The objectives of this project are to understand the role of the plant hormone auxin during pathogen infection and disease development and to elucidate the mechanisms by which the plant bacterial pathogen Pseudomonas syringae modulates auxin synthesis and signaling in plants. The investigators will use a combination of genetic, molecular and biochemical approaches to identify the biosynthetic pathways used by P. syringae to synthesize auxin and to investigate how P. syringae virulence factors alter host auxin physiology to promote pathogenesis. The proposed experiments are innovative and relevant because they integrate studies of the pathogen, plant host and auxin synthesis and signaling within the host and should yield improved understanding of the role(s) of auxin (and potentially other plant hormones) and hormone signaling during pathogen infection and disease development. The work will benefit society at large through a better understanding of pathogen virulence and disease susceptibility, and may lead to novel control methods and development of plants with increased disease resistance. The project will provide stimulating and valuable research and educational opportunities for high school, undergraduate, graduate and postdoctoral students in the investigators' laboratories, and both laboratories work with educational programs to attract students from under-represented groups to become involved in the research.
The Kunkel Lab studies virulence strategies of plant pathogens. Recent studies indicate that modulation of plant hormone physiology is an important virulence strategy for several plant pathogens, including the bacterial pathogen Pseudomonas syringae. We are currently focusing on how P. syringae strain DC3000 manipulates auxin signaling in Arabidopsis thaliana. This project had three specific aims: 1) Investigate whether PstDC3000 produces auxin as a virulence factor, 2) Investigate the mode of action of the P. syringae virulence factor AvrRpt2 in modulating host auxin physiology, and 3) Investigate the roles of auxin during PstDC3000 pathogenesis. We worked collaboratively with the group of Dr. Libo Shan at Texas A&M University on Aim 2. Research Findings 1. PstDC300 produces auxin by an unknown pathway. Many P. syringae strains, including PstDC3000, synthesize the auxin indole-3-acetic acid (IAA) in culture. However, the biosynthetic pathways used by PstDC3000 to produce IAA are not known. Further, whether IAA production contributes to PstDC3000 virulence has not been investigated. We are elucidating the PstDC3000 IAA biosynthetic pathway so that we can generate mutants impaired for IAA synthesis. Our feeding studies and genetic analysis revealed that, contrary to what had been proposed, PstDC3000 does not use the indole-3-acetamide (IAM) pathway to synthesize IAA. Rather, we found that PstDC3000 utilizes either the Indole-3-pyruvate( IPyA) pathway or synthesizes IAA directly from IAAld (Figure 1). As the PstDC3000 genome doesn’t encode an obvious IPDC enzyme, we favor the latter hypothesis. The genes encoding enzymes involved in this pathway are not known, and we are working to identify candidate genes using bioinformatic and genetic approaches. 2. The cysteine protease activity of AvrRpt2 is required for enhancing auxin sensitivity in plant. In collaboration with the Shan lab, we demonstrated that the P. syringae virulence protein AvrRpt2, which is injected into plant cells via the Type III secretion system, alters host auxin physiology. To understand how AvrRpt2 accomplishes this we investigated whether the ability of AvrRpt2 to promote auxin-responsiveness depends on its known cysteine protease activity. Biochemical studies demonstrated that the cysteine protease activity is required for stimulating degradation of the AUX/IAA transcriptional repressor proteins. However, the AUX/IAA proteins are not the direct targets of AvrRpt2 protease activity. Thus AvrRpt2 may cleave one or more negative regulators of AUX/IAA degradation. These findings are reported in Cui, et al., 2013. Plant Physiol. The Kunkel lab’s contribution to this aim was to generate transgenic Arabidopsis plants expressing wild-type or mutant versions of AvrRpt2, to assess the importance of AvrRpt2 cysteine protease activity on auxin signaling and virulence activity in whole plants. We found that the cysteine protease activity is required for conferring enhanced auxin sensitivity to transgenic plants. We also observed that, while the cysteine protease activity is required for full AvrRpt2 virulence activity, AvrRpt2 may also promote disease via a mechanism independent of the protease activity. 3. Auxin promotes disease development via a mechanism that does not involve suppression of salicylic acid-mediated defenses. When we started this work, a favorite hypothesis was that auxin promotes pathogen growth in plant tissue by suppressing host defense responses mediated by salicylic acid (SA). We investigated this by examining the interaction of auxin and SA in the context of infection in plants with elevated levels of auxin. To do this we took advantage of transgenic plants overexpressing the YUCCA1 auxin biosynthesis gene and demonstrated that elevated IAA promotes susceptibility to PstDC3000 without suppressing SA-dependent defenses. This work was published in 2013 (Mutka et al, Plant J.). This suggests that IAA promotes other changes in the plant that render host tissue more suitable for pathogen growth. Alternatively, or in addition, IAA may directly impact the pathogen, for example by stimulating expression of virulence genes (Figure 2). Broader impacts The above findings are important contributions to the understanding of pathogen virulence strategies and the roles of the plant hormone IAA in host-pathogen interactions. This benefits society and may lead to development of plants with increased disease resistance. This project also provided scientific training for a variety of students. During the funding period of this NSF award the Kunkel lab trained 5 WUSTL undergraduates, 3 of whom are authors on published papers or manuscripts in preparation (S. Fawley*, T. Tsao*, L. Katin-Grazzini, A. Choi, and J. Mason*, * = authorship), one visiting undergraduate (J. Mungin, Tuskegee U.) and 2 graduate students (A. Mutka, S. McClerklin). All students received one-on-one mentoring from Dr. Kunkel, with emphasis on developing into creative and critical and scientists able to design and execute hypothesis-driven, well-designed experiments, and who can interpret their data in the context of the dynamic fields of plant-microbe interactions, hormone physiology and signal transduction. They also received training on preparing and presenting oral presentations and writing research proposals and articles. The graduate students also gained significant mentoring experiences, as they each supervised at least two undergraduate students.
