Validation of new salicylic acid binding proteins using a photoactivable analogue

There are several hormones in plants such as ethylene, auxin and salicylic acid (SA) that regulate many physiological processes. SA is an essential signaling molecule in plant development. It is also critical for plant innate immunity and the defense against pathogens. SA is required for the expression of pathogen-related genes and the development of systemic acquired resistance. Despite this central role of SA in plants, very few SA binding proteins (SABPs) are currently known. In an effort to identify new SABPs, the Klessig lab used Arabidopsis protein microarrays to probe for them. My project aimed to validate that those putative SABPs do indeed bind SA. This validation will help understand the effectiveness and limitations of the protein microarray method to find small molecule binding proteins.

My Experience

I transiently expressed and purified Arabidopsis proteins in N. benthaminana. Using molecular tools such as SDS-Polyacrilamide gel electrophoresis and western blots, I confirmed the purity of the extracted proteins. To test for SA binding, I used an SA analog, 4-azidoSA, which is able to crosslink the putative SABP to SA. The protein-SA complex is then probed using an anti-SA antibody. This method allows us to differentiate true SABPs from false positives resulting from the protein microarray screening. We managed to validate 4 SABPs as true positives.