Date of this Version
UCARE Poster session, University of Nebraska-Lincoln Research Fair, April 2016, Lincoln, NE.
I had the great privilege of working with Dr. Karin van Dijk, through UCARE, researching the specific pathways through which histone deacetylation occurs in plants during the 2015-2016 academic year. My first year conducting research helped me develop my technical skills and learn about experimental protocols and how to conduct them. I would like to continue my research during the 2016-2017 academic year and now that I have a year of research experience, be able to further develop my skills and learn more about my research project.
Pseudomonas syringae is a bacterial pathogen that is well known for causing bacterial speck disease in various hosts, including the economically relevant crops, tomato and soybean. One of the primary mechanisms used by P. syringae to cause disease is the injection of a plethora of effector proteins via the type 3 secretion system (T3SS) into the plant host cells. Although we know these proteins collectively enable the pathogen to cause disease by primarily disabling or subverting immune defenses, the specific details of how each effector protein does this is not very well understood for the majority of effector proteins. However, we do know that very quickly after infection, changes in host gene expression can be detected. Studies in the laboratory of Dr. Karin van Dijk found that there is a rapid deacetylation of host histone H3 lysine 9 (H3K9) upon infection with P. syringae. The deacetylation of H3k9ac was found to be located along a number of innate immune genes, and a correlative reduction of gene expression was observed as well. This reduced acetylation was found to depend on a functional T3SS and the effectors traveling through it. We believe this effector-driven deacetylation of H3K9 is involved in the impairment of the plants immune response to the pathogen, which enables the pathogen to cause disease. The purpose of this research is to investigate the molecular mechanism by which the effector-dependent deacetylation occurs. It is possible the type III effectors (T3E) trigger an expression change in one or more known histone acetyltransferases (HATs) and/or histone deacetylases (HDACs). To begin to explore this, we have performed qRT-PCR on plant samples exposed to the pathogen or not to measure expression levels of several known HATs and HDACs. We found several HDACs that are upregulated in plants exposed to the pathogen and will focus our research on these. This research is important because it will allow us to help identify the mechanism by which P. syringae suppresses immunity and thus cause disease in staple crops like soybeans. This may help aid in the development of products to protect these plants from infection.
We hypothesize that the effector-driven deacetylation occurs through upregulation of a plant-encoded Histone deacetylase that deacetylases H3K9ac. I will be working on this research in cooperation with another undergraduate student, who is approaching this research from the perspective of bacterial effector proteins. I will not work directly with her, but our two research questions overlap and the findings from each study will help in understanding our own projects.
For this research I will use T-DNA lines of Arabidopsis defective in specific HDACs. I will analyze these plants for their ability/inability to deacetylate H3K9ac along innate immune genes once exposed to the pathogen. I will also use these plants in pathogenicity assays to determine their susceptibility to P. syringae. I will begin this experiment by growing wildtype and mutant Arabidopsis plants under similar conditions to allow for as little growth deviation as possible. Once the plants are fully grown, I will separate the plants into different groups. One group will be the control group and will get infiltrated with a buffer solution. Another group will be the group of plants exposed to wild type P. syringae. The last group will be exposed to a mutant strain of P. syringae unable to produce the T3SS. If used in pathogenicity assays, growth of the bacterial strains will be followed over a 6-day period by plating leaf samples on media selective for P. syringae. To analyze chromatin deacetylation, we will use two different techniques, immunoblotting and Chromatin Immunoprecipitation combined with quantitative PCR (ChIP-qPCR). After exposure, the leaves from each of the plants in each group will be harvested. For the samples that will be analyzed by immunoblotting, we will flash freeze these leaves in liquid nitrogen and store the tissue at -80 until use. For the ChIP-qPCR analysis, Chromatin will be cross-linked with formaldehyde and the leaves flash frozen and stored at -80 until use.
For the data analysis, we will use immunoblot analysis of leaf tissue to determine if the mutant plants can still deacetylate H3K9ac upon P. syringae infection. We will separate proteins isolated from leaf tissue on SDS-PAGE gels, blot the proteins onto membranes and use anti-H3K9ac antibodies and anti-H3 antibodies to detect H3 acetylateion at K9 and total H3 acetylation, respectively. We will use densitometry to determine relative acetylation of H3K9. This will allow us to compare acetylation levels between the different Arabidopsis lines. I will use ChIP-qPCR in order to determine if deacetylation happened along innate immune genes. To do so, chromatin will be prepared from frozen cross-linked plant tissues. Next the chromatin will be sheared into small fragments (averaging 500 bp) and precipitated using either anti-H3K9ac or anti-H3 antibodies. Precipitated chromatin will be decross-linked and the released DNA will be analyzed with qPCR using primers to amplify specific innate immune genes. By comparing relative PCR product amounts between strains, I will be able to determine if there is a change in the H3K9 acetylation along these innate immune genes in the mutant Arabidopsis lines relative to the wildtype.
- Plant Arabidopsis plants
- Conduct library research/literature review
- Conduct research (gather data)
- Inject plants with P. syringae
- Collect leaves
- Prepare pathogenicity assays
- Perform immunoblot analysis on the three groups of leaves
- Use ChIP-qPCR on the three leaf groups
- Analyze data
- Determine acetylation of H3K9ac after infection of P. syringae in all three plant groups using densitrometry
- Determine if deacetylation occurred from ChIP-qPCR
- Present findings at Nebraska Academy of Science
- Present findings at UCARE presentation
Some of the benchmarks mentioned above have been achieved during the 2015-2016 academic year through UCARE, such as conducting research by injecting plants with P. syringae, collecting leaves, preparing pathogenicity assays, performing immunoblot analysis on the three groups of leaves, and also determining the acetylation of H3K9ac after infection of P. syringae in all three plant groups using densitrometry. During the academic year I hope to continue with my research by performing ChIP-qPCR on our leaf samples and analyzing the data to determine if deacetylation has occurred.