Isolamento, caracterização e regulação de genes que codificam pectato liase em Crinipellis perniciosa, agente etiológico da vassoura-de-bruxa do cacaueiro (Theobroma cacao)

Abstract

In this work, genes encoding the pectinolytic enzyme pectate lyase were isolated from the genome of the fungus Crinipellis perniciosa, etiological agent of the witches ́ broom disease of cocoa. Pectinolytic enzymes have been correlated with the pathogenicity of some phytopathogenic fungi. Preliminary database searches of the Genome Project “Witches ́ broom”, revealed the presence of sequences, in the C. perniciosa genome, with similarity to the pectate lyases of different fungi. Two sequences with similarity to the Aspergillus niger pectate lyase A and the Fusarium solani f. sp. pisi pectate lyase D, were used for the construction of specific oligonucleotides. The DNA fragments amplified were used as homologous probe in the copy number determination of these genes in the C. perniciosa genome and in the isolation of genes encoding pectate lyase from C. perniciosa genomic library constructed in the λEMBL3 bacteriophages. Hybridization analysis with C. perniciosa total DNA cleaved by the enzymes BamHI, EcoRI e XbaI, revealed the presence of at least 2 copies of the gene encoding pectate lyase with similarity to the A. niger pectato liase A and one copy of the gene encoding pectate lyase with similarity to the F. solani f. sp. pisi pectate lyase D. Based on this initial characterization three genes, pec1A, pec1B and pec2, were isolated from the C. perniciosa genomic library and characterized. The pec1A and pec1B genes have, respectively, open read frames of 1141 and 1346 pb and are both interrupted by 7 introns. The pec2 gene has one open read frame of 936 pb and is interrupted by only 3 introns. The complete promoter analysis was not possible; however, some cis-elements involved in the regulation of genes could be identified in pec1B and pec2 genes. The proteins encoding by pec1A, pec1B and pec2 have respectively, 244, 307 and 253 amino acids. The proteins PEL1A and PEL1B presented 60% identities among themselves, but lower identities, 4,3 and 4,6%, respectively, with PEC2. When compared with other fungi pectate lyases, PEC1A and PEC1B presented high identities with A. niger and A. fumigatus pectate lyase A. The PEC2 enzyme presented the highest identities with proteins from a new pectate lyase group, represented by the multigenic family encoding pectate lyases in F. solani. f. sp. pisi. Multiple alignments between the C. perniciosa and the different phytopathogenics fungi pectate lyases, as well as the phylogenetics analyses, suggest that PEC2 really belongs to a new class of pectate lyase, represented by the F. solani f. sp. pisi pectate liases. The pec1A and pec1B genes were expressed in all carbon resources evaluated, as in pectin, pectin and glucose, glucose and cocoa pulp, as well as in pectin in pH 4,0, 6,8 e 8,0. However, differences in the level of expression of these genes were detected, as the highest expression of the pec1A and pec1B genes in pH values equal to 8,0. The pec2 gene was not expressed in any of the evaluated conditions. This is the first report of isolation and characterization of genes encoding pectate lyase in basidiomycets.