Functional characterization of Rvi12_Cd5 against apple scab via transgenesis and cisgenesis approaches

The Rvi12 (Vb) region, originating from the Siberian crab apple Malus. baccata ‘Hansen’s baccata #2’ (HB2), confers apple scab resistance to the fungal pathogen Venturia inaequalis. Previously, gene prediction and in silico characterization identified a single putative candidate resistance gene, named as Rvi12_Cd5 at Rvi12 (Vb) locus. The constitutive expression of Rvi12_Cd5 in’ HB2’, together with its structural similarity to known resistance genes, makes it the most likely candidate for Rvi12 scab resistance in apple. In this study, the cloning and functional characterization of Rvi12_Cd5 was performed to confirm its functional role in Rvi12 based scab resistance. The gene was cloned with 35S promoter and inserted into the susceptible apple cultivar ‘Gala’ via agrobacterium-mediated transformation. Inoculated transgenic ‘Gala’ lines, overexpressing Rvi12_Cd5 were able to induce high to partial resistance against mixed inoculum of V. inaequalis with range of resistance symptoms (class 0-3b without class 1) strongly suggesting that the candidate gene Rvi12_Cd5 is the functional Rvi12 (Vb) gene. This is the third cloned gene for apple scab resistance to date. In an overview of Rvi12_Cd5 natural promoter sequence in-silico analysis revealed transcription factor binding sites (TFBSs) corresponding to six major plant defense related transcription factors such as AP2/ERF, bHLH, TGA/bZIP, MYB, NAC, and WRKY. To generate cisgenic lines, Rvi12_Cd5 under the control of its own promoter was transferred to scab susceptible apple cultivar ‘Gala’. A vector designed with three molecular mechanisms for cisgene expression, kanamycin resistance for selection of transgenic apple and T-DNA excision system by heat inducible site specific FLP/FRT recombinase system respectively, was used in Agrobacterium-mediated transformation. The candidate gene Rvi12_Cd5 was able to induce high to partial resistance against V. inaequalis under its own promoter. After transformation, all transformed lines were analyzed to assess copy number, integration site, mRNA expression level and resistance to apple scab. All of these lines induced low mRNA expression of Rvi12_Cd5 compared with the natural expression of ‘Hansen’s baccata # 2’ except for one cisgenic apple line. Even a low expression of Rvi12_Cd5 was sufficient to induce plant reaction and reduce fungal growth compared with the scab-susceptible ‘Gala’ except two transformed lines. The site specific FLP/FRT recombinase system was efficace to eliminate completely the T-DNA cassette in five independent apple cisgenic lines carrying a single insertion of Rvi12_Cd5. These lines need another layer of validation for T-DNA insertion sites and exogenous DNA removal by PCR and sequencing.