The role of Vfa1, Vfa2 and Vfa4 genes in resistance to venturia inaequalis in transgenic 'McIntosh' apple plants

Apple scab caused by the ascomycete Venturia inaequalis is the most important disease of the cultivated apple (Malus X domestica Borkh.). The gene for resistance to V. inaequalis most frequently used in apple breeding is Vf, derived from Malus floribunda 821. A cluster of four resistance gene paralogs (Vfa1, Vfa2, Vfa3, and Vfa4) encoding proteins with leucine-rich repeats and transmembrane domains was isolated and cloned from the scab-resistant apple cultivar 'GoldRush'. Vfa3 was determined to be truncated, and was not used in this study. Intact genomic candidate genes (Vfa1, Vfa2, and Vfa4) were separately cloned into the binary vector pCAMBIA2301 to include the intact ORF, at least 2 kb of the native promoter of the gene, and the 3'-UTR region. Each of the three constructs was separately introduced into the scab-susceptible apple cultivar 'McIntosh' via Agrobacterium-mediated transformation. Transgenic lines grown in a growth chamber were subjected to scab inoculation using a mixture of five races (1 to 5) of V. inaequalis. Disease symptoms were assessed for individual plants from each of the transgenic lines for quality of resistance reactions and amount of sporulation. Transgenic lines expressing the Vfa4 gene were as, or more susceptible, than the control (non-transgenic 'McIntosh'). In contrast, Vfa1 and Vfa2 increased resistance to V. inaequalis in transgenic lines when compared to the control. Fewer leaves were infected, and amount of sporulation was less in Vfa1 and Vfa2 plants. Microscopic studies supported the macroscopic evidence of increased resistance in Vfa1 and Vfa2 plants. Although inoculations must be repeated, the results obtained so far clearly indicate that Vfa1 and Vfa2 are involved in apple scab resistance. Determination of levels of Vfa transgene expression are in progress using RT-PCR and western blot analysis on leaves inoculated or not inoculated with V. inaequalis.