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 RGA5HMA2 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.
A novel glycoside hydrolases family 57 gene (gh-57) was found from a metagenomic fosmid library constructed from a black smoker chimney sample 4143-1 from the Mothra hydrothermal vent at the Juan de Fuca Ridge. Sequence and homology analysis using BLAST revealed that it had high similarity to gh-57 family. Conserved domain research revealed that the novel gh-57 contained a Glyco-hydro-57 domain and five conserved regions, including two putative catalytic residues Glu¹⁵⁴ and Asp²⁶³. The three-dimensional features of the protein and its homologue from Pyrococcus horikoshii OT3 known as α-amylase were generated by homology modeling. The gh-57 gene was cloned, expressed, and purified in Escherichia coli using pQE system. Enzyme activity revealed that the recombinant protein could hydrolyze soluble starch and demonstrated amylase activity. It showed an optimal pH of 7.5, an optimal temperature of 90 °C, and its thermostability at 90 °C could remain over 50% enzyme activity for 4 h. The enzyme activity could be increased by DTT and Mg²⁺ while an inhibitory effect was observed with EDTA, ATP, and Ca²⁺. These results showed that the gh-57 gene was a novel thermostable amylase from oceanic microorganisms.
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