2013 Annual Report
1a.Objectives (from AD-416):
1: Describe key bacterial pathways involved in disease establishment and progression.
1A: Characterize the role of ncRNAs and the RNA binding proteins Crc and Hfq in pathogenesis.
1B: Identify ECF sigma factor regulons and determine their role(s) in plant interactions.
2: Identify bacterial transcriptional responses to plant signals and defense systems in planta.
2A: In vitro, examine bacterial transcriptional responses to conditions thought to be relevant in planta.
2B: Examine bacterial transcription during plant infection.
2C: Evaluate each gene in P. syringae for its contribution to fitness in plants.
2D: Develop systems models of pathogen-plant interactions.
1b.Approach (from AD-416):
Bacterial plant pathogens are responsible for major losses in nearly all crops. Attempts to develop resistance in host plant species have been hindered by a lack of understanding of the complex network of plant-microbe interactions. A central problem is that the pathways used by bacteria to sense and respond to the environment inside the plant are largely unknown. Building upon our previous work in Pseudomonas syringae pv. tomato DC3000, we will address this problem by analyzing non-coding RNAs and ECF sigma factors, two classes of regulatory factors that are known linchpins of gene regulation. We will use established deep sequencing methods, such as ChIP-Seq, RNA-Seq and RNA 5'-end capture, and novel methods, such as genomic footprinting and in planta RNA-Seq, to monitor bacterial gene expression as it occurs during infection. The synthesis of the data sets from these experiments will reveal many key regulatory pathways involved in pathogenesis and virulence.
The preparation for and eventual implementation of sequestration had a substantial impact on the project's ability to meet its milestones. In particular, milestones 4 (subobjective 2A) and 7 (subobjective 2D) were not met because there were insufficient funds to hire personnel to perform the tasks. Unless the project receives additional funding, these subobjectives will be abandoned.
Nevertheless, substantial progress was made on the other milestones. In particular, the project continues to develop reagents and software pipelines for performing novel high-throughput experiments, such as RIP-Seq and TnSeq, performing experiments using apoplastic fluid extracted from both unconditioned and inoculated plants, and performing experiments in planta to study the bacteria’s response to the plant environment and defenses. Overall, the project is on track to perform high-throughput sequencing on a number of libraries in FY2014.
Not surprising, as the work has progressed several unexpected issues have arisen. For instance, as part of subobjective 1A, we had proposed to perform RIP-Seq in order to better understand the RNA-binding activity of the Crc protein. However, in the laboratory, we have been unable to confirm the RNA-binding activity of Crc that was previous reported. Coincidently, a recent report suggests that Crc's previously reported RNA-binding activity was actually the result of contamination by Hfq, and that Crc may not have RNA-binding activity. We are performing additional experiments to determine whether it is worth performing RIP-Seq on Crc.
Regulating iron uptake in Pseudomonas syringae. ARS researchers at Ithaca, New York used high-throughput sequencing methods to identify the genes in Pseudomonas syringae pv. tomato DC3000 regulated by the iron starvation (IS) sigma factors, PSPTO_0444, PSPTO_1209, and PSPTO_1286. These three sigma factors were found to regulate a number of genes involved with the detection and uptake of siderophores, protein complexes used to sequester and transport iron into the cell where it is used for essential functions. The presence of so many different uptake mechanisms suggests that DC3000 is capable of utilizing a wide variety of different siderophores. This likely helps DC3000 flourish in the presence of a variety of other environmental bacteria.
Filiatrault, M.J., Stodghill, P., Wilson, J.M., Butcher, B.G., Chen, H., Meyers, C.R., Cartinhour, S.W. 2013. CrcZ and CrcX regulate carbon utilization in Pseudomonas syringae pathovar tomato strain DC3000. RNA Biology. 10(2):243-253.
Worley, J.N., Russell, A.B., Wexler, A.G., Bronstein, P., Kvitko, B.H., Krasnoff, S., Munkvold, K.R., Swingle, B.M., Gibson, D.M., Collmer, A. 2013. Pseudomonas syringae pv. tomato DC3000 CmaL (PSPTO4723), a DUF1330 family member, is needed to produce L-allo-isoleucine, a precursor for the phytotoxin coronatine. Journal of Bacteriology. 195(2):287-296.
Swingle, B.M., Markel, E.J., Butcher, B.G., Myers, C.R., Stodghill, P., Cartinhour, S.W. 2013. Regulons of the Pseudomonas syringae pv. tomato DC3000 iron starvation sigma factors PSPTO_0444, PSPTO_1209 and PSPTO_1286. Applied and Environmental Microbiology. 79(2):725-727.