Roles of dynamin-related protein network in plant immune responses and development
Authors:   , Ekanayake Gayani 
Published: December 2023

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI-COLUMBIA AT REQUEST OF AUTHOR.] At the host-pathogen interphase, the PM's protein composition has a critical role in how plants perceive and respond to the invading pathogenic attacks. However, the intracellular molecular machinery that modulates the PM proteome for signal initiation and attenuation of responses to biotic stimuli remains mostly undefined. PM localized pattern recognition receptors (PRRs) are capable of recognizing their cognate ligands known as pathogen-associated molecular patterns (PAMPs). In our model plant, Arabidopsis thaliana, FLAGELLIN SENSING 2 (FLS2) is a well-characterized PRR that recognizes the bacterial flagellin or its 22 amino acid flg22 peptide in the extracellular space and thereby initiate the cascade of intracellular immune responses culminating in PAMPtriggered immunity (PTI). Constitutive activation of these immune responses can be detrimental to plant growth and development. Therefore, to tightly regulate the immune responses, the PRRs need to be at the PM in the correct abundance. A dynamic and complex plant vesicular trafficking network, which includes secretion and endocytosis, contributes to fine-tune the PM protein composition. These trafficking processes add or remove PM cargo proteins (PRRs, transporters, and other proteins with immune functions), including FLS2, to or from the PM via membrane-bound small vesicles. clathrin-coated vesicles (CCV) is one of the best-studied vesicle types in eukaryotes, which form at the PM, trans-golgi network (TGN)/early endosomes (EE) and cell plate in plants. As the model PRR in plant immunity, FLS2 undergoes ligand-induced endocytosis via CCVs utilizing a process referred to as clathrin-mediated endocytosis (CME). Increasing evidence indicates that ligand-induced CME of FLS2 removes the activated form of this receptor from the site of flg22-perception (the PM) as a means to attenuate immune signaling. In addition to stimulus dependent CME, FLS2 is internalized from the PM via constitutive endocytosis that occurs in the absence of any stimulus, likely as part of general quality control of the PM. Considering the complexity of CCV-dependent cargo trafficking in plants, relatively few components are known to be functioning in flg22-induced signaling and endocytosis of FLS2 from the PM. A previous study from our lab has identified a novel immune role of Arabidopsis DYNAMIN RELATED PROTEIN 2B (DRP2B) in immunity against Pto DC3000 bacteria. Furthermore, drp2b mutant exhibits non-canonical flg22-signaling defects and a 20 percent reduction of ligand-induced endocytosis of FLS2 from the PM. To delineate the dynamin-related protein network functioning with DRP2B at the PM, our preliminary evidence identified the plant specific DRP1A to co-immunoprecipitate with DRP2B. As supported by previous studies, DRP1A and DRP2B interact biochemically and colocalize at CCV sites at the PM. In Arabidopsis, DRP2B encodes for a large GTPase that colocalizes and interacts with CCV components at the PM. DRP2B belongs to a family of 16 Arabidopsis DRPs that are separated into six different subfamilies (AtDRP1-6) based on their predicted domain structure and functions in fission of diverse membranes vesicles and/or organelles. Of these subfamilies, only Arabidopsis DRP1 and DRP2 family members are known to participate as CCV accessory proteins in CME from the PM and cell plate. DRP1A is one of five plant-specific dynamins that lack the pleckstrin homology (PH)-domain and proline rich domain (PRD) that are present in the DRP2 family and responsible for binding to target membrane and protein interaction binding, respectively. In this Ph.D. thesis, I provide evidence that loss of DRP1A resulted in similar non-canonical flg22-signaling defects as drp2b, which indicated that DRP1A and DRP2B likely operated by affecting a shared pathway during PTI, potentially through governing PM abundance of the same proteins with immune function. I also discovered that DRP1A had an additional role in plant immunity because drp1a mutants exhibited increased effector delivery and significantly increased susceptibility against Pto DC3000 compared to drp2b. Consistent with DRP1A colocalizing and interacting biochemically with DRP2B, DRP1A and DRP2B genetically interacted a) synergistically in plant growth, resulting in seedling lethal drp1a drp2b double mutants; and b) additively in ligand-induced endocytosis resulting in a block of ligand-induced endocytosis of FLS2. The combinatorial loss of DRP1A and DRP2B also led to hyperaccumulation of FLS2 at the PM in the absence of any stimulus indicative of their synergistic role in governing constitutive endocytosis of FLS2. I further discovered and then delineated the combinatorial roles of DRP1A and DRP2B in constitutive activation of immune responses utilizing drp1a drp2b double mutant seedlings. In drp1a drp2b double mutant seedlings, alleviation of elevated levels of the plant defense hormone salicylic acid (SA) mitigated only increased mRNA accumulation of the SA marker gene PR. However, hyperaccumulated levels of the cell death reactive oxygen species H2O2 and two cell wall polymers, namely callose and lignin, were independent of SA. I also provide evidence that under basal conditions, increased callose accumulation was dependent on the callose synthase POWDERY MILDEW RESISTANT 4 (PMR4). In contrast, all other activated immune responses that I evaluated in the drp1a drp2b double mutant seedlings were independent of PMR4. I also reported that loss of both DRP1A and DRP2B resulted in hyperaccumulation of FLS2 at the PM; but constitutively activation of SA, PR1 mRNA, callose and H2O2 accumulation were independent of FLS2. Taken these results together, my studies expanded our limited knowledge of vesicular trafficking proteins that modulate callose and lignin accumulation, potentially through modulating the PM abundance and/or activity of PMR4 and an elusive S-monolignol transporter, respectively. My studies also allowed me to separate the role(s) of DRP1A and DRP2B in growth and development from those in plant immunity because neither additional loss of FLS2, PMR4 or the SALICYLIC-INDUCED DEFICIENCY2 (SID2), encoding a key enzyme in SA biosynthesis, could mitigate the stunted growth defect. My work also defined new roles in plant immunity for Arabidopsis DRP2A, which is the only other member of the AtDRP2 subfamily and shares 93 percent amino acid sequence identity to its paralogue DRP2B. Previous studies show that DRP2A has no apparent role in early flg22-induced signaling and FLS2-GFP endocytosis compared to wild-type. Here, I provide evidence that drp2a mutant showed impaired late flg22-induced immune responses (callose, PR1 mRNA) in a non-canonical manner. However, the signaling defects of drp2a were different from those observed in drp2b, indicating that DRP2A and DRP2B may modulate the same set of proteins with function in late flg22-signaling but affected proteins with roles in early flg22-responses differently. Collectively, this work advanced our limited understanding of the roles of the plant CCV assessory proteins DRP1A, DRP2B, and DRP2A in modulating multiple plant cellular processes, including their non-canonical roles in flg22-signaling and immunity against Pto DC3000 bacterial strains. Utilizing genetics, my studies also enabled to uncouple combinatorial roles of DRP1A and DRP2B in growth and development from those in immune functions. I also added FLS2 and identified potential PM candidate proteins with roles in modulating the plant cell wall to the short list of cargo proteins that utilize DRP1A and DRP2B for ligand-induced and/or constitutive CME.