Engineering of Sr33 and Sr50 plant immune receptors to alter recognition specificity and autoactivity

Abstract

Plants possess cytoplasmic immune receptors called nucleotide-binding leucine-rich repeat receptors (NLRs) that recognize the presence of a pathogen through a range of mechanisms: direct binding of effectors or indirect recognition of effector actions. The direct binding of effectors has been shown to be mediated through the NLR’s leucine-rich repeat (LRR) domain. Accurate prediction of amino acids involved in these direct interactions can greatly enhance understanding of effector recognition and inform efforts to engineer new resistance. In this study, we utilized two homologous NLR resistance genes from wheat, Sr33 and Sr50, that recognize distinct effectors by directly binding to them through their LRR domain. While the effector recognized by Sr50 is known and described as AvrSr50, the effector recognized by Sr33 remains unknown. Through a combination of phylogenetics, allele diversity analysis in the LRR and structural modeling, we identified the amino acids in Sr50 likely to physically interact with its effector. Mutation of these sites helped identify 12 amino acids we hypothesized to be sufficient to mediate effector binding in Sr50. Changing these 12 corresponding amino acids in Sr33, we showed AvrSr50-dependent initiation of cell death in wheat protoplasts and Nicotiana benthamiana. Furthermore, we were able to pinpoint and change amino acid residues that govern autoactivity of Sr50 in the wheat protoplast cell death assay. These findings are a major advance towards the successful engineering of new effector recognition specificities in direct binder NLRs.

The effector recognition by synthetic sensor NLR receptors requires the concerted action of multiple interfaces within and outside the integrated domain

“…Pikm1 HMA ID with nanobodies of fluorescent proteins, which could recognize antigen fluorescent proteins and trigger immunity in N. benthamiana 25 . Tamborski et al created a mutant of wheat NLR receptor Sr33 capable of recognizing the avirulence effector AvrSr50 from stem rust by switching amino acid residues in the LRR domain with the AvrSr50-binding residues in Sr50 26 . However, the functionality of these designer NLRs remains to be verified in their host plants.…”

Majority of the highly variable NLRs in maize share genomic location and contain additional target-binding domains

“…These specificity-determining residues characteristically cluster on the concave side of the LRR domain. Targeted modification of these residues might lead to derivation of novel immune specificities in the native maize NLRs, as demonstrated for wheat disease resistance genes Sr33, Sr50 (Tamborski et al, 2022), and Sr35 (Förderer et al, 2022) thus overcoming the relative lack of natural diversity. Despite the differences in exact positions of the candidate binding residues among hvNLRs, our results agree with the inference by Förderer et al that target binding on the inside of the LRR favors an open NB-ARC conformation in an activation mechanism shared among distantly related direct-recognition NLRs.…”

Direct recognition of pathogen effectors by plant NLR immune receptors and downstream signalling

“…A wheat homolog of MLA, Sr50, also directly recognizes its corresponding effector AvrSr50 from the wheat stem rust fungus [ 39 ]. Some AvrSr50 variants from virulent rust isolates escape Sr50 binding via one or a few mutations on the effector surface [ 40 ], while a central region of the LRR is the main contributor to its recognition specificity [ 41 ]. Likewise, recognition between the Arabidopsis NLR RPP1 and the downy mildew effector ATR1 involves interaction of effector surface residues with the C-terminal region of RPP1 including the LRR [ 42 , 43 ].…”