Identification and functional characterization of secreted effector proteins of the hemibiotrophic fungus Colletotrichum higginsianum

The hemibiotrophic ascomycete fungus Colletotrichum higginsianum causes anthracnose on cruciferous crops and the model plant Arabidopsis thaliana. Successful infection of wild-type plants requires sequential development of specialized infection structures, including melanized appressoria for initial penetration, and bulbous biotrophic hyphae formed inside living epidermal cells. It was hypothesized that appressoria and biotrophic hyphae secrete effector proteins that permit the fungus to evade or disarm host defence responses and to reprogram host cells. This study aimed to define the repertoire of fungal effectors expressed during plant infection and to characterise their biological activity. Discovery of Colletotrichum higginsianum Effector Candidates (ChECs) was accomplished by computational mining collections of infection stage-specific expressed sequence tags (ESTs) for genes encoding solubly secreted proteins with either no homology to known proteins or resembling presumed effectors from other pathogens. Fungal cell types and infection stages sampled for cDNA generation and pyrosequencing included developing and mature in vitro appressoria, early invasive growth in planta, biotrophy and late necrotrophy. After assembling contiguous sequences, analysis of their EST composition allowed the identification of putative plant-induced genes and the definition of a set of 69 ChEC genes that are preferentially expressed at biotrophy-relevant stages. In relation to other infection stages, the early host invasion transcriptome was enriched for genes encoding higginsianum-specific proteins and plant-induced secreted proteins, including ChECs. This suggests that the initial establishment of biotrophy requires the highest proportion of stage-specific effectors and diversified genes. One further ChEC was identified using a complementary proteomic analysis of secreted proteins produced by conidial germlings developing in vitro. Expression analysis showed that transcription of most of the ChECs chosen for further study were highly stage-specific, with ChEC3, ChEC3a, ChEC4 and ChEC6 all being plant-induced. Targeted gene replacement showed that neither ChEC1 nor ChEC2 contribute measurably to fungal virulence. Upon transient expression in tobacco, ChEC3, ChEC3a or ChEC5 all suppressed plant cell death evoked by a C. higginsianum homologue of Necrosis and Ethylene-inducing Peptide1-like proteins, but not by the Phytophthora infestans elicitin INF1, suggesting that there is functional redundancy between C. higginsianum effectors. ChEC4 was found to contain a functional nuclear localization signal and signal peptide, and was shown to be secreted by the fungus during plant infection using fluorescent protein-tagging. This raises the possibility that ChEC4 is translocated into the host nucleus for transcriptional re-programming