Selective deployment of virulence effectors correlates with host specificity in a fungal plant pathogen.

The hemibiotrophic fungal plant pathogen Colletotrichum orbiculare is predicted to secrete hundreds of effector proteins when the pathogen infects cucurbit crops, such as cucumber and melon, and tobacco (Nicotiana benthamiana), a distantly related Solanaceae species. Here, we report the identification of sets of C. orbiculare effector genes that are differentially required for fungal virulence to two phylogenetically distant host species. Through targeted gene knockout screening of C. orbiculare 'core' effector candidates defined based on in planta gene expression, we identified: four host-specific virulence effectors (named effector proteins for cucurbit infection, or EPCs) that are required for full virulence of C. orbiculare to cucurbit hosts, but not to the Solanaceae host N. benthamiana; and five host-nonspecific virulence effectors, which collectively contribute to fungal virulence to both hosts. During host infection, only a small subset of genes, including the host-specific EPC effector genes, showed preferential expression on one of the hosts, while gene expression profiles of the majority of other genes, including the five host-nonspecific effector genes, were common to both hosts. This work suggests that C. orbiculare adopts a host-specific effector deployment strategy, in addition to general host-blind virulence mechanisms, for adaptation to cucurbit hosts.

Multiple Colletotrichum species commonly exhibit focal effector accumulation in a biotrophic interface at the primary invasion sites in their host plants.

Fungal plant pathogens deploy a suite of secreted proteins, called effectors, to facilitate successful infection. Several fungal pathogens have been reported to secrete and accumulate their effector proteins in the host-pathogen interfacial spaces. Previously, we reported that the strain 104-T of the cucurbit anthracnose pathogen Colletotrichum orbiculare secretes and accumulates mCherry-tagged effectors along with the formation of ring-shaped fluorescence signals beneath the appressoria. However, it was unclear whether these effector accumulation patterns occur in other C. orbiculare isolates and other species belonging to the Colletotrichum genus. Here, we investigated the effector localization during host infection of C. orbiculare MAFF306589, C. trifolii MAFF305078, which infects alfalfa, and C. higginsianum MAFF305635, which infects Brassicaceae plants. We generated effector-reporter lines of each species, which constitutively expressed mCherry-tagged CoDN3 effector (CoDN3:mCherry). Immunoblotting analysis of the liquid culture fluids of the generated lines detected CoDN3:mCherry, which confirmed secretion of CoDN3:mCherry by fungal cells. Via inoculation assays in the corresponding host plants, we detected ring-shaped CoDN3:mCherry fluorescence around the appressorial invasion sites in all tested reporter lines. These results suggest that pathogens in the Colletotrichum genus have evolutionarily conserved the trait of effector secretion in the infection stage irrespective of differences in their hosts.