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

Abstract

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) 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 defence responses that are deleterious to the plant. This may be due to plant NLRs associating in highly complex signalling networks that co-evolve 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 co-evolved, and mismatching between non-co-evolved 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 co-evolved 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.

Development of an NLR-ID Toolkit and Identification of Novel Disease-Resistance Genes in Soybean

“…The NLR-ID gene has become a key target for NLR engineering due to its ability to recognize various pathogen effectors via their varying IDs. While the HMA domain represents the model ID in plant immunity [ 82 , 83 ] and has been engineered to mediate broad pathogen resistance [ 84 , 85 , 86 ], the mining and detailed analysis of other IDs in atypical NLR proteins could help us to design more specialized NLRs to cope with rapidly evolving pathogens. Therefore, our study provides a toolkit for the functional study of the IDs in plant NLR proteins, and the broad-spectrum antiviral gene SRZ4 reported here might have significant application value.…”

A root-specific NLR network confers resistance to plant parasitic nematodes

“…Cell death phenotypes were scored with a range from 0 (no visible necrosis) to 7 (fully confluent necrosis) according to Adachi et al, 2019a). Quantification and statistical analysis was performed by using the besthr R library (MacLean, 2019) and plotted using a script described in Bentham et al, 2023. Scoring for all experiments can be found in Supplementary Dataset 4.…”

Bioengineering a plant NLR immune receptor with a robust binding interface towards a conserved fungal pathogen effector

“…Structure-guided bioengineering of the effector-binding regions of NLRs is a promising mechanism to modify receptor recognition specificity (4)(5)(6)(7)(8). Editing or domain-swapping of non-canonical integrated domains found in some NLRs has been particularly effective for either expanding or altering the effector response profiles of these immune receptors (4,7,(9)(10)(11)(12)(13).…”

“…The RGA5-HMA domain has been engineered to gain binding to non-corresponding MAX effectors including AVR-PikD and AVR-Pib (4,9,12,13). Importantly, to achieve full resistance to AVR-Pib in cereals, the engineered full-length RGA5/RGA4 receptor pair required additional modification of the C-terminal region that directly follows the HMA domain in the RGA5 receptor (12,13).…”