Fernando: Getting One Step Closer to Sclerotinia Control Through Cultivar Resistance and Biological Applications
Date: May 2016
Term: 3 years
Status: Completed
Researcher(s): Dilantha Fernando, Mark Belmonte, Teresa de Kievit, Ian Girard, Kelly Duke, Sanjay Saikia, Philip Walker and Jenna Millar, University of Manitoba
SaskCanola Investment: n/a
Total Project Cost: n/a
Funding Partners: n/a
Project Summary
Canola, one of the world’s most valuable oilseeds, is under constant pressure by the fungal pathogen, Sclerotinia sclerotiorum, the causal agent of white stem rot. Researchers at the University of Manitoba conducted a three-year multi-study project to investigate the structural, molecular, and physiological defense response of B. napus to S. sclerotiorum in susceptible and tolerant hosts. Through RNA sequencing, new genes and novel regulators of plant defense were identified, including potential resistance genes. Bioformatics tools have been developed to analyze large-scale RNA sequencing datasets of canola, including canola leaf tissues infected with Sclerotinia, and comparisons of biocontrol treatments, which will serve as a valuable resource for researchers interested in developing resistant lines and biocontrol options.
Canola (Brassica napus) is one of the world’s most valuable oilseeds and is under constant pressure by the necrotrophic fungal pathogen, Sclerotinia sclerotiorum, the causal agent of white stem rot. Developing new strategies to combat yield losses in B. napus requires a thorough understanding of the genes and gene regulatory networks underlying the plant defense response, especially in existing tolerant breeding cultivars.
Researchers at the University of Manitoba conducted a three-year multi-study project to investigate the structural, molecular, and physiological defense response of B. napus to S. sclerotiorum in susceptible and tolerant hosts. The overarching goal of this research was to identify novel defense genes and underlying molecular framework of the defense response in canola to: i) sclerotinia infection, ii) identification of cell-specific defense molecules in tolerant and susceptible canola cultivars to sclerotinia infection, and iii) application of the biocontrol bacterium PA23 in the presence and absence of sclerotinia.
Aim 1. To identify changes in global gene expression in susceptible versus tolerant canola cultivars in response to Sclerotinia infection (whole leaf analysis).
Researchers used next generation RNA sequencing on control and infected leaves of the tolerant B. napus genotype Zhongyou821 (ZY821), and the susceptible Westar using a petal inoculation technique to accurately mimic field conditions of Sclerotinia infection. The results showed that both susceptible and control cultivars undergo large-scale transcriptional reprogramming following foliar infection of Sclerotinia. Using RNA sequencing, researchers identified over 1200 previously undiscovered genes associated with the plant defense. New biological processes not previously associated with this pathosystem including protein modification and epigenetic control may contribute to the explanation of tolerant phenotype. Novel regulators of the plant defense processes responsible for providing tolerance to S. sclerotiorum were identified, including a potential quantitative trait locus for resistance.
Aim 2. To elucidate and identify tissue-specific defense responses to Sclerotinia infection that occurs in Westar (susceptible) versus Zhongyou821 or ZY821 (tolerant) cultivars.
In this study, researchers compared leaf tissues of Westar (susceptible) and ZY821 (tolerant) cultivars. For each cultivar, three leaf tissue types, epidermis, mesophyll and vasculature were collected. Infected and non-infected leaf tissues were collected at 24 hours post inoculation with Sclerotinia-infected petals (almost 30,000 histological sections from at least 24 samples). Although the leaf tissue processing and microtome sectioning presented many challenges, researchers successfully developed well-established and optimized protocols for leaf tissues in canola. Using laser capture microdissection, enough cells for RNA sequencing were successfully collected, with over 1.10 million reads to analyze with more on the way. Bioinformatics tools have been developed to analyze large-scale RNA sequencing datasets in canola.
The results confirmed that Sclerotinia infection induces tissue specific defense responses in susceptible leaves of B. napus plants. Vascular tissues are transcriptomically distinct compared to epidermis and mesophyll, which may be a result of the transport functions of the vasculature compared to the actively photosynthesizing mesophyll or the protective function of the epidermis. The distribution of shared and unique genes among the tissue types of susceptible leaves suggests gene activity tends to be tissue specific where few genes are shared or overlap between tissues. A large number of genes are unique to vascular tissue, while a small number of genes are shared among tissue types. This data will provide a high resolution, tissue specific atlas of the plant defense response immediately following infection of mature leaves with Sclerotinia and will serve as a valuable resource for researchers interested in developing resistant lines.
Aim 3. To elucidate changes in gene expression and physiology occurring in canola plants in response to the biocontrol bacterium PA23 in the presence and absence of S. sclerotiorum.
In this study, researchers sprayed B. napus plants with solutions of the biocontrol agent Pseudomonas chlororaphis or PA23 only, S. sclerotiorum ascospores only, or PA23 24 hours prior to S. sclerotiorum ascospores and incubated under humid conditions for 72 hours to confirm PA23’s efficacy at preventing fungal infection in plants. Leaf tissue from these plants was collected and used for follow-up experiments. Using this infection model, leaf necrosis was visible under lesion-forming petals as soon as 24 hours post infection with S. sclerotiorum in plants receiving pathogen treatment only.
The results showed that the application of PA23 to the aerial surfaces of canola plants protects canola from Sclerotinia and reduces lesion formation by 90%. RNA sequencing showed the biocontrol agent PA23 induces global changes in gene activity within the canola leaf in the presence and absence of Sclerotinia. By itself, PA23 activated unique defense networks indicative of defense priming, and likely operates through modulation of defense responses in the plant and through the induction and activation of unique gene expression patterns directly at the host pathogen interface. Understanding these interactions will aid in the development of biocontrol systems as an alternative to chemical pesticides for protection of important crop systems.
Scientific publications.
Duke, K, Becker MG, Girard IJ, Millar, JL, W. G. Fernando WDG, Belmonte, MF, and de Kievit T. 2017. The biocontrol agent Pseudomonas chlororaphis PA23 primes Brassica napus defenses through distinct gene networks. BMC Genomics 18:467.
Girard IJ, Tong C, Mao X, Becker MG, de Kievit T, Fernando WDG, Li G, Belmonte MF. 2016. RNA sequencing of Brassica napus reveals cellular redox control of Sclerotinia infection. Journal of Experimental Botany. Vol. 68, No. 18 pp. 5079–5091.
Girard IJ, Mcloughlin AG, de Kievit T, Fernando WGD, Belmonte MFB. 2016. Integrating large-scale data and RNA technology to protect crops from fungal pathogens. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2016.00631
Selin C, de Kievit TR, Belmonte MF, and Fernando WGD. 2016. Elucidating the role of effectors in plant-fungal interactions: progress and challenges. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2016.00600
Girard IJ, Tong C, Mao X, Becker MG, de Kievit T, Fernando WDG, Li G, Belmonte MF. Global RNA profiling of the initial Sclerotinia sclerotiorum – Brassica napus infection process reveals cross talk between redox, hormone and carbon metabolism pathways. To be submitted to Molecular Plant, May 2016.