C-Type LECTIN receptor-like kinase 1 and ACTIN DEPOLYMERIZING FACTOR 3 are key components of plasmodesmata callose modulation.

Plasmodesmata (PDs) are intercellular organelles carrying multiple membranous nanochannels that allow the trafficking of cellular signalling molecules. The channel regulation of PDs occurs dynamically and is required in various developmental and physiological processes. It is well known that callose is a critical component in regulating PD permeability or symplasmic connectivity, but the understanding of the signalling pathways and mechanisms of its regulation is limited. Here, we used the reverse genetic approach to investigate the role of C-type lectin receptor-like kinase 1 (CLRLK1) in the aspect of PD callose-modulated symplasmic continuity. Here, we found that loss-of-function mutations in CLRLK1 resulted in excessive PD callose deposits and reduced symplasmic continuity, resulting in an accelerated gravitropic response. The protein interactome study also found that CLRLK1 interacted with actin depolymerizing factor 3 (ADF3) in vitro and in plants. Moreover, mutations in ADF3 result in elevated PD callose deposits and faster gravitropic response. Our results indicate that CLRLK1 and ADF3 negatively regulate PD callose accumulation, contributing to fine-tuning symplasmic opening apertures. Overall, our studies identified two key components involved in the deposits of PD callose and provided new insights into how symplasmic connectivity is maintained by the control of PD callose homoeostasis.

Secretory membrane traffic in plant-microbe interactions.

Plant defense responses include the extracellular release of defense-related molecules, such as pathogenesis-related proteins and secondary metabolites, as well as cell wall materials. This primarily depends on the trafficking of secretory vesicles to the plasma membrane, where they discharge their contents into the apoplastic space via soluble N-ethylmaleimide sensitive factor attachment protein receptor-assisted exocytosis. However, some pathogenic and symbiotic microbes have developed strategies to manipulate host plant exocytic pathways. Here, we discuss the mechanisms by which plant exocytic pathways function in immunity and how microbes have evolved to manipulate those pathways.

CCOAOMT1, a candidate cargo secreted via VAMP721/722 secretory vesicles in Arabidopsis.

We previously found that VAMP721/722 SNARE proteins guide secretory vesicles to pathogen-attacking sites during immune responses in Arabidopsis, which suggests that these vesicles should deliver immune molecules. However, the lethality of vamp721 vamp722 double null mutant makes it difficult to understand the nature of cargo transported via VAMP721/722 vesicles. Since VAMP721/722-depleted (VAMP721+/-VAMP722-/- and VAMP721-/-VAMP722+/-) plants show compromised resistance to extracellular pathogens, we assume that an immune protein secreted through the VAMP721/722-engaged exocytosis would be remained more in VAMP721/722-depleted plants than WT. By comparing intracellular proteins between WT and VAMP721/722-depleted plants, we found caffeoyl-CoA O-methyltransferase 1 (CCOAOMT1) involved in the lignin biosynthesis was more abundantly detected in both VAMP721/722-depleted lines than WT. Plants are well-known to deposit secondary cell walls as physical barriers at pathogen-attempting sites. Therefore, extracellular detection of CCOAOMT1 and impaired resistance to Pseudomonas syringae DC3000 in ccoaomt1 plants suggest that plants secrete cell wall-modifying enzymes at least including CCOAOMT1 to reinforce the secondary cell walls for immunity.

Endoplasmic reticulum stress-induced accumulation of VAMP721/722 requires CALRETICULIN 1 and CALRETICULIN 2 in Arabidopsis.

Excessive demand for translation and protein folding in the endoplasmic reticulum (ER) can cause ER stress in plants. Here, we show that CALRETICULIN 1 (CRT1) and CRT2 are critical components in the accumulation of VESICLE-ASSOCIATED MEMBRANE PROTEIN 721 (VAMP721) and VAMP722 during ER stress responses. We show that CRT2 interacts with VAMP722 and that CRT1/2 post-translationally maintain elevated VAMP721/722 levels under ER stress. The greater growth inhibition in VAMP721/722-deficient plants, induced by tunicamycin, suggests that plants under ER stress maintain physiological homeostasis, at least in part, by regulating VAMP721/722 levels, as VAMP721/722 are known to participate in various biological processes.

Synaptotagmin 1 Negatively Controls the Two Distinct Immune Secretory Pathways to Powdery Mildew Fungi in Arabidopsis.

PEN1, one of the plasma membrane (PM) syntaxins, comprises an immune exocytic pathway by forming the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex with SNAP33 and VAMP721/722 in plants. Although this secretory pathway is also involved in plant growth and development, how plants control their exocytic activity is as yet poorly understood. Since constitutive PEN1 cycling between the PM and endocytosed vesicles is critical for its immune activity, we studied here the relationship of PEN1 to synaptotagmin 1 (SYT1) that is known to regulate endocytosis at the PM. Interestingly, syt1 plants showed enhanced disease resistance to the Arabidopsis-adapted Golovinomyces orontii fungus, and elevated protein but not transcript levels of PEN1 Calcium-dependent promotion of PEN1-SYT1 interaction suggests that SYT1 controls defense activities of the PEN1-associated secretory pathway by post-translationally modulating PEN1. Increased PEN1-SYT1 interaction and inhibited PEN1 SNARE complex induction by G. orontii additionally suggest that the adaption of phytopathogens to host plants might partly result from effective suppression of the PEN1-related secretory pathway. Further genetic analyses revealed that SYT1 also regulates the atypical peroxisomal myrosinase PEN2-associated secretory pathway.

Syntaxin of plant proteins SYP123 and SYP132 mediate root hair tip growth in Arabidopsis thaliana.

Root hairs are fast-growing tubular protrusions on root epidermal cells that play important roles in water and nutrient uptake in plants. The tip-focused polarized growth of root hairs is accomplished by the secretion of newly synthesized materials to the tip via the polarized membrane trafficking mechanism. Here, we report the function of two different types of plasma membrane (PM) Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), SYP123 and SYP132, in the growth of root hair in Arabidopsis. We found that SYP123, but not SYP132, localizes in the tip region of root hairs by recycling between the brefeldin A (BFA)-sensitive endosomes and the PM of the expanding tip in an F-actin-dependent manner. The vesicle-associated membrane proteins VAMP721/722/724 also exhibited tip-focused localization in root hairs and formed ternary SNARE complexes with both SYP123 and SYP132. These results demonstrate that SYP123 and SYP132 act in a coordinated fashion to mediate tip-focused membrane trafficking for root hair tip growth.

Co-option of a default secretory pathway for plant immune responses.

Cell-autonomous immunity is widespread in plant-fungus interactions and terminates fungal pathogenesis either at the cell surface or after pathogen entry. Although post-invasive resistance responses typically coincide with a self-contained cell death of plant cells undergoing attack by parasites, these cells survive pre-invasive defence. Mutational analysis in Arabidopsis identified PEN1 syntaxin as one component of two pre-invasive resistance pathways against ascomycete powdery mildew fungi. Here we show that plasma-membrane-resident PEN1 promiscuously forms SDS-resistant soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complexes together with the SNAP33 adaptor and a subset of vesicle-associated membrane proteins (VAMPs). PEN1-dependent disease resistance acts in vivo mainly through two functionally redundant VAMP72 subfamily members, VAMP721 and VAMP722. Unexpectedly, the same two VAMP proteins also operate redundantly in a default secretory pathway, suggesting dual functions in separate biological processes owing to evolutionary co-option of the default pathway for plant immunity. The disease resistance function of the secretory PEN1-SNAP33-VAMP721/722 complex and the pathogen-induced subcellular dynamics of its components are mechanistically reminiscent of immunological synapse formation in vertebrates, enabling execution of immune responses through focal secretion.

A bacterial type III effector targets plant vesicle-associated membrane proteins.

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.

Epigenetic control of abiotic stress signaling in plants.

BACKGROUND: Although plants may be regularly exposed to various abiotic stresses, including drought, salt, cold, heat, heavy metals, and UV-B throughout their lives, it is not possible to actively escape from such stresses due to the immobile nature of plants. To overcome adverse environmental stresses, plants have developed adaptive systems that allow appropriate responses to diverse environmental cues; such responses can be achieved by fine-tuning or controlling genetic and epigenetic regulatory systems. Epigenetic mechanisms such as DNA or histone modifications and modulation of chromatin accessibility have been shown to regulate the expression of stress-responsive genes in struggles against abiotic stresses. OBJECTIVE: Herein, the current progress in elucidating the epigenetic regulation of abiotic stress signaling in plants has been summarized in order to further understand the systems plants utilize to effectively respond to abiotic stresses. METHODS: This review focuses on the action mechanisms of various components that epigenetically regulate plant abiotic stress responses, mainly in terms of DNA methylation, histone methylation/acetylation, and chromatin remodeling. CONCLUSIONS: This review can be considered a basis for further research into understanding the epigenetic control system for abiotic stress responses in plants. Moreover, the knowledge of such systems can be effectively applied in developing novel methods to generate abiotic stress resistant crops.

Synaptotagmin 4 and 5 additively contribute to Arabidopsis immunity to Pseudomonas syringae DC3000.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are essential for vesicle trafficking in plants. Vesicle-associated membrane protein 721 and 722 (VAMP721/722) are secretory vesicle-localized R-SNAREs, which are involved in a variety of biological processes in plants. Compared to VAMP721/722, a VAMP721/722-interacting plasma membrane (PM)-localized Qa-SNARE is engaged in a rather specific physiological process. This indicates that an in vivo regulator controls an interaction between a Qa-SNARE and VAMP721/722 for a specific cellular activity. We previously reported that synaptotagmin 5 (SYT5) modulates the interaction between SYP132 PM Qa-SNARE and VAMP721/722 for Arabidopsis resistance to Pseudomonas syringae DC3000. In this study, we show that defense against P. syringae DC3000 is compromised in SYT4-lacking plants, which belongs to the same subclade as SYT5. Further elevation of bacterial growth in syt4 syt5-2 plants compared to either syt4 or syt5-2 single mutant suggests that SYT4 and SYT5 play additive roles in Arabidopsis immunity to P. syringae DC3000.

Synaptotagmin 5 Controls SYP132-VAMP721/722 Interaction for Arabidopsis Immunity to Pseudomonas syringae pv tomato DC3000.

Vesicle-associated membrane proteins 721 and 722 (VAMP721/722) are secretory vesicle-localized arginine-conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) to drive exocytosis in plants. They are involved in diverse physiological processes in plants by interacting with distinct plasma membrane (PM) syntaxins. Here, we show that synaptotagmin 5 (SYT5) is involved in plant defense against Pseudomonas syringae pv tomato (Pst) DC3000 by regulating SYP132-VAMP721/722 interactions. Calcium-dependent stimulation of in vitro SYP132-VAMP722 interaction by SYT5 and reduced in vivo SYP132-VAMP721/722 interaction in syt5 plants suggest that SYT5 regulates the interaction between SYP132 and VAMP721/722. We interestingly found that disease resistance to Pst DC3000 bacterium but not to Erysiphe pisi fungus is compromised in syt5 plants. Since SYP132 plays an immune function to bacteria, elevated growth of surface-inoculated Pst DC3000 in VAMP721/722-deficient plants suggests that SYT5 contributes to plant immunity to Pst DC3000 by promoting the SYP132-VAMP721/722 immune secretory pathway.

SNAREs in Plant Biotic and Abiotic Stress Responses.

In eukaryotes, membraneous cellular compartmentation essentially requires vesicle trafficking for communications among distinct organelles. A donor organelle-generated vesicle releases its cargo into a target compartment by fusing two distinct vesicle and target membranes. Vesicle fusion, the final step of vesicle trafficking, is driven intrinsically by complex formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNAREs are well-conserved across eukaryotes, genomic studies revealed that plants have dramatically increased the number of SNARE genes than other eukaryotes. This increase is attributed to the sessile nature of plants, likely for more sensitive and harmonized responses to environmental stresses. In this review, we therefore try to summarize and discuss the current understanding of plant SNAREs function in responses to biotic and abiotic stresses.

Reactive-oxygen-species-mediated mechanism for photoinduced antibacterial and antiviral activities of Ag3PO4.

Cubic-shaped Ag3PO4 crystals with a mean size of 1 µm were synthesized by a precipitation method from a mixed solution of AgNO3, Na2HPO4, and triethanolamine. The antibacterial activities against Escherichia coli, Listeria innocua, and Pseudomonas syringae DC3000 in both the absence and presence of Ag3PO4 under dark conditions and in the presence of Ag3PO4 under red-light (625 nm) and blue-light (460 nm) irradiation were examined. The concentrations of reactive oxygen species (ROS) were also measured in the antibacterial action of the Ag3PO4 against Escherichia coli. The photoinduced enhancement of the Ag3PO4 antibacterial activity under blue-light irradiation is explained by the formation of ROS during the antibacterial action of the Ag3PO4. Moreover, the antiviral activity of Ag3PO4 against amphotropic 10A1 murine leukemia virus enhanced under blue-light irradiation via ROS production. These results provide an insight into extended bio-applications of Ag3PO4.

Regulation of cellular VAMP721/722 abundance in arabidopsis.

Sessile plants are continuously threatened by biotic and abiotic environmental stresses. Since stress responses are in general accompanied by growth retardation, plants in nature should tightly control timing and duration of their stress responses for sustained growth. We previously reported that vesicle-associated membrane protein (VAMP) 721 and 722 are required for growth/development and stress responses in plants. It is suggested that plants regulate expression of VAMP721/722 and/or drive VAMP721/722 to form distinct SNARE complexes with different plasma membrane (PM)-residing SNARE proteins in response to distinct stimuli. We here report that immune signaling triggered by the bacterial flg22 elicitor elevates VAMP721/722 levels in calreticulin 1 and 2 (CRT1/2)-lacking plants. Since VAMP721/722 amounts were reported not to be increased by an ER stress inducer, tunicamycin in crt1/2 plants, our results suggest that ER stress and immune signalings distinctly control cellular abundance of VAMP721/722.

Vesicle trafficking in plant immunity.

To defend against extracellular pathogens, plants primarily depend on cell-autonomous innate immunity due to the lack of the circulatory immune system including mobile immune cells. To extracellularly restrict or kill the pathogens, plant cells dump out antimicrobials. However, since antimicrobials are also toxic to plant cells themselves, they have to be safely delivered to the target sites in a separate vesicular compartment. In addition, because immune responses often requires energy otherwise used for the other metabolic processes, it is very important to properly control the duration and strength of immune responses depending on pathogen types. This can be achieved by regulating the sensing of immune signals and the delivery/discharge of extracellular immune molecules, all of which are controlled by membrane trafficking in plant cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are now considered as the minimal factors that can merge two distinct membranes of cellular compartments. Hence, in this review, known and potential immune functions of SNAREs as well as regulatory proteins will be discussed.

Arabidopsis immune secretory pathways to powdery mildew fungi.

Innate immune responses in host plants begin with the recognition of pathogen-specific nonself molecules and terminate with the secretion of immune molecules. In the dicotyledonous model plant, Arabidopsis thaliana, two distinct secretory pathways required for disease resistance to powdery mildew fungi have been identified so far. One is an exocytic pathway consisting of PEN1, SNAP33 and VAMP721/722 SNARE proteins, but the other is an efflux-mediated one composed of PEN2 atypical myrosinase and PEN3 ABC transporter. Based on the conservation of the mechanically same exocytic pathway in the monocotyledonous plant barely, the former is regarded as an ancient secretory pathway, whereas the latter is considered as a newly evolved one in the Brassicaceae family including Arabidopsis. We recently identified synaptotagmin 1 as an additional regulator of these two secretory pathways. With current results, we discuss how these two secretory pathways contribute to Arabidopsis immunity depending on fungal adaptedness to Arabidopsis.

Dual Effect of the Cubic Ag3PO4 Crystal on Pseudomonas syringae Growth and Plant Immunity.

We previously found that the antibacterial activity of silver phosphate crystals on Escherichia coli depends on their structure. We here show that the cubic form of silver phosphate crystal (SPC) can also be applied to inhibit the growth of a plant-pathogenic Pseudomonas syringae bacterium. SPC pretreatment resulted in reduced in planta multiplication of P. syringae. Induced expression of a plant defense marker gene PR1 by SPC alone is suggestive of its additional plant immunity-stimulating activity. Since SPC can simultaneously inhibit P. syringae growth and induce plant defense responses, it might be used as a more effective plant disease-controlling agent.

Interplay between ABA and GA Modulates the Timing of Asymmetric Cell Divisions in the Arabidopsis Root Ground Tissue.

In multicellular organisms, controlling the timing and extent of asymmetric cell divisions (ACDs) is crucial for correct patterning. During post-embryonic root development in Arabidopsis thaliana, ground tissue (GT) maturation involves an additional ACD of the endodermis, which generates two different tissues: the endodermis (inner) and the middle cortex (outer). It has been reported that the abscisic acid (ABA) and gibberellin (GA) pathways are involved in middle cortex (MC) formation. However, the molecular mechanisms underlying the interaction between ABA and GA during GT maturation remain largely unknown. Through transcriptome analyses, we identified a previously uncharacterized C2H2-type zinc finger gene, whose expression is regulated by GA and ABA, thus named GAZ (GA- AND ABA-RESPONSIVE ZINC FINGER). Seedlings ectopically overexpressing GAZ (GAZ-OX) were sensitive to ABA and GA during MC formation, whereas GAZ-SRDX and RNAi seedlings displayed opposite phenotypes. In addition, our results indicated that GAZ was involved in the transcriptional regulation of ABA and GA homeostasis. In agreement with previous studies that ABA and GA coordinate to control the timing of MC formation, we also confirmed the unique interplay between ABA and GA and identified factors and regulatory networks bridging the two hormone pathways during GT maturation of the Arabidopsis root.

Characterization of single stranded telomeric DNA-binding proteins in cultured soybean (Glycine max) cells.

We have identified and characterized a protein factor in soybean (Glycine max) nuclear extracts that binds to plant single stranded telomeric DNA repeats. A single DNA-protein complex was detected in gel retardation assays using synthetic telomeres and nuclear extracts. The protein forming this complex was designated soy-bean (Glycine max) single stranded telomeric DNA-binding protein (Gm-STBP). Gm-STBP binds to single stranded telomeric DNA containing more than two repeats. It does not bind to Tetrahymena, human or mutated plant telomere sequences, and its binding activity is not affected by RNase treatment. Gm-STBP activity gradually decreased after suspension cultures entered stationary phase. A slower migrating band was formed with extracts of earlier and later phases of soybean suspension cultures. Our findings suggest that binding of Gm-STBP to plant single stranded telomeric DNA may play a role in the proper functioning of telomeres during development.

Identification and characterization of an auxin-inducible protein kinase, VrCRK1, from mungbean.

An auxin-inducible protein kinase, VrCRK1, was isolated by a differential reverse transcriptase-polymerase chain reaction, using mRNAs extracted from auxin-treated mungbean hypocotyls. VrCRK1 exhibits high homology with plant CDPKs over catalytic domains, however, it does not have any calcium-binding EF-hand which is typically shown in plant CDPKs. Auxin treatment increased the expression level of VrCRK1. However, the increased level was reduced to basal level by treatment with PCIB, an auxin inhibitor. When extracts of mungbean hypocotyls were immunoprecipitated and the resultant immunoprecipitates were used as the enzyme source, kinase activity of VrCRK1 was found, and such activity was also increased by auxin treatment. In transgenic tobacco plants that express VrCRK1, the transcript levels of some auxin-dependent genes were elevated as much as those in wild type plants treated with auxin. These results indicate that gene expression of VrCRK1 is specifically induced by auxin, and that VrCRK1 may play a role in auxin signaling via protein phosphorylation.