Identification of a key gain-of-function residue for effector binding by in vitro shuffling of barley Mla NLR genes

Natural plant populations maintain high resistance ( R ) gene diversities that provide effective pathogen resistance; however, such diversity has been reduced significantly by the genetic bottlenecks associated with plant domestication and breeding. 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. The barley NLRs MLA13 and MLA7 confer resistance to different strains of the barley powdery mildew pathogen through direct interaction with the pathogen effectors AVR _A13 and AVR _A7 respectively. Using DNA shuffling, we generated a variant library by recombining the Mla7 and Mla13 genes in vitro . The variant library was cloned into yeast generating ∼4,000 independent clones and was screened for interaction with AVR _A13 and AVR _A7 using a yeast-two-hybrid (Y2H) assay. This yielded a number of clones that encode NLR proteins that interacted with AVR _A13 . Sequence analysis showed that the interacting MLA proteins 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 facilitated 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 rationale design. We suggest these findings represent a step towards evolving novel recognition capabilities rapidly in vitro .