UKRI/BBSRC-NSF/BIO Determining the Roles of Fusarium Effector Proteases in Plant Pathogenesis

Small proteins called effectors secreted by plant infecting pathogens are a focus of research in numerous laboratories in the USA, UK and elsewhere, because their study sheds light on how pathogenic microbes cause plant disease. Often these effector studies also reveal the identity of key host proteins in the attacked plants that regulate immunity. However, our knowledge of effectors produced by microscopic filamentous fungi that cause different plant diseases is still quite limited, even though fungal pathogens represent very serious risks to global food security and in some cases human and animal health. One such pathogen is Fusarium graminearum (Fg), which causes Fusarium Head Blight (FHB) on wheat and barley, but which also infects dozens of other plant species, including the import global crop species maize, rice, soybean and the model experimental species Arabidopsis.

FHB disease in cereal crops occurs just after crop flowering (anthesis) and goes on to reduce grain yield and grain quality. Particularly concerning is that this disease contaminates grain with toxic compounds called mycotoxins, with the most common being deoxynivalenol (DON). Current FHB control measures are complex but inadequate, involving deploying partially resistant cultivars, using partially effective fungicides and altering agricultural practices. As a consequence, strict legislation and the removal/reduction of mycotoxin contaminated grains post-harvest and in processor chains is needed to ensure food and feed are safe for grain consumers (i.e., humans, farmed animals and birds).
To meet expected food demand in 2050, when the world is projected to have an additional 2 billion people, it is imperative that we reduce crop losses to FHB. The goal of this project is to develop genetic-based resistance in wheat and barley to Fusarium species that cause FHB disease. Plants have the ability to detect disease-causing pathogens and then activate a robust defence response that ultimately leads to localised cell death to stop the invader. To detect pathogens, plants use sensor proteins that are modified by enzymes that pathogens secrete during the infection process. This project focuses on identifying enzymes secreted by the fungus Fg that are required for infection of wheat and barley floral tissues. Once such enzymes are identified, sensor proteins will be designed that can activate defence responses in wheat and barley upon modification by these Fg enzymes. Such a system would thus confer resistance to infection by Fusarium species that cause FHB disease without the use of costly and environmentally damaging pesticides. This approach to Fusarium control should be transferrable to a wide array of important crop plants that are damaged by other Fusarium species.

This collaborative US-UK project will involve multidisciplinary teams at Rothamsted Research, UK, Indiana University, Indiana, USA and the USDA-ARS laboratory at Purdue University, Indiana, USA.

Additional proteases secreted by Fg will be tested for their contribution to virulence through the generation of gene deletion strains and in planta assessments. Fg protease translocation into plant cells will be evaluated using the split GFP expression system and confocal microscopy.
The target sites of proteases that contribute to virulence will be identifying in N. benthamiana leaves and wheat protoplasts using biotin proximity labelling. Cleavage sites in the putative targets will be identified using mass-spectrometry.

In parallel, we will determine the optimal cleavage sequence for each Fg 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 dozens of sequences that can be cleaved by each protease can be recovered and the consensus cleavage sequence determined.
The consensus cleavage sequence for a given Fg protease will be inserted into wheat PBL11 and tested for cleavage by the protease and subsequent activation of PBR1 using transient expression in N. benthamiana and in wheat protoplasts. Functional cleavage sequences will be introduced into the wheat genome using CRISPR-Cas9 mediated exon replacement. GE wheat lines will be made for 3 different PBL11 decoy constructs and 1 nonmodified control construct. The T1 generation will be tested for resistance to Fg infection, Fg biomass burden and mycotoxin accumulation.

For proteases that localise to the apoplast, we will generate extracellular decoy substrates that are anchored to the plasma membrane. For these substrates, protease cleavage will release a flg22 peptide 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 to simultaneous activate PTI/ETI.