Periodic Reporting for period 1 - transcriPTIon (Understanding transcriptional regulation in plant PAMP-triggered immunity)
Berichtszeitraum: 2016-09-21 bis 2018-09-20
Like animals, plants possess an immune system, capable of fending off most pathogens. However, like animals, the plant immune system can fail to suppress some infections, leading to disease. In the case of agricultural crop production, these diseases reduce yields, sometimes estimated up to 25% worldwide. Agricultural pathogens and the insects that spread them can be controlled by chemical sprays, but these sprays can have detrimental effects on the ecosystem and frequently are quickly overcome by resistant pathogens. Integrated pest management systems seek to work with the environment and the plant’s own immune system, but in order to effectively manage and even improve plants’ immune systems, we must understand why and how they work.
Significant work towards this problem has already been done, revealing an intricate system in which plants first recognize the presence of microbes through plant cell membrane-bound pattern recognition receptors (PRRs), which recognize conserved core microbial signatures, pathogen-associated molecular patterns (PAMPs). This recognition triggers a series of responses in the plant, which culminate in increased resistance to infection, called pattern-triggered immunity (PTI). Although some pathogens can suppress PTI, leading to further plant-microbe biochemical warfare, PTI is frequently enough to reduce or eliminate disease appearance in plants.
One of the core aspects of PTI is a massive transcriptional reprogramming, in which the plant changes which genes are expressed/not expressed, and thus which proteins are present/not present. Although this transcriptional reprogramming has been studied for over a decade, many questions remain unanswered, such as the complement and nature of the transcription factors which control it. The transcriPTIon project was conceived to identify core PTI-regulating TFs, the upstream mechanisms by which they themselves were regulated, and the specific downstream genes each controlled.
I have investigated these questions through a large RNAseq experiment, using an unprecedented diversity of PAMPs and resolution of timing. Through this I have identified previously unexplored aspects of PTI-associated transcription, including identification of several transcription factors proven or implicated to have key roles in PTI.
Significant work towards this problem has already been done, revealing an intricate system in which plants first recognize the presence of microbes through plant cell membrane-bound pattern recognition receptors (PRRs), which recognize conserved core microbial signatures, pathogen-associated molecular patterns (PAMPs). This recognition triggers a series of responses in the plant, which culminate in increased resistance to infection, called pattern-triggered immunity (PTI). Although some pathogens can suppress PTI, leading to further plant-microbe biochemical warfare, PTI is frequently enough to reduce or eliminate disease appearance in plants.
One of the core aspects of PTI is a massive transcriptional reprogramming, in which the plant changes which genes are expressed/not expressed, and thus which proteins are present/not present. Although this transcriptional reprogramming has been studied for over a decade, many questions remain unanswered, such as the complement and nature of the transcription factors which control it. The transcriPTIon project was conceived to identify core PTI-regulating TFs, the upstream mechanisms by which they themselves were regulated, and the specific downstream genes each controlled.
I have investigated these questions through a large RNAseq experiment, using an unprecedented diversity of PAMPs and resolution of timing. Through this I have identified previously unexplored aspects of PTI-associated transcription, including identification of several transcription factors proven or implicated to have key roles in PTI.
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.
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.
TranscriPTIon has already expanded our understanding of plant responses to pathogens and stands poised to reveal more beyond the end of the project. Specifically, the extent and timing of the common PTI- and abiotic-stress responses has never more clearly been defined. This has implications in basic science for understanding how plants understand their environment and make the transition from the general stress response to stress-specific responses. In a similar vein, the PTI-specific response and elicitor-specific responses could not have been identified by previous approaches. I am currently testing the importance of PTI-specific genes in plant resistance induced by elicitors, and preliminary results suggest that at least some PTI-specific genes are required for plant elicitor-induced resistance. This has implications both in basic science for the understanding of which biochemical pathways underlie plant disease resistance, but also for practical applications, to tune these pathways for improved plant resistance. Finally, the identification of transcription factors regulating general, PTI-specific, and elicitor-specific responses, and the roles of these in plant immunity represents both an avenue to better understand how plant immune responses are mediated, and a central lever to access and improve plant disease resistance, agricultural robustness, and food yield for society.