Although I originally proposed a screen in the model plant Arabidopsis thaliana of lines, which inducibly overexpress a single transcription factor each, this did not prove feasible. However, I was able to investigate the same questions through alternate means; namely by a broad-scale and fine-resolution assay of transcriptional responses to elicitors. Elicitors include both the PAMPs described above and damage-associated molecular patterns, both of which can be associated with disease. Previous work has suggested that many elicitors induce similar responses in plants, but as most studies focus on one elicitor and time point, it wasn’t clear whether elicitor differences were real or the result of different experimental conditions, and the timing of response was similarly not defined. The transcriptional work I performed allowed me to address these questions as well as, through promoter analysis, identifying transcription factors both known and novel which seem to play important roles in the regulation of elicitor responses.
During my MSCA Fellowship, I conducted a time-course experiment examining transcriptional responses to seven different elicitors over a range of time points within three hours post elicitor treatment. These elicitors represent a range of different classes, including elicitors derived from different types of pathogens and elicitors recognized by different mechanisms within the plant. Despite the diversity of elicitors, I found that all induced a large core set of genes, particularly at the earliest assayed time points. Indeed, at early time points the plant’s response to elicitors was similar to its early response to many abiotic environmental stresses like heat, extreme light, or drought. This indicates that the plant’s rapid response is dominated by a general stress response – a similar phenomenon has been shown in yeast, and explored in plants, though not with this degree of temporal resolution. In contrast, at late time points, responses become more specific. One elicitor induced a large number of genes not induced by any other tested elicitors, indicating perhaps a specific response or a greater sensitivity to this treatment. Additionally, even at later time points, I could identify a small core set of genes induced by all elicitors, and by no abiotic stresses, potentially indicating a PTI-universal and PTI-specific response. For all these classes of response, I used publically-available databases of transcription factor binding sites to identify transcription factors that preferentially bind the genes upregulated in each pattern. I am currently testing these implicated TFs, and have already discovered at least one regulator of the general stress response that is indeed necessary for plant resistance triggered by elicitor perception.
The RNAseq approach was slower to identify transcription factors than the originally proposed screen, and accordingly did not allow sufficient time for the more specific study proposed for transcription factor regulation and specific targets. However, the large-scale RNAseq experiment necessitated the development and refinement of several protocols for RNA extraction, sequencing library preparation, and data analysis, and I am currently preparing a protocol paper describing this process in collaboration with another lab at TSL. Finally, I have begun to investigate the effect of known PTI signaling mechanisms on the identified general stress, PTI-specific, and elicitor-specific gene sets, and this will continue past the Fellowship period, to complete a story for publication.
