Identification of a Key Gain-of-Function Residue for Effector Binding by In Vitro Shuffling of Barley Mla NLR Genes.

Abstract

Natural plant populations maintain high resistance (R) gene diversities that provide effective pathogen resistance; however, agricultural crops typically contain limited R gene diversity so resistance is often short-lived as pathogens evolve rapidly to evade recognition. The Mildew resistance locus A (Mla) R gene family of barley and wheat represents a rich source of natural genetic variation that is ideal for mining disease resistance specificities. Mla R genes encode immune receptor proteins of the nucleotide-binding leucine-rich repeat (NLR) class that recognise unrelated plant pathogens by binding secreted virulence proteins termed effectors. Using DNA shuffling, we generated a variant library by recombining the barley Mla7 and Mla13 genes in vitro. The variant library was cloned into yeast generating ~4,000 independent clones and was screened for interaction with corresponding barley powdery mildew effectors AVR_A13 and AVR_A7 using a yeast-two-hybrid assay. This yielded a number of MLA protein variants that interacted with AVR_A13. Sequences of the interacting MLA variants can be clustered into three groups, all of which contain a critical residue from MLA13. While MLA13 and MLA7 differ by 30 residues across the LRR domain, the replacement of leucine to serine at this position in MLA7 is necessary and sufficient for interaction with AVR_A13 in yeast and AVR_A13-dependent immune signalling in planta. We have established a pipeline that evolves MLAs to recognise distinct pathogen effectors without the requirement for protein structural knowledge and the use of rational design. We suggest these findings represent a step towards evolving novel recognition capabilities rapidly in vitro.