The Actin-Pathogen Connection

The Actin-Pathogen Connection: Linking the cytoskeleton to immunity and virulence

The eukaryotic actin cytoskeleton is comprised of a dynamic and tightly regulated pool of globular (G)- and filamentous (F)-actin whose activities and organization are linked to a diverse array of cellular functions. Actin, together with the more than 75 actin-binding proteins identified in plants, regulates key signaling processes, including those required for stomatal movement and immunity. While the function and activity of actin is well described for its numerous roles in defining cell shape and powering movement of organelles and macromolecules, the function of the actin cytoskeleton as a component of the eukaryotic immune system is not well-defined. In both plants and animals, the actin cytoskeleton has been demonstrated to respond to a variety of pathogen-derived elicitors, however, the definition of the mechanism(s) underpinning this function is lacking.

Current work in our lab is focused on 3 aspects of actin-pathogen dynamics during immune signaling, described below.

Project_ Actin_1Pathogen targeting of the host actin cytoskeleton

The link between the actin cytoskeleton and the plant immune system has been best defined through the analysis of PTI. For example, numerous early studies have shown that perturbation to the host cell through the application of pharmacological agents that modify the stochastic behavior of the actin cytoskeleton (e.g., cytochalasin-D, jasplakinolide) also impact immune signaling and pathogen infection. Collectively, these studies have not only demonstrated a requirement for cytoskeletal rearrangement at the site of pathogen penetration, but that actin filament organization is required for the function of numerous intercellular processes, including trafficking, the production of metabolites associated with resistance signaling, and response to mechanical force. Additional work has further demonstrated that following perturbation to the naïve cycling of the plant actin cytoskeleton, many of the early events required for the activation of PTI, including attenuation of signaling through endocytosis of the PRR, MAPK signaling, the generation of reactive oxygen, callose deposition, and the activation of transcription, are blocked.

Our recent work has identified a suite of Pseudomonas syringae type III effector (T3E) proteins that specifically target the Arabidopsis actin cytoskeleton. We are now working to define 1) the basal function of these specific effector targets/processes, and 2) the impact of pathogen targeting on the disruption of homeostatic cellular processes requiring actin. Our ultimate goal is to not only use pathogen T3Es as molecular and cellular probes to better define immune signaling, but to use these effectors to interrogate the function and regulation of these processes in the absence of pathogen infection.

Relevant publications:

Shimono, M*., Lu, Y*., Porter, K., Kvitko, B., Creason, A., Henty-Ridilla, J., He, S.Y., Chang, J.H., Staiger, C., and Day, B. (2016). The Pseudomonas syringae type III effector HopG1 induces actin filament remodeling in Arabidopsis in association with disease symptom development. Plant Physiol. 171: 2239-2255. (* Equal contribution).

Li, J., Henty-Ridilla, J.L., Staiger, B.H., Day, B., and Staiger, C.J. (2015). Capping protein integrates multiple MAMP signaling pathways to modulate actin dynamics during plant innate immunity. Nature Comm.  28: 7206. doi: 10.1038/ncomms8206.

Henty-Ridilla, J.L., Li, J., Day, B., and Staiger, C.J. (2014). ADF4 regulates actin dynamics during innate immune signaling. Plant Cell. 26: 340-352

Porter, K., Shimono, M., Tian, M., and Day, B. (2012). Arabidopsis Actin-depolymerizing Factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics. PLoS Pathogens, 8(11): e1003006. doi:10.1371/journal.ppat.1003006

Tian, M., Chaudhry, F., Ruzicka, D.R., Meagher, R.B., Staiger, C.J., and Day, B. (2009). Arabidopsis actin depolymerizing factor AtADF4 mediates defense signal transduction triggered by the Pseudomonas syringae effector AvrPphB. Plant Physiol. 150: 815-824.

Project_ Actin_2Regulation of actin cytoskeletal organization

The eukaryotic actin cytoskeleton is required for the function of numerous cellular processes, including response to stress, injury, and disease. At present, we know almost nothing about the link(s) between changes in actin cytoskeletal dynamics and the regulation of gene expression. Our long-term goals are to define how pathogens disrupt the stochastic behavior of the host actin cytoskeleton to cause disease. To achieve this, we are using pathogens, and their associated virulence factors, to probe and define the mechanisms associated with the cellular organization of the actin cytoskeleton, including the immune signaling processes linked to actin cytoskeletal dynamics. Using this approach, we will not only gain insight into the cellular processes required for immunity, but the mechanisms by which perturbations in          cytoskeletal function give rise to disease.

Previous work from my research group has uncovered the actin-based mechanisms used by plants and pathogens to resist infection and to cause disease, respectively. Based on our published and preliminary data, we can propose the following central hypothesis: Pathogens directly target the host actin cytoskeleton to disrupt the nucleocytoplasmic movement of actin and members of the ADF/cofilin family of proteins. In support of this hypothesis, we have identified two key events that underpin the transition from the naïve homeostatic function of the cytoskeleton to an immune-activated signaling platform. First, we have identified a bacterial pathogen effector protein that cleaves and inactivates the ADF4 kinase, CPK3. Second, we have identified an interaction within the nucleus between ADF4 and 4 members of a stress-associated transcription factor family. To define the immunity and pathogen virulence mechanisms associated with these events, we are pursuing three complementary areas of research to characterize the nucleocytoplasmic regulation of actin: (1) Characterize ADF4 and CPK3 phosphorylation dynamics during immune signaling; (2) Define the organization dynamics of the actin cytoskeleton; and (3) Characterize the function and activity of actin and ADF4 immune-associated regulons.

This work is currently funded by the National Institutes of Health, 1R01GM125743.

Relevant publications:

Li, P. and Day, B. (2019). Battlefield Cytoskeleton: Cytoskeletal regulation, and pathogen targeting, of plant immunity. Mol Plant-Microbe Interact. In Press.

Porter, K., Shimono, M., Tian, M., and Day, B. (2012). Arabidopsis Actin-depolymerizing Factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics. PLoS Pathogens, 8(11): e1003006. doi:10.1371/journal.ppat.1003006

Henty-Ridilla, J.L., Li, J., Day, B., and Staiger, C.J. (2014). ADF4 regulates actin dynamics during innate immune signaling. Plant Cell. 26: 340-352

Henty, J.L., Shimono, M., Li, J., Chang, J.H., Day, B.*, and Staiger, C.J.* (2013). The plant actin cytoskeleton is a novel component of innate immunity. PLoS Path. 9: e1003290. doi:10.1371/journal.ppat.1003290. *co-corresponding authors.

Day, B., Henty, J., Porter, K., and Staiger, C. (2011). The pathogen-actin connection: A platform for defense signaling in    plants. Ann. Rev. Phytopathol. 49: 483-506.

 

Project_ Actin_3Stomatal Immunity: The actin cytoskeleton at the interface of host-pathogen-environment signaling.

The current favored model for the regulation of actin dynamics posits that actin filaments are constantly rearranged via polymerization, severing, and depolymerization, processes largely governed by the activity of profilin and actin depolymerizing factors (ADFs), respectively. However, the purpose(s) of this incessant re-modeling are largely unknown. Our working hypothesis is: Actin remodeling is important for mediating plant immunity against pathogens. To test this, we are using a combination of quantitative cell biology, an analysis of the convergence of hormone and immune signaling, and classical plant pathology-based approaches to define 1) How the organization of the actin cytoskeleton support the spatiotemporal dynamics of stomatal immunity, 2) How immune hormone signaling nodes linked, 3) How pathogens target cytoskeletal regulation to defeat immunity, and finally, 4) How plants sense cytoskeletal perturbation to activate immunity. 

Recent studies have established that stomatal closure is an important output of innate immunity in plants. In collaboration with Dr. Sheng Yang He, we are using pathogen associated molecular patterns (PAMPs) and live bacteria, including plant pathogenic bacteria (e.g., Pseudomonas syringae pv. tomato [Pst] DC3000) to elucidate the core signaling components involved in stomatal immunity – PAMP recognition and signaling associated with stomata closure and blocking of pathogen invasion and growth. While significant knowledge gaps still remain, our data indicate that actin is linked to many events associated with the activation of stomatal immunity, including hormone signaling associated with the perception of abiotic and biotic stress. We are developing the stomatal guard cells as a paradigm for the study, and further dissection, of the molecular-genetic links between actin dynamics and the pathogen response.

This work is currently funded by the National Science Foundation, IOS-1557437.

Relevant publications:

Shimono, M., Higaki, T., Kaku, H., Shibuya, N., Hasezawa, S., and Day, B. (2016). Quantitative evaluation of stomatal cytoskeletal patterns during the activation of immune signaling in Arabidopsis thalianaPLoS One. 11:e0159291.