Virus-induced gene silencing in hairy roots to test root pathogen resistance
Term: 3 years, beginning in 2023
Status: Ongoing
Researcher(s): Chris Todd, University of Saskatchewan
SaskCanola Investment: $168,935
Total Project Cost: $397,870
Funding Partners: TBC
Objectives
Establish protocols for virus induced gene silencing (VIGS) in Plasmodiophora brassicae infected canola hairy roots.
Use VIGS to silence canola genes that are identified as interacting partners of clubroot effector proteins.
Evaluate Host Induced Gene Silencing (HIGS) as a functional genomics tool for clubroot research, and the potential for plants to down-regulate clubroot pathogen genes to interrupt the infection process.
Project Description
The proposed research we propose builds on an approach that promises to significantly enhance current knowledge of the mechanisms by which the clubroot pathogen causes disease and provides a new functional genomics tool to the research community. Beyond the immediate results, the advent of CRISPR-based precision engineering now makes integration of desired genetic changes into existing germplasm a very real opportunity. Having a mechanism to quickly survey potential candidates in advance should increase effectiveness of these approaches. This is early-phase research aimed at understanding the fundamental mechanisms involved in the establishment and proliferation of an economically important problem for canola producers. Application of this approach to clubroot, and to other soil borne pathogens, will accelerate the development of disease resistant cultivars and thereby reduce yield loss to disease.
Clubroot is a serious soil-borne disease of cruciferous crops worldwide. The causal agent of clubroot is P. brassicae, a pathogenic protist that exploits an intracellular, obligate biotrophic parasitism to colonize host root tissues. It is a difficult pathogen to work with, requiring culture on host plant tissue, and suffers from a lack of available functional genomics tools. It cannot be transformed, limiting researchers’ ability to express foreign genes to track it or to edit its genome. Lack of these tools make it both a difficult pathosystem to work in and slows research progress. Despite the economic importance of clubroot disease to the canola industry, we still know little about the molecular mechanisms involved in the establishment of P. brassicae infection and subsequent disease progression. Since P. brassicae is a protist, rather than a fungal or bacterial pathogen, it is reasonable to expect that it has evolved one or more infection strategies not shared with other plant pathogens. Thus far, management of clubroot disease in canola has been using clubroot-resistant germplasm, that to date has relied on single-gene resistance. In the long term, clubroot resistance based on multiple genes, operating through several different pathways and mechanisms, is likely to provide more effective and durable resistance compared to single gene-for-gene interactions that provide high selective pressure for any new pathotype variants to rapidly overcome single source resistance.
One of the difficulties of working with root pathogens is that it is more difficult to engage in direct manipulation of the host tissues in a “normal” soil environment that is conducive to pathogen infection. Additionally, techniques like Virus Induced Gene Silencing (VIGS) have been underutilized because it typically relies on the aboveground inoculation of silencing constructs and variation in delivery of these from leaves to roots. Using this system, we will first develop hairy root tissues containing these constructs and subsequently infect them. In the long term we wish to be able to use VIGS as a functional genomics tool to determine the role of both plant and pathogen genes that play roles in the establishment of clubroot infection. We aim to identify multiple different molecular mechanisms that may be able to be manipulated to provide clubroot resistant germplasm.
Virus Induced Gene Silencing (VIGS) is a technique to silence genes in plants. Similar to RNAi, it relies on the host plant’s response to invading viruses, triggering an RNA-mediated destruction of viral transcripts. By incorporating the host plant specific DNA sequences into an invading virus endogenous plant genes can be silenced together with viral RNAs. VIGS is a rapid method to evaluate phenotypic effects of gene silencing within a single plant generation. It is amenable to virtually any gene target and is particularly useful as a functional genomics tool in species that do not have a large mutant collection. Similarly, it allows silencing of genes that might not be viable as mutants, such as those that are embryonic lethal. The goal of this project is to use the VIGS to test gene function related to clubroot establishment and develop it to study the infection of canola roots by P. brassicae. If successful, in the longer term we expect that these gene targets could be widely adopted by the breeding community to accelerate the translation of genomic information into real-world advances in clubroot resistance. This strategy will involve the use of Agrobacterium-mediated transformation of canola roots with constructs containing clubroot pathogen genes or canola host genes in an Apple Latent Spherical Virus (ALSV) vector, which is an RNA virus known to work well in Brassicas. Using a similar strategy, the 3rd objective will be to attempt to do host-induced gene silencing (HIGS) of key clubroot pathogen genes.