A proteomics-based approach towards identifying host and pathogen proteins critical to clubroot establishment in canola

Project Summary

Background

Clubroot is a devastating, soil borne disease of cruciferous plants that poses a serious threat to the productivity and economic stability of the Canadian canola industry. The disease is caused by the biotrophic protist Plasmodiophora brassicae, which after infection of a host plant, exhibits an obligate intracellular life cycle in colonizing host root tissues. Currently, very little is known about the virulence mechanism(s) of P. brassicae, particularly at the stage of primary infection and in the transition to secondary infection that leads to clubroot establishment and the subsequent development of root galls. The proposed research aims to identify key molecular events that facilitate the establishment and development of clubroot disease.

Objectives

Objective 1a – Identification of Plasmodiophora brassicae effectors using a BioID-based method in Arabidopsis.
Objective 1b – Screen of putative Plasmodiophora brassicae effectors in transgenic Arabidopsis.
Objective 2a – Targeted analysis of root hair cell (RHC) and root cortical cell (RCC) proteomes of clubroot resistant and susceptible canola.
Objective 2b – Assessment of differentially expressed canola proteins for association with resistance/susceptibility to clubroot.

Project benefit

Knowledge gained from using the Arabidopsis-Plasmodiophora pathosystem model is integral to informing our understanding of host and pathogen biology during disease progression. Functional characterization of P. brassicae effector proteins and their interacting plant partners from specific cell types and subcellular compartments will tell us exactly how the pathogen uses these virulence factors to promote disease. With this knowledge, we open up the possibility of CRISPR-based precision engineering of canola to alter host ‘susceptibility genes’ and convert them into clubroot resistance genes. Effectors may also be used as tools to accelerate the identification of resistance genes in breeding programs (in a process referred to as ‘effectoromics’ (1)), and these proteins have been more recently exploited to identify immune receptors that act to perceive pathogens (2).
In the Brassicaceae-P. brassicae pathosystem, several resistance genes (R-genes) have been identified and cloned (eg., (3)). However, to date, no corresponding P. brassicae avirulence genes (Avr-genes)
have been identified, nor do we understand the molecular basis for resistance. Using a BioID-based approach in clubroot-resistant and clubroot-susceptible canola cultivars, we will identify key differences in cell- and/or organelle-specific proteomes expressed at even the earliest stages of infection. Once identified, these sequences may be exploited to develop novel approaches towards decreasing infection and subsequent clubroot establishment and progression.
In summary, the proposed research will deliver knowledge and tools to improve utilization of existing clubroot resistant cultivars and to accelerate the discovery of new clubroot resistance genes, with the anticipation of exploring broad-spectrum and durable clubroot resistance that will be highly beneficial to breeders and growers of canola and other Brassica crops.

Literature cited

1. D. I. Kim et al., Proc. Natl. Acad. Sci. U. S. A. (2014), doi:10.1073/pnas.1406459111.
2. K. J. Roux, D. I. Kim, B. Burke, D. G. May, Curr. Protoc. protein Sci. (2013), doi:10.1002/cpps.51.
3. M. G. Miltenburg, thesis (2019).