Roles of post-Golgi vesicular trafficking components in plant growth and immunity
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[EMBARGOED UNTIL 12/1/2024] Delineating the molecular mechanisms of plant immunity can help inspire novel ways to engineer more resistant plants which could contribute to reducing crop loss due to pathogenic infection. The plasma membrane (PM) is a crucial contact point for plant defense as various proteins localized to the PM function in the perception of pathogens within the apoplast, initiation and attenuation of immune signaling, and production of defense related components. Thus, during the preparation and execution of plant defense responses, the PM must be tightly controlled to ensure an effective immune response. One method of modulating the PM composition is through vesicular trafficking via clathrin-coated vesicles (CCVs). Vesicular trafficking is the coordinated movement of cargo from one organelle to another via membrane-bound vesicles. CCVs function in post-Golgi trafficking and form at both the PM and trans-Golgi network/ early endosome (TGN/EE). The TGN/EE serves as an important sorting station for CCV trafficking by ensuring that proteins are directed to the correct functional location. However, the roles of TGN/EE-localized vesicular trafficking components in coordinating cellular and biological processes remain largely unexplored. Here, we examine the role of four vesicular trafficking components using reverse genetics and biochemical approaches to examine whether individual or combinatorial loss of these components impacts plant growth or immunity in the model plant Arabidopsis. Firstly, I presented a protocol for the staining and automated quantification of callose deposition in Arabidopsis seedlings and mature leaves after elicitation. Callose deposition is a defense response that is widely utilized to investigate defects in the plant immune signaling pathway. For instance, I used this technique throughout Chapter 4 to investigate a preformed callose deposition defect in Arabidopsis mutant seedlings. Additionally, the quantification methodology is valuable to the broader research community as it could be applied to quantify other cellular features. Our lab previously identified the CCV component EPSIN1 (EPS1) as a positive regulator of plant immunity against the pathogenic bacterium Pseudomonas syringae pv tomato (Pto) DC3000. EPS1 is a monomeric clathrin adaptor localized to the TGN/EE implicated in trafficking to the PM and vacuole. However, we found that the TGN/EE- localized adaptor MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1) does not have an apparent function in bacterial immunity against Pto DC3000. EPS1 and MTV1 are Epsin N-terminal Homology (ENTH)-domain containing adaptors that are predicted to form a conserved tertiary structure typical of the ENTH domain. Thus, we attribute diverse biological functions in immunity likely to differences in protein primary structure demonstrated by the low amino acid similarity. Previous results from our lab confirmed that EPS1 was in-complex with the clathrin coat component, CLATHRIN HEAVY CHAIN2 (CHC2). Additionally, CHC2 and EPS1 displayed a synergistic genetic interaction in preformed callose deposition that was independent of elevated salicylic acid. Here, I further investigated the preformed defense responses in chc2 eps1 double mutant seedlings by finding that, in contrast to the upregulation of the immune marker gene PATHOGENESIS-RELATED 1 (PR1), the preformed callose deposition was also independent of continuous light during growth. Notably, we uncovered that the preformed callose deposition was dependent on CALLOSE SYNTHASE12 (CALS12)/POWDERY MILDEW RESISTANT4 (PMR4), which may be linked to increased PMR4 protein at the PM of chc2 eps1 seedlings. We also found that preformed callose deposition, independent of elevated PR1 mRNA, did not contribute to bacterial immunity against Pto DC3000, further elucidating the role of preformed callose deposition. EPS1 also biochemically interacts with VESICLE TRANSPORT V-SNARE 11 (VTI11)/ZIG, so we isolated the eps1 zig double mutant to explore the genetic interaction in plant growth and immunity. We discovered that EPS1 and VTI11/ZIG behaved independently or redundantly depending on the different stages of growth or plant organ. Furthermore, EPS1 and VTI11/ZIG displayed a synergistic interaction in plant immunity against the pathogenic bacterium Pto DC3000. Notably, we uncovered a novel role for VTI11/ZIG in modulating the total protein abundance of the immune receptor FLAGELLIN SENSING 2 (FLS2), that will be explored further in future studies. Lastly, I generated tools that will be essential for investigating the physiological role of the ENTH domain of EPS1 in addition to examining biochemical interactions with EPS1. We successfully generated an antibody against the ENTH domain of EPS1 that will be useful for in planta Arabidopsis studies and potentially to confirm an orthologous maize EPS1 mutant. Additionally, I developed recombinant full-length and variant proteins of EPS1, VTI11, and VTI12 that can be utilized to investigate biochemical interactions after optimizing or redesigning the attempted pull-down assays. Altogether, this work advanced the limited understanding of TGN/EE-localized vesicular trafficking components in modulating various plant processes including plant growth, bacterial immunity, and alteration of the PM proteome.
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Ph. D.