Resistance in plants against biotic stress through forward and reverse genetics

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Resistance in plants can be either qualitative, defined as absence of disease, or quantitative, defined as reduced disease severity, but the distinction is not always apparent. Based on wheat/barley-fusarium head blight and potato-late blight we have evidenced that the resistance is mainly due to metabolites. Following inoculation, the pathogens produce microbe associated molecular patterns that are recognized by host receptors, which trigger downstream genes to regulate resistance genes that biosynthesize metabolites that are antimicrobial and/or deposited to enforce the cell walls to contain the pathogen to initial infection. Some of the transcription factors and hub genes in the metabolic pathway appear to control significant amounts of resistance, as proved based on transient gene silencing. The commercial cultivars also contain metabolic pathway specific hierarchies of these genes but one or a few may be nonfunctional. These missing link genes can be replaced with resistance genes identified here to recover their ability to reproduce the lost metabolites, thus the resistance. We hypothesize that by pyramiding a few significant effect genes, regulating specific metabolic pathways, in a commercial susceptible cultivar the resistance against pathogen stress can be significantly increased. In the present proposal several resistant potato genotypes and a cultivar Russet Burbank susceptible to late blight will be RNA sequenced and metabolites profiled. Candidate metabolites with high fold change will be used to identify the candidate biosynthetic and regulatory genes. The candidate genes that are functional in resistant and nonfunctional in Russet Burbank will be silenced to prove the resistance effects. These gens or DNA segments will be replaced in Russet Burbank based on genome editing tools. Protoplasts will be produced, CRISPR-Cas9 system constructs of resistance genes developed and introduced to protoplasts to replace the non-functional genes with functional genes identified here, regenerated into calli, genes verified and regenerated into plants. This cisgenic (gene transfer between sexually compatible plants) Russet Burbank cultivar will be checked for gene replacement in the expected location. Superiority of cisgenic over the non-cisgenic cultivar in resistance proved under greenhouse and field conditions, registered as a new cultivar and commercialized. Russet Burbank covers more than 40% of potato produced in North America, and all original producers are expected to use cisgenic Russet Burbank cultivar. Technology developed here can be adopted to improve other plant-pathogen systems.