Extracellular niche establishment by plant pathogens.

The plant extracellular space, referred to as the apoplast, is inhabited by a variety of microorganisms. Reflecting the crucial nature of this compartment, both plants and microorganisms seek to control, exploit and respond to its composition. Upon sensing the apoplastic environment, pathogens activate virulence programmes, including the delivery of effectors with well-established roles in suppressing plant immunity. We posit that another key and foundational role of effectors is niche establishment - specifically, the manipulation of plant physiological processes to enrich the apoplast in water and nutritive metabolites. Facets of plant immunity counteract niche establishment by restricting water, nutrients and signals for virulence activation. The complex competition to control and, in the case of pathogens, exploit the apoplast provides remarkable insights into the nature of virulence, host susceptibility, host defence and, ultimately, the origin of phytopathogenesis. This novel framework focuses on the ecology of a microbial niche and highlights areas of future research on plant-microorganism interactions.

Arabidopsis Dynamin-Related Protein AtDRP2A Contributes to Late Flg22-Signaling and Effective Immunity Against Pseudomonas syringae Bacteria.

In eukaryotes, dynamins and dynamin-related proteins (DRPs) are high-molecular weight GTPases responsible for mechanochemical fission of organelles or membranes. Of the six DRP subfamilies in Arabidopsis thaliana, AtDRP1 and AtDRP2 family members serve as endocytic accessory proteins in clathrin-mediated endocytosis. Most studies have focused on AtDRP1A and AtDRP2B as critical modulators of plant pattern-triggered immunity (PTI) against pathogenic, flagellated Pseudomonas syringae pv. tomato DC3000 bacteria and immune signaling in response to the bacterial flagellin peptide flg22. Much less is known about AtDRP2A, the closely related paralog of AtDRP2B. AtDRP2A and AtDRP2B are the only classical, or bona fide, dynamins in Arabidopsis, based on their evolutionary conserved domain structure with mammalian dynamins functioning in endocytosis. AtDRP2B but not AtDRP2A is required for robust ligand-induced endocytosis of the receptor kinase FLAGELLIN SENSING2 for dampening of early flg22 signaling. Here, we utilized Arabidopsis drp2a null mutants to identify AtDRP2A as a positive contributor to effective PTI against P. syringae pv. tomato DC3000 bacteria, consistent with reduced PATHOGEN RELATED1 (PR1) messenger RNA accumulation. We provide evidence that AtDRP2A is a novel modulator of late flg22 signaling, contributing positively to PR1 gene induction but negatively to polyglucan callose deposition. AtDRP2A has no apparent roles in flg22-elicited mitogen-activated protein kinase defense marker gene induction. In summary, this study adds the evolutionary conserved dynamin AtDRP2A to a small group of vesicular trafficking proteins with roles as non-canonical contributors in immune responses, likely due to modulating one or both the localization and activity of multiple different proteins with distinct contributions to immune signaling. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A method for quantitation of apoplast hydration in Arabidopsis leaves reveals water-soaking activity of effectors of Pseudomonas syringae during biotrophy.

The plant apoplast has a crucial role in photosynthesis and respiration due to its vital function in gas exchange and transpiration. The apoplast is also a dynamic environment that participates in many ion and nutrient transport processes via plasma membrane-localized proteins. Furthermore, diverse microbes colonize the plant apoplast, including the hemibiotrophic bacterial pathogen, Pseudomonas syringae pv. tomato (Pto) strain DC3000. Pto DC3000 initiates pathogenesis upon moving through stomata into the apoplast and then proliferating to high levels. Here we developed a centrifugation-based method to isolate and quantify the apoplast fluid in Arabidopsis leaves, without significantly damaging the tissue. We applied the simple apoplast extraction method to demonstrate that the Pto DC3000 type III bacterial effectors AvrE1 and HopM1 induce hydration of the Arabidopsis apoplast in advance of macroscopic water-soaking, disruption of host cell integrity, and disease progression. Finally, we demonstrate the utility of the apoplast extraction method for isolation of bacteria proliferating in the apoplast.

Novel roles for Arabidopsis dynamin-related proteins DRP1A and DRP2B in resistance against Botrytis cinerea fungal infection.

Arabidopsis DYNAMIN-RELATED PROTEIN1A (AtDRP1A) and AtDRP2B are large GTPases that function together in endocytosis for effective cytokinesis, cell enlargement and development. A recent study shows that these DRPs contribute to ligand-induced endocytosis of the immune receptor FLAGELLIN SENSING2 (AtFLS2) to modulate flg22-immune signaling, and they are required for immunity against Pseudomonas syringae pv. tomato bacteria. Here, we demonstrate that atdrp1a and atdrp2b single mutants showed increased susceptibility to Botrytis cinerea indicating that AtDRP1A and AtDRP2B are necessary for effective resistance against this necrotrophic fungus. Thus, we expanded our limited understanding of clathrin endocytic accessory proteins in immunity against plant pathogens.

Dynamic nutrient acquisition from a hydrated apoplast supports biotrophic proliferation of a bacterial pathogen of maize.

Plant pathogens perturb their hosts to create environments suitable for their proliferation, including the suppression of immunity and promotion of water and nutrient availability. Although necrotrophs obtain water and nutrients by disrupting host-cell integrity, it is unknown whether hemibiotrophs, such as the bacterial pathogen Pantoea stewartii subsp. stewartii (Pnss), actively liberate water and nutrients during the early, biotrophic phase of infection. Here, we show that water and metabolite accumulation in the apoplast of Pnss-infected maize leaves precedes the disruption of host-cell integrity. Nutrient acquisition during this biotrophic phase is a dynamic process; the partitioning of metabolites into the apoplast rate limiting for their assimilation by proliferating Pnss cells. The formation of a hydrated and nutritive apoplast is driven by an AvrE-family type III effector, WtsE. Given the broad distribution of AvrE-family effectors, this work highlights the importance of actively acquiring water and nutrients for the proliferation of phytopathogenic bacteria during biotrophy.

DYNAMIN-RELATED PROTEIN DRP1A functions with DRP2B in plant growth, flg22-immune responses, and endocytosis.

Ligand-induced endocytosis of the immune receptor FLAGELLIN SENSING2 (FLS2) is critical for maintaining its proper abundance in the plasma membrane (PM) to initiate and subsequently down regulate cellular immune responses to bacterial flagellin or flg22-peptide. The molecular components governing PM abundance of FLS2, however, remain mostly unknown. Here, we identified Arabidopsis (Arabidopsis thaliana) DYNAMIN-RELATED PROTEIN1A (DRP1A), a member of a plant-specific family of large dynamin GTPases, as a critical contributor to ligand-induced endocytosis of FLS2 and its physiological roles in flg22-signaling and immunity against Pseudomonas syringae pv. tomato DC3000 bacteria in leaves. Notably, drp1a single mutants displayed similar flg22-defects as those previously reported for mutants in another dynamin-related protein, DRP2B, that was previously shown to colocalize with DRP1A. Our study also uncovered synergistic roles of DRP1A and DRP2B in plant growth and development as drp1a drp2b double mutants exhibited severely stunted roots and cotyledons, as well as defective cell shape, cytokinesis, and seedling lethality. Furthermore, drp1a drp2b double mutants hyperaccumulated FLS2 in the PM prior to flg22-treatment and exhibited a block in ligand-induced endocytosis of FLS2, indicating combinatorial roles for DRP1A and DRP1B in governing PM abundance of FLS2. However, the increased steady-state PM accumulation of FLS2 in drp1a drp2b double mutants did not result in increased flg22 responses. We propose that DRP1A and DRP2B are important for the regulation of PM-associated levels of FLS2 necessary to attain signaling competency to initiate distinct flg22 responses, potentially through modulating the lipid environment in defined PM domains.

Staining and automated image quantification of callose in Arabidopsis cotyledons and leaves.

Callose is a ß-1,3-glucan polysaccharide that is deposited at discrete sites in the plant cell wall in response to microbial pathogens, likely contributing to protection against pathogen infection. Increased callose deposition also occurs in response to the 22-amino acid peptide flg22, a pathogen-associated molecular pattern (PAMP) derived from bacterial flagellin protein. Here, we provide protocols for callose staining using aniline blue in cotyledon and leaf tissue of the model plant Arabidopsis thaliana. Aniline blue stain utilizes a fluorochrome that complexes with callose for its visualization by microscopy using an ultraviolet (UV) filter. For robust quantification of callose deposits, we outline an automated image analysis workflow utilizing the freely available Fiji (Fiji Is Just ImageJ; NIH) software and a Trainable Weka Segmentation (TWS) plugin. Our methodology for automated analysis of large batches of images can be easily adapted to quantify callose in other tissues and plant species, as well as to quantify fluorescent structures other than callose.

EPSIN1 Modulates the Plasma Membrane Abundance of FLAGELLIN SENSING2 for Effective Immune Responses.

The plasma membrane (PM) provides a critical interface between plant cells and their environment to control cellular responses. To perceive the bacterial flagellin peptide flg22 for effective defense signaling, the immune receptor FLAGELLIN SENSING2 (FLS2) needs to be at its site of function, the PM, in the correct abundance. However, the intracellular machinery that controls PM accumulation of FLS2 remains largely undefined. The Arabidopsis (Arabidopsis thaliana) clathrin adaptor EPSIN1 (EPS1) is implicated in clathrin-coated vesicle formation at the trans-Golgi network (TGN), likely aiding the transport of cargo proteins from the TGN for proper location; but EPS1's impact on physiological responses remains elusive. Here, we identify EPS1 as a positive regulator of flg22 signaling and pattern-triggered immunity against Pseudomonas syringae pv tomato DC3000. We provide evidence that EPS1 contributes to modulating the PM abundance of defense proteins for effective immune signaling because in eps1, impaired flg22 signaling correlated with reduced PM accumulation of FLS2 and its coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1). The eps1 mutant also exhibited reduced responses to the pathogen/damage-associated molecular patterns elf26 and AtPep1, which are perceived by the coreceptor BAK1 and cognate PM receptors. Furthermore, quantitative proteomics of enriched PM fractions revealed that EPS1 was required for proper PM abundance of a discrete subset of proteins with different cellular functions. In conclusion, our study expands the limited understanding of the physiological roles of EPSIN family members in plants and provides novel insight into the TGN-associated clathrin-coated vesicle trafficking machinery that impacts plant PM-derived defense processes.

Never Walk Alone: Clathrin-Coated Vesicle (CCV) Components in Plant Immunity.

At the host-pathogen interface, the protein composition of the plasma membrane (PM) has important implications for how a plant cell perceives and responds to invading microbial pathogens. A plant's ability to modulate its PM composition is critical for regulating the strength, duration, and integration of immune responses. One mechanism by which plant cells reprogram their cell surface is vesicular trafficking, including secretion and endocytosis. These trafficking processes add or remove cargo proteins (such as pattern-recognition receptors, transporters, and other proteins with immune functions) to or from the PM via small, membrane-bound vesicles. Clathrin-coated vesicles (CCVs) that form at the PM and trans-Golgi network/early endosomes have emerged as the prominent vesicle type in the regulation of plant immune responses. In this review, we discuss the roles of the CCV core, adaptors, and accessory components in plant defense signaling and immunity against various microbial pathogens.