Yang Liu, Xin Zhang, Guixin Yuan, Dongli Wang, Yangyang Zheng, Mengqi Ma, Liwei Guo, Vijai Bhadauria, You-Liang Peng, Junfeng Liu

Proc Natl Acad Sci U S A (PNAS); 2021 Nov 2; 118(44): e2110751118.  

doi: 10.1073/pnas.2110751118.

Abstract

Plant nucleotide-binding and leucine-rich repeat (NLR) receptors recognize avirulence effectors directly through their integrated domains (IDs) or indirectly via the effector-targeted proteins. Previous studies have succeeded in generating designer NLR receptors with new recognition profiles by engineering IDs or targeted proteins based on prior knowledge of their interactions with the effectors. However, it is yet a challenge to design a new plant receptor capable of recognizing effectors that function by unknown mechanisms. Several rice NLR immune receptors, including RGA5, possess an integrated heavy metal-associated (HMA) domain that recognizes corresponding Magnaporthe oryzae Avrs and ToxB-like (MAX) effectors in the rice blast fungus. Here, we report a designer rice NLR receptor RGA5^HMA2 carrying an engineered, integrated HMA domain (RGA5-HMA2) that can recognize the noncorresponding MAX effector AvrPib and confers the RGA4-dependent resistance to the M. oryzae isolates expressing AvrPib, which originally triggers the Pib-mediated blast resistance via unknown mechanisms. The RGA5-HMA2 domain is contrived based on the high structural similarity of AvrPib with two MAX effectors, AVR-Pia and AVR1-CO39, recognized by cognate RGA5-HMA, the binding interface between AVR1-CO39 and RGA5-HMA, and the distinct surface charge of AvrPib and RAG5-HMA. This work demonstrates that rice NLR receptors with the HMA domain can be engineered to confer resistance to the M. oryzae isolates noncorresponding but structurally similar MAX effectors, which manifest cognate NLR receptor-mediated resistance with unknown mechanisms. Our study also provides a practical approach for developing rice multilines and broad race spectrum-resistant cultivars by introducing a series of engineered NLR receptors.

See https://pubmed.ncbi.nlm.nih.gov/34702740/

Fig. 3.

RGA5-HMA2 recognizes AvrPib in vitro and in vivo. (A) Y2H assays showing the specific interactions of RGA5-HMA domain (residues 982 to 1,116) with AVR-Pia and RGA5-HMA2 domain (residues 982 to 1,116) with AvrPib. (B) MBP pull-down assays showing the specific interaction of the RGA5-HMA domain with AVR-Pia and the RGA5-HMA2 domain with AvrPib. The recombinant proteins MBP-RGA5-HMA, MBP-RGA5-HMA2, HA-AvrPib, and HA-AVR-Pia purified from E. coli were used for the MBP pull-down analysis. The fusion proteins were detected using the anti-HA and anti-MBP antibodies. (C) MST analysis showing the dissociation constants of AvrPib and AVR-Pia with the RGA5-HMA or RGA5-HMA2 domain. The experiment was repeated three times. Bars ± SD (n = 3). (D) Co-IP of RGA5^HMA2 (full-length RGA5 with the integrated engineered HMA domain [HMA2]) with AvrPib. HA-RGA5^HMA2 and GFP-AvrPib were transiently coexpressed in N. benthamiana leaves, and the proteins extracted from the leaves were incubated with GFP beads and detected separately by the anti-HA and anti-GFP antibodies.