Coordination of Phospholipid-Based Signaling and Membrane Trafficking in Plant Immunity

Coordination of phospholipid-based signaling and membrane trafficking connects perception of pathogens with the activation of efficient defense reactions in plant immunity.Exploring the spatiotemporal organization of phospholipids and lipid–protein interactions provides crucial information for elucidating this coordination.The functions of phospholipids and their derivatives include: acting as lipid landmarks of various membranes; promoting membrane deformation; regulating cytoskeletal dynamics; and recruiting trafficking regulators.Phospholipid-derived molecules have crucial roles in regulating endocytosis, exocytosis, and the exo/endocytosis balance. During biotic stress, feedback between phospholipid-based signaling and membrane trafficking coordinates signal recognition and activation of immune responses. In plants, defense-associated signal transduction involves key membrane-related processes, such as phospholipid-based signaling and membrane trafficking. Coordination of these processes occurs in the lipid bilayer of plasma membrane (PM) and luminal/extracellular membranes. Deciphering the spatiotemporal organization of phospholipids and lipid–protein interactions provides crucial information on the mechanisms that link phospholipid-based signaling and membrane trafficking in plant immunity. In this review, we summarize recent advances in our understanding of these connections, including deployment of key enzymes and molecules in phospholipid pathways, and roles of lipid diversity in membrane trafficking. We highlight the mechanisms that mediate feedback between phospholipid-based signaling and membrane trafficking to regulate plant immunity, including their novel roles in balancing endocytosis and exocytosis. In plants, defense-associated signal transduction involves key membrane-related processes, such as phospholipid-based signaling and membrane trafficking. Coordination of these processes occurs in the lipid bilayer of plasma membrane (PM) and luminal/extracellular membranes. Deciphering the spatiotemporal organization of phospholipids and lipid–protein interactions provides crucial information on the mechanisms that link phospholipid-based signaling and membrane trafficking in plant immunity. In this review, we summarize recent advances in our understanding of these connections, including deployment of key enzymes and molecules in phospholipid pathways, and roles of lipid diversity in membrane trafficking. We highlight the mechanisms that mediate feedback between phospholipid-based signaling and membrane trafficking to regulate plant immunity, including their novel roles in balancing endocytosis and exocytosis. Phospholipids are components of the membrane bilayer and function in signal transduction [1.Takac T. et al.Recent advances in the cellular and developmental biology of phospholipases in plants.Front. Plant Sci. 2019; 10: 362Crossref PubMed Scopus (24) Google Scholar]. Phospholipid-based signaling (see Glossary) involves the activation of phospholipases (PLs) and lipid kinases, the production of lipid signals, and the binding to downstream targets via lipid–protein interactions [2.Hong Y. et al.Plant phospholipases D and C and their diverse functions in stress responses.Prog. Lipid Res. 2016; 62: 55-74Crossref PubMed Scopus (193) Google Scholar]. Based on its biochemical and regulatory properties, each PL or lipid kinase heads a specific phospholipid-based signaling pathway. These pathways alter cellular and physiological processes, including membrane deformation, membrane trafficking events, endomembrane organization, and cytoskeletal rearrangement [3.Pleskot R. et al.Regulation of cytoskeletal dynamics by phospholipase D and phosphatidic acid.Trends Plant Sci. 2013; 18: 496-504Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 4.Takenawa T. Phosphoinositide-binding interface proteins involved in shaping cell membranes.Proc. Jpn. Acad. B Phys. 2010; 86: 509-523Crossref PubMed Scopus (22) Google Scholar, 5.Zhang Q. et al.Phosphatidic acid regulates microtubule organization by interacting with MAP65-1 in response to salt stress in Arabidopsis.Plant Cell. 2012; 24: 4555-4576Crossref PubMed Scopus (168) Google Scholar]. Plant have evolved a sophisticated immune system that perceives and fights pathogenic microbes [6.Dodds P.N. Rathjen J.P. Plant immunity: towards an integrated view of plant-pathogen interactions.Nat. Rev. Genet. 2010; 11: 539-548Crossref PubMed Scopus (1919) Google Scholar]. Membrane trafficking has an essential role in this fight by delivering defense-related proteins to the plasma membrane (PM) or other endomembrane compartments [7.Wang W.M. et al.Protein trafficking during plant innate immunity.J. Integr. Plant Biol. 2016; 58: 284-298Crossref PubMed Scopus (31) Google Scholar]. During membrane trafficking, the rearrangement of lipid composition at the PM and endomembrane system occurs continuously, which may serve as a platform for protein sorting and trafficking machineries, defining the fate of vesicles or proteins inside the cell [8.Boutte Y. Lipids at the crossroad: shaping biological membranes heterogeneity defines trafficking pathways.PLoS Biol. 2018; 16e2005188Crossref PubMed Scopus (6) Google Scholar]. As the bilayer-forming structural components of membranes, phospholipids and their associated signaling transduction have important roles in this rearrangement [9.Noack L.C. Jaillais Y. Precision targeting by phosphoinositides: how PIs direct endomembrane trafficking in plants.Curr. Opin. Plant Biol. 2017; 40: 22-33Crossref PubMed Scopus (61) Google Scholar]. In this review, we discuss recent advances in understanding the regulatory mechanisms of phospholipid-based signal transduction in membrane trafficking, including the spatiotemporal distribution of lipid signals, membrane deformation, cytoskeletal dynamics, and recruitment of trafficking regulators. We highlight the roles of phospholipid-derived regulators in endocytosis, exocytosis, and in the exocytosis–endocytosis balance. We also discuss how phospholipids and their derivatives participate in the coordination of signal transduction and membrane trafficking to regulate plant immunity. Membrane phospholipids regulate the perception of extracellular signals in the establishment of plant defense; therefore, understanding phospholipid-based signaling is essential to elucidate the regulatory role of phospholipids in plant immunity [10.Meijer H.J. Munnik T. Phospholipid-based signaling in plants.Annu. Rev. Plant Biol. 2003; 54: 265-306Crossref PubMed Scopus (475) Google Scholar]. PLs and lipid kinases generate different intracellular messengers by cleaving or phosphorylating different bonds in phospholipids, thus affecting a range of physiological processes in plants [11.Tang Y. et al.Arabidopsis type II phosphatidylinositol 4-kinase PI4Kγ5 regulates auxin biosynthesis and leaf margin development through interacting with membrane-bound transcription factor ANAC078.PLoS Genet. 2016; 12e1006252Crossref PubMed Scopus (27) Google Scholar] (Figure 1). Plant PLs can be grouped into three families (PLA, PLC, and PLD) with distinct enzymatic activities and substrate specificities. PLAs catalyze the hydrolysis of acyl groups from the sn-1 and sn-2 positions. PLCs cleave the proximal phosphodiester bonds of phospholipids, and PLDs cleave the terminal phosphodiester bonds [12.Ryu S.B. Phospholipid-derived signaling mediated by phospholipase A in plants.Trends Plant Sci. 2004; 9: 229-235Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 13.Chen G. et al.Plant phospholipase A: advances in molecular biology, biochemistry, and cellular function.Biomol. Concepts. 2013; 4: 527-532Crossref PubMed Scopus (29) Google Scholar, 14.Pokotylo I. et al.The plant non-specific phospholipase C gene family. Novel competitors in lipid signalling.Prog. Lipid Res. 2013; 52: 62-79Crossref PubMed Scopus (76) Google Scholar, 15.Chen G. et al.Biology and biochemistry of plant phospholipases.Crit. Rev. Plant Sci. 2011; 30: 239-258Crossref Scopus (68) Google Scholar]. PLAs (including PLA1, secretory PLAs, and patatin-like PLAs) hydrolyze phosphatidylcholine (PC), phosphatidylethanolamine (PE), monogalactosyldiacylglycerol (MGDG), digalactosyl-diacylglycerol (DGDG), and triacylglycerol (TAG). PLCs are subdivided into phosphoinositide (PI)-specific PLCs (PI-PLCs) and PC-specific PLCs (PC-PLCs). Plant PC-PLCs act on diverse substrates, including PC, PE, and phosphatidylserine (PS). PI-PLCs cleave phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol-4-phosphate [PtdIns(4)P], and phosphatidylinositol [14.Pokotylo I. et al.The plant non-specific phospholipase C gene family. Novel competitors in lipid signalling.Prog. Lipid Res. 2013; 52: 62-79Crossref PubMed Scopus (76) Google Scholar,16.Abd-El-Haliem A.M. Joosten M.H. Plant phosphatidylinositol-specific phospholipase C at the center of plant innate immunity.J. Integr. Plant Biol. 2017; 59: 164-179Crossref PubMed Scopus (21) Google Scholar]. PLDs are a diverse family and have a range of substrates containing PE, PC, PS, PI, phosphatidylglycerol (PG), and N-acyl phosphatidylethanolamine (NAPE) [15.Chen G. et al.Biology and biochemistry of plant phospholipases.Crit. Rev. Plant Sci. 2011; 30: 239-258Crossref Scopus (68) Google Scholar]. Similar to PLCs, PI kinases (including PI3K, PI4K, PI5K, PI3P 5-kinase, PI4P 5-kinase, and PI5P 4-kinases) also act on PI, phosphorylating the inositol ring on the D3, D4, or D5 position to generate different phosphorylated phosphatidylinositol (PtdIns) isomers [11.Tang Y. et al.Arabidopsis type II phosphatidylinositol 4-kinase PI4Kγ5 regulates auxin biosynthesis and leaf margin development through interacting with membrane-bound transcription factor ANAC078.PLoS Genet. 2016; 12e1006252Crossref PubMed Scopus (27) Google Scholar]. Plants have three isomers of PtdIns [PtdIns(3)P, PtdIns(4)P, and PtdIns(5)P] and two isomers of PIP2 [PtdIns(3,5)P2 and PtdIns(4,5)P2] [17.Nakamura Y. Plant phospholipid diversity: emerging functions in metabolism and protein-lipid interactions.Trends Plant Sci. 2017; 22: 1027-1040Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar] (Figure 1). Generally, the function of PLs and lipid kinases are related to the phospholipid molecules that they produce [18.Zhao J. Phospholipase D and phosphatidic acid in plant defence response: from protein-protein and lipid-protein interactions to hormone signalling.J. Exp. Bot. 2015; 66: 1721-1736Crossref PubMed Scopus (103) Google Scholar]. Free fatty acids (FFAs) and lysophospholipids (LPLs) are the main products of PLAs [18.Zhao J. Phospholipase D and phosphatidic acid in plant defence response: from protein-protein and lipid-protein interactions to hormone signalling.J. Exp. Bot. 2015; 66: 1721-1736Crossref PubMed Scopus (103) Google Scholar] (Figure 1). FFAs are reported to be involved in jasmonic acid biosynthesis, and other products, such as lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), participate in a range of signaling pathways, such as defense and wound responses, cell growth and senescence, and polar tip growth [18.Zhao J. Phospholipase D and phosphatidic acid in plant defence response: from protein-protein and lipid-protein interactions to hormone signalling.J. Exp. Bot. 2015; 66: 1721-1736Crossref PubMed Scopus (103) Google Scholar,19.Yang W.Y. et al.AtPLAI is an acyl hydrolase involved in basal jasmonic acid production and Arabidopsis resistance to Botrytis cinerea.J. Biol. Chem. 2007; 282: 18116-18128Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 20.Kim H.J. et al.Endoplasmic reticulum- and Golgi-localized phospholipase A2 plays critical roles in Arabidopsis pollen development and germination.Plant Cell. 2011; 23: 94-110Crossref PubMed Scopus (59) Google Scholar, 21.Lee O.R. et al.Phospholipase A2 is required for PIN-FORMED protein trafficking to the plasma membrane in the Arabidopsis root.Plant Cell. 2010; 22: 1812-1825Crossref PubMed Scopus (75) Google Scholar]. PLCs are major membrane phospholipases and generate signals such as diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) (Figure 1). In contrast to animal systems, plant cells lack IP3 receptors and counterparts of animal protein kinase C (PKC). Thus, some derivatives of DAG and IP3, such as phosphatidic acid (PA), diacylglycerol pyrophosphate (DGPP), and hexakisphosphate (IP6), function as second messengers in plant signaling pathways [22.van Leeuwen W. et al.Visualization of phosphatidylinositol 4,5-bisphosphate in the plasma membrane of suspension-cultured tobacco BY-2 cells and whole Arabidopsis seedlings.Plant J. 2007; 52: 1014-1026Crossref PubMed Scopus (135) Google Scholar,23.Xue H. et al.Involvement of phospholipid signaling in plant growth and hormone effects.Curr. Opin. Plant Biol. 2007; 10: 483-489Crossref PubMed Scopus (89) Google Scholar]. PLDs are similar to other PLs (such as PLAs and PLCs), in which their functions are often carried out through their lipid products. The PLD-derived PA can alter the enzyme activity of PLD, the distribution of PA-targeted proteins, and membrane–cytoskeleton interactions regulated by PLDs [24.Yao H. Xue H. Phosphatidic acid plays key roles regulating plant development and stress responses.J. Integr. Plant Biol. 2018; 60: 851-863Crossref PubMed Scopus (58) Google Scholar]. In addition to being produced by PLDs, PA is synthesized through phosphorylation of DAG by DAG kinase [3.Pleskot R. et al.Regulation of cytoskeletal dynamics by phospholipase D and phosphatidic acid.Trends Plant Sci. 2013; 18: 496-504Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar]. PtdIns isomers are produced by PI kinases and have emerged as key components in several trafficking events, acting as membrane-docking targets for proteins containing specific lipid-binding domains, such as the zinc-finger-like domain of Fab1p, YOTB, Vac1p, EEA1 (FYVE), phagocyte oxidase (PX), zinc finger domain (ZFD), zinc- and calcium-binding domain (ZAC), and PHOX (PH) domains [10.Meijer H.J. Munnik T. Phospholipid-based signaling in plants.Annu. Rev. Plant Biol. 2003; 54: 265-306Crossref PubMed Scopus (475) Google Scholar]. Moreover, PtdIns isomers have specific spatial and temporal localizations in the PM or endomembrane system, which makes them important in many cellular processes, including clathrin-mediated endocytosis (CME), polarized trafficking, endosomal sorting, and vacuolar biogenesis [9.Noack L.C. Jaillais Y. Precision targeting by phosphoinositides: how PIs direct endomembrane trafficking in plants.Curr. Opin. Plant Biol. 2017; 40: 22-33Crossref PubMed Scopus (61) Google Scholar]. The plant immune system relies on the membrane trafficking network to quickly transport defense-associated molecules to specific subcellular compartments [7.Wang W.M. et al.Protein trafficking during plant innate immunity.J. Integr. Plant Biol. 2016; 58: 284-298Crossref PubMed Scopus (31) Google Scholar]. The orchestration of phospholipid signaling and membrane trafficking is essential for plant immune responses. PLs, lipid kinases, and lipid molecules ensure the correct spatiotemporal distribution of defense-associated molecules by modulating membrane deformation, regulating cytoskeletal rearrangement, and recruiting trafficking regulators. During the plant defense response, the spatiotemporal dynamics and translocation of PLs usually lead to the activation of PLs and the recruitment of defense regulators; this process is involved in transporting defense regulators [2.Hong Y. et al.Plant phospholipases D and C and their diverse functions in stress responses.Prog. Lipid Res. 2016; 62: 55-74Crossref PubMed Scopus (193) Google Scholar] (Figure 1). For example, in arabidopsis (Arabidopsis thaliana), the PL sPLA2-α shuttles from cytoplasmic vesicles to the nucleus by interacting with the transcription factor AtMYB30, a positive regulator of plant hypersensitive response. The translocation of AtsPLA2-α may activate AtMYB30 and signal trafficking into the nucleus [25.Froidure S. et al.AtsPLA2-alpha nuclear relocalization by the Arabidopsis transcription factor AtMYB30 leads to repression of the plant defense response.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 15281-15286Crossref PubMed Scopus (61) Google Scholar] (Figure 1 and Table 1). In another example, PLDδ relocalizes from the PM to papillae in extracellular spaces during infection by the fungus Blumeria graminis f. sp hordei (Bgh) (Figure 1). PA generated by PLDδ accumulates in papillae and recruits effector proteins, such as protein kinases, phosphatases, and NADPH oxidases, thereby initiating PA-related plant defense signaling [26.Xing J. et al.Secretion of phospholipase Ddelta functions as a regulatory mechanism in plant innate immunity.Plant Cell. 2019; 31: 3015-3032Crossref PubMed Scopus (31) Google Scholar]. Thus, PLs may serve as the vesicle targeting signal and their translocation may be related to export of defense proteins during pathogen attack.Table 1Candidates in the Coordination of Phospholipid-Based Signaling and Membrane Trafficking in Plant ImmunityLipidsRelated enzymesInteracting proteinsFunctionsRefsFatty acidssPLA2MYB30Relocalization of MYB30 in plant hypersensitive response[25.Froidure S. et al.AtsPLA2-alpha nuclear relocalization by the Arabidopsis transcription factor AtMYB30 leads to repression of the plant defense response.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 15281-15286Crossref PubMed Scopus (61) Google Scholar]PAPLDsAtPEN1SNARE-mediated secretion in penetration resistance[26.Xing J. et al.Secretion of phospholipase Ddelta functions as a regulatory mechanism in plant innate immunity.Plant Cell. 2019; 31: 3015-3032Crossref PubMed Scopus (31) Google Scholar]NPR1Translocation of NPR1 to nucleus in plant defense responses[28.Janda M. et al.Phospholipase D affects translocation of NPR1 to the nucleus in Arabidopsis thaliana.Front. Plant Sci. 2015; 6: 59Crossref PubMed Scopus (19) Google Scholar]DAGPI-PLCStimulating defense metabolite synthesis[83.Vasconsuelo A. et al.Involvement of the PLC/PKC pathway in Chitosan-induced anthraquinone production by Rubia tinctorum L. cell cultures.Plant Sci. 2003; 165: 429-436Crossref Scopus (58) Google Scholar]PtdIns(4)PAtPI4Kβ1/β2AtPUB13, AtRabA4BTrafficking of PRR FLAGELIN-INSENSITIVE2[62.Antignani V. et al.Recruitment of PLANT U-BOX13 and the PI4Kbeta1/beta2 phosphatidylinositol-4 kinases by the small GTPase RabA4B plays important roles during salicylic acid-mediated plant defense signaling in Arabidopsis.Plant Cell. 2015; 27: 243-261Crossref PubMed Scopus (59) Google Scholar]PtdIns(3)PPI3KPE and PtdIns(3)P-enriched endosomes for building large replication compartments of virus[33.Feng Z. et al.Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus.PLoS Pathog. 2019; 15e1007530Crossref PubMed Scopus (28) Google Scholar]PR1Secreting defense protein PR1[34.Pecenkova T. et al.Subcellular localization of Arabidopsis pathogenesis-related 1 (PR1) protein.Int. J. Mol. Sci. 2017; 18: 825Crossref Scopus (27) Google Scholar]StREM1.3, BIK1PM–MVB tethering[88.Tao K. et al.Tethering of multi-vesicular bodies and the tonoplast to the plasma membrane in plants.Front. Plant Sci. 2019; 10: 636Crossref PubMed Scopus (7) Google Scholar]SH3P2, ATG8Regulating autophagosome formation in plant response to viral infection[89.Zhuang X. et al.A BAR-domain protein SH3P2, which binds to phosphatidylinositol 3-phosphate and ATG8, regulates autophagosome formation in Arabidopsis.Plant Cell. 2013; 25: 4596-4615Crossref PubMed Scopus (153) Google Scholar]PtdIns(4,5)P2PIP5KExo70Increasing exocytic membrane supply of EIHM for successful infection[86.Ivanov S. Harrison M.J. Accumulation of phosphoinositides in distinct regions of the periarbuscular membrane.New Phytol. 2019; 221: 2213-2227Crossref PubMed Scopus (10) Google Scholar] Open table in a new tab In yeast, decreased levels of PA on the endoplasmic reticulum (ER) lead to the relocalization of the transcriptional repressor Opilp from the ER to the nucleus, repressing target gene expression [27.Loewen C.J. et al.Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid.Science. 2004; 304: 1644-1647Crossref PubMed Scopus (363) Google Scholar]. In plants, we lack evidence that the spatiotemporal localization of PA is linked to membrane trafficking in pathogen defense responses. However, n-butanol interferes with the translocation of NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) to the nucleus in response to plant defense and the accumulation of PLDδ at the fungus entry sites accompanied by defense-related vesicles trafficking, probably through the distribution of PAs [26.Xing J. et al.Secretion of phospholipase Ddelta functions as a regulatory mechanism in plant innate immunity.Plant Cell. 2019; 31: 3015-3032Crossref PubMed Scopus (31) Google Scholar,28.Janda M. et al.Phospholipase D affects translocation of NPR1 to the nucleus in Arabidopsis thaliana.Front. Plant Sci. 2015; 6: 59Crossref PubMed Scopus (19) Google Scholar] (Table 1). In addition, PtdIns serve as biochemical landmarks for different membranes in the endomembrane system: PtdIns(4)P localizes to the Golgi apparatus, trans-Golgi network (TGN), late endosome, and PM; PtdIns(4,5)P2 localizes to the PM; and PtdIns(3)P localizes to the late endosome and tonoplast [29.Gerth K. et al.Guilt by association: A phenotype-based view of the plant phosphoinositide network.Annu. Rev. Plant Biol. 2017; 68: 349-374Crossref PubMed Scopus (45) Google Scholar,30.Simon M.L. et al.A multi-colour/multi-affinity marker set to visualize phosphoinositide dynamics in Arabidopsis.Plant J. 2014; 77: 322-337Crossref PubMed Scopus (150) Google Scholar] (Figure 1). In animal cells, PtdIns(4,5)P2 is the major cellular PI; by contrast, PtdIns4P is predominant in plant cells [31.Li L. et al.A phosphoinositide-specific phospholipase C pathway elicits stress-induced Ca2+ signals and confers salt tolerance to rice.New Phytol. 2017; 214: 1172-1187Crossref PubMed Scopus (62) Google Scholar]. However, PtdIns(4,5)P2, not PtdIns(4)P, is recruited to the extrahaustorial membrane (EHM) in response to powdery mildew infection [32.Qin L. et al.Specific recruitment of phosphoinositide species to the plant-pathogen interfacial membrane underlies Arabidopsis susceptibility to fungal infection.Plant Cell. 2020; 32: 1665-1688Crossref PubMed Scopus (19) Google Scholar] (Figure 1). The different subcellular distributions of PIs suggest their involvement in coordinating membrane recruitment and regulating trafficking. More importantly, studies showing that PE- and PtdIns(3)P-enriched endosomes were hijacked by viruses to build large replication compartments in plant cells supported the role of PtdIns in membrane trafficking [33.Feng Z. et al.Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus.PLoS Pathog. 2019; 15e1007530Crossref PubMed Scopus (28) Google Scholar] (Table 1). In arabidopsis, the secretion of the defense protein PATHOGENESIS-RELATED 1 (PR1) occurs through an unconventional pathway that starts from the ER and proceeds to transport PR1 from PtdIns(3)P-positive late endosome/multivesicular body (LE/MVB)-like vesicles instead of Golgi [34.Pecenkova T. et al.Subcellular localization of Arabidopsis pathogenesis-related 1 (PR1) protein.Int. J. Mol. Sci. 2017; 18: 825Crossref Scopus (27) Google Scholar] (Figure 2, Key Figure, and Table 1). Membrane fusion is an essential step for membrane trafficking, and the formation of the fusion pore involves considerable membrane bending [35.Chernomordik L.V. Kozlov M.M. Mechanics of membrane fusion.Nat. Struct. Mol. Biol. 2008; 15: 675-683Crossref PubMed Scopus (694) Google Scholar]. Phospholipids and their derivatives change membrane topology to promote membrane fusion and modulate exocytosis and endocytosis [36.Bader M.F. Vitale N. Phospholipase D in calcium-regulated exocytosis: lessons from chromaffin cells.Biochim. Biophys. Acta. 2009; 1791: 936-941Crossref PubMed Scopus (59) Google Scholar,37.Liu Y. et al.Phosphatidic acid-mediated signaling.Adv. Exp. Med. Biol. 2013; 991: 159-176Crossref PubMed Scopus (75) Google Scholar] (Figure 3). Different lipids form membranes of different curvatures: membranes comprising two-tailed PCs form nearly flat lipid monolayers, membranes comprising cone-shaped PA and DAG have a negative spontaneous curvature, and membranes comprising inverted cone-shaped lipids, such as PtdIns(4,5)P2, one-tailed lipids, LPC, and lysophosphatidic acid (LPA), have a positive spontaneous curvature [38.Zhukovsky M.A. et al.Phosphatidic acid in membrane rearrangements.FEBS Lett. 2019; 593: 2428-2451Crossref PubMed Scopus (49) Google Scholar] (Figure 3A). Therefore, their distinct molecular shapes allow various lipids to form structures of different curvatures and to have fundamental roles in the regulation of membrane fusion. PA is the simplest cellular glycerophospholipid and its cone shape indicates a likely central role in regulating membrane fusion [39.Zeniou-Meyer M. et al.Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage.J. Biol. Chem. 2007; 282: 21746-21757Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar]. In animals, SNARE proteins recruit PA to fusion sites to promote fusion [40.Lam A.D. et al.SNARE-catalyzed fusion events are regulated by Syntaxin1A-lipid interactions.Mol. Biol. Cell. 2008; 19: 485-497Crossref PubMed Scopus (124) Google Scholar]. PA likely fulfils a similar role in plants. In the antifungal defense response known as penetration resistance, PA accumulates and colocalizes with PLDδ and the PENETRATION1 soluble N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) complex at the pathogen entry site [26.Xing J. et al.Secretion of phospholipase Ddelta functions as a regulatory mechanism in plant innate immunity.Plant Cell. 2019; 31: 3015-3032Crossref PubMed Scopus (31) Google Scholar] (Figure 3B). The localization of PLDδ may control the polar secretion of antimicrobial compounds via PA production to promote vesicle fusion [26.Xing J. et al.Secretion of phospholipase Ddelta functions as a regulatory mechanism in plant innate immunity.Plant Cell. 2019; 31: 3015-3032Crossref PubMed Scopus (31) Google Scholar,41.Pinosa F. et al.Arabidopsis phospholipase ddelta is involved in basal defense and nonhost resistance to powdery mildew fungi.Plant Physiol. 2013; 163: 896-906Crossref PubMed Scopus (76) Google Scholar]. In addition, PA-derived DAG and LPA influence membrane fusion. For example, increased levels of DAG on the inner leaflet of the synaptic membrane enhance membrane fusion [42.Chernomordik L.V. Kozlov M.M. Membrane hemifusion: crossing a chasm in two leaps.Cell. 2005; 123: 375-382Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar]. In human cells, DAG stimulates exocytosis of TRPC6-containing vesicles that are predocked to the PM; it targets these vesicles to the C1 domain of Munc13, which facilitates SNARE complex assembly [43.Xie J. et al.Munc13 mediates klotho-inhibitable diacylglycerol-stimulated exocytotic insertion of pre-docked TRPC6 vesicles.PLoS ONE. 2020; 15e0229799PubMed Google Scholar] (Figure 3B). However, the roles of DAG and LPA in membrane fusion and membrane topology have not been thoroughly investigated in plant cells. PtdIns(4,5)P2 is another key regulator that acts during fusion in vesicle trafficking [44.De Craene J.O. et al.Phosphoinositides, major actors in membrane trafficking and lipid signaling pathways.Int. J. Mol. Sci. 2017; 18: 634Crossref PubMed Scopus (108) Google Scholar,45.Martin T.F. PI(4,5)P2-binding effector proteins for vesicle exocytosis.Biochim. Biophys. Acta. 2015; 1851: 785-793Crossref PubMed Scopus (82) Google Scholar]. In mammalian cells, docked vesicles colocalize with membrane sites enriched in PtdIns(4,5)P2, suggesting a direct effect of PI on membrane curvature and tension [45.Martin T.F. PI(4,5)P2-binding effector proteins for vesicle exocytosis.Biochim. Biophys. Acta. 2015; 1851: 785-793Crossref PubMed Scopus (82) Google Scholar]. Several studies in plant cells confirmed a similar role for PtdIns(4,5)P2 in establishing sites for vesicle trafficking. In endocytosis, PtdIns(4,5)P2 colocalizes with clathrin and affects the formation of clathrin foci at the PM [46.Ischebeck T. et al.Phosphatidylinositol 4,5-bisphosphate influences PIN polarization by controlling clathrin-mediated membrane trafficking in Arabidopsis.Plant Cell. 2013; 25: 4894-4911Crossref PubMed Scopus (120) Google Scholar,47.Konig S. et al.Salt-stress-induced association of phosphatidylinositol 4,5-bisphosphate with clathrin-coated vesicles in plants.Biochem. J. 2008; 415: 387-399Crossref PubMed Scopus (94) Google Scholar]. PtdIns(4,5)P2 interacts with the exocyst subunit SEC3 and contributes to the efficiency of SEC3a recruitment to specific domains on the PM, locally increasing tethering and fusion of secretory vesicles [48.Bloch D. et al.Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth.Plant Physiol. 2016; 172: 980-1002PubMed Google Scholar]. Although increasing evidence suggests an essential role for PtdIns(4,5)P2 in promoting membrane fusion, the mechanisms remain to be demonstrated in plants. In response to pathogen infection, actin filaments and microtubules facilitate the rapid relocalization of immune cargo, host organelles, and proteins [49.Li P. Day B. Battlefield cytoskeleton: turning the tide on