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Retooling plant immunity for resistance to blast fungi

Periodic Reporting for period 2 - BLASTOFF (Retooling plant immunity for resistance to blast fungi)

Reporting period: 2019-03-01 to 2020-08-31

Plant NLR-type immune receptors tend to have a narrow spectrum of pathogen recognition, which is currently limiting their value in agriculture. NLRs can recognize pathogen effectors through unconventional domains that have evolved by duplication of an effector target followed by fusion into the NLR. One NLR with an integrated domain is the rice resistance protein Pik-1, which binds an effector of the blast fungus Magnaporthe oryzae via its Heavy-Metal Associated (HMA) domain. We solved the crystal structure of the HMA domain of Pik-1 in complex with a blast fungus effector and gained an unprecedented level of detail of the molecular interactions that define pathogen recognition. This led to the overall aim of this proposal to generate a complete picture of the biophysical interactions between blast fungus effectors and HMA-containing cereal proteins to guide the retooling of the plant immune system towards resistance to blast diseases. M. oryzae is a general cereal killer that infects wheat, barley and rice, which are staple food for a majority of the world population. The central hypothesis of the proposed research is that mutations in cereal HMA-containing proteins will result in broad-spectrum resistance to blast fungi.

To achieve our goal, we are pursuing the following objectives:

1. BIOPHYSICS. Define the biophysical properties that underpin binding of M. oryzae effectors to HMA-containing proteins of cereal crops.

2. RECEPTOR ENGINEERING. Develop Pik-1 receptors that respond to a wide-spectrum of M. oryzae effectors.

3. GENOME EDITING. Mutate HMA domain-containing genes in cereal genomes to confer broad-spectrum blast resistance.

We aim to generate a thorough understanding of the biophysical properties of pathogen effector binding to cereal HMA proteins, and deliver traits and non-transgenic cultivars for breeding blast disease resistance in cereal crops.
Magnaporthe oryzae is a multihost pathogen that infects >50 species of grasses, including the cereal crops wheat, barley and rice. This pathogen deploys an arsenal of secreted proteins called effectors to infect its host plants and counteract plant defences. Three effectors, AVR-Pia, AVR1-CO39 and AVR-Pik are known to bind sHMA proteins but can also be recognized by NLR resistance proteins with integrated HMA domains. We have completed our survey of M. oryzae effectors that interact with sHMA proteins and determined the diversification of these effectors in host-specific lineages of M. oryzae. The pipeline included 185 M. oryzae effector candidates, which we publicly released as a community bioresource. We have tested these candidates for their ability to interact with sHMA proteins using yeast two-hybrid screens. We have now identified and validated several new effector-HMA interacting pairs.

We made significant progress in understanding the biophysical properties that define binding of a family of proteins related to AVR-Pik (APikL; AVR-Pik like) with HMA proteins. APikL display amino acid polymorphisms at the HMA binding interface. We have resolved crystal structures of particular effector target complexes (APikL2 in complex with a Setaria italica HMA-Domain) and identified a common binding interface between both, AVR-PikD and APikL2 with the target HMA-Domain. We also conducted a gain-of-binding mutant screen in which we replaced polymorphic residues in APikL2 with their counterparts of AVR-PikD to understand which residues contribute to the broader target spectrum of AVR-PikD. Interestingly, alleles of the conserved effector APikL2 (from wheat and rice isolates) show differential binding. This leads to a gain of binding of the APikL2_Ta allele (from wheat isolate) to one of the HMA binding partners. Based on the crystal structure we predicted amino acid polymorphisms that could contribute to this difference in binding. Using single residue mutants we can show that a derived mutation in the APikL2_Ta allele enhances binding. We thus hypothesize that APikL2 has evolved a larger binding spectrum in wheat blast isolates.

We demonstrated that protein engineering expands the effector recognition profile of a rice NLR immune receptor. We used structure-guided engineering to expand the response profile of Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an effector-binding interface of the integrated Pikp–HMA domain increased the binding affinity for AVR-Pik variants in vitro and in vivo. This translates to an expanded cell-death response to AVR-Pik variants previously unrecognized by Pikp in planta. The structures of the engineered Pikp–HMA in complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors where direct interaction between effectors and NLRs is established, particularly where this interaction occurs via integrated domains.

We made some progress towards the development of novel blast resistant wheat using genome editing of susceptibility genes. We focused on members of the sHMA family that are regulators of plant immunity responses and encode host proteins that are required by pathogens to help facilitate their entry and spread within plant tissue. The homologous S-genes were then identified in wheat and barley by computational approaches using combination of similarity searches and phylogenetic analyses to select the most likely wheat functional homologues of the rice sHMA genes. We designed CRISPR single guide RNAs (sgRNA) that can simultaneously target all copies of every sHMA gene in the polyploid wheat genome. We generate deletions from ~50 to 500 bp in the targeted genes by using 2 to 4 sgRNA sequences per gene construct. We propagated most lines to T1 generation and beyond. We are now starting phenotypic screen to identify deletion lines with enhanced resistance to various isolates of the blast fungus.
• A clone resource of Magnaporthe oryzae effectors that share sequence and structural similarities across host-specific lineages
• New effectors from the multihost blast fungus Magnaporthe oryzae that target HMA domain containing host proteins
• Biophysical properties that define binding of pathogen effectors to a family of host proteins
• Understanding effector adaptation in Magnaporthe oryzae after a host jump
• Reconstructing the evolution of immune receptors towards binding pathogen effectors
• Mechanistic understanding of plant NLR immune receptor function
• Cross-reactivity of a NLR immune receptors to distinct effectors
• Protein engineering expands the effector recognition profile of a rice NLR immune receptor
• Gene editing in wheat and barley of fungal susceptibility genes