Generation of miRNA160 and miR166 endogenous Target Mimicry (eTM) transgenic lines in potato and their assessment against Phytophthora interactions.

Abstract

One of the great challenges for researchers is to overcome the late blight disease of potato caused by the oomycete pathogen, Phytophthora infestans. Recent studies suggest involvement of several microRNAs during plant-pathogen interaction. microRNAs 160 (miR160) and miR166 are highly conserved miRNAs among different plant species that are known to play various developmental roles. Studies in our lab indicate involvement of miR160 and miR166 during potato – Phytophthora interaction. In order to further investigate the role of these miRNAs in plant immunity, we generated knockdown lines of miR160 and miR166 using endogenous target mimicry (eTM) approach. miR160, miR166 and their target genes were successfully detected in our model system Solanum tuberosum spp. Desiree. Using Agrobacterium-mediated transformation technique, we successfully generated endogenous target mimicry (eTM) lines for miR160 and miR166. Representative eTM lines of both these miRNAs have been confirmed by RT-PCR analysis. To assess the level of knockdown in these lines, real-time PCR based approach was attempted. Our results indicate successful reduction in levels of miR160 by ~ 66% in eTM160 clone-1 and by ~91% in eTM160 clone-2, whereas reduction of miR166 level was ~ 96% in eTM166 clone 1 and by ~91% in eTM166 clone 2. All the four clones were subsequently transferred to soil and were gradually hardened. These lines are presently grown in growth chamber conditions and are being subjected to Phytophthora infection to understand their role in immunity.