Objective
The overall objective of this project is to develop canola lines with targeted modifications in gene expression of BnCLIP (2 lipase genes) and BnABC1K6 (4 kinase gene copies) (these 6 genes are part of a novel Jasmonic Acid disease response pathway recently discovered by Y. Xu), evaluate their resistance to Sclerotinia infection, and identify molecular markers associated with resistance (ie other genes affected by overexpression of BnCLIP-3 and -4 and knocking out the 4 copies of BnABC1K6 kinase genes). The generated canola lines identified molecular markers, and mechanistic insights can then be applied in breeding programs and marker-assisted selection to enhance SSR resistance. The long-term goal is to diversify canola cultivars by introducing Sclerotinia resistance genes and collaborating with breeders and industry to implement molecular markers for these traits, ultimately improving disease resistance across canola crops.
Project Description
Canola productivity is increasingly threatened by sclerotinia stem rot (SSR) resulting in yield losses of over 50% in severely infected fields. In Western Canada, average SSR disease incidence typically ranges from 14-30% and can cause substantial yield losses. For example, in 2016, more than 90% of surveyed fields were affected by SSR, resulting in 7-15% yield loss. Beyond yield reduction, SSR also reduces seed quality, leading to downgrading at delivery points, reduced export potential, and substantial economic losses for growers and the canola industry. Its long-lived sclerotia can persist in soil for years, undermining crop rotation and posing recurring threats to canola and other crops in prairie cropping systems. This persistence makes SSR management particularly challenging for Canadian growers, where crop rotations frequently include susceptible pulse and oilseed crops.
The control of these necrotrophic pathogens is challenging as management strategies rely largely on chemical control while cultural practices such as crop rotation have been used with limited success. These approaches are further constrained by environmental variability and potential negative impacts on soil health, which can ultimately reduce canola seed production and threaten long-term food security. Consequently, genetic resistance is regarded as the most sustainable and environmentally friendly strategy for disease management. However, no qualitative resistance to S. sclerotiorum has been identified in canola. While several sources of quantitative resistance, primarily quantitative trait loci (QTLs), have been reported, none provides complete protection, and resistance levels vary depending on the environment and developmental stage. Therefore, identifying novel genes and molecular markers underlying quantitative resistance and developing more robust resistant canola varieties to widely spread SSR disease is a critical need. Jasmonic acid (JA) is a key plant hormone that regulates defense responses to necrotrophic pathogens like S. sclerotiorum. When plants detect such infections, JA biosynthesis is rapidly triggered, leading to the accumulation of signaling molecules such as methyl jasmonate. These compounds activate a cascade of gene expression that strengthens the plant defenses. The first step in JA biosynthesis involves the enzymatic release of α-linolenic acid from chloroplast membrane lipids, a process carried out by lipases, particularly the phospholipase A1 (PLA1) family. Among them, DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1) and a group of DAD1-like lipases (DALLs) have been implicated in wound-induced JA production. Downregulating these lipases reduces disease resistance in Arabidopsis, while enhancing JA signaling through lipase overexpression has been shown to improve resistance against pathogen infection in Arabidopsis and rice, highlighting their biotechnological potential to enhance disease resistance in Brassica napus, a close relative species to Arabidopsis. Despite their importance, the molecular mechanisms that regulate lipase activity in JA biosynthesis remain poorly understood. Recent work in the Xu lab uncovered a novel lipase-kinase regulatory network involved in JA biosynthesis. Using advanced proximity labeling and proteomics approaches, we identified a list of protein interactors of a canola chloroplast DALL lipase (BnCLIP1), including a poorly characterized kinase (ABC1K6) previously implicated in plastoglobule regulation. Notably, we observed that the subcellular localization of this kinase shifted from a diffuse distribution in chloroplasts to co-localizing with DALL lipases when co-expressed, suggesting a direct interaction and functional association. In silico analysis identified a few predicted phosphorylation sites for DALL lipases which are at the surface of enzyme and can be accessible by kinase. Sequence analysis suggests that the canola contains 4 copies of highly conserved BnCLIPs and 4 copies of highly conserved BnABC1K6, which also share very high sequence similarity with corresponding Arabidopsis DALL lipases and kinases. Interestingly, expression profiling revealed that BnCLIP lipases especially BnCLIP3 and 4 were upregulated in response to S. sclerotiorum infections in the resistant canola line compared to the susceptible line. In contrast, the BnABC1K6 kinases displayed an opposing expression trend (downregulated in response to S. sclerotiorum infections in the resistant canola line compared to the susceptible line), implying they may function antagonistically in pathogen responses. Together, our findings suggest a putative lipase-kinase regulatory complex for JA biosynthesis, presenting new targets for enhancing disease resistance in canola plants.
Building on this discovery, we aim to enhance canola resistance to S. sclerotiorum by creating targeted modifications in the lipase-kinase complex. Specifically, we will generate BnCLIP3 and BnCLIP4 overexpression lines, as well as a BnABC1K6-1, -2, -3, -4 quadruple CRISPR/Cas9 knockout lines. By analyzing transcriptional responses and lipid profiles of these modified lines following S. sclerotiorum infection, we will identify additional resistance-associated genes and lipid markers. These molecular markers will then be validated across a diverse set of canola genotypes to facilitate the development of genomic markers and selection of resistant lines, which can subsequently inform the development of breeding tools. The resulting canola lines, particularly the non-GM CRISPR knockouts, along with the validated markers, will support the creation of new canola varieties with enhanced disease resistance.
