Genes determinantes de patogenicidade e virulência e análise parcial do genoma mitocondrial de Colletotrichum lindemuthianum, agente causal da antracnose do feijoeiro comum

• Ano de defesa: 2007
• Autor(a) principal: Soares, Marcos Antônio
• Orientador(a): Queiroz, Marisa Vieira de
• Banca de defesa: Rodrigues, Fabrício de ávila , Souza, Elaine Aparecida
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Microbiologia Agrícola
• Departamento: Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse
• País: BR
• Palavras-chave em Português: Colletotrichum lindemuthianum; Genes de patogenicidade e virulência; DNA mitocondrial; PacC
• Palavras-chave em Inglês: Colletotrichum lindemuthianum; Pathogenicity and virulence genes; Mitochondrial DNA; PacC
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS
• Link de acesso: http://locus.ufv.br/handle/123456789/1573

Colletotrichum lindemuthianum is one of the main pathogens affecting common bean (Phaseolus vulgaris) in tropical regions. In the present work we describe three mutants of C. lindemuthianum obtained by insertional mutagenesis using the REMI technique (Restriction Enzyme-Mediated Integration). The mutants were selected based on their lower virulence/pathogenicity and the partial characterization of the mutated genes is presented. In addition, the mitochondrial genome of this phytopathogen was partially characterized. The results show that the gene pacC1 plays a role in vegetative growth and pathogenicity. The use of the restriction enzyme NarI in the transformation of C. lindemuthianum increased approximately 10-fold the transformation efficiency using plasmid pAN7.1. Mutant strains mut5, mut29, and mut65 were isolated from a total of 580 transformants based on their altered ability to infect susceptible bean plants. Strains mut29 and mut65 failed to penetrate hypocotyl tissues of the common bean, but caused anthracnose symptoms on mature leaves. This result indicates that strains mut29 and mut65 show a phenotype that varies according to the type of vegetal tissue used in the infectivity tests. The sequence of the plasmids recovered from strains mut29 and mut65 showed that the integration of vector pAN7.1 was indeed mediated by the restriction enzyme NarI, thus characterizing REMI events. Sequence analysis showed that the gene coding for the enzyme tetrahydrofolate synthase (tlsCl) was interrupted by the transformation vector in strain mut65. On the other hand, a gene putatively coding for a phospholipase C (plcCl) was tagged in strain mut29. In addition, strain mut29 showed a second integration event at the 3 terminal region of a gene showing sequence similarity to a MSF transporter (msfCl). This constitutes the first report of these genes in C. lindemuthianum. The third mutant, utpac2, was tagged to an interruption of gene pacCl, showing sequence similarity to a transcription factor of the family PacC/Rim101 of fungi and yeast. This family of transcription factors is responsible for gene activation or repression in response to extracellular pH. The activation of these transcription factors depends on a signal transduction pathway capable of detecting external pH variations and then transmits the signal until the activation of the transcription factor. The cDNA sequence of gene pacCl was obtained from a C. lindemuthianum EST data bank. Its predicted protein has 581 amino acids and shows high sequence similarity to proteins from the family of transcription regulators PacC/Rim101; its N-terminal region has three Zinc-finger motifs of the type Cys2Hys2. We show that the level of pacCl transcript accumulation increased with more alkaline extracellular pH. Growth of the pacCl null mutant, mutpac2, was inhibited in alkaline environments and secreted small amounts of lipases. This mutant proved unable to macerate host tissues, thus indicating the role of this gene in the pathogenesis of C. lindemuthianum. Trascription of pacCl was detected by RT-PCR in the wild-type strain 18 hours after the inoculation of susceptible host plants and was progressively increased accompanying fungal biomass during infection kinetics. Another two cDNAs coding for proteins homologous to members of the protein family PalA/Rim20 and PalF/Rim8 of fungi and yeast were described as well. These proteins also participate in signal transduction pathways. The detection of genes positively regulated by PacCl during the transition from the biotrophic to the necrotrophic stages of the C. lindemuthianum s life cycle may aid in understanding the molecular mechanisms involved in the switch process of hemibiotrophic fungi, and may consequently lead to the discovery of alternative control methods for anthractnose. Using a technique of differential hybridization able to identify highly repetitive DNA, we isolated an sequenced a 9,456 bp fragment corresponding to part of the mitochondrial genome of C. lindemuthianum. This fragment showed a high A+T content (66%) and contained the genes cox1, cox3, nad6, rnl, rns, and 12 tRNA genes. The genes coding for proteins in this fragment showed preferential codon usage. Only the gene cox1 was interrupted by a group I intron containing an ORF for an endonuclase of the type LAGLIDADG, involved in the homing process. A synteny comparison of the genes present in this fragment and their phylogenetic analysis using three concatenated mitochondrial proteins of several fungal species grouped C. lindemuthianum with other fungi of the order Phylachoralles.