Genome wide functional analysis of Plasmodiophora brassicae effectors and the management of clubroot disease

Date: November 30, 2021
Term: 5 years
Status: Completed
Researchers: Peta Bonham-Smith, Christopher Todd and Yangdou Wei (University of Saskatchewan)
SaskCanola Investment: $106,088
Total Project Cost: $382,838
Funding Partners: ADF

Grower Benefits

• One of the first studies to characterize effector proteins with key roles in clubroot infection and disease progression.

• Successful identification of P. brassicae effector proteins and their functions in planta during disease progression.

• Identification of the host target proteins of these effectors providing an initial characterization of the molecular and cellular mechanisms underlying P. brassicae virulence and host resistance.

• Effectors and host target proteins characterized in this study will provide a valuable resource for the identification of new R-genes that will benefit both breeders and producers of canola and other crops in Canada.

Project Summary

Clubroot disease in canola and other brassicas is caused by the pathogen Plasmodiophora brassicae. Researchers at the University of Saskatchewan conducted a five-year study to identify and characterize effector proteins and their role in the establishment and progression of clubroot disease. Various genomic and molecular approaches were used for screening and identifying putative P. brassicae effectors, their proteins and function. Overall, this study is one of the first to characterize effector proteins with key roles in clubroot pathogenicity. Researchers have produced a substantial annotation of P. brassicae effector proteins and their functions in planta during the infection process. The effectors and their host targets characterized in this study will provide a valuable resource for the identification of new R-genes that will benefit both breeders and producers of canola and other crops in Canada.

Clubroot disease in canola and other brassicas is caused by the pathogen Plasmodiophora brassicae. Successful infection relies on effector proteins that are able to suppress the host immune system as well as induce morphological changes in the plant that can increase infection rate. Although effector proteins have a high potential to play an important role in P. brassicae infection and clubroot development and are therefore potential targets for future breeding strategies, their function and roles in clubroot pathogenicity are not well understood.

Researchers at the University of Saskatchewan initiated a five-year study in 2016 to develop a genome wide identification and in planta expression profiling of the Plasmodiophora brassicae secretome, a set of proteins expressed and secreted by the pathogen. Other objectives were to conduct a functional analysis of P. brassicae effectors, and analysis of virulence mechanisms of P. brassicae on host plants. Effector proteins are mostly secretory proteins that enable plant infection by manipulating the host response. They may also activate defense strategies in resistant plant genotypes. Researchers also explored the genetic diversity of some of the genes that encode these small secreted effector proteins. 

The model plant Arabidopsis thaliana ecotype Columbia, which is susceptible to clubroot disease, was used in this study for most of the experiments. The Brassica napus canola cultivar Westar was used to generate resting spores of P. brassicae. Canola plants were inoculated at the four-leaf stage, and at 35 days post inoculation (dpi) fresh galls were processed to collect the resting spores. For species comparisons, several Brassica species were used, including: Arabidopsis, canola (B. napus), arugula (Eruca sativa), broccoli (B. oleracea var. italica), cabbage (B. oleracea var. oleracea) and wild mustard (B. kaber), as well as Nicotiana benthamiana. Shoot and roots of infected plants at appropriate dpi, and non-infected samples at the same time points were collected for RNA-seq analysis to identify up- or down-regulated plant genes and expressed P. brassicae genes. Various genomic and molecular approaches were used for screening and identifying putative P. brassicae effectors, their structure and function. 

In this study, RNA-Seq analysis was used to identify genes coding putative small, secreted P. brassicae proteins that were up-regulated during infection of the model plant Arabidopsis thaliana at 17, 20 and 24 days dpi and from a 35 dpi canola root gall cDNA library. Researchers identified and partially characterized 32 (from Arabidopsis) and 52 (from canola) P. brassicae putative effectors, with a more in-depth functional characterization of some effectors, identifying important roles in the P. brassicae infection process that may prove to be potential targets for future breeding strategies. Researchers were able to successfully establish the probable function and role in P. brassicae infection and disease progression for a select number of effectors. Very little variability of some of these candidate effectors was described among pathotypes.

Overall, this study is one of the first to characterize effector proteins with key roles in clubroot pathogenicity. Researchers have produced a substantial annotation of P. brassicae effector proteins and their functions in planta during infection and disease progression. They have also identified some host targets of P. brassicae effectors that has allowed an initial characterization of the molecular and cellular mechanisms underlying P. brassicae virulence and host resistance. These effectors and their host targets characterized in this study provide a valuable resource for the identification of new R-genes that will benefit both breeders and producers of canola and other crops in Canada.           

Scientific Publications: 

Hossain, Md. M., Pérez-López, E., Todd, C. D., Wei, Y., & Bonham-Smith, P. C. (2021). Endomembrane-targeting Plasmodiophora brassicae effectors modulate PAMP triggered immune responses in plants. Frontiers in Microbiology, 12, 651279. https://doi.org/10.3389/fmicb.2021.651279 

Pérez-López, E., Hossain, M. M., Wei, Y., Todd, C. D., & Bonham-Smith, P. C. (2021). A clubroot pathogen effector targets cruciferous cysteine proteases to suppress plant immunity. Virulence, 12(1), 2327–2340 https://doi.org/10.1080/21505594.2021.1968684 

Muirhead, K., & Pérez-López, E. (2020). ClubrootTracker: Helping to plan a clubroot-free farm. Plant Health Progress, 21(3), 185-187 https://doi.org/10.1094/PHP-01-20- 0003-RS. (Editor’s Pick) 

Pérez-López, E., Hossain, M., Tu, J., Waldner, M., Kusalik, A. J., Wei, Y., Todd, C.D. & Bonham-Smith, P.C. (2020) Genome-wide transcriptomic analysis identified putative small-secreted Plasmodiophora brassicae proteins. Journal of Eukaryotic Microbiology, 67(3), 337-351 https://doi.org/10.1111/jeu.12784 (A top cited article published between 1 Jan 2021-Dec 2021) 

Botero, A., García, C., Gossen, B.D., Strelkov, S.E., Todd, C.D., Bonham-Smith, P.C. & Pérez-López, E. (2019) Clubroot disease in Latin America: distribution and management strategies. Plant Pathology, 68(5), 827-833 https://doi.org/10.1111/ppa.13013 (Most downloaded paper of 2019) 

Irani, S., Todd, C.D., Wei Y. & Bonham-Smith PC (2019). Changes in phenylpropanoid pathway gene expression in roots and leaves of susceptible and resistant Brassica napus lines in response to Plasmodiophora brassicae inoculation, Physiological & Molecular Plant Pathology, 106, 196-203. https://doi.org/10.1016/j.pmpp.2019.02.007 

Pérez-López, E., Waldner, M., Hossain, M., Kusalik, A.J., Wei, Y., Bonham-Smith, P.C. & Todd, C.D. (2018) Identification of Plasmodiophora brassicae effectors – A challenging goal. Virulence, 9(1), 1344-1355 https://doi.org/10.1080/21505594.2018.1504560