A pectato liase codificada pelo gene pecCl1 é importante para agressividade de Colletotrichum lindemuthianum

• Autor(a) principal: Fassoni, Andréia Cnossen
• Data de Publicação: 2012
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/5364
• topic: Colletotrichum lindemuthianum; Pectato liase; Fase necrotrófica; Antracnose; Split-marker; Patogenicidade; Colletotrichum lindemuthianum; Pectate lyase; Necrotrophic phase; Anthracnose; Split-marker; Pathogenicity; CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOSSANIDADE::MICROBIOLOGIA AGRICOLA

Colletotrichum lindemuthianum is the causal agent of common bean anthracnose. Genes that encode cell wall-degrading enzymes are essential for the development of this disease. The pectinases are characterized as the most important group of cell wall- degrading enzymes produced by phytopathogen fungi. The gene coding for pectate lyase, pecCl1, was previously identified in a suppressive subtractive library of bean infected with C. lindemuthianum. Isolation of the gene pecCl1 made it possible to obtain mutants and to analyze the regulation of this gene during development of anthracnose, determining whether the pectate lyase is a pathogenic factor. Thus, the aim of our study was structurally and functionally characterize the gene encoding pectate lyase in C. lindemuthianum. Initially, was performed the structural analysis of the gene pecCl1. The complete nucleotide sequence of the gene pecCl1 was deposited in Genbank with accession number JX270683. The analysis of the promoter region revealed some putative cis-elements and potential binding motifs of transcription factors involved in the regulation of pectate lyase gene expression. The deduced amino acid sequence of pecCl1 showed sequence identity with the pectate lyase F of Colletotrichum higginsianum and the pectate lyase C of Glomerella graminicola M1.001. Furthermore, it was found putative conserved domain pfam03211 of the pectate lyases superfamily. The gene pecCl1 is represented by a single copy in the C. lindemuthianum genome. However, into the genome of Colletotrichum graminicola, three sequences encoding pectate lyase showed sequence identity with the gene pecCl1 of C. lindemuthianum, and into the genome of C. higginsianum seven sequences encoding pectate lyase showed sequence identity with the gene pecCl1 of C. lindemuthianum, indicating that the C. lindemuthianum genome can possess other genes encoding pectate lyase. Phylogenetic analysis of pectate lyase amino acid sequences of filamentous fungi exhibited the formation of two distinct groups which are grouped on the basis of members of the pectate lyases multigene family. The Split-Marker technique was effective in C. lindemuthianum pecCl1 gene inactivation, allowing the study of pecCl1 function in a mutant by specific integrations and without ectopic integrations. The pecCl1 gene inactivation did not lead to complete loss of the pectate lyase activity, and consequently only decreased anthracnose symptoms in its host, which is consistent with the presence of other genes coding pectate lyase, allowing greater flexibility in pathogen aggressiveness. The analysis of differential expression of gene pecCl1 by qPCR was performed at different stages of bean infection and were observed expression levels of pecCl1 at all stages of development of the fungus in the plant, but a significant increase was observed five days after infection, in the onset of necrotrophic stage. At this stage, secondary hyphae cause extensive degradation of plant cell wall through the secretion of wide range of depolymerases, among these, the pectate lyase. Thus, the pectate lyase encoded by the gene pecCl1 is important to aggressiveness of C. lindemuthianum. The analysis of pectate lyases in C. lindemuthianum can not only assist in understanding the disease, but may also lead to discovery of one more target for disease control.