This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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Take-All is a pathogen infecting the roots of wheat caused by the fungus Gaeumannomyces tritici (Gt). Gt colonises and damages the vascular tissues which impairs water and nutrient uptake. This leads to stunted growth, premature grain ripening, blackened stem bases and black lesions on the roots, all contributing to a reduced grain yield and quality. During an average year, take-all disease causes a 5-20% reduction in yields but in a high disease level year, reductions of up to 50% have been observed. The milder, wetter conditions predicted to come with climate change are favoured by fungal pathogens that threaten food security. Therefore, being the most important root disease of wheat, research to understand the infection mechanisms of Take-All as well as potential control methods is imperative.
First I investigated the virulence of 6 different strains of Gt of type A (19d1, 8d and 19e1) and type B (4e, 23d and 14LH10). Through phenotyping of adult Triticum aestivum, characterisation of each strain’s impact on plant health was observed. Measurements of plant height, ear number, flag leaf length, ear length, tiller number, root system length, root number, dry biomass of root system and infection severity (measured through the Take-All index) showed stronger virulence among type A strains compared to type B strains (Figure 1). Statistical analysis revealed that plants infected with type A isolates showed significantly stunted growth (P=0.045) and shorter root systems than the control (P=0.002) and had greater levels of infection than both the control and type B infected plants (P<0.001). However, the strain 4e showed that it had lost virulence with a phenotype similar to the control.
Figure.1. (A) Take-All infection severity, (B) height and (C) root systemS length of wheat infected with different strains of Gaeumannomyces tritici where differing letters above represent significant differences in the Take-All index between treatments.
Additionally, Virus Induced Gene Silencing (VIGS) was used to characterise the function of genes with the potential to improve resistance to Take-All disease. The candidate genes had been identified though past experiments involving the beneficial fungus Gaeumannomyces hyphopodiodies (Gh). Gh is a non-pathogenic relative of Gt which is unable to penetrate through the root cortex and therefore, unlike Gt, does not cause to damage to the plant’s vasculature. Infection with Gh has been shown to be beneficial in reducing Take-All infection and, while little is known about the mechanism underlying this, reduced infection was correlated with the down regulation of several genes. Here VIGS was used to identify whether silencing of these genes could reduce infection severity in plants inoculated with Gt. The first gene investigated was Cellulose Synthase 7 (CESA7) which is required for secondary cell wall synthesis in Arabidopsis and mutations in this gene have previously been associated with improved resistance to the soil-borne bacterium Ralstonia solanacearum and the necrotrophic fungus Plectosphaerella cucumerina. The other gene investigated was β-1,3-Glucan synthase 1 (Gls1) which is involved in fungal cell wall synthesis. Through Host Induced Gene Silencing (HIGS), down regulation of Gls1 has been shown to protect wheat from Fusarium Head Blight (FHB) and wheat lines transformed with the Gls1 construct also showed reduced FHB symptoms.
Agrobacterium was transformed with binary vectors carrying the target sequences of the genes of interest using electroporation and inoculation solutions prepared. This was used to agroinfiltrate 4-week-old Nicotiana bethamiana. Leaf sap from infiltrated leaves 5-7 days post infiltration was used to inoculate leaves of Wheat seedlings (10 days old). The bottom of the pots were then cut off and placed on top of another pot containing Gt inoculum and covered by a mesh. This allowed separation of new and old root growth to see whether the gene silencing strength differed between new and old tissue. Seedlings were grown for three weeks before imaging and assessing the root’s disease levels. Seedlings then imaged and assessed for Take-All infection severity.
Scanning of the root systems using the WinRhizo software revealed that CESA7 targeted samples had significantly longer total root length (P=0.004) (Figure 2).
Figure.2. Total root length of new root growth
Visual assessment of Take-All infection showed a lower percentage of roots with lesions than the control in both Gls1 and CESA7 silenced plants, although this was not significant (Figure 3). For those plants showing no Gt infection, DNA was extracted, and a Gt housekeeping gene amplified. This confirmed that there was no infection in all but 2 samples and suggests that the lack of lesions was due to resistance to Gt.
Figure.3. Percentage of roots showing Take-All lesions in wheat seedlings.
Following this, RNA extraction from root samples of plants showing virus symptoms and real-time quantitative PCR (q_PCR) was used to confirm whether the genes had been successfully silenced. This showed reduced expression of CESA7 compared to the control both above and below the mesh, suggesting successful gene silencing (Figure 4). Additionally, this result correlates with the reduced infection levels observed in the CESA7 samples. However, a greater level of silencing was observed below the mesh.
Figure.4. The relative quantity of RNA detected through qPCR for CESA7 and the control MCS4D showing successful silencing of CESA7.
To identify whether the beneficial effect of Gh was systemic, seedlings were grown with their roots split between two pots (Figures 5 and 6). In one treatment, one pot contained Gt and the other Gh, another had Gh and Gt in the same pot and PDA in the other. A control with PDA in one pot and Gt in the other was used to provide a comparison for infection severity. Following three weeks, assessment of infection severity revealed a significantly lower level of infection in the pots containing both Gh and Gt compared to those with Gh in one pot and Gt in the other (P = 0.0064). This result clearly shows the ability of Gh to reduce Take-All infection severity but also suggests that the response is localised to the roots infected with Gh and there is therefore no systemic effect, which is consistent with the results of the previous experiment.
Figure 5. The setup of the split root experiment whereby the roots of wheat seedlings were split between two pots containing either Gh and Gt in the same pot or Gh on one side and Gt on the other.
Figure 6. The percentage of roots showing Take-All infection where the roots were split between two pots containing either Gh and Gt in the same pot or Gh on one side and Gt on the other.
Overall, this project has given me the opportunity to develop my practical skills, both in the lab and the field. It has provided me with an insight into how a research lab runs as well as what a future PhD in plant pathology would entail. I have thoroughly enjoyed my time at Rothamsted and would like to thank the Take-All team, especially Wanxin Chen and Javier Palma-Guerrero for their guidance throughout the project as well as BSPP for the opportunity.