The Pseudomonas syringae cysteine protease AvrPphB activates the Arabidopsis resistance protein RPS5 by cleaving a second host protein, PBS1. AvrPphB induces defense responses in other plant species, but the genes and mechanisms mediating AvrPphB recognition in those species have not been defined. Here, we show that AvrPphB induces defense responses in diverse barley cultivars. We also show that barley contains two PBS1 orthologs, that their products are cleaved by AvrPphB, and that the barley AvrPphB response maps to a single locus containing a nucleotide-binding leucine-rich repeat (NLR) gene, which we termed AvrPphB Response 1 (Pbr1). Transient coexpression of PBR1 with wild-type AvrPphB but not with a protease inactive mutant triggered defense responses, indicating that PBR1 detects AvrPphB protease activity. Additionally, PBR1 coimmunoprecipitated with barley and Nicotiana benthamiana PBS1 proteins, suggesting mechanistic similarity to detection by RPS5. Lastly, we determined that wheat cultivars also recognize AvrPphB protease activity and contain two putative Pbr1 orthologs. Phylogenetic analyses showed, however, that Pbr1 is not orthologous to RPS5. Our results indicate that the ability to recognize AvrPphB evolved convergently and imply that selection to guard PBS1-like proteins occurs across species. Also, these results suggest that PBS1-based decoys may be used to engineer protease effector recognition–based resistance in barley and wheat.

The Pseudomonas syringae cysteine protease AvrPphB activates the Arabidopsis resistance protein RPS5 by cleaving a second host protein, PBS1. AvrPphB induces defense responses in other plant species, but the genes and mechanisms mediating AvrPphB recognition in those species have not been defined. Here, we show that AvrPphB induces defense responses in diverse barley cultivars. We show also that barley contains two PBS1 orthologs, that their products are cleaved by AvrPphB, and that the barley AvrPphB response maps to a single locus containing a nucleotide-binding leucinerich repeat (NLR) gene, which we termed AvrPphB Resistance 1 (Pbr1). Transient co-expression of PBR1 with wild-type AvrPphB, but not a protease inactive mutant, triggered defense responses, indicating that PBR1 detects AvrPphB protease activity. Additionally, PBR1 co-immunoprecipitated with barley and N. benthamiana PBS1 proteins, suggesting mechanistic similarity to detection by RPS5. Lastly, we determined that wheat cultivars also recognize AvrPphB protease activity and contain a Pbr1 ortholog. Phylogenetic analyses showed however that Pbr1 is not orthologous to RPS5. Our results indicate that the ability to recognize AvrPphB evolved convergently, and imply that selection to guard PBS1-like proteins is ancient. Also, the results suggest that PBS1-based decoys may be used to engineer protease effector recognition-based resistance in barley and wheat.

Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems.