Plant protoplast-based assay to screen for salicylic acid response-modulating bacteria

Abstract

Leaves host remarkably diverse microbes, collectively referred to as the leaf microbiota. While many beneficial functions have been attributed to the plant microbiota, the individual contributions of leaf-colonising bacteria range from pathogenic to mutualistic interactions. Omics approaches demonstrated that some leaf-colonising bacteria evoke substantial changes in gene expression and metabolic profiles in the plant host, including plant immunity. While omic approaches provide a system level view on cellular functions, they are costly and laborious, thereby severely limiting the throughput of the number of bacterial strains that can be tested in planta. To enable cost-effective high-throughput screens, we have developed a plant protoplast-based assay to measure real-time target gene expression changes following bacterial inoculation. Here, protoplasts were isolated from leaves of stable transgenic plants containing a pPR1:eYFP-nls construct. Changes in yellow fluorescence were captured for up to 96 treatments using a plate reader. This allowed the monitoring of changes in the salicylic acid-dependent plant immune response over time. Protoplast isolation per se evoked mild fluorescence responses, likely linked to endogenous salicylic acid production. This is advantageous in a bacterial assay, as bidirectional changes in PR1 expression can be measured. Plate reader-generated data were validated via fluorescence microscopy and RT-qPCR. Fluorescence microscopy further demonstrated heterogeneity in the response of individual protoplasts, which is potentially linked to differences in cell-type. In summary, the protoplast assay is an affordable and easily up-scalable way of measuring changes in target gene expression to bacterial colonisation.