Performing Department
(N/A)
Non Technical Summary
The overall goal of this project is to use genome editing to generate wheat and barley lines that have enhanced resistance to Fusarium Head Blight (FHB), which is caused by Fusarium graminearum (Fg). FHB disease reduces yield and grain quality, and contaminates grain with mycotoxins, most commonly deoxynivalenol. FHB disease threatens wheat production worldwide and is predicted to become even more problematic in the future due to climate change. Currently, this disease is most commonly controlled using heavy applications of fungicides, which causes collateral damage to the environment.To develop FHB-resistant wheat and barley, we are taking advantage of an endogenous surveillance system that activates immune responses upon cleavage of specific signalling proteins (protein kinases) by proteases secreted by pathogens. This multidisciplinary project arises from discoveries made by three laboratories, two based in the USA, one led by Roger Innes at Indiana University (RI), the second by Matthew Helm (MH) with the USDA-ARS located at Purdue University, USA, and the third based in the UK, led by Kim Hammond-Kosack Rothamsted Research, Herts (KHK). We initiated a three-pronged collaborative project ~ 2.5 years ago involving training of undergraduate and early graduate researchers with funding from the US Wheat and Barley Scab Initiative (USWBSI) and the BBSRC SWBioDTP rotation project scheme. Specifically, the UK team focused on identifying proteases secreted by Fg by searching the newly available, but unpublished, pan-genome sequence of Fg. Three of these proteases were then then tested for their contribution to virulence towards wheat spikes and Arabidopsis floral tissues, which revealed that at least one made a significant contribution. The Innes team focused on developing genetic screens in yeast and E. coli to identify the cleavage sites of the selected Fg proteases. Identification of such cleavage sites is a prerequisite for generating decoy substrates of these proteases. The Helm team focused on functionally characterising the plant disease resistance protein, PBR1, from wheat and barley. Specifically, the Helm team showed that activation of PBR1-mediated immune responses is dependent on the proteolytic cleavage of a host protein from barley known as HvPBL11. Intriguingly, the closest homolog of barley PBL11 in wheat, TaPBL11, also activated PBR1 when cleaved, demonstrating that the mechanism of activation of PBR1 is conserved between wheat and barley.The proposed project has the following six specific objectives:1. Determine which additional Fg proteases contribute to the disease-causing ability (virulence) of F. graminearum (KHK & MH labs)2. Determine which Fg proteases are translocated from the fungus into the host plant cells (KHK lab)3. Identify host targets of Fg proteases (MH lab)4. Identify cleavage sites of Fg proteases (RI lab)5. Generate and test decoy substrates for activation of PBR1 (RI & MH labs)6. Generate stably transformed Arabidopsis and wheat lines expressing decoy substrates (KHK, MH & RI labs)The overall ambition of this project is to generate genetically enhanced wheat and barley lines that are resistant to FHB disease.Therefore, we will engage with a range of commercial cereal breeding companies based in the USA and Europe to explain this project and to establish and maintain an open dialogue as the project proceeds.Our other important objective is to devise and participate in a range of highly targeted outreach activities involving US school pupils and US/UK cereal based agricultural communities as well as more general scientific outreach activities at Rothamsted interacting with the general public.
Animal Health Component
0%
Research Effort Categories
Basic
60%
Applied
(N/A)
Developmental
40%
Goals / Objectives
The primary goal of this project is to understand how proteases secreted by fungal pathogens contribute to disease, with a specific focus on the fungal pathogen Fusarium graminearum (Fg), causal agent of Fusarium Head Blight (FHB), a major disease of wheat and barley. We have identified a set of 8 secreted proteases that are highly upregulated during early stages of Fg infection in wheat, and which are conserved across diverse Fusarium species. This conservation and expression pattern indicates that they play a central role in pathogenesis. To understand this role, we must determine their site of action within the host, their specific targets, and their cleavage site specificities. We must also understand how loss of these proteases, either singly, or in combination, affects the infection process of Fg. Analysis of single gene deletants has revealed that at least one of these proteases, TPP1, contributes to virulence. TPP1 contains a predicted chloroplast targeting motif and transient expression of TPP1-GFP in N. benthamiana revealed localization to chloroplasts and cytoplasm. The presence of a functional chloroplast targeting motif in TPP1 strongly suggests that it is translocated into host cells during infection. To identify targets of TPP1 and any other Fg proteases that are found to contribute to virulence, we will use biotin-based proximity labeling. Targeting will be confirmed by demonstrating cleavage of putative targets by the corresponding protease. We have developed innovative approaches for accomplishing each of these tasks, including the use of a split GFP assay that enables detection of protease translocation into host cells, and the creation of a high throughput genetic screen in E. coli to identify optimal cleavage sites for fungal proteases. These cleavage sites will be used to generate decoy substrates that activate defense responses upon cleavage, and thus confer resistance to FHB. For proteases that are restricted to the apoplast, we propose a novel substrate that releases the bacterial-derived peptide flg22, which will induce pattern-triggered immunity (PTI) upon release. For translocated proteases, we will use the PBS1-like protein PBL11, which we have shown induces effector-triggered immunity (ETI) in barley and wheat upon cleavage.Our specific objectives are:1) Determine which Fg proteases contribute to virulence2) Determine which Fg proteases are translocated into host cells3) Identify host targets of Fg proteases4) Identify cleavage sites of Fg proteases5) Generate and test decoy substrates for activation of PBR16) Generate Arabidopsis and wheat lines expressing decoy substratesThe methods that will be developed in this project will greatly enhance our ability to identify and characterize translocated effector proteins. The proposed experiments will also test the efficacy of a novel extracellular protease detection system and will assess whether simultaneous detection of extracellular and translocated pathogen proteases will synergistically activate defenses.
Project Methods
Proteases secreted by Fusarium graminearum (Fg) will be tested for their contribution to virulence through the generation of single and higher order gene deletion strains and then assessing these mutants using floral infections of wheat and Arabidopsis. Fg protease translocation into plant cells will be evaluated using the split GFP expression system. Briefly, Fg stably transformed with different protease-GFP11 tag constructs will be inoculated onto various tissue types (i.e., cotyledons, petals, sepals) of homozygous Arabidopsis 35S:GFP1-10 line and confocal microscopy used to directly visualise protease translocation and the subsequent spatial and temporal localisation patterns.Proteases that contribute to infection will then be further analysed by identifying their targets in N. benthamiana leaves and wheat protoplasts using biotin proximity labelling. Putative targets will then be evaluated for cleavage by these proteases. Cleavage sites will then be identified using mass-spectrometry. In parallel to identification and characterization of protease targets, we will determine the optimal cleavage sequence for each protease using an E. coli based genetic screen. This system enables screening of random heptamer amino acid sequences for cleavage by a protease of interest. Cleavage leads to de-repression of a kanamycin resistance gene. By selecting for growth on kanamycin that is dependent on protease expression, we will be able to quickly identify dozens of sequences that can be cleaved by each protease and thus determine a consensus cleavage sequence for each.Once a consensus cleavage sequence for a given Fg protease is identified, we will insert this sequence into wheat PBL11 (a receptor-like cytoplasmic kinase) and test for cleavage by the protease and subsequent activation of the disease resistance protein PBR1. These assays will be performed using transient expression in N. benthamiana and in wheat protoplasts. Cleavage sequences that are shown to be functional in these transient assays will then be introduced into the wheat genome using CRISPR-Cas9 mediated exon replacement. Our goal is to generate genome-edited wheat lines for three different PBL11 decoy constructs and one nonmodified control construct. These will be tested in the T1 generation for resistance to Fg infection, progression of macroscopic FHB disease symptoms, Fg biomass burden in different tissue types and mycotoxin accumulation in grains.For proteases that localize to the apoplast, we will generate extracellular decoy substrates that are anchored to the plasma membrane. For these substrates, cleavage by the protease will release a peptide (flg22) that induces pattern-triggered immunity (PTI). Ideally, we will generate transgenic wheat plants that express both cell surface and intracellular decoy substrates that detect two or more Fg proteases, as simultaneous activation of pattern-triggered immunity (PTI) by cell surface receptors and effector-triggered immunity (ETI) by intracellular receoptors has shown to be confer especially robust immunity.