GENE INACTIVATION IN THE PLANT PATHOGEN GLOMERELLA-CINGULATA - 3 STRATEGIES FOR THE DISRUPTION OF THE PECTIN LYASE GENE PNLA

The feasibility of performing routine transformation-mediated mutagene sis in Glomerella cingulata was analysed by adopting three one-step ge ne disruption strategies targeted at the pectin lyase gene pnlA. The e fficiencies of disruption following transformation with gene replaceme nt- or gene truncation-disruption vectors were compared. To effect rep lacement-disruption, G. cingulata was transformed with a vector carryi ng DNA from the pnlA locus in which the majority of the coding sequenc e had been replaced by the gene for hygromycin B resistance. Two of th e 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 trunc ation-disruption vectors was also assessed. Both vectors carried a 5'a nd 3'truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the se lectable marker differed in the two vectors. In one vector the homolog ous G. cingulata gpdA promoter controlled hygromycin B phosphotransfer ase expression (homologous truncation vector), whereas in the second v ector promoter elements were from the Aspergillus nidulans gpdA gene ( heterologous truncation vector). Following transformation with the hom ologous truncation vector, nine transformants were analysed by Souther n 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 w as not detected by Northern hybridisation in pnlA(-) transformants. pn lA(-) transformants failed to produce a PNLA protein with a pI identic al to one normally detected in wild-type isolates by silver and activi ty staining of isoelectric focussing gels. Pathogenesis on Capsicum an d apple was unaffected by disruption of the pnlA gene, indicating that the corresponding gene product, PNLA, is not essential for pathogenic ity. Gene disruption is a feasible method for selectively mutating def ined loci in G. cingulata for functional analysis of the corresponding gene products.