Transposon tagging is a powerful tool for identifying functional genes of phytopathogenic fungi. We used the transposon impala of Fusarium oxysporum to tag pathogenicity genes of Diaporthe helianthi, the agent of sunflower stem canker. An Agrobacterium tumefaciens-mediated transformation system was developed to obtain mutants to be screened for changes in pathogenicity. The impala transposon was inserted in the niaD gene encoding nitrate reductase of Aspergillus nidulans. Both the niaD and a hygromycin resistance cassette, based on the hph gene of E. coli were introduced in the T-DNA region of a binary vector to give plasmid pBI121. A. tumefaciens LBA 4404 was used as a helper strain to transform a stable highly pathogenic D. helianthi niaDmutant, derived from a French strain of D. helianthi. Stable transformants developed within 10-20 days of incubation on the selection medium. The presence of the hph gene, conferring resistance to hygromycin, was confirmed by PCR analysis. A phenotypic excision test will be used to test impala activity in the genome of D. helianthi. niaD:impala transformants will be inoculated on a medium containing nitrate as sole nitrogen source to select niaD+ revertants. The reversion to nitrate prototrophy will be considered diagnostic for impala excision. Avirulent revertants will then be used to isolate impala-tagged genes essential for pathogenicity.
Agrobacterium-mediated transformation of Diaporthe helianthi.
FinettiSialer MM;
2003
Abstract
Transposon tagging is a powerful tool for identifying functional genes of phytopathogenic fungi. We used the transposon impala of Fusarium oxysporum to tag pathogenicity genes of Diaporthe helianthi, the agent of sunflower stem canker. An Agrobacterium tumefaciens-mediated transformation system was developed to obtain mutants to be screened for changes in pathogenicity. The impala transposon was inserted in the niaD gene encoding nitrate reductase of Aspergillus nidulans. Both the niaD and a hygromycin resistance cassette, based on the hph gene of E. coli were introduced in the T-DNA region of a binary vector to give plasmid pBI121. A. tumefaciens LBA 4404 was used as a helper strain to transform a stable highly pathogenic D. helianthi niaDmutant, derived from a French strain of D. helianthi. Stable transformants developed within 10-20 days of incubation on the selection medium. The presence of the hph gene, conferring resistance to hygromycin, was confirmed by PCR analysis. A phenotypic excision test will be used to test impala activity in the genome of D. helianthi. niaD:impala transformants will be inoculated on a medium containing nitrate as sole nitrogen source to select niaD+ revertants. The reversion to nitrate prototrophy will be considered diagnostic for impala excision. Avirulent revertants will then be used to isolate impala-tagged genes essential for pathogenicity.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
