Abstract
Colletotrichum higginsianum is a widely distributed fungus attacking many cruciferous species. To investigate pathogenic mechanisms of the pathogen on the host Arabidopsis thaliana, we screened and obtained a virulence-deficient mutant Ch-1-T513 in a T-DNA insertion mutant library of C. higginsianum. The mutant Ch-1-T513 produced yellow colony centers with distorted multi-branching hyphal tips as well as producing few conidia. Heavily swollen hyphae in the mutant could be observed, and intra-hyphal hyphae were found to be formed in the balloon-shaped hyphae. The mutant failed to produce lesions on 12-day-old Arabidopsis seedlings, and invasive hyphae did not differentiate into large primary and thin secondary hyphae after appressorial formation on Arabidopsis leaves, but formed abundant bulbous hyphae in epidermal cells. Southern blot analysis showed Ch-1-T513 had double-site T-DNA integrations. The mutant had insertions upstream of genes for a major facilitator superfamily (MFS) transporter, ChMfs1 and an aldo/keto reductase, ChAkr. Complementation experiments by transforming genomic sequences from a wild-type strain into the insertion mutant demonstrated that ChMfs1 is involved in the Ch-1-T513 phenotype. The complementation strain C-ChMfs1-1 exhibited normal hyphal morphology, conidiation, and pathogenicity identical to the wild-type. The results demonstrate that ChMfs1 is involved in intra-hyphal hyphae production, conidiation, and pathogenicity in C. higginsianum. To our knowledge, this is the first report of a MFS transporter gene in a phytopathogenic fungus associated with intra-hyphal hyphae formation, playing a key role in infection of its plant host.
Highlights
Colletotrichum higginsianum as a hemibiotrophic fungal pathogen causes anthracnose disease on many cruciferous plants, including economically important species, Brassica spp. as well as Arabidopsis thaliana (O’Connell et al, 2004)
In separate complementation studies for these two genes corresponding to the insertions, we demonstrated that the major facilitator superfamily (MFS) transporter named ChMfs1 is responsible for the mutant Ch-1-T513 phenotype
This study demonstrates that ChMfs1 is involved in pathogenicity and formation of intra-hyphal hyphae during infection phases of C. higginsianum and complements a novel function of MFS transporters
Summary
Colletotrichum higginsianum as a hemibiotrophic fungal pathogen causes anthracnose disease on many cruciferous plants, including economically important species, Brassica spp. as well as Arabidopsis thaliana (O’Connell et al, 2004). Anthracnose conidia typically produce germination hyphae that differentiate into melanized appressoria, which in turn produce. ChMfs in Colletotrichum higginsianum penetration pegs and enter into plant cell. Once inside the plant cell, the fungus forms swollen biotrophic primary hyphae which proliferate by invading living epidermal cells. These necrotrophic secondary hyphae spread more quickly across host cells causing chlorosis and necrosis. A. thaliana is an important model host with a completely sequenced genome available and ease of genetic analysis as well as an extensive mutant collection (O’Connell et al, 2004). The C. higginsianum–Arabidopsis pathosystem is an attractive model system for studying molecular mechanisms of plant–microbe interactions (O’Connell et al, 2004; Huser et al, 2009)
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