| Popis: |
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 testedin 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. |
