Molecular Cloning and Functional Characterization of Brassica UBC13 Genes

Abstract

Lysine63 (K)-linked polyubiquitination of target proteins is a fundamentally different process from conventional K48-linked polyubiquitination that targets proteins for degradation via the 26S proteosome. Lys63-linked polyubiquitination regulates numerous cellular processes. The unique feature of Ubc13 compared to other ubiquitin-conjugating enzymes (Ubcs) is its ability to form a stable complex with a Ubc-E2 variant (Uev), which promotes the formation of Lys63-linked polyubiquitination. Ubc13 functions in DNA damage tolerance in budding yeast and is involved in several pathways in mammalian cells. Arabidopsis contains two UBC13 genes and four UEV1 genes that are involved in various developmental processes and stress responses including DNA damage response, root development and immunity. Recent studies imply that AtUbc13s contribute to plant susceptibility against soil-borne pathogen such as clubroot, a major disease in Brassica napus. However, there is no published information regarding characterization of B. napus Ubc13s (BnUbc13s). This project aims to understand functions of Ubc13 and Ubc13-Uev1 complexes in canola. As canola is a polyploid and often contains many homologous genes, this study aims to provide guidelines to selectively target a subset of homologous genes by gene editing to protect from clubroot disease. Twelve BnUBC13 genes were identified through genomic data analysis, eight of which encode proteins different from AtUbc13s were cloned and characterized. All eight BnUbc13s were able to physically interact with AtUev1 to form stable complexes. Furthermore, BnUBC13 genes functionally complemented the yeast ubc13 null mutant defects, suggesting that BnUBC13s can replace yeast UBC13 in DNA damage tolerance. Furthermore, a CRIPSR/Cas9 construct was designed to simultaneously target five BnUBC13 genes and was used to transform B. napus cv. Westar (DH12075). Twenty-eight out of thirty regenerated lines were found to contain homozygous or heterozygous mutations in 5 targeted BnUBC13 genes, validating our genomic editing approach in canola. In addition, BnUBC13 transcript levels in resistant and susceptible canola before and after clubroot infection were analyzed based on the in-house RNA-seq data and were found to not fluctuate drastically. This study provides convincing data to support notions that B. napus Ubc13s promotes Ly63-linked polyubiquitination, that BnUbc13s are involved in error-free DNA damage tolerance and that BnUBC13s are housekeeping genes.