An exquisite and intricate crosstalk exists between bacterial phytopathogens and the host cells and tissues they invade. New information suggests that a dynamic network of filaments, the cytoskeleton, is essential for both resistance and susceptibility to plant pathogens. However, exactly which molecules from the bacterium stimulate cytoskeletal responses, and which plant proteins are necessary for these responses remains poorly understood. In this project, the investigators will dissect the genetic basis for crosstalk between bacterial effector proteins and the plant host cytoskeleton. Using both molecular genetics tools and advanced imaging technologies, the investigators will test the hypothesis that specific pathogen effector proteins perturb plant actin-binding proteins (ABPs), which in turn leads to alterations in the plant cytoskeleton during defense signaling. A toolbox of actin and actin-binding protein mutants will be developed, and a delivery system for introducing bacterial effectors into host cells will be exploited. Through this work, the PIs aim to identify the function of bacterial effector proteins and 39 plant genes/proteins in order to uncover the signaling network. Lead PI Staiger will coordinate activities between the groups and exploit state-of-the-art imaging modalities to evaluate the defects in actin dynamics in the ABP collection and in response to effector proteins. The Day and Chang laboratories will use genetic approaches to dissect whether crosstalk involves pathogen-associated molecular pattern recognition responses (PTI) or effector-triggered immunity (ETI), or both. These two labs will also use a novel bacterial delivery system to associate changes in actin dynamics to a corresponding bacterial effector protein. The two labs will also prepare constructs for protein-protein interaction assays and subcellular localization. All project data and resources created in this work will be disseminated in a timely fashion at a joint website: http://actinpathogen-network.msu.edu. The broader impact of this work will contribute to a deep understanding of host?pathogen signaling and will enable crop modifications to protect against diseases that devastate agricultural productivity. As a unique outreach component, we will establish a summer workshop to train all project scientists and other interested parties, including local high school students, in advanced methods for imaging cellular dynamics.
The processes that regulate immune signaling in plants and animals are typically linked to broader, essential, physiological processes, including development, growth, cell division, and death. In the current project funded by the National Science Foundation, research demonstrated that the actin cytoskeleton - a macromolecular structure required for fundamental processes that define and regulate cell shape and movement - is a key component of the immune signaling network. Using the cellular organization of actin as a key readout, the impact of pathogen infection on basic cytoskeletal features (e.g., bundling, shape, direction) was quantified using a series of quantitative parameters developed through the funded project. From these analyses, the work supported by this project showed that not only is actin required for immunity, but that the actin cytoskeleton is specifically targeted by pathogens as a mechanism to promote infection, and ultimately, to cause disease. This work provided the first evidence that plant pathogens target the actin cytoskeleton to block immune signaling. In Year 1 of the funding period, we demonstrated that a key regulator of actin dynamics, actin depolymerizing factor-4 (ADF4) is required for immunity to the plant pathogen Pseudomonas syringae. From this first result, we further demonstrated the basal activity of ADF4 is required for numerous fundamental plant processes, including the regulation of actin filament length, rates of severing, and the degree of actin polymerization. To link these quantitative measures to broader physiological processes, we next showed (Year 2) that by impacting the basal function of the actin cytoskeleton, we also altered numerous other processes, including gene expression and stomata movement. This observation provided a foundation for future and continuing work to investigate the link between processes required for immunity and those processes known to be key components of host cell processes required for survival. Among these include the function of stomata (required for gas exchange), the expression of genes associated with circadian cycling, and the ability of plants to sustain extended periods of environmental stress (i.e., drought tolerance). In total, these data fit the primary tenant that plants have evolved the use of required processes to survey against stress and injury, and in parallel, pathogens have evolved strategies to manipulate these processes to cause disease. In the final year of the project (Year 3), we extended these studies to define the precise mechanism(s) that pathogens use to cause disease, a goal that has broad application to the study of plant and animal immune signaling pathways. This work has proved to be the most impactful of the funded work, and has yielded the identity of 2 virulence strategies pathogens use to cause disease. In the first, pathogens induce processes in plants typically associated with death. In short, the pathogen activates specific signaling processes normally initiated only during cell death signaling, thus "tricking" the plant to initiate programmed cell death processes, turning off immunity. In the second strategy, the research supported by the NSF revealed that pathogens deploy a secreted virulence factor to shut down actin dynamics, effectively disarming immunity by turning off basic physiological processes. In total, the work supported by funding from the NSF has linked the regulation of basic cellular processes to the activity of the actin cytoskeleton, a key virulence target of pathogens. As a function of host cell performance, linking key processes to a ubiquitous cellular component (i.e., the actin cytoskeleton) has obvious advantages; first and foremost, it provides a seamless mechanism to continually survey for stress and injury. However, in doing so, it provides an "Achilles Heel" for pathogen manipulation, and if successful, as was demonstrated in the current funded research, if a pathogen can uncover the central regulatory node, it not only gains access to the immune system, but to the broader control of basic physiological processes that a plant (or animal) requires for survival.
