Identification of soluble secreted proteins from appressoria of Colletotrichum higginsianum by analysis of expressed sequence tags

Abstract

The hemibiotrophic ascomycete Colletotrichum higginsianum causes anthracnose disease on brassica crops and the model plant Arabidopsis. Melanized appressoria pierce the host cuticle and cell wall to form specialized biotrophic hyphae inside living epidermal cells. To identify proteins secreted by appressoria that may function as virulence effectors, a cDNA library was prepared from mature appressoria formed in vitro. Bidirectional sequencing of 980 clones generated 1442 high-quality expressed sequence tags (ESTs), comprising 518 unique sequences. BLASTX analysis showed that 353 (68 %) of these had significant similarity to entries in the NCBI non-redundant protein database, of which 49 were also homologous to experimentally verified fungal pathogenicity genes. ORFs were predicted ab initio from the unique sequences and screened for potential signal peptides using SignalP. Fifty-three unique sequences (10 %) were predicted to encode proteins entering the secretory pathway, of which 26 were likely to be soluble secreted proteins. For a selected subset of these, RT-PCR showed that seven genes that encode secreted proteins of unknown function, including two Colletotrichum-specific genes, are upregulated in appressoria and expressed early during plant infection, and therefore represent candidate effectors. INTRODUCTIONThe ascomycete genus Colletotrichum comprises one of the most economically damaging groups of plant-pathogenic fungi, causing anthracnose diseases and blights on an enormous range of dicot and monocot crop plants in temperate, tropical and subtropical regions (Bailey & Jeger, 1992). The crucifer anthracnose pathogen Colletotrichum higginsianum has a wide host range, attacking many cultivated forms of Brassica and Raphanus as well as wild crucifers, including the model plant Arabidopsis thaliana (Narusaka et al., 2004;O'Connell et al., 2004). C. higginsianum employs a two-stage, hemibiotrophic infection strategy: after melanized appressoria breach the host cuticle and cell wall, the fungus initially grows biotrophically inside living epidermal cells, before entering a destructive necrotrophic phase in which host tissues are killed and extensively macerated by cell-wall-degrading enzymes. During the biotrophic phase, C. higginsianum differentiates specialized intracellular primary hyphae similar to haustoria, which expand and locally modify the host plasma membrane (Shimada et al., 2006). However, in contrast to obligately biotrophic pathogens of Arabidopsis, Colletotrichum can be cultured axenically and stably transformed using a variety of methods; it is also a haploid organism with uninucleate conidia, which facilitates mutational analyses. This pathosystem therefore provides an attractive model for studying biotrophy, in which both partners in the interaction are genetically tractable.Whole-genome sequencing projects have revealed that fungal and oomycete plant pathogens possess large repertoires of secreted proteins, which can play diverse roles in pathogenicity and interactions with host cells (Dean...

Sequential Delivery of Host-Induced Virulence Effectors by Appressoria and Intracellular Hyphae of the Phytopathogen Colletotrichum higginsianum

“…ChEC6:mRFP and ChEC36:mRFP fusions were expressed and targeted to this pore before any ingression into the host cell had occurred. Given that in vitro and in planta appressoria are morphologically indistinguishable [29] and that these effectors were not expressed by in vitro appressoria, this suggests that host-derived signals, rather than developmental cues, induce ChEC expression. Moreover, these signals must be sensed before penetration hyphae emerge, presumably via the wall-less pore region.…”

“…(c). A control gene encoding a non-secreted calpain protease, although absent from most sequenced ascomycete genomes [29], is conserved in all species tested. The ethidium bromide-stained agarose gel before blotting is shown below as loading control.…”

EST mining identifies proteins putatively secreted by the anthracnose pathogen Colletotrichum truncatum

“…Studies conducted to date have investigated the secretome at different stages of the infection process, including penetration (appressorial morphogenesis), biotrophic and necrotrophic stages. Kleeman et al [13] identified 26 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 (< 400 amino acids in length when mature), presence of cysteine residues and of a putative SP [14].…”

Proteomic studies of phytopathogenic fungi, oomycetes and their interactions with hosts

“…Biocomputational algorithms are used to scan Nterminal signal peptides in predicted proteins from whole-genome sequences or expressed sequence tags (ESTs). Using this approach, dozens of secreted proteins were identified from P. infestans (Torto et al 2003;Tian et al 2004), Colletotrichum higginsianum (Kleemann et al 2008), and Venturia inaequalis (Brown et al 2009). The signal sequence trap is a yeast-based genetic screen for signal peptide encoding cDNA.…”