Jk. Bowen et al., GENE INACTIVATION IN THE PLANT PATHOGEN GLOMERELLA-CINGULATA - 3 STRATEGIES FOR THE DISRUPTION OF THE PECTIN LYASE GENE PNLA, MGG. Molecular & general genetics, 246(2), 1995, pp. 196-205
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e had been replaced by the gene for hygromycin B resistance.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.