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

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-1OsHIPP43). 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.

The activity of the RGA5 sensor NLR from rice requires binding of its integrated HMA domain to effectors but not HMA domain self-interaction.

The rice nucleotide-binding (NB) and leucine-rich repeat (LRR) domain immune receptors (NLRs) RGA4 and RGA5 form a helper NLR/sensor NLR (hNLR/sNLR) pair that specifically recognizes the effectors AVR-Pia and AVR1-CO39 from the blast fungus Magnaporthe oryzae. While RGA4 contains only canonical NLR domains, RGA5 has an additional unconventional heavy metal-associated (HMA) domain integrated after its LRR domain. This RGA5HMA domain binds the effectors and is crucial for their recognition. Investigation of the three-dimensional structure of the AVR1-CO39/RGA5HMA complex by X-ray crystallography identified a candidate surface for effector binding in the HMA domain and showed that the HMA domain self-interacts in the absence of effector through the same surface. Here, we investigated the relevance of this HMA homodimerization for RGA5 function and the role of the RGA5HMA effector-binding and self-interaction surface in effector recognition. By analysing structure-informed point mutations in the RGA5HMA -binding surface in protein interaction studies and in Nicotiana benthamiana cell death assays, we found that HMA self-interaction does not contribute to RGA5 function. However, the effector-binding surface of RGA5HMA identified by X-ray crystallography is crucial for both in vitro and in vivo effector binding as well as effector recognition. These results support the current hypothesis that noncanonical integrated domains of NLRs act primarily as effector traps and deepen our understanding of the sNLRs' function within NLR pairs.

Insight into the structure and molecular mode of action of plant paired NLR immune receptors.

The specific recognition of pathogen effectors by intracellular nucleotide-binding domain and leucine-rich repeat receptors (NLRs) is an important component of plant immunity. NLRs have a conserved modular architecture and can be subdivided according to their signaling domain that is mostly a coiled-coil (CC) or a Toll/Interleukin1 receptor (TIR) domain into CNLs and TNLs. Single NLR proteins are often sufficient for both effector recognition and immune activation. However, sometimes, they act in pairs, where two different NLRs are required for disease resistance. Functional studies have revealed that in these cases one NLR of the pair acts as a sensor (sNLR) and one as a helper (hNLR). The genes corresponding to such resistance protein pairs with one-to-one functional co-dependence are clustered, generally with a head-to-head orientation and shared promoter sequences. sNLRs in such functional NLR pairs have additional, non-canonical and highly diverse domains integrated in their conserved modular architecture, which are thought to act as decoys to trap effectors. Recent structure-function studies on the Arabidopsis thaliana TNL pair RRS1/RPS4 and on the rice CNL pairs RGA4/RGA5 and Pik-1/Pik-2 are unraveling how such protein pairs function together. Focusing on these model NLR pairs and other recent examples, this review highlights the distinctive features of NLR pairs and their various fascinating mode of action in pathogen effector perception. We also discuss how these findings on NLR pairs pave the way toward improved plant disease resistance.

New recognition specificity in a plant immune receptor by molecular engineering of its integrated domain.

Plant nucleotide-binding and leucine-rich repeat domain proteins (NLRs) are immune sensors that recognize pathogen effectors. Here, we show that molecular engineering of the integrated decoy domain (ID) of an NLR can extend its recognition spectrum to a new effector. We relied for this on detailed knowledge on the recognition of the Magnaporthe oryzae effectors AVR-PikD, AVR-Pia, and AVR1-CO39 by, respectively, the rice NLRs Pikp-1 and RGA5. Both receptors detect their effectors through physical binding to their HMA (Heavy Metal-Associated) IDs. By introducing into RGA5_HMA the AVR-PikD binding residues of Pikp-1_HMA, we create a high-affinity binding surface for this effector. RGA5 variants carrying this engineered binding surface perceive the new ligand, AVR-PikD, and still recognize AVR-Pia and AVR1-CO39 in the model plant N. benthamiana. However, they do not confer extended disease resistance specificity against M. oryzae in transgenic rice plants. Altogether, our study provides a proof of concept for the design of new effector recognition specificities in NLRs through molecular engineering of IDs.

A novel robust and high-throughput method to measure cell death in Nicotiana benthamiana leaves by fluorescence imaging.

Assessing immune responses and cell death in Nicotiana benthamiana leaf agro-infiltration assays is a powerful and widely used experimental approach in molecular plant pathology. Here, we describe a reliable high-throughput protocol to quantify strong, macroscopically visible cell death responses in N. benthamiana agro-infiltration assays. The method relies on measuring the reduction of leaf autofluorescence in the red spectrum upon cell death induction and provides quantitative data suitable for straightforward statistical analysis. Two different well-established model nucleotide-binding and leucine-rich repeat domain proteins (NLRs) were used to ensure the genericity of the approach. Its accuracy and versatility were compared to visual scoring of the cell death response and standard methods commonly used to characterize NLR activities in N. benthamiana. A discussion of the advantages and limitations of our method compared to other protocols demonstrates its robustness and versatility and provides an effective means to select the best-suited protocol for a defined experiment.