Plant diseases caused by pathogens and pests are a constant threat to global food security. Direct crop losses and the measures used to control disease (e.g. application of pesticides) have significant agricultural, economic, and societal impacts. Therefore, it is essential that we understand the molecular mechanisms of the plant immune system, a system that allows plants to resist attack from a wide variety of organisms ranging from viruses to insects. Here, we provide a roadmap to plant immunity, with a focus on cell-surface and intracellular immune receptors. We describe how these receptors perceive signatures of pathogens and pests and initiate immune pathways. We merge existing concepts with new insights gained from recent breakthroughs on the structure and function of plant immune receptors, which have generated a shift in our understanding of cell-surface and intracellular immunity and the interplay between the two. Finally, we use our current understanding of plant immunity as context to discuss the potential of engineering the plant immune system with the aim of bolstering plant defenses against disease.

Bioengineering of plant immune receptors has emerged as a key strategy for generating novel disease resistance traits to counteract the expanding threat of plant pathogens to global food security. However, current approaches are limited by rapid evolution of plant pathogens in the field and may lack durability when deployed. Here, we show that the rice nucleotide-binding, leucine-rich repeat (NLR) immune receptor Pik-1 can be engineered to respond to a conserved family of effectors from the multihost blast fungus pathogen Magnaporthe oryzae . We switched the effector binding and response profile of the Pik NLR from its cognate rice blast effector AVR-Pik to the host-determining factor pathogenicity toward weeping lovegrass 2 (Pwl2) by installing a putative host target, OsHIPP43, in place of the native integrated heavy metal–associated domain (generating Pikm-1 ^OsHIPP43 ). This chimeric receptor also responded to other PWL alleles from diverse blast isolates. The crystal structure of the Pwl2/OsHIPP43 complex revealed a multifaceted, robust interface that cannot be easily disrupted by mutagenesis, and may therefore provide durable, broad resistance to blast isolates carrying PWL effectors in the field. Our findings highlight how the host targets of pathogen effectors can be used to bioengineer recognition specificities that have more robust properties compared to naturally evolved disease resistance genes.

Factors including climate change and increased global exchange are set to escalate the prevalence of plant diseases, posing an unprecedented threat to global food security and making it more challenging to meet the demands of an ever-growing population. As such, new methods of pathogen control are essential to help with the growing danger of crop losses to plant diseases. The intracellular immune system of plants utilizes nucleotide-binding leucine-rich repeat (NLR) receptors to recognize and activate defense responses to pathogen virulence proteins (effectors) delivered to the host. Engineering the recognition properties of plant NLRs toward pathogen effectors is a genetic solution to plant diseases with high specificity, and it is more sustainable than several current methods for pathogen control that frequently rely on agrochemicals. Here, we highlight the pioneering approaches toward enhancing effector recognition in plant NLRs and discuss the barriers and solutions in engineering the plant intracellular immune system.

Plant NLR immune receptors are multidomain proteins that can function as specialized sensor/helper pairs. Paired NLR immune receptors are generally thought to function via negative regulation, where one NLR represses the activity of the second and detection of pathogen effectors relieves this repression to initiate immunity. However, whether this mechanism is common to all NLR pairs is not known. Here, we show that the rice NLR pair Pikp-1/Pikp-2, which confers resistance to strains of the blast pathogen Magnaporthe oryzae (syn. Pyricularia oryzae) expressing the AVR-PikD effector, functions via receptor cooperation, with effector-triggered activation requiring both NLRs to trigger the immune response. To investigate the mechanism of Pikp-1/Pikp-2 activation, we expressed truncated variants of these proteins, and made mutations in previously identified NLR sequence motifs. We found that any domain truncation, in either Pikp-1 or Pikp-2, prevented cell death in the presence of AVR-PikD, revealing that all domains are required for activity. Further, expression of individual Pikp-1 or Pikp-2 domains did not result in cell death. Mutations in the conserved P-loop and MHD sequence motifs in both Pikp-1 and Pikp-2 prevented cell death activation, demonstrating that these motifs are required for the function of the two partner NLRs. Finally, we showed that Pikp-1 and Pikp-2 associate to form homo- and hetero-complexes in planta in the absence of AVR-PikD; on co-expression the effector binds to Pikp-1 generating a tri-partite complex. Taken together, we provide evidence that Pikp-1 and Pikp-2 form a fine-tuned system that is activated by AVR-PikD via receptor cooperation rather than negative regulation.

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 rice blast fungus Magnaporthe oryzae secretes a battery of effector proteins to facilitate host infection. Among these effectors, pathogenicity toward weeping lovegrass 2 (Pwl2) was originally identified as a host specificity determinant for the infection of weeping lovegrass (Eragrostis curvula) and is also recognized by the barley (Hordeum vulgare) Mla3 resistance protein. However, the biological activity of Pwl2 remains unknown. Here, we showed that the Pmk1 MAP kinase regulates PWL2 expression during the cell-to-cell movement of M. oryzae at plasmodesmata-containing pit fields. Consistent with this finding, we provided evidence that Pwl2 binds to the barley heavy metal–binding isoprenylated protein HIPP43, which results in HIPP43 displacement from plasmodesmata. Transgenic barley lines overexpressing PWL2 or HIPP43 exhibit attenuated immune responses and increased disease susceptibility. In contrast, a Pwl2SNDEYWY variant that does not interact with HIPP43 fails to alter the plasmodesmata localization of HIPP43. Targeted deletion of 3 PWL2 copies in M. oryzae resulted in a Δpwl2 mutant showing gain of virulence toward weeping lovegrass and barley Mla3 lines, but reduced blast disease severity on susceptible host plants. Taken together, our results provide evidence that Pwl2 is a virulence factor that suppresses host immunity by perturbing the plasmodesmatal deployment of HIPP43.