Objective
1. Characterize the virulence of 18 A1/D1 isolates from AB, SK, and MB in a greenhouse experiment.
2. Develop Laser Capture Microdissection (LCM) as a cost-effective tool to profile V. longisporum gene expression across different stages of infection (7, 14, 21, 28 days post-inoculation).
3. Use LCM and single cell RNA sequencing to profile gene expression of virulent and avirulent A1/D1 isolates during root and stem infection (ie early and late infection stages).
Project Description
Verticillium longisporum is a collection of three diploid lineages (A1/D1, A1/D2, and A1/D3) derived from three distinct hybridizations between different parental haploid Verticillium species (A1 with D1, D2, or D3). All highly virulent isolates of V. longisporum found on Brassica napus across Canada and Europe belong to the A1/D1 lineage, though not all A1/D1 isolates exhibit high virulence. In response to invading phytopathogens plants have a two-layered immune system that restricts the movement of detected microorganisms. Through the secretion of diverse proteins called effectors which disrupt or disarm plant defense responses, many phytopathogens can suppress or evade the plant immune system. Effectors function in many ways, including inhibiting host enzymes, modulating plant immune responses, and targeting host gene-silencing mechanisms. For example, many fungal phytopathogens deploy LysM effectors, which bind to derivatives of fungal cell walls with high affinity, effectively sequestering them from detection by the plant immune system. Fungal phytopathogens secrete hundreds of small proteins, believed to be effectors, during the process of host colonization. The specific set of effectors produced by a phytopathogen plays a key role in determining its host specificity and significantly influences the success of the infection. For example, in the Verticillium dahliae strain VdLs17, the loss of a single LysM effector Vd2LysM, which is highly expressed during plant infection, leads to a drastic reduction of virulence on tomato but no other host plant species. During the early stages of infection, V. longisporum enters canola plants through lateral roots and root hairs. At this point, it functions as a biotrophic pathogen, drawing nutrients from living plant cells while evading or suppressing the host’s immune responses. As the disease progresses, V. longisporum spreads throughout the mature plant, including roots, stems, and leaves, and shifts to a necrotrophic lifestyle, extracting nutrients from dead or dying tissue. This transition is marked by increased plant senescence and the formation of microsclerotia. Thus, the V. longisporum genes expressed during successful infection will likely vary between early and late infection stages. Genetic analysis of members of the virulent A1/D1 lineage from Canada and Europe indicate that isolates are closely related despite showing large differences in pathogenicity on canola. This presents a unique opportunity to uncover the genetic and molecular determinants of pathogenicity among A1/D1 isolates (i.e. effector repertoire), which exhibit different levels of virulence despite being very closely related. Characterizing virulence-associated effectors is essential for both understanding how pathogens cause disease and supporting crop improvement strategies. In plant breeding, these effectors can be used not only to identify resistance genes in new cultivars but also to pinpoint susceptibility loci in vulnerable varieties. Additionally, tracking these key effectors helps monitor the emergence and spread of virulence across pathogen populations. We propose to use laser capture microdissection (LCM) to identify effector genes uniquely expressed by virulent V.
longisporum isolates during different infection stages. Current state-of-the-art techniques such as single cell RNA-sequencing, whereby RNA is isolated and profiled from single cells within a sample, are still limited by interference of plant cells and the inability to assign gene expression profiles of individual pathogen cells to different structures found within infected tissues. Furthermore, single cell RNA sequencing is cost prohibitive and only feasible with pliable tissues, limiting its use for many applications LCM overcomes all these limitations and represents a powerful but underutilized tool in plant pathology because it can isolate pathogen cells of interest for analysis of expressed genes or proteins. This project is complementary to ongoing work performed by the involved research team. Dr. Chawla is conducting comparative genomic analyses on a diverse set of V. longisporum isolates collected from across the Prairies. This work includes assessing their pathogenicity, as well as characterizing their genetic diversity, population structure, and sequence-level variation. The expression data collected here of virulent and avirulent V. longisporum isolates will
complement the comparative genomic data by refining the list of genes involved in virulence. Drs. Hwang and Strelkov are investigating yield loss, disease development, evaluating canola genotypes for resistance to verticillium stripe and determining the interaction between verticillium stripe and blackleg. This work has also generated a large collection of V. longisporum isolates from across the Prairies and developed improved inoculation techniques. The proposed work will leverage these resources, allowing us to discover the molecular determinants of virulence in V. longisporum.
