![]() This RGA5HMA domain binds the effectors and is crucial for their recognition. While RGA4 contains only canonical NLR domains, RGA5 has an additional unconventional heavy metal‐associated (HMA) domain integrated after its LRR domain. 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. Two independently purified protein samples were used to test the binding of the AVR-PikD effector to the different HMAs. Bars and error bars represent the mean and average deviation calculated for the %Rmax values estimated from n = 2 independent experiments carried out on two different days (open green and red circles) wit n = 1 to n = 4 technical replicates per experiment. d Comparison of the binding response (bound fraction) at 1 µM of AVR effectors, expressed as the percentage of the theoretical maximum response (%Rmax) normalized for the amount of MBP:HMA immobilized on the chip and corrected for the MBP-tag contribution. The binding curves obtained with the wild-type and an inactive variant of AVR1-CO39 (a) or AVR-Pia (b) are shown in the top and lower insets, respectively. Superimposed sensorgrams are shown for wild-type RGA5_HMA (green), RGA5_HMAm1 (black), RGA5_HMAm1m2 (blue), Pikp-1_HMA (red), as well as for MBP alone (gray) that serves as a negative control. a-c AVR1-CO39 (a), AVR-Pia (b), and AVR-PikD (c) were injected (black arrows) at 1 µM for 2 min on the different MBP:HMA fusion proteins captured by anti-MBP antibody immobilized on the chip. Here the authors show that the integrated decoy domain of the rice NLR RGA5 can be engineered to trigger immune responses upon binding a non-cognate effector.Įngineered RGA5_HMAs bind strongly to AVR-PikD in vitro. Plant NLR proteins trigger immune responses upon recognition of pathogen effectors. Altogether, our study provides a proof of concept for the design of new effector recognition specificities in NLRs through molecular engineering of IDs. However, they do not confer extended disease resistance specificity against M. 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. By introducing into RGA5_HMA the AVR-PikD binding residues of Pikp-1_HMA, we create a high-affinity binding surface for this effector. Both receptors detect their effectors through physical binding to their HMA (Heavy Metal-Associated) IDs. 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. Here, we show that molecular engineering of the integrated decoy domain (ID) of an NLR can extend its recognition spectrum to a new effector. Plant nucleotide-binding and leucine-rich repeat domain proteins (NLRs) are immune sensors that recognize pathogen effectors. ![]()
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