Understanding the clubroot disease at the single cell level
Term: 2 years
Status: Ongoing
Researcher(s): Edel Perez Lopez, Elisa Fantino, Soham Mukhopadhyay, Florent Sylvestre, University Laval; Bruno Guillotin, Centre national de la recherche scientifique (CNRS)
SaskOilseeds Investment: $129,375
Total Project Cost: $172,500
Funding Partners: ACPC
Objective
Study the clubroot disease at the cellular level in susceptible and resistant Arabidopsis ecotypes infected with a resistance-breaking P. brassicae isolate.
Study the clubroot disease at the cellular level in susceptible and resistant canola lines infected with a resistance-breaking P. brassicae isolate.
Project Description
Clubroot is a major disease that threatens Canadian canola industry. Developing strong resistance and understanding the clubroot pathogen are top priorities for CARP research, which will lead to increase productivity.
In the canola industry, using resistant varieties has been a key strategy to avoid the damage caused by the clubroot pathogen. However, over the years, this resistance has been quickly overcome by new isolates of Plasmodiophora brassicae, emphasizing the need to identify durable sources of resistance. Recently, two research groups identified the gene RPB1 or WTS as a strong candidate for developing clubroot-resistant cultivars, claiming that Arabidopsis thaliana ecotypes bearing this gene were resistant to the European pathotype P1+ and several Chinese field isolates. However, unpublished results from our lab show that most of these resistant Arabidopsis ecotypes are susceptible to Canadian P. brassicae isolates, such as Pb3A, used as a model isolate in our studies. Pb3A can also break down Mendel resistance in canola cultivars, a phenomenon observed widely in Canada. Additionally, we found new Arabidopsis ecotypes resistant to Canadian isolates of the clubroot pathogen, but after evaluating their full life cycle, we determined that what we observed was not true resistance but tolerance, as only a few plants survived until flowering.
Two questions arise from this:
1. What are the genetic and molecular mechanisms behind the ability of Canadian Plasmodiophora brassicae isolates to overcome previously identified resistance genes RPB1 in Arabidopsis or Mendel in canola?
2. What factors contribute to the observed tolerance rather than resistance in certain Arabidopsis thaliana ecotypes when exposed to Canadian isolates of Plasmodiophora brassicae?
Single-cell transcriptomics (scRNA-seq) represents a revolutionary approach in molecular biology, allowing for the resolution of gene expression at the individual cell level. Traditional RNA sequencing averages gene expression across thousands of cells, potentially masking critical cellular heterogeneity and specific responses to infection. This is crucial because the clubroot infection process is initiated in the root hair, and slowly the pathogen invades the cortex and the pericycle to establish a nutrient sink over a period of 20 days. Thus, the transcriptomic responses between different cell types within the root are spatio-temporally regulated, and traditional bulk RNA sequencing fails to dissect them. To make the matter more complex, plant roots are continuously exposed to microbial communities present in the rhizosphere. Root cells must distinguish between friends and foes, and recent studies have shown that different root zones respond differently to MAMP stimuli. Therefore, it is vital to understand how plant hosts respond to a protist pathogen, which also carries chitin on its spores, a well-known MAMP that triggers immunity, and how the amplitude of the defense response is regulated at various time points post-infection.
This project aims to answer the previously stated questions using scRNA-seq, pinpointing key regulatory genes and pathways involved in the defense against P. brassicae. Since the infected cells are full of dividing pathogen cells, we can also gauge the transcriptomic landscape of the pathogen, allowing us to shed light on the strategies employed by the pathogen to manipulate host cellular processes. This includes identifying groups of effector proteins secreted by P. brassicae at different stages of the lifecycle, as well as the host cellular machinery hijacked during infection, all at the single cell scale, never studied before because the technology was not available.