Candidate genes for disease resistance in Petunia hybrida
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Susceptibility genes are plant genes targeted by pathogens to establish infection. To develop genetic resistance to pathogens in Petunia hybrida, the genes for mildew locus O (PhMlo1) and eukaryotic translation initiation factor 4E (PheIF4E) were identified and characterized. The genomic sequence of petunia Mlo and eIF4E was determined using petunia EST sequences with homology to tomato SlMlo1 and SleIF4E. Two approaches were taken to examine whether eliminating PhMlo1 expression could confer powdery mildew resistance. First, an EMS-mutagenized population of P. hybrida ‘Mitchell’ was developed after optimizing EMS exposure levels. High-resolution melting (HRM) analysis was used to screen DNA from M2 plants for PhMlo1 variation. Two SNPs were identified that resulted in the missense mutations S130L and G176E. The G176E substitution was predicted by Provean software analysis to be deleterious to PhMlo1 function. The second approach involved the reduction of PhMlo1 expression through RNA interference (RNAi). A PhMlo1 RNAi construct was developed and introduced into petunia by Agrobacterium-mediated transformation. We examined whether resistance to powdery mildew (Podosphaera xanthii) could be obtained in P. hybrida by knocking down the susceptibility gene PhMlo1. Ten independently-transformed shoots were rooted and the regenerated T0 plants were propagated vegetatively. Real time-PCR analysis found that four PhMlo1-RNAi lines had reduced levels of PhMlo1 expression compared to transgenic controls. In two experiments with whole plants, leaves of PhMlo1-RNAi lines and controls were inoculated with P. xanthii conidia. The time course of infection and the infected leaf area were examined over a 16-day incubation in a growth chamber. RNAi lines showed delayed infection and a reduced area of infection compared to transgenic controls. The transgenic lines were self-pollinated and T1 progeny were produced and examined for powdery mildew resistance and pleiotropic effects on growth. Powdery mildew resistance was observed in the progeny of transgenic petunia, but the knockdown of Mlo had a stronger effect on growth than seen in other plant species.