Convergent Evolution of Effector Protease Recognition by Arabidopsis and Barley.

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.

Clonal Amplification-Enhanced Gene Expression in Synthetic Vesicles.

In cell-free gene expression, low input DNA concentration severely limits the phenotypic output, which may impair in vitro protein evolution efforts. We address this challenge by developing CADGE, a strategy that is based on clonal isothermal amplification of a linear gene-encoding dsDNA template by the minimal Φ29 replication machinery and in situ transcription-translation. We demonstrate the utility of CADGE in bulk and in clonal liposome microcompartments to boost up the phenotypic output of soluble and membrane-associated proteins, as well as to facilitate the recovery of encapsulated DNA. Moreover, we report that CADGE enables the enrichment of a DNA variant from a mock gene library via either a positive feedback loop-based selection or high-throughput screening. This new biological tool can be implemented for cell-free protein engineering and the construction of a synthetic cell.

Imaging Flow Cytometry for High-Throughput Phenotyping of Synthetic Cells.

The reconstitution of basic cellular functions in micrometer-sized liposomes has led to a surge of interest in the construction of synthetic cells. Microscopy and flow cytometry are powerful tools for characterizing biological processes in liposomes with fluorescence readouts. However, applying each method separately leads to a compromise between information-rich imaging by microscopy and statistical population analysis by flow cytometry. To address this shortcoming, we here introduce imaging flow cytometry (IFC) for high-throughput, microscopy-based screening of gene-expressing liposomes in laminar flow. We developed a comprehensive pipeline and analysis toolset based on a commercial IFC instrument and software. About 60 thousands of liposome events were collected per run starting from one microliter of the stock liposome solution. Robust population statistics from individual liposome images was performed based on fluorescence and morphological parameters. This allowed us to quantify complex phenotypes covering a wide range of liposomal states that are relevant for building a synthetic cell. The general applicability, current workflow limitations, and future prospects of IFC in synthetic cell research are finally discussed.