Stacking potato NLR genes activates a calcium-dependent protein kinase and confers broad-spectrum disease resistance to late blight

Late blight, caused by the oomycete plant pathogen Phytophthora infestans, is a destructive disease that leads to significant yield loss in potatoes and tomatoes. The introgression of disease resistance (R) genes, which encode nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs), into cultivated potatoes, is highly effective in controlling late blight. Here, we generated transgenic 2R and 3R potato lines by stacking R genes Rpi-blb2/Rpi-vnt1.1 and Rpi-vnt1.1/RB/R8, respectively, in the susceptible cv. Desiree background. The resulting 2R and 3R transgenic potato plants showed resistance to highly virulent P. infestans field isolates. We hypothesized that stacking R genes either resulted in up-regulation of a broader range of immune-related genes, or, more importantly, increase in the fold change of gene expression. To test our hypotheses, we performed transcriptome analysis and identified a subset of core immune-related genes that are induced in response to P. infestans in transgenic lines carrying single R genes versus lines carrying stacks of multiple R genes. In our analysis, stacking R genes resulted not only in the induction of a broader range of defense-associated genes but also a global increase in gene expression fold change, caused by the pathogen. We further demonstrated that the calcium-dependent protein kinase 16 (StCDPK16) gene significantly contributed to resistance to a virulent P. infestans strain, in the R gene background, in a kinase activity-dependent manner. Thus, our data suggest that stacking the R genes enhances late blight resistance through modulating the expression of a broader range of defense-related genes and highlights CDPK16 as a novel player in potato R gene-mediated resistance.