Screening Of Pathogenicity-deficient Mutants From A T-DNA Insertional Library Of Colletotrichum Higginsianum And Characterized Function Analysis Of Pathogenesis-Associate Genes

To investigate the molecular and genetic mechanisms underlying virulence of Colletotrichum higginsianum on Arabidopsis thaliana, a T-DNA insertion mutant library of C. higginsianum, the causal agent of crucifer anthracnose, was established using Agrobacterium tumefaciens-mediated transformation. Among875transformants tested for virulence on Arabidopsis, six mutants with altered virulence, including an appressorial melanin-deficient mutant Ch-1-T734, a mutant Ch-1-T45defective penetration, three mutants, Ch-1-T679, Ch-1-T732and Ch-1-T801, that cause hypersensitive reactions on host Arabidopsis, and a mutant Ch-1-T513which was impaired in the switch to necrotrophy, were obtained.Southern blot analysis indicated that the mutants Ch-1-T732and Ch-1-T734harbored single-site T-DNA integrations, while Ch-1-T513harbored two T-DNA insertions. Border flanking sequences of T-DNAs from these mutants were recovered by inverse polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR. Sequence analyses revealed that single T-DNA insertions in mutant Ch-1-T734targeted the coding region of a gene with unknown function, and in mutant Ch-1-T732targeted a gene encoding a copper amine oxidase. The two T-DNA insertion sites in mutant Ch-1-T513had insertions upstream of genes for a major facilitator superfamily transporter and an aldo/keto reductase. None of these genes has previously been implicated in virulence of the phytopathogenic fungi.Among these avirulent mutants, Ch-1-T734showed altered color in colony growth and produced melanin-deficient, albino appressoria. The T-DNA insertion in Ch-1-T734was detected in the coding region of a gene named C. higginsianum melanin-deficiency gene (Ch-MEL1), which is highly similar to a gene encoding a hypothetical protein in Collectotrichum gloeosporioides (GenBank ELA33048). To validate whether the Ch-MEL1gene was associated with virulence of the mutant Ch-1-T734, targeted gene disruption and complementation experiments were carried out The appressoria of△Ch-mell null mutants were defective in melanization and failed to penetrate the host epidermal cells. When inoculated onto the wounded leaf tissues, the△Ch-mell mutants grew on host tissues but failed to cause lesions beyond the wound site. In contrast, both the complement C△Ch-mel1-2and the wild type produced melaninized appressoria and caused necrosis on leaves of Arabidopsis. Ch-MEL1is required for both appressorial melanin production in C. higginsianum and post-invasive growth in host tissues.Ch-1-T513showed abnormal bulbous hyphae during the separate infection phases, and had two T-DNA insertions, one insertion was located of82bp upstream of a gene showing high similarity to major facilitator superfamily (MFS) transporter in Coccidioides posadasii (GenBank EER28508), and the other insertion was at411bp upstream of a gene with high similarity to an aldo/keto reductase of Glomerella graminicola (GenBank EFQ31134). Complementation studies using the wild-type genomic regions corresponding to the insertions showed that one is responsible for the Ch-1-T513phenotype. The corresponding gene was designated Ch-MFS1(for C. higginsianum major facilitator superfamily transporter). The study demonstrate that Ch-MFS1was a major facilitator superfamily multidrug transporter involved in the growth of hyphae, conidiation, and pathogenicity in C. higginsianum.With identification of new avirulent mutants and their associated genes, this study provides novel insights into molecular mechanisms underlying pathogenicity of the hemibiotroph, C. higginsianum.