, oligosaccharides and antibiotics. contig was similar to glucose-regulated protein in that it is not an extracellular protein. grp is a subgroup of heat shock protein and located in the er lumen where it assists in the post-translational import and folding of proteins as well as the reversible binding of misfolded and underglycosylated proteins. clone ct21- was matched to a toxb precursor of p. tritici-repentis. toxb is a host specific chlorosis toxin and was identified as a potential pathogenicity factor in p. tritici-repentis, the causal agent of tan spot of wheat. several enzymes were found to possess transmembrane domains and hence were classified as ceaps. the cu/zn superoxide dismutase is a key enzyme in the dismutation of superoxide radicals resulting from cellular oxidative metabolism into hydrogen peroxide. clone ct21- matched an enzyme called peptidyl-prolyl cis-trans isomerase b. ppiase possesses a chaperone activity to fold proteins into active configuration by catalyzing cis/trans isomerization on proline-peptide bonds. an enzyme chitin deacetylase involved in modifying fungal cell wall was also identified. two unigenes, ct21- and ct21- encoded metalloreductase and ascorbate peroxidase oxidoreductases, respectively. the metalloreductase contained a transmembrane domain, hence was classified as ceap, whereas the ascorbate peroxidase lacked a transmembrane domain, and was therefore included under the category of other proteins. candidate effector proteins it is assumed that fungal effectors are likely to be small, cysteine-rich and soluble extracellular proteins that do not become cross linked into fungal cell walls. with the exponential rise in the number of fungal phytopathogen genomes being sequenced, the likelihood of finding effector orthologs in other species is increasing. for example, c. fulvum lysm effector ecp has orthologs from filamentous fungal species, including plant pathogens. we identified secreted proteins that met characteristic criteria, including proteins homologous to functionally uncharacterized proteins from fungal species. the predicted proteins encoded by these unigenes shared the characteristics of being small, lacking a transmembrane domain, and being relatively rich in cysteine. the functionally annotated homologs to two additional clones also shared these features. these candidate effector proteins were predicted to possess cysteine disulfide bridges that may aid protein stability in the extracellular milieu and inside the host cell to protect them from degradation. putative disulfide bond spacing was conserved between predicted orfs and similar proteins available in the public domain. among the functionally annotated proteins, two discrete unigenes were similar to c. fulvum lysm effector protein ecp induced during the colonization of tomato. however, unlike ecp contig and ct21- contained and lysm, respectively. contig exhibited similarity to an eliciting plant response-like protein that contained a cp domain. cp proteins from ceratocystis fimbriata are surface proteins that induce necrosis and elicit phytoalexin synthesis in plants. a unigene was matched to a small secreted protein from nectria haematococca with cysteine residues in the mature protein. contig was similar to a hr-inducing protein from ceratocystis ulmi. homologs to unigenes were hypothetical proteins. other proteins the predicted peptides of a group of unigenes showed similarities to diverse proteins from fungal genome sequences. the encoded peptide of clone ct21- was similar to a chloroperoxidase related protein. chloroperoxidase catalyzes the insertion of chlorine, bromine and iodine atoms into organic acceptor molecules. est clone ct21- was aligned with glutaminase, an enzyme catalyzing the hydrolysis of glutamine to glutamic acid. glutaminases play a role in the acquisition of nitrogen from less preferred sources. the oxidoreductase enzyme l-ascorbate peroxidase catalyzes the chemical reaction l-ascorbate + h2o ↔dehydroascorbate + h2o. the remaining unigenes could not be classified by homology search into known or putative proteins. however, sequence analyses of predicted peptides revealed that most of these unigenes were rich in glycine or leucine. ct21- was identified as a nudix/mutt family protein nudix hydrolase. nudix hydrolases hydrolyze substrates of general structure nucleoside diphosphate linked to another moiety, x to yield nucleotide monophosphate and p-x. time-course expression analysis of genes encoding putative effector proteins and ceaps using semiquantitative rt-pcr, we examined transcript levels of genes encoding candidate effectors and ceaps identified from the est library in different cell types formed in vitro, viz. ungerminated conidia, mycelia and mycelia grown in minimal media supplemented with lentil leaflet cell walls. expression was also analyzed in planta at various stages of plant infection, namely appressorial penetration, primary hyphae and secondary hyphae. time-course expression profiles were obtained for candidate effector encoding genes. when considering the in planta infection process, two kinds of expression patterns could be distinguished. est clones contig ct21- and ct21- were expressed at both in planta biotrophic- and necrotrophic-developmental stages. expression at lower level was also detected in the appressorial penetration stage for two clones, contigs and three clones, contigs and ct21- were expressed constitutively in all in vitro cell types. clones, contig and ct21- identified as homologs of lysm effector ecp, were expressed in mycelia, whereas they were undetectable in conidia and cell wall treated mycelia, suggesting the possible involvement of these effectors genes in vegetative growth. transcripts from four genes were exclusively accumulated during the necrotrophic phase of the infection process. among them, two genes also showed expression in the cell wall treated mycelia, however, at a lower level than during the in planta necrotrophic phase, whereas contig appeared to be expressed in mycelia and cell wall treated mycelia. clone ct21- showed constitutive expression in all cell types tested. another subclass of ceaps tested for expression was cih protein encoding unigenes. all three were constitutively expressed in all cell types tested, indicating their role in vegetative development of the pathogen. in addition, their transcripts were also accumulated at both biotrophic- and necrotrophic- in planta developmental stages, suggesting that cih glycoprotein is a basal component of the cell wall of c. truncatum as described previously. functional analysis of selected candidate effectors by pvx agroinfiltration assay to investigate if candidate effectors have elicitor activity, we expressed candidate effector proteins and m. oryzae cp protein by mean of agroinfiltration in tobacco leaves. est analysis of contig revealed a cdna of 784-bp with an orf of 417-bp encoding a 139-aa preprotein. a sp of 15-aa with a cleavage site in between glycine- and serine- was predicted in the preprotein, and cysteine residues were found in the mature protein. using disulfind server, three cystine disulfide bridges were predicted in the mature protein at a confidence level of >. this protein showed no homology to any functionally characterized protein. the cdna of contig was 775-bp in size with an orf of 414-bp encoding a 138-aa preprotein. a sp of 21-aa with a cleavage site between valine- and serine- and a cp domain spanned from 23-aa to 138-aa were predicted at the n- and c-termini, respectively. the cp domain contains cysteine residues, which are known to form two disulphide bonds. proteins belonging to the cp family share common characteristics, such as a small size, an n-terminal sp for secretion and a conserved cysteine residue pattern. protein encoded by unigene contig possessed all features of a cp protein, hence we termed it ctcp sequence alignment of ctcp with homologues from other fungal species revealed that ctcp showed significant similarity with fungal proteins of cp family like m. oryzae sm, hypocrea atroviridis eliciting plant response like protein, hypocrea virens snodprot and gibberella pulicaris snodprot-fs. based on sequence features, ctcp belongs to epl cluster of cp proteins. recently, a m. oryzae cp protein mgsm was shown to induce hr and confer broad-spectrum disease resistance in arabidopsis. est analysis of contig revealed a cdna of 686-bp with an orf of 426-bp encoding a 142-aa preprotein. a sp of 17-aa with a cleavage site between alanine- and isolecine- was predicted at the n-terminus of the preprotein, and cysteine residues were predicted in the mature protein. this protein showed significant homology to the hr inducing protein of ceratocystis ulmi and contained 4-cysteine residues. the orfs of three candidate effectors from c. truncatum and mgsm from m. oryzae with and without sp were cloned into pvx vector pgr and transformed into agrobacterium tumefaciens strain gv we used the empty vector pgr as negative control, which upon transient expression showed no micro- and/or macroscopic phenotype. we also incorporated two r/avr protein pairs as positive controls that have been shown to interact and trigger cell death in tobacco: pto/avrpto and cf9/avr. agroinfiltration of recombinant a. tumefaciens strains carrying pgr106-mgsm11- in tobacco leaves resulted in macroscopic cell death days after infiltration, whereas the rest of the strains were unable to cause any macroscopic phenotype. transient co-expression of the genes that encode avr/r protein pairs elicited a spotted faint glazing on the tobacco leaf surface h post infiltration, which progressed to tissue collapse and became confluent days post infiltration. to investigate whether ctcp protein causes any microscopic cell death, we collected leaf tissues from the edges of pgr pgr106-ctcp11- pgr106-ctcp122- pgr106-mgsm11- and pgr106-mgsm122- infiltration zones for polyphenolic compound- associated autofluorescence under ultraviolet light. the pgr pgr106-ctcp11- pgr106-ctcp122- and pgr106-mgsm122- edges had no autofluorescence under uv light, whereas pgr106-mgsm edges showed strong autofluorescence under uv light. discussion the morphological and nutritional transition of a hemibiotrophic pathogen from biotrophy to necrotrophy plays a critical role in disease development. during this switch, fungal phytopathogens may secrete large repertoires of secretory proteins to manipulate host innate immunity in concert with a destructive arsenal of cwdes that manifest the disease phenotype. our strategy in this study was to catalogue genes encoding secretory proteins from c. truncatum by constructing a directional cdna library representing the biotrophy-necrotrophy switch and then to exploit biocomputational tools to scan resulting fungal ests for proteins containing canonical n-terminal sps. to date, cdna library- and est-based studies have been conducted to identify secretory protein encoding genes from haustoria of puccinia striiformis f.sp. tritici, c. higginsianum appressoria, in planta infection stages of venturia inaequalis and mycelia of the corn pathogen c. graminicola grown in vitro under different secretion inducing conditions. there has been no previous est-based analysis of the biotrophy-necrotrophy switch for any fungal pathogen. our library contained fungal unigenes encoding secretory proteins. functional classification of the secretory proteins based on homology searches and sequence analyses revealed the presence of four potential groups: hydrolytic enzymes, ceaps, candidate effector proteins and other proteins. hemibiotrophs orchestrate a physiological switch from asymptomatic infection to massive cell death and tissue dissolution, presumably resulting from the coordinated secretion of cell-death elicitors and hydrolytic enzymes. with of unigenes belonging to ec classes and hydrolases and lyases are a major group of enzymes identified at the biotrophy-necrotrophy switch. in general, fungal pathogens secrete a battery of cwdes capable of hydrolyzing host cell wall components to accommodate a necrotrophic life style. these cwdes include carbohydrate esterases, glycosyl hydrolases, polysaccharide lyases and peptidases, which the fungal pathogens utilize to degrade the host plant cell wall for nutrition. the glycosidases like galactosidase and arabinosidase may liberate sugar moieties that can be used as a nutritional source for c. truncatum during the necrotrophic growth. glycosidases may also have a role in decreasing the steric hinderance for other cwdes by removing protruding side chains from the backbone of polymers and providing increased access for endo forms of cwdes. pectinases weaken the cell wall and expose cell wall components to other enzymes, such as cellulases and hemicellulases. genome sequencing has revealed that the hemibiotrophic phytopathogen m. oryzae is predicted to possess glycosyl hydrolases, polysaccharide lyases, and carbohydrate esterases. our library retrieved glycosyl hydrolases, polysaccharide lyases, and carbohydrate esterases. another class of identified hydrolases was peptidases that may have a function in the extracellular processing of fungal secreted proteins, degradation of proteinaceous components involved in the plant response against pathogens or degradation of host tissue. esterases, glycosyl hydrolases, lyases, and peptidases could also weaken host cell walls, allowing for an easier access to nutrients. the interaction between a pathogen and its host is to a large extent orchestrated by the proteins that are secreted or localized to or at the cell membrane or cell wall. the ceaps are secreted through the endoplasmic reticulum pathway and tethered either to the fungal cell membrane or wall. some ceaps lack a transmembrane domain but possess c-terminal gpi anchor addition signal. gpi anchoring is an important mechanism to tether extracellular proteins to the plasma membrane or cell wall. the carboxyl termini of these proteins have a sequence motif that is recognized by a protein complex located in the er, known as the gpi transamidase. the gpi transamidase complex cleaves the protein at a position within this motif, termed the omega site, and transfers the gpi anchor en bloc to the newly generated c terminus of the protein. a genome-wide inventory of secretory proteins of p. sojae and p. ramorum revealed putative secretomes of and proteins, respectively, of which a subset of approximately proteins contain putative gpi anchor addition signals. the majority of elicitins from phytophthora possess a gpi anchor. components of the cell envelope of pathogens can be rapidly acclimatized by a changing environment. the ability to respond to a changing milieu is critical for pathogens to effectively evade, tolerate and/or suppress host immune responses. therefore, the structure and composition of the cell envelope is dynamic and changes during different stages of in planta growth. we observed an abrogation or a reduction in transcript levels of genes encoding ceaps during the biotrophic phase and an abrupt induction in the necrotrophic phase. the switch from voluminous biotrophic hyphae to thin necrotrophic hyphae involves an increased surface area to volume ratio, therefore may require elevated transcription of some of these cell surface protein encoding unigenes. this ratio and thinner cell walls of the secondary hyphae would be advantageous for both efficient nutrient uptake and secretion of wall degrading enzymes, and possibly toxins, during the necrotrophic growth of hemibiotrophic fungal pathogens. the predicted products of three discrete unigenes ct21- ct21- and ct21- resembled the biotrophy associated protein cih from c. lindemuthianum. the cih was shown to be secreted into the cell wall of invasive biotrophic hyphae and the plant-pathogen interface. in the present study, we found that the c. truncatum cih homolog was expressed constitutively in all in vitro cell types tested and both during the biotrophic- and necrotrophic- phases. this result was contradictory to studies with c. higginsianum where the cih protein was specifically expressed in the biotrophic phase. it suggests either the transcription level of cih does not reflect the protein level, or c. truncatum may employ a different approach to host invasion that is unlike that of other species of colletotrichum. based on the in planta transcript profiling of nine genes encoding putative effector proteins, we identified two types of expression patterns: biotrophic/necrotrophic-specific and necrotrophic-specific. transient expression of three candidate effectors in tobacco leaves revealed that none of them was able to elicit an hr response. surprisingly, our data on ctcp were in contrast of that recently published on m. oryzae cp protein, mgsm mgsm upon transient expression, stimulated hr and conferred broad-spectrum resistance to arabidopsis against botrytis cinerea and pseudomonas syringae pv. tomato. the predicted products of two discrete unigenes showed homology to extracellular protein from c. fulvum. lysms are n-acetyl d-glucosamine binding modules that have been found in mammalian and plant pathogenic fungi as well as in saprophytes. therefore, they may also have a role in counter defense by protecting fungal cell walls against plant chitinases. bolton et al. demonstrated that ecp was induced by the plant pathogenic fungus c. fulvum during the initial infection of tomato and acted as a virulence factor. during infection, lysm domain-containing protein ecp sequesters chitin oligosaccharides that are released from the cell walls of invading hyphae to avert the elicitation of host immunity. this sequestering may represent a common strategy of host immune suppression by fungal pathogens, because lysm effectors are widely conserved in the fungal kingdom. we identified several ests that may encode novel effectors. using bioinformatics tools, we identified soluble secreted proteins of unknown function. three of them were not expressed in the biotrophic phase while induced during the necrotrophic phase. their expression was also detected either in some or all of the in vitro cell types. such genes may be important for establishing fungal necrotrophy. one was constitutively expressed during all in planta developmental stages except appressorial penetration and in all in vitro cell types, suggesting its general role in both vegetative development and pathogenesis.  we catalogued proteins putatively secreted by c. truncatum and several candidate effectors had been mined from the est library. it was observed that the transcript levels of candidate effector genes were either induced during the necrotrophic growth of the pathogen compared with that during the biotrophic growth or elevated during both phases. through in planta expression of candidate effectors, we demonstrated the functional role of one effector. in summary, our study suggests that suppression of host defense during the biotrophic phase and the subsequent unmasking of pamps or elicitors during the necrotrophic phase may be involved in pathogenesis of c. truncatum. 
sequence analyses of unigenes revealed four potential groups: hydrolases, cell envelope associated proteins, candidate effectors and other proteins. for a selected subset of ceaps and candidate effectors, semiquantitative rt-pcr showed that these transcripts were either expressed constitutively in both in vitro and in planta or induced during plant infection. eleven candidate effector genes were identified based on features common to characterized fungal effectors, i.e. c. truncatum likely secretes an arsenal of proteins that are implicated in maintaining a compatible interaction with its host. some of these proteins may have a role in establishing compatible interaction with the host plant. using potato virus x based transient expression assays, we showed that one of the candidate effectors, i. e. contig that encodes a cerato-platanin domain containing protein, unlike cp proteins from other fungal pathogens was unable to elicit a hypersensitive response. nearly 39% of the ests were predicted to encode proteins of fungal origin and among these, ests were predicted to contain n-terminal signal peptides in their deduced open reading frames. a directional cdna library was constructed from mrna isolated from infected lens culinaris leaflet tissues displaying the biotrophy-necrotrophy switch of c. truncatum and expressed sequence tags with an average read of > bp from the 5-prime end were generated. the current study catalogues proteins putatively secreted at the in planta biotrophy-necrotrophy transition of c. truncatum. transition from biotrophy to a destructive necrotrophic phase called the biotrophy-necrotrophy switch is critical in symptom development.
 colletotrichum truncatum is a haploid, hemibiotrophic, ascomycete fungal pathogen that causes anthracnose disease on many economically important leguminous crops. this pathogen exploits sequential biotrophic- and necrotrophic- infection strategies to colonize the host. transition from biotrophy to a destructive necrotrophic phase called the biotrophy-necrotrophy switch is critical in symptom development. c. truncatum likely secretes an arsenal of proteins that are implicated in maintaining a compatible interaction with its host. some of them might be transition specific.  a directional cdna library was constructed from mrna isolated from infected lens culinaris leaflet tissues displaying the biotrophy-necrotrophy switch of c. truncatum and expressed sequence tags with an average read of > bp from the 5-prime end were generated. nearly 39% of the ests were predicted to encode proteins of fungal origin and among these, ests were predicted to contain n-terminal signal peptides in their deduced open reading frames. the sequences could be assembled into tentative unigenes comprising contigs and singletons. sequence analyses of unigenes revealed four potential groups: hydrolases, cell envelope associated proteins, candidate effectors and other proteins. eleven candidate effector genes were identified based on features common to characterized fungal effectors, i.e. they encode small, soluble, cysteine-rich proteins with a putative sp. for a selected subset of ceaps and candidate effectors, semiquantitative rt-pcr showed that these transcripts were either expressed constitutively in both in vitro and in planta or induced during plant infection. using potato virus x based transient expression assays, we showed that one of the candidate effectors, i. e. contig that encodes a cerato-platanin domain containing protein, unlike cp proteins from other fungal pathogens was unable to elicit a hypersensitive response.  the current study catalogues proteins putatively secreted at the in planta biotrophy-necrotrophy transition of c. truncatum. some of these proteins may have a role in establishing compatible interaction with the host plant. 