Allelic compatibility in plant immune receptors facilitates engineering of new effector recognition specificities.

Engineering the plant immune system offers genetic solutions to mitigate crop diseases caused by diverse agriculturally significant pathogens and pests. Modification of intracellular plant immune receptors of the nucleotide-binding leucine-rich repeat (NLR) receptor superfamily for expanded recognition of pathogen virulence proteins (effectors) is a promising approach for engineering disease resistance. However, engineering can cause NLR autoactivation, resulting in constitutive defense responses that are deleterious to the plant. This may be due to plant NLRs associating in highly complex signaling networks that coevolve together, and changes through breeding or genetic modification can generate incompatible combinations, resulting in autoimmune phenotypes. The sensor and helper NLRs of the rice (Oryza sativa) NLR pair Pik have coevolved, and mismatching between noncoevolved alleles triggers constitutive activation and cell death. This limits the extent to which protein modifications can be used to engineer pathogen recognition and enhance disease resistance mediated by these NLRs. Here, we dissected incompatibility determinants in the Pik pair in Nicotiana benthamiana and found that heavy metal-associated (HMA) domains integrated in Pik-1 not only evolved to bind pathogen effectors but also likely coevolved with other NLR domains to maintain immune homeostasis. This explains why changes in integrated domains can lead to autoactivation. We then used this knowledge to facilitate engineering of new effector recognition specificities, overcoming initial autoimmune penalties. We show that by mismatching alleles of the rice sensor and helper NLRs Pik-1 and Pik-2, we can enable the integration of synthetic domains with novel and enhanced recognition specificities. Taken together, our results reveal a strategy for engineering NLRs, which has the potential to allow an expanded set of integrations and therefore new disease resistance specificities in plants.

The allelic rice immune receptor Pikh confers extended resistance to strains of the blast fungus through a single polymorphism in the effector binding interface.

Arms race co-evolution drives rapid adaptive changes in pathogens and in the immune systems of their hosts. Plant intracellular NLR immune receptors detect effectors delivered by pathogens to promote susceptibility, activating an immune response that halts colonization. As a consequence, pathogen effectors evolve to escape immune recognition and are highly variable. In turn, NLR receptors are one of the most diverse protein families in plants, and this variability underpins differential recognition of effector variants. The molecular mechanisms underlying natural variation in effector recognition by NLRs are starting to be elucidated. The rice NLR pair Pik-1/Pik-2 recognizes AVR-Pik effectors from the blast fungus Magnaporthe oryzae, triggering immune responses that limit rice blast infection. Allelic variation in a heavy metal associated (HMA) domain integrated in the receptor Pik-1 confers differential binding to AVR-Pik variants, determining resistance specificity. Previous mechanistic studies uncovered how a Pik allele, Pikm, has extended recognition to effector variants through a specialized HMA/AVR-Pik binding interface. Here, we reveal the mechanistic basis of extended recognition specificity conferred by another Pik allele, Pikh. A single residue in Pikh-HMA increases binding to AVR-Pik variants, leading to an extended effector response in planta. The crystal structure of Pikh-HMA in complex with an AVR-Pik variant confirmed that Pikh and Pikm use a similar molecular mechanism to extend their pathogen recognition profile. This study shows how different NLR receptor alleles functionally converge to extend recognition specificity to pathogen effectors.

Multiple variants of the fungal effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19, providing a foundation to engineer plant defense.

Microbial plant pathogens secrete effector proteins, which manipulate the host to promote infection. Effectors can be recognized by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik effector from the rice blast fungus Magnaporthe oryzae is recognized by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a noncanonical integrated heavy-metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defenses. The host targets of AVR-Pik are also HMA-domain-containing proteins, namely heavy-metal-associated isoprenylated plant proteins (HIPPs) and heavy-metal-associated plant proteins (HPPs). Here, we demonstrate that one of these targets interacts with a wider set of AVR-Pik variants compared with the Pik-1 HMA domains. We define the biochemical and structural basis of the interaction between AVR-Pik and OsHIPP19 and compare the interaction to that formed with the HMA domain of Pik-1. Using analytical gel filtration and surface plasmon resonance, we show that multiple AVR-Pik variants, including the stealthy variants AVR-PikC and AVR-PikF, which do not interact with any characterized Pik-1 alleles, bind to OsHIPP19 with nanomolar affinity. The crystal structure of OsHIPP19 in complex with AVR-PikF reveals differences at the interface that underpin high-affinity binding of OsHIPP19-HMA to a wider set of AVR-Pik variants than achieved by the integrated HMA domain of Pik-1. Our results provide a foundation for engineering the HMA domain of Pik-1 to extend binding to currently unrecognized AVR-Pik variants and expand disease resistance in rice to divergent pathogen strains.

Gene Duplication and Mutation in the Emergence of a Novel Aggressive Allele of the AVR-Pik Effector in the Rice Blast Fungus.

Higher yield potential and greater yield stability are common targets for crop breeding programs, including those in rice. Despite these efforts, biotic and abiotic stresses continue to impact rice production. Rice blast disease, caused by Magnaporthe oryzae, is the most devastating disease affecting rice worldwide. In the field, resistant varieties are unstable and can become susceptible to disease within a few years of release due to the adaptive potential of the blast fungus, specifically in the effector (avirulence [AVR]) gene pool. Here, we analyzed genetic variation of the effector gene AVR-Pik in 58 rice blast isolates from Thailand and examined the interaction between AVR-Pik and the cognate rice resistance gene Pik. Our results reveal that Thai rice blast isolates are very diverse. We observe four AVR-Pik variants in the population, including three previously identified variants, AVR-PikA, AVR-PikD, and AVR-PikE, and one novel variant, which we named AVR-PikF. Interestingly, 28 of the isolates contained two copies of AVR-Pik, always in the combination of AVR-PikD and AVR-PikF. Blast isolates expressing only AVR-PikF show high virulence to rice cultivars encoding allelic Pik resistance genes, and the AVR-PikF protein does not interact with the integrated heavy metal-associated domain of the Pik resistance protein in vitro, suggesting a mechanism for immune evasion.

Effector target-guided engineering of an integrated domain expands the disease resistance profile of a rice NLR immune receptor.

A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effector's host targets. Direct binding of effectors to these integrated domains activates plant defenses. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defenses. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide the mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants was resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to-nature disease resistance in crops.