Discovery of Pathogenicity Genes in the Crucifer Anthracnose Fungus Colletotrichum higginsianum, Using Random Insertional Mutagenesis

Abstract

Agrobacterium tumefaciens-mediated transformation (ATMT) was used for random insertional mutagenesis to identify pathogenicity genes in the hemibiotrophic fungus Colletotrichum higginsianum. A high-throughput primary infection assay on Arabidopsis thaliana seedlings allowed the rapid screening of 8,850 transformants. Forty mutants showing reproducible pathogenicity defects on Arabidopsis and Brassica plants were obtained, and their infection phenotypes were characterized microscopically. Six mutants were impaired in appressorial melanization, fifteen had reduced penetration ability, 14 induced host papillae or hypersensitive cell death, and five were affected in the transition from biotrophy to necrotrophy. Southern blot analysis showed 58% of the transformants had single-site T-DNA integrations. Right-border flanking sequences were recovered from 12 mutants by inverse polymerase chain reaction (PCR) or thermal asymmetric interlaced PCR and were used to isolate the tagged genes from a genomic library. The putative pathogenicity genes encoded homologs of a major facilitator superfamily phosphate transporter, importin-beta2, ornithine decarboxylase, beta-1,3(4)-glucanase, ATP-binding endoribonuclease, carbamoyl-phosphate synthetase, and the polyprotein precursor of N-acetylglutamate kinase and N-acetylglutamyl-phosphate reductase. Six further loci were homologous to proteins of unknown function. None of these genes were previously implicated in the pathogenicity of any Colletotrichum species. The results demonstrate that ATMT is an effective tool for gene discovery in this model pathogen.

Plant Defense Mechanisms Are Activated during Biotrophic and Necrotrophic Development of Colletotricum graminicola in Maize

“…This observation is further supported by the high susceptibility of primary hyphae to plant defenses. Mutants of C. graminicola, Colletotrichum lindemuthianum, and C. higginsianum, impaired in the switch from biotrophy to necrotrophy, only produced primary hyphae and were not able to colonize more than a limited number of cells in the host, and no anthracnose development was evident (Dufresne et al, 2000;Thon et al, 2002;Huser et al, 2009). In those cases, plant defenses do successfully restrict the growth of C. graminicola and C. higginsianum mutants arrested in the biotrophic stage.…”

One stop shop: backbones trees for important phytopathogenic genera: I (2014)

“…The pathogenicity genes of C. higginsianum were discovered by random mutagenesis (Huser et al 2009). Genomes and transcriptomes of C. higginsianum and C. graminicola were studied through the use of two different infection strategies by O'Connell et al (2012).…”

Entry Mode–Dependent Function of an Indole Glucosinolate Pathway in Arabidopsis for Nonhost Resistance against Anthracnose Pathogens

“…This demonstrated that molecular components of preinvasion immune responses differ between different ascomycete fungal parasites, even though these employ the same infection strategy of direct entry into host epidermal cells mediated by an appressorium. Recently, PEN2 was found to be involved in basal preinvasion resistance of Arabidopsis against the host-adapted pathogen C. higginsianum (Huser et al, 2009). However, it remained unknown whether the PEN2-dependent indole glucosinolate metabolism pathway contributes to nonhost resistance against Colletotrichum species.…”