Modulation of expression of the apple scab resistance gene HcrVf2 in classically bred and genetically engineered scab-resistant apple cultivars
Final Report Abstract
Cisgenesis is more and more used to add a single trait such as apple scab resistance or fire blight resistance. In transgenics a point of discussion and controversy is the expression level of the transgene and possible negative side effects of “high” expression. In cisgenics this controversy can find true answers, as expression in the GM-plants can be compared to classical bred plants with the same gene. Therefore the aim of this project was to quantify HcrVf2expression levels in classically bred scab Vf-resistant genotypes and in various GM-resistant genotypesunder varying outer conditions. I wanted to understand if differences are due to different constitutive HcrVf2 expression or to modulation differences and if they are finally related to the phenotype. It is relevant to understand how this resistance gene is transcribed and modulated during infection in natural cultivars in order to implement this knowledge for developing further cisgenic lines and may serve as model for any cisgenic GM, additionally it will spark discussion of what is a desired/acceptable level of expression. Modulation of expression was analyzed as a function of time and stress, using quantitative real-time PCR.Validation of reference genes revealed that β-actin, RNA polymerase II, and UBC(encoding ubiquitin-conjugating enzyme) are most suitable. These reference genes should be the choice when performing qPCR experiments on Malus × domestica. In the present project nine apple genotypes were tested under several biotic and abiotic stresses and it could be demonstrated that inoculation with the apple scab fungus Venturiainaequalis does not lead to significantly higher HcrVf2 expression, neither in classically bred cultivars nor in trans- or cisgenic 'Gala' lines. It is the first time, that HcrVf2 expression is investigated in this extend and under diverse conditions. The results give a deeper insight into the plant–pathogen recognition system between Malus × domestica andV.inaequalis. Comparable gene expression studies of the second scab resistance gene Vr2 have been done. These results will be compared to the HcrVf2expression data.
