The CRISPR/Cas-based techniques represent a major breakthrough in targeted genome editing of living organisms. In the case of wheat, the technology has already enabled the specific reconstruction of previously characterized mutations, resulting in economic benefits. In terms of implementation, the most efficient method is to integrate the gene encoding the Cas enzyme responsible for DNA cleavage and the guideRNA's gene that designates the target sequence, into the plant genome and then to remove it after mutagenesis. However, as a result of transgenic integration, small and untraceable pieces of DNA can be incorporated into chromosomes, which are difficult to identify. Therefore, various DNA-free transient techniques have been developed, such as the biolistic delivery of the Cas-guideRNA ribonucleoprotein complex into tissue cultures, but its mutation frequency is very low. Our aim is to develop a more efficient genome editing method for wheat that does not require transformation of the target genome. To achieve this, barley is transformed with a wheat genome-specific CRISPR system and then, via sexual crossing, this barley genome is introduced into the wheat egg cell. As a result, the wheat genome can be edited throughout the life cycle of the hybrid plant. Thereafter, the wheat genome is once again introduced by backcrossing with the original parent, and after self-fertilization coupled with paternal genome elimination, edited wheat progeny without barley chromosomes is selected. Our preliminary results demonstrate that this system is capable of generating specific gene mutations in the wheat genome very efficiently.

Creating highly efficient resistance against wheat dwarf virus in barley by employing CRISPR/Cas9 system

Genome-wide identification of RNA silencing-related genes and their expressional analysis in response to heat stress in barley (Hordeum vulgare l.)

RNA interference (RNAi) is indispensable regulatory mechanism present in almost all eukaryotes controlling developmental processes, stress responses and genome integrity. The ARGONAUTE1 (AGO1) is the main effector component of the RNA-induced silencing complex (RISC) predominantly responsible for micro RNA (miRNA) mediated repression of target mRNAs. In our previous work we revealed the presence of high level of free miRNA species in the cytoplasm, unbound to AGO1. We also demonstrated that distinct miRNA precursors are responsible for the altered AGO1 loading of various miRNAs. In the proposed work we would like to elucidate the fine molecular mechanism of this newly identified regulatory system which determines the biological activity of miRNAs by sorting only a subset of the produced miRNA pools into the effector complexes. MiR168, targeting AGO1 mRNA, typically accumulates in AGO1 unbound, free, forms in the cytoplasm. In our experiments, we will use modified precursor structures to map structural feature regulating the loading efficiency of miR168 into AGO1. We will also investigate the biological role various miRNA biogenesis proteins in regulating the accumulation/production of protein unbound miRNA species. We also intend to translate our results to economically important plants to extend our understanding to the practical utilization of the gained data. As a result of our research we hope to uncover the action of a pivotal regulatory mechanism in the miRNA pathway which can help to improve the artificial miRNA-based research tools and provide data about the action of miRNAs determining important traits of crop plants.

Controlled RISC loading efficiency of miR168 defined by miRNA duplex structure adjusts ARGONAUTE1 homeostasis

AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading