Gene inactivation in the plant pathogen Glomerella cingulata: three strategies for the disruption of the pectin lyase gene pnlA

• Published in: Molecular & general genetics Vol. 246; no. 2; p. 196
• Main Authors: Bowen, J.K, Templeton, M.D, Sharrock, K.R, Crowhurst, R.N, Rikkerink, E.H.A. (New Zealand Ltd., Auckland (New Zealand). Horticulture and Food Research Inst.. Molecular Genetics Group)
• Format: Journal Article
• Language: English
• Published: Germany 20.01.1995
• Subjects: Ascomycota - genetics; Ascomycota - pathogenicity; Base Sequence; Capsicum - microbiology; GENE; Gene disruption; GENES; Genes, Plant - genetics; Genetic Vectors; GLOMERELLA CINGULATA; Isoelectric Focusing; LIASAS; LYASE; Molecular Sequence Data; Pathogenicity; PATOGENICIDAD; Pectin lyase; PECTINAS; PECTINE; Pectins - metabolism; Pflanzenzuechtung; Plant Diseases - etiology; Plants, Medicinal; Polysaccharide-Lyases - genetics; POUVOIR PATHOGENE; SECUENCIA NUCLEOTIDICA; SEQUENCE NUCLEOTIDIQUE; Transformation, Genetic
• ISSN: 0026-8925; 1432-1874

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnlA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA-); both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried a 5'and 3'truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA(-) transformants.