Clonagem e caracterização dos genes que codificam endo-xilogalacturonana hidrolase, o fator de transcrição PacC e a proteína PalA em Crinipellis perniciosa, agente causal da vassoura-de-bruxa em Theobroma cacao

• Autor(a) principal: Silva, Gilvan Ferreira da
• Data de Publicação: 2007
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1554
• topic: PacC; Crinipellis perniciosa; Vassoura-de-bruxa; Theobroma cacao; PacC; Crinipellis perniciosa; Theobroma cacao; CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS

Crinipellis perniciosa is the causal agent of witches broom disease in cocoa plants as well as in other species of Theobroma, with important economic impact in both cocoa and cupuaçu crops. We report the isolation and characterization of the genes encoding endo-xylogalacturonan hydrolase (Xgh), PalA and PacC. The xghCp gene contains a 1,251 base pairs coding region interrupted by four putative introns. The deduced protein displays 417 amino acid residues and is coded by a single-copy gene (xghCp). Gene transcription has been analyzed by semi-quantitative RT-PCR, and the effects of either pH or the carbon sources glucose and citric pectin have been evaluated. Gene transcription was not repressed by 1% glucose. In the presence of pectin, xghC transcripts were detected from pH 4,0 to pH 8,0. Phylogenetic analysis of the deduced protein, XghCp, grouped it with endo- xylogalacturonan hydrolase of both Aspergillus tubingensis and Aspergillus niger, as well as with two Xgh similar enzymes of Aspergillus fumigatus. Multiple alignment revealed that XghCp presents a GIK sequence (Gly-lle-Lys), common to all endoxylogalacturonan known hydrolases, in the region corresponding to the polygalacturonase substrate binding site. This is the first report of an endoxylogalacturonan hydrolase in a basidiomycete. Cloning and characterization of palA and pacC, both related to signal transduction in response to pH in C. perniciosa, were also achieved. palACp transcription was not regulated by pH, and the deduced protein showed a conserved BRO1 domain. The phylogenetic analysis showed it to be a widely conserved protein, from fungi to human beings. A total of 4,347 bp corresponding to pacCCp, which displayed an ORF containing 2,472 bp interrupted by 8 putative introns, has been sequenced. Three possible sites of PacC cis-elements (5 GCCAG3 ) have been localized in the promoter region, suggesting a self-induction system for this gene under alkaline pH. Transcripts of pacCCp were detected by RT-PCR at pH 6.8 and pH 8.0 at 8, 18, and 32 hours after induction. At pH 4.0, basal transcription was observed from 8 to 32 hours. The deduced sequence exhibited 824 amino acid residues with zinc fingers domains highly conserved in yeasts and filamentous fungi. YPXL/l protein-protein recognition motifs, which interact with PalA, were localized in the C-terminal region of PacCCp between amino acid residues 637 and 742. pacC and palA identification suggests that C. perniciosa presents a signaling cascade in response to pH, which would allow PacCCp transcription factor to act as a regulator of environmental pH controlled virulence factors