Periodic Reporting for period 4 - BLASTOFF (Retooling plant immunity for resistance to blast fungi)
Reporting period: 2022-03-01 to 2023-08-31
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.
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.
The research project has empowered the BLASTOFF Team to establish a platform for bioengineering disease resistance genes, combining structural and evolutionary biology with synthetic biology. This effort has culminated in the development of novel blast resistant rice and wheat through genome editing of susceptibility genes, alongside expanding the effector recognition profile of a rice NLR immune receptor via protein engineering.
Our study focused on the multihost pathogen Magnaporthe oryzae, which infects over 50 species of grasses, including key cereal crops like wheat, barley, and rice. We completed a comprehensive survey of M. oryzae effectors that interact with small Heavy Metal-Associated (sHMA) proteins, identifying several new effector-HMA interacting pairs through yeast two-hybrid screens. This work has provided valuable insights into the diversification of M. oryzae effectors and their interactions with host proteins.
The project helped the development of Pikobodies, namely bioengineered immune system proteins that exploits antibodies' uniquely flexible immune systems, enabling plants the ability to fight off emerging pathogens.
Significant progress was made in elucidating the binding properties of multiple M. oryzae effector proteins to HMA domains, revealing multiple binding interfaces and the impact of amino acid polymorphisms on effector specificity. Through structure-guided mutagenesis and protein engineering, we have successfully modified the rice NLR immune receptor Pikp to recognize and respond to previously elusive variants of the AVR-Pik effector, demonstrating the potential of targeted molecular interventions to broaden plant disease resistance.
Concurrently, our work on genome editing in rice and wheat aimed at knocking out susceptibility genes has progressed, employing CRISPR technology to target the sHMA gene family. This has led to the generation of lines with targeted deletions for enhanced resistance to various blast fungus isolates. These combined efforts underscore the potential of integrating structural biology, synthetic biology, and genome editing to create crops with improved resistance to devastating diseases.
Our study focused on the multihost pathogen Magnaporthe oryzae, which infects over 50 species of grasses, including key cereal crops like wheat, barley, and rice. We completed a comprehensive survey of M. oryzae effectors that interact with small Heavy Metal-Associated (sHMA) proteins, identifying several new effector-HMA interacting pairs through yeast two-hybrid screens. This work has provided valuable insights into the diversification of M. oryzae effectors and their interactions with host proteins.
The project helped the development of Pikobodies, namely bioengineered immune system proteins that exploits antibodies' uniquely flexible immune systems, enabling plants the ability to fight off emerging pathogens.
Significant progress was made in elucidating the binding properties of multiple M. oryzae effector proteins to HMA domains, revealing multiple binding interfaces and the impact of amino acid polymorphisms on effector specificity. Through structure-guided mutagenesis and protein engineering, we have successfully modified the rice NLR immune receptor Pikp to recognize and respond to previously elusive variants of the AVR-Pik effector, demonstrating the potential of targeted molecular interventions to broaden plant disease resistance.
Concurrently, our work on genome editing in rice and wheat aimed at knocking out susceptibility genes has progressed, employing CRISPR technology to target the sHMA gene family. This has led to the generation of lines with targeted deletions for enhanced resistance to various blast fungus isolates. These combined efforts underscore the potential of integrating structural biology, synthetic biology, and genome editing to create crops with improved resistance to devastating diseases.
• 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
• 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