Anthracnose In Common Bean Research Articles

A GAL4-like Protein Is Involved in the Switch between Biotrophic and Necrotrophic Phases of the Infection Process of Colletotrichum lindemuthianum on Common Bean

Random insertional mutagenesis was conducted with the hemibiotrophic fungus Colletotrichum lindemuthianum, causal agent of common bean anthracnose. Nine mutants that were altered in their infection process on the host plant were generated. One of these, H433 is a nonpathogenic mutant able to induce necrotic spots on infected leaves rapidly. These spots are similar to those observed during the hypersensitive reaction. Cytological observations showed that the development of the mutant H433 is stopped at the switch between the biotrophic and the necrotrophic phases. This mutant carries two independent insertions of the transforming plasmid pAN7-1. Complementation studies using the wild-type genomic regions corresponding to the two insertions showed that one is responsible for the H433 phenotype. Sequencing analysis identified a single open reading frame that encoded a putative transcriptional activator belonging to the fungal zinc cluster (Zn[II](2)Cys(6)) family. The corresponding gene was designated CLTA1 (for C. lindemuthianum transcriptional activator 1). Expression studies showed that CLTA1 is expressed in low amounts during in vitro culture. Targeted disrupted strains were generated, and they exhibited the same phenotype as the original mutant H433. Complementation of these disrupted strains by the CLTA1 gene led to full restoration of pathogenicity. This study demonstrates that CLTA1 is both a pathogenicity gene and a regulatory gene involved in the switch between biotrophy and necrotrophy of the infection process of a hemibiotrophic fungus.

Clk1, a serine/threonine protein kinase-encoding gene, is involved in pathogenicity of Colletotrichum lindemuthianum on common bean.

A random insertional mutagenesis in Colletotrichum lindemuthianum, the causal agent of common bean anthracnose, generated four mutants that showed altered pathogenicity when tested on intact seedlings, excised leaves, and/or excised hypocotyls. One of these mutants, H290, produced very few lesions on bean leaves and appeared affected in its ability to penetrate the leaf cuticle. Molecular analyses showed that the border sequences of the unique integration site of the disrupting pAN7-1 plasmid in the mutant exhibited homology with conserved domains of serine/threonine protein kinases. The corresponding wild-type sequences were cloned and a gene replacement vector with a mutated copy harboring a selection marker constructed. Transformation of the wild-type pathogen produced a strain with a phenotype identical to the original mutant. Genomic and cDNA sequences indicated that the disrupted gene is a member of the serine/threonine protein kinase family. The gene, called clk1 (Colletotrichum lindemuthianum kinase 1), was weakly expressed in the mycelium of the wild-type strain grown on rich and minimal synthetic media but was undetectable during the infection even when a sensitive reverse transcriptase-polymerase chain reaction methodology was used. This study represents the first characterization of altered pathogenicity mutants in C. lindemuthianum produced by random mutagenesis and demonstrates the involvement of a member of the serine/threonine kinase gene family in the early steps of the infection process.