Use of Rna-based technologies for targeted gene silencing in cereals
Gene silencing techniques are essential to study gene function and produce crops with desired agronomic traits. Over the past decade, RNA interference (RNAi)-based methods involving experimental modulation of gene expression at the post-transcriptional level have only allowed partial gene silencing. Recently, new biotechnological tools, in ... particular CRISPR/Cas-based technologies, have become available for precise gene editing. The RNA-directed Cas9 nuclease introduces heritable precise insertions and deletions into the eukaryotic genome that can result both in altered gene function or complete disruption of the codon reading frame. In this work, an in-depth analysis of the technical aspects and applications of CRISPR/Cas9 and RNAi technologies is performed on the cereal model plant barley (Hordeum vulgare). In addition to further evidence for the potential application of RNAi and Cas9-mediated gene silencing, the work also uncovered new roles of Non-expressor of PR1 (NPR1) and the Microrchidia (MORC) protein family in the interaction of barley with microbial pathogens. Specifically, CRISPR/Cas9 was established for precise gene editing in barley and applied to two members of the epigenetically active MORC family. Whereas, the RNAi tool was used to generate barley plants with partial immunodeficiency driven by NPR1 knockdown. After Agrobacterium-mediated transformation, the MORC and NPR1 mutants were functionally characterized and their effects were investigated in plant-microbe interaction and modulation of plant fitness. In CRISPR/Cas9-mediated MORC knockout mutants, we elucidated role of different MORC family members in regulating plant immunity to a broad range of plant pathogens and their role in genome stability. In RNAi-silenced NPR1 plants, its function was investigated during the establishment of the mutualistic symbiosis between barley and the beneficial Alphaproteobacterium Rhizobium radiobacter F4 and in induced systemic resistance (ISR). The results presented here suggest that MORC proteins and NPR1 are involved in modulating disease resistance and plant fitness. This reveals potential gene candidates that may contribute to the development of new breeding strategies for higher-yielding and more resistant barley varieties.