This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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Wheat is a major crop globally. As the global population increases, it is essential to increase wheat yields to meet demand. Improving disease resistance will reduce yield loss caused by pathogenic microbes. However, how improved disease resistance might compromise interactions with beneficial microbes is often overlooked. Arbuscular mycorrhizal fungi (AMF) form a beneficial symbiosis with around 80% of land plants in the soil. The plant exchanges carbon for mineral nutrients from the fungus, thereby improving the plant’s nutrient acquisition and reducing reliance on environmentally detrimental chemical fertilisers. This summer, I studied wheat WRKY10 transcription factor that regulates the signalling of the essential defence hormone, jasmonic acid. A 2021 study found that virus-induced gene silencing (VIGS) of WRKY10 increased resistance to Septoria tritici Blotch (STB) disease, the primary threat to wheat yields. Caused by the fungal pathogen Zymoseptoria tritici, STB accompanies chlorosis and necrosis symptoms in wheat leaves (Figure 1), which leads to over 40% yield loss when untreated.
Regarding AMF, jasmonic acid has been found to regulate colonisation, although effects vary based on the species and methods used. This interaction has not yet been studied in wheat. During this summer placement, I aimed to validate the role of WRKY10 in STB disease resistance by using WRKY10 RNAi and overexpression lines. I also investigated CIB1 RNAi and overexpression lines. As a positive regulator of WRKY10, CIB1 binds to WRKY10’s promoter. I hypothesised was that silencing WRKY10 and CIB1 would recapitulate improved resistance to STB, but may decrease AMF colonisation levels. To investigate whether changes in WRKY10 and CIB1 expression affect STB resistance, spores of an infectious strain of Zymoseptoria tritici were inoculated to various wheat lines (Figure 2).
The control cultivar, KWS Cashel, known to be highly susceptible to STB, already showed severe infection symptoms. However, I could not evaluate the genotype effect because the disease symptoms were not fully developed in other lines. In the second experiment, I inoculated plants with spores of AM fungi, Rhizophagus irregularis. After six weeks, the plants were harvested, and a colonisation level was quantified microscopically. I significantly improved the Trypan Blue staining protocol for wheat, testing various temperatures, incubation times and concentrations. Overall, there was no significant difference in the AMF colonisation levels in these mutants, although the WKRY10 RNAi and overexpression lines displayed slightly reduced AM colonisation. Therefore, if any of the mutants have improved STB resistance, it may not be at the cost of AMF colonisation. However, the high colonisation levels at the point of harvest (over 90% in wild-type) may have obscured the chance to quantify potential differences. CIB1 overexpressing plants showed significantly lower root fresh weight (RFW) than wild-type upon AM fungal colonisation, while WRKY10 overexpression mutants showed increased RFW in both non-inoculated and AM colonised plants compared to wild-type. These contradictory effects warrant further investigation of signalling gene expression analyses.
Overall, this project has been an incredible experience. I have tried many techniques and learned many skills which will be invaluable to my future career. I have enjoyed seeing the applications of plant pathology research and hope to continue researching plant-microbe interactions further in the future. I would like to thank everyone at the Cambridge Crop Science Centre and NIAB, especially Dr Jeongmin Choi, Abi Brock and Dr Stéphanie Swarbreck, for all their help throughout this experience and the BSPP for the funding.
Sophie Perryman
University of Cambridge
Figure 1 Fungal structures in wheat. UPPER) Leaves of wheat line, KWS Cashel, was infected for 17 days with Septoria fungal pathogen, Zymoseptoria tritici. The inoculated leaf (bottom) shows necrotic lesions and pycnidia (black dots), the reproductive structure of the pathogen, compared to the uninfected leaf (top). LOWER) Wheat root stained with Trypan Blue to show arbuscular mycorrhizal fungal colonisation. The arrow indicates a fungal structure called arbuscule, which provides high surface area for nutrient exchange.
Figure 2 Experimental design. a) Study of STB resistance. Symptom assessment involved quantifying coverage of inoculated leaf with necrosis, chlorosis and pycnidia, and calculating severity of disease. b) Study of AMF colonisation. Once harvested, root samples were stained with Trypan Blue to quantify AMF colonisation. Samples were taken for RNA extraction, to perform qRT-PCR for WRKY10, CIB1, and marker genes of AM symbiosis.
