Final Report Summary - NGRB (Next generation disease resistance breeding in plants) Outbreaks caused by filamentous plant pathogens (fungi and oomycetes) have increased in frequency and are a chronic threat to global food security. Plants, similar to animals, rely on immune receptors to ward off pathogens. One class of immune receptors are nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins that respond to invading pathogens, detect specific pathogen proteins known as effectors and activate immune responses. How plant NLR-type immune receptors respond to pathogens is poorly understood. In particular, plant NLRs tend to have a narrow spectrum of pathogen recognition, which is currently limiting their value in agriculture. We undertook various mutagenesis and functional screens to improve plant immune receptors. We have discovered that we can generate mutants of NLR immune receptors with expanded response to hitherto unrecognized pathogen effectors. We showed that such mutations can be transferred across plant species to related immune receptors to target additional pathogens. This work contributed to understanding how NLR receptor activity can be modulated. In particular, we now know that mutations across particular domains of NLR proteins, can yield sensitized receptors that are “trigger-happy” with markedly lower thresholds of activation. Although such NLR mutants do not always translate into an effective resistance response against virulent races of pathogens, this is a major step forward in our capacity to engineer improved immune receptors. Together with knowledge of pathogen effector diversity, this strategy can be exploited to develop synthetic immune receptors.In a complementary strategy, we developed new methods to introduce beneficial mutations in crop plant genomes using genome editing. We demonstrated the applicability of the CRISPR/Cas9 technology for the purposes of targeted mutagenesis in plants. We applied the CRISPR/Cas9 technology to introduce knock-out mutations into candidate susceptibility genes or creating constitutively active forms of positive regulators of disease resistance, for example by deleting inhibitory domains. In a proof-of-concept study, we reported on generating, in less than ten months, Tomelo, a non-transgenic tomato variety resistant to the powdery mildew fungal pathogen using the CRISPR/Cas9 technology. We used whole genome sequencing to show that Tomelo does not carry any foreign DNA sequences but only carries a deletion that is indistinguishable from naturally occurring mutations. We also presented evidence for CRISPR/Cas9 being a highly precise tool, as we did not detect off-target mutations in Tomelo. Using this pipeline, mutations can be readily introduced into elite or locally adapted tomato varieties in less than a year with relatively minimal effort and investment.Our work highlights the application of genome editing as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. This approach generates plant varieties that are free of recombinant DNA (no exogenous transgenic sequences) and that only vary from the original cultivar in a few nucleotide substitutions, making them essentially indistinguishable from naturally occurring mutations. We hope that genome edited plant varieties, such as Tomelo, are adopted world-wide with similar regulatory burden as traditionally bred varieties to meet the food demands of the growing world’s population and promote competitiveness of the agrobiotech sector by reducing chemical input.
