Molecular insights into the role of VRS5 in tillering and lateral spikelet development in barley

In barley, the transcription factor VRS5, regulates both tiller development and lateral spikelet development. Both of these processes are important yield related traits and their genetic connection makes it difficult to manipulate these traits independently. The main objective in this thesis was to provide molecular insights that can help untangle these traits, with the focus on VRS5. As a transcription factor, VRS5 can directly bind to DNA and regulate the expression of downstream targets. Understanding what these targets are, can help to better understand how VRS5 exactly functions and regulates these different processes. In Chapter 2, we identified these direct targets in barley by combining two techniques. Using DNA-affinity purification followed by sequencing (DAP-seq), we identified the genome-wide binding sites of VRS5. Next to that, we compared gene expression in vrs5 mutants to wild-type at four developmental stages using RNA sequencing. By combining the genes that are differentially expressed in vrs5 and the loci where VRS5 binds, we obtained a set of direct VRS5 targets that are thus bound by and regulated by VRS5. Next, we looked in more detail at VRS5 as a protein in Chapter 3. We found that in barley two different protein isoforms of VRS5 are formed; VRS5alpha and VRS5beta. We wanted to know if these isoforms function differently, and by CRISPR mutagenesis we made mutations causing only one of the protein isoforms to be formed. After investigating these lines, we found that it is possible to uncouple the tillering from the row-type function. While complete knock-out lines of vrs5 have both an increased number of tillers and six-rowed to intermediate spike phenotype, the vrs5alpha mutants only show increased tillering, but a two-rowed spike. We showed that these phenotypical differences are likely caused by differences in DNA binding of the isoforms in complex with another transcription factor, TB2. One of the direct targets of VRS5 identified in Chapter 2, VRS1 was already known as thus far the most downstream regulator of lateral spikelet development. Despite this role in such an agronomically important trait, little is known of the molecular function of VRS1. In Chapter 4, we identified the consensus DNA binding motif of VRS1, using DAP-seq. In combination with an already available RNA-seq dataset of a VRS1 allele, we also identified some potential direct downstream targets of VRS1. One of these targets was SOC1-like, of which we made CRISPR mutants. These mutants showed significantly smaller lateral spikelets compared to WT. Many of the experiments performed in this thesis would be impossible without having an efficient CRISPR based mutagenesis protocol. In Chapter 5, we showed how we greatly improved the editing efficiency of Cas9 in barley by combining several methods shown to improve editing efficiency. With this improved system we were able to generate multiple knock-out lines and even double knock-outs in primary transformants already. In conclusion, with the molecular insights provided in this thesis, I showed several potential ways to help untangle yield-related traits in barley, specifically via VRS5.