Bonham-Smith: Genomics of Clubroot Disease Development in Canola and Development of in planta RNAi to Impart Novel Resistance
Date: February 2016
Term: n/a
Status: Completed
Researcher(s): Peta Bonham-Smith and Yangdou Wei, University of Saskatchewan; Gary Peng and Bruce Gossen, Agriculture and Agri-Food Canada, Saskatoon SK
SaskCanola Investment: n/a
Total Project Cost: n/a
Funding Partners: ADF
Project Summary
Clubroot, resulting from an infection by Plasmodiophora brassicae is a devastating disease of canola. Researchers initiated a five-year study in 2010 to investigate the cellular and molecular mechanisms of P. brassicae infection, and to develop molecular markers and tools for utilizing pathogen protein targets for improved resistance to clubroot disease in canola. As a result of the project, a cDNA library was constructed, from which over 2000 P. brassicae sequences were isolated. A transcriptome analysis of infected roots was also established and will be used to identify and develop molecular markers for commercial canola resistance breeding programs. The results from this work will be of use to the clubroot research community in the utilisation of pathogen protein targets for improved resistance to clubroot disease in canola.
Clubroot, resulting from an infection by Plasmodiophora brassicae is a devastating disease of canola (Brassica napus). While the emphasis in research over the past few years has led to the successful identifications of a few resistance (R) genes in canola and other Brassica species, the matching avirulence (Avr) genes in P. brassicae have still to be identified. There is a need to better understand the molecular biology of P. brassicae infection, clubroot disease development and the mode of action of secreted effector proteins and R genes, in order to develop molecular tools and strategies for imparting long term durable resistance into canola breeding programs.
Researchers at the University of Saskatchewan, in collaboration with Agriculture and Agri-Food Canada, initiated a five-year study in 2010 to investigate the cellular and molecular mechanisms of P. brassicae infection, and to develop molecular markers and tools for utilizing pathogen protein targets for improved resistance to clubroot disease in canola. At the start of the project, researchers established a clubroot disease nursery using the most common P. brassicae pathotype 3, a dual culture system for studying pathogen-host interactions, and labelling techniques for in planta visualisation of the pathogen.
The first project objective was the construction of a cDNA library in order to develop detailed molecular pathways of both the host and pathogen responses to clubroot development, and identify new resistance possibilities in canola to clubroot disease. The library was developed from P. brassicae-infected canola (Westar) main roots, lateral roots and gall tissues at 35 days post inoculation (dpi). From the library, 20,000 clones were isolated and10,500 clones sequenced, resulting in a 50:50 split between canola and P. brassicae sequences. Over 2000 putative and unique P.brassicae sequences (many full length cDNAs) were isolated from the cDNA library, increasing the number of known P. brassicae sequences approximately 20-fold. As well, no pathogenesis-related (PR) genes were identified so far in the library, suggesting that B. napus roots have a weak stress/defensive response to a P. brassicae infection.
In an attempt to develop plants with P. brassicae resistance, a second main objective of the project was to generate transgenic B. napus plants with RNAi targeting specific P. brassicae sequences. Next generation high throughput sequencing was conducted to profile gene expression of both the host, including both a related wildtype species Arabidopsis and canola, and the pathogen P. brassicae during different disease developmental stages. Once completed, the analysis of the transcriptome data for infected and control, above and below ground tissues will provide the understanding of the whole plant networked response to infection, together with the pathogen effector profile at various stages of infection and disease progression. The findings will be used to identify and develop molecular markers for commercial canola resistance breeding programs.
The third objective of the project was to identify P. brassicae effector proteins involved in colonization of canola roots and the identification and characterization of Avr genes. Prevalent races were catalogued and characterized based on the presence of Avr genes triggering a plant hypersensitive response (HR). From the cDNA library, 15 putative effector proteins (pathogen secreted proteins that aid pathogen infectivity) were identified as not being expressed in resting spores but expressed at later stages of infection and disease progression. Of these, four were associated with plant Programmed Cell Death (a plant process to inhibit pathogen spread) and are being studied further as possible tools for improved plant resistance. Subsequently in 2015, the P. brassicae genome was published and provided the project with a reference genome against which infected versus non-infected transcriptome data could be mapped. While still being analysed, these data are providing the necessary insights into the plant-pathogen interaction networks that highlight the pathogen's successful ability to escape detection, throughout its complete intracellular life cycle, by the plant1.
The final objective of the project was to deploy an in planta RNAi approach to impart resistance to clubroot in commercially grown, high-yielding Canadian canola varieties. Based on initial RNAi trial results, this technology could be used in the establishment of improved resistance to clubroot in canola. Overall, the results from this work are still at an early stage of 'usefulness' to the producer/industry but will be of use to the clubroot research community in the utilisation of pathogen protein targets for improved resistance to clubroot disease in canola. The identification of putative effector proteins is continuing through a second ADF grant (ADF Project #20160138).
Scientific publications.
1. Irani S, Trost B, Waldner M, Nayidu N, Tu J, Kusalik, AJ, Todd CD, Wei Y, Bonham-Smith PC 2018 Transcriptome analysis of response to Plasmodiophora brassicae infection in the Arabidopsis shoot and root, BMC Genomics 19(1): 23.