Effect of disruption of a cutinase gene (cutA) on virulence and tissue specificity of Fusarium solani f. sp. cucurbitae race 2 toward Cucurbita maxima and C. moschata.
A 3.9-kb genomic DNA fragment from the cucurbit pathogen Fusarium solani f. sp. cucurbitae race 2 was cloned. Sequence analysis revealed an open reading frame of 690 nucleotides interrupted by a single 51-bp intron. The nucleotide and predicted amino acid sequences showed 92 and 98% identity, respectively, to those of the cutA gene of the pea pathogen F. solani f. sp. pisi. A gene replacement vector was constructed and used to generate cutA- mutants that were detected with a polymerase chain reaction (PCR) assay. Seventy-one cutA- mutants were identified among the 416 transformants screened. Vector integration was assessed by Southern analysis in 23 of these mutants. PCR and Southern analysis data showed the level of homologous integration was 14%. Disruption of the cutA locus in mutants was confirmed by RNA gel blot hybridization. Neither virulence on Cucurbita maxima cv. Delica at any of six different inoculum concentrations, nor pathogenicity on intact fruit of four different species or cultivars of cucurbit or hypocotyl tissue of C. maxima cv. Crown, was found to be affected by disruption of the cutA gene.
Crowhurst
R N
RN
Molecular Genetics Group, Horticulture and Food Research Institute of New Zealand Ltd., Auckland. RCrowhurst@hort.cri.nz
Binnie
S J
SJ
Bowen
J K
JK
Hawthorne
B T
BT
Plummer
K M
KM
Rees-George
J
J
Rikkerink
E H
EH
Templeton
M D
MD
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002265:Carboxylic Ester Hydrolases / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004950:Esterases / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity*; D008969:Molecular Sequence Data; D010641:Phenotype; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014170:Transformation, Genetic; D014675:Vegetables / Q000382:microbiology*
D018076:DNA, Complementary; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C019952:cutinase