REPOSITORY > RESULTS

Abstract

The increasing population of the world and its food demands require focused efforts for the improvement of crop plants to ensure sustainable food production. Small RNAs (sRNAs) are emerging as promising targets for genetic engineering to enhance crop yields by improving plant´s performance under various environmental stress conditions, including more efficient response against pathogen attack. Potato virus Y (PVY) is one of the most important viruses affecting potato that causes substantial economic losses in worldwide potato production. We aimed at unraveling the roles of sRNAs in the complex immune signaling network controlling the establishment of tolerant (cv. Désirée and cv. Pentland Squire) and hypersensitive response (HR) of potato (cv. Rywal) to the virus. Employing high-throughput sequencing technology, we discovered more than 200 already annotated potato microRNAs (miRNAs), 199 novel potato miRNAs and more than 9000 potato phased interfering siRNAs (phasiRNAs). Expression patterns of these identified small RNAs (sRNAs) were characterized and compared between PVY-infected and healthy potato plants. In addition, this information was linked to expression profiles of their target transcripts to link sRNA level responses to physiological processes. Besides the already described regulation of R gene transcripts, we have discovered an interesting novel sRNAs-gibberellin regulatory circuit being activated in tolerant plants of cv. Désirée. Increased levels of potato miR167e and phasiRNA931 were reflected in decreased levels of transcripts involved in gibberellin (GA) biosynthesis. Moreover, decreased concentration of GA confirmed this regulation. We further showed that this regulation is salicylic acid (SA)-dependent as the miR167e and phasiRNA931 levels were diminished in SA depleted NahG-Désirée expressing severe disease symptoms. By comparing sRNA´s responses to PVY infection between cv. Désirée and cv. Pentland Squire, we identified six commonly responding sRNAs. One of these potato sRNAs, miR390a, was shown to represent an additional regulatory potential of plants to downregulate GA signaling in order to promote immune responses against PVY. In resistant cv. Rywal, the levels of several sRNAs targeting immune receptor transcripts declined from lesions to surrounding tissue in response to PVYN-Wi to modulate the immune responses. In addition, potato miR398c was shown to be important for ROS homeostasis during HR in cv. Rywal. We also optimized sRNA RT-qPCR protocol for high-quality sRNA expression studies from homogenized leaf material of potato, and showed that this protocol can be used as a validation tool for sRNA sequencing results. In addition, we also established a protocol for parallel extraction of both short sRNAs and long RNAs from low amounts of plant material (i.e. small tissue sections) as well as protocol for efficient DNA removal and RNA purification providing high-quality RNA/sRNAs suitable for RT-qPCR and sequencing of both sRNAs and mRNAs. Finally, we also optimized protocols for bioinformatics analysis of sRNA sequencing data using ShortStack tool. We showed that ShortStack pipeline allows faster, more stringent but reliable prediction for novel miRNA and MIR loci discovery.