The Conserved Colletotrichum spp. Effector Candidate CEC3 Induces Nuclear Expansion and Cell Death in Plants

Plant pathogens secrete proteins, known as effectors, that promote infection by manipulating host cells. Members of the phytopathogenic fungal genus Colletotrichum collectively have a broad host range and generally adopt a hemibiotrophic lifestyle that includes an initial biotrophic phase and a later necrotrophic phase. We hypothesized that Colletotrichum fungi use a set of conserved effectors during infection to support the two phases of their hemibiotrophic lifestyle. This study aimed to examine this hypothesis by identifying and characterizing conserved effectors among Colletotrichum fungi. Comparative genomic analyses using genomes of ascomycete fungi with different lifestyles identified seven effector candidates that are conserved across the genus Colletotrichum. Transient expression assays showed that one of these putative conserved effectors, CEC3, induces nuclear expansion and cell death in Nicotiana benthamiana, suggesting that CEC3 is involved in promoting host cell death during infection. Nuclear expansion and cell death induction were commonly observed in CEC3 homologs from four different Colletotrichum species that vary in host specificity. Thus, CEC3 proteins could represent a novel class of core effectors with functional conservation in the genus Colletotrichum.

Comparative Genome Analyses of 18 Verticillium dahliae Tomato Isolates Reveals Phylogenetic and Race Specific Signatures

Host resistance is one of the few strategies available to combat the soil borne pathogenic fungus Verticillium dahliae. Understanding pathogen diversity in populations is key to successfully deploying host resistance. In this study the genomes of 18 V. dahliae isolates of races 1 (n = 2), 2 (n = 4), and 3 (n = 12) from Japan, California, and North Carolina were sequenced and mapped to the reference genome of JR2 (from tomato). The genomes were analyzed for phylogenetic and pathogen specific signatures to classify specific strains or genes for future research. Four highly clonal lineages/groups were discovered, including a lineage unique to North Carolina isolates, which had the rare MAT1-1 mating type. No evidence for recombination between isolates of different mating types was observed, even in isolates of different mating types discovered in the same field. By mapping these 18 isolates genomes to the JR2 reference genome, 193 unique candidate effectors were found using SignalP and EffectorP. Within these effectors, 144 highly conserved effectors, 42 mutable effectors (truncated or present in some isolates but absent in others), and 7 effectors present in highly variable regions of the chromosomes were discovered. Of the 144 core effectors, 21 were highly conserved in V. alfalfae and V. longisporum, 7 of which have no known function. Within the non-core effectors 30 contained large numbers of non-synonymous mutations, while 15 of them contained indels, frameshift mutations, or were present on highly variable regions of the chromosome. Two of these highly variable region effectors (HVREs) were only present in race 2 isolates, but not in race 3 isolates. The race 1 effector Ave1 was also present in a highly variable region. These data may suggest that these highly variable regions are enriched in race determinant genes, consistent with the two-speed genome hypothesis.

Conserved fungal effector suppresses PAMP-triggered immunity by targeting plant immune kinases

Plant pathogens have optimized their own effector sets to adapt to their hosts. However, certain effectors, regarded as core effectors, are conserved among various pathogens, and may therefore play an important and common role in pathogen virulence. We report here that the widely distributed fungal effector NIS1 targets host immune components that transmit signaling from pattern recognition receptors (PRRs) in plants. NIS1 from two Colletotrichum spp. suppressed the hypersensitive response and oxidative burst, both of which are induced by pathogen-derived molecules, in Nicotiana benthamiana. Magnaporthe oryzae NIS1 also suppressed the two defense responses, although this pathogen likely acquired the NIS1 gene via horizontal transfer from Basidiomycota. Interestingly, the root endophyte Colletotrichum tofieldiae also possesses a NIS1 homolog that can suppress the oxidative burst in N. benthamiana. We show that NIS1 of multiple pathogens commonly interacts with the PRR-associated kinases BAK1 and BIK1, thereby inhibiting their kinase activities and the BIK1-NADPH oxidase interaction. Furthermore, mutations in the NIS1-targeting proteins, i.e., BAK1 and BIK1, in Arabidopsis thaliana also resulted in reduced immunity to Colletotrichum fungi. Finally, M. oryzae lacking NIS1 displayed significantly reduced virulence on rice and barley, its hosts. Our study therefore reveals that a broad range of filamentous fungi maintain and utilize the core effector NIS1 to establish infection in their host plants and perhaps also beneficial interactions, by targeting conserved and central PRR-associated kinases that are also known to be targeted by bacterial effectors.

Genome-Enabled Phylogeographic Investigation of the Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 and Screening for Sources of Resistance Against Its Core Effectors

Phylogeographic studies inform about routes of pathogen dissemination and are instrumental for improving import/export controls. Genomes of 17 isolates of the bacterial wilt and potato brown rot pathogen Ralstonia solanacearum race 3 biovar 2 (R3bv2), a Select Agent in the United States, were thus analyzed to get insight into the phylogeography of this pathogen. Thirteen of fourteen isolates from Europe, Africa, and Asia were found to belong to a single clonal lineage while isolates from South America were genetically diverse and tended to carry ancestral alleles at the analyzed genomic loci consistent with a South American origin of R3bv2. The R3bv2 isolates share a core repertoire of 31 type III-secreted effector genes representing excellent candidates to be targeted with resistance genes in breeding programs to develop durable disease resistance. Toward this goal, 27 R3bv2 effectors were tested in eggplant, tomato, pepper, tobacco, and lettuce for induction of a hypersensitive-like response indicative of recognition by cognate resistance receptors. Fifteen effectors, eight of them core effectors, triggered a response in one or more plant species. These genotypes may harbor resistance genes that could be identified and mapped, cloned, and expressed in tomato or potato, for which sources of genetic resistance to R3bv2 are extremely limited.