Mol. Cells 2021; 44(9): 670-679
Published online September 10, 2021
https://doi.org/10.14348/molcells.2021.0100
© The Korean Society for Molecular and Cellular Biology
Correspondence to : hsyun@konkuk.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
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.
Keywords plant immunity, Pst DC3000, SYP132, SYT5, VAMP721/722
Soluble
The first identified SNARE components to form a biologically relevant SNARE complex in plants are the plasma membrane (PM)-localized SYP121 (syntaxin of plant 121, also called PEN1) Qa-SNARE, the PM-attached SNAP33 Qbc-SNARE and the vesicle-residing VAMP721/722 (vesicle-associated membrane proteins 721 and 722) R-SNAREs (Collins et al., 2003; Kwon et al., 2008). The SYP121-SNAP33-VAMP721/722 ternary SNARE complex drives an immune exocytosis. Interestingly, while SYP121 function is limited to defense against fungal pathogens, VAMP721/722 are additionally required for resistance to oomycete pathogens (Kwon et al., 2008). VAMP721/722 are also required for growth, cell division and abiotic stress responses (El Kasmi et al., 2013; Ichikawa et al., 2014; Kim et al., 2019; Kwon et al., 2008; Yi et al., 2013; Yun et al., 2013). Promiscuous SNARE complex formation of VAMP721/722 with distinct Qa-SNAREs such as SYP121, SYP111 (KNOLLE), SYP123 and SYP132 (El Kasmi et al., 2013; Ichikawa et al., 2014; Kwon et al., 2008; Yun et al., 2013) strongly suggests that at least one regulatory protein should control the SNARE complex formation of VAMP721/722 with a specific Qa-SNARE in a particular biological process in plants. This can be additionally supported by promiscuous SNARE complex formation of SYP121 with members in the VAMP72 R-SNARE group (Kwon et al., 2008).
In plants, three groups of regulatory proteins such as Sec1/Munc18 (SM), small GTPase and synaptotagmin (SYT) have been studied for modulating the trafficking functionality of SNAREs. The KEULE (also called SEC11) SM protein controls the SNARE complex formation of VAMP721/722 with KNOLLE for cytokinesis (Karnahl et al., 2018; Park et al., 2012), but with SYP121 for potassium uptake and growth (Karnik et al., 2013; 2015; Waghmare et al., 2019; Zhang et al., 2019) by binding to the N-terminus of respective Qa-SNARE. Recently, a KEULE paralog, SEC1B, was found to be predominantly engaged in regulating the SYP132-dependent secretion during pollen growth (Karnahl et al., 2018). The plant-specific ARA6 GTPase is regarded to switch the interaction of SYP121 between VAMP721/722 and VAMP727 (Ebine et al., 2011). SYP121 is known to continuously cycle between the PM and endosomes (Reichardt et al., 2011). Focal accumulation of endocytosed SYP121 or its barley ortholog, ROR2, to fungal entry sites was found to require the GNOM ADP-ribosylation factor-guanine nucleotide exchange factor (ARF-GEF) in Arabidopsis or ARFA1b/c GTPases in barely, respectively (Bohlenius et al., 2010; Nielsen et al., 2012). SYT1 preferentially residing in the endoplasmic reticulum (ER) (Levy et al., 2015; Perez-Sancho et al., 2015) was originally found to repair damaged PM by abiotic stresses and control endocytosis often resulting in promoting cell-to-cell movement of viral movement proteins (Lewis and Lazarowitz, 2010; Schapire et al., 2008; Yamazaki et al., 2008). SYT1 together with SYT5 was recently found to regulate the intactness and rearrangement of ER-PM contact sites for stress responses to rare earth elements and viral pathogens in plants (Ishikawa et al., 2020; Lee et al., 2020).
We previously found that SYT1 additionally down-regulates SYP121 abundance possibly via endocytosis to control plant disease resistance to fungal pathogens (Kim et al., 2016). We therefore examined whether SYT5 also functions in plant immunity. Based on specific impairment of resistance to
Plants used for experiments were grown at 22°C with 10-h light/14-h dark photoperiod. To isolate T-DNA-inserted
To express recombinant proteins, cDNAs corresponding to
To test the interaction between SYP132 and SYT5, equimolar recombinant HA-SYP132 and GST-SYT5∆TM were incubated in the absence or presence of 1 mM CaCl2. To assess the effect of Ca2+ on SYT5-SYP132 interaction, 1 mM EDTA was added during incubation. To test the interaction between SYP132 and VAMP722, equimolar recombinant HA-SYP132 and GST-VAMP722 were mixed together with GST-free SYT5∆TM in the absence or presence of 1 mM CaCl2. To analyze whether the Ca2+-promoted SYT5∆TM-SYP132 interaction affects SYP132-VAMP722 interaction, 1 mM EDTA was added during incubation. Interacted proteins were then precipitated with glutathione-Sepharose 4B and the precipitates were analyzed by immunoblot with anti-HA antibody to detect HA-SYP132 in the precipitates.
Proteins were extracted from the indicated genotype plants by suspending ground plant materials in 1× phosphate-buffered saline (PBS) containing 1% Triton X-100. Protein amounts were measured by the Bio-Rad protein assay (Bio-Rad, USA). Protein extracts were first pre-cleared with Protein A/G-agarose beads (Santa Cruz Biotechnology, USA) and incubated with anti-SYT5 or anti-VAMP721/722 antibody. Anti-SYT5 antibody-bound or anti-VAMP721/722 antibody-bound proteins were then retrieved by precipitation with Protein A/G-agarose beads. The immunoprecipitates were finally analyzed by immunoblot with anti-SYP132 antibody. A part (3%) of protein extracts used for immunoprecipitation were subject to immunoblot with anti-SYT5, anti-SYP121, anti-SYP132, anti-VAMP721/722 antibody, or anti-SNAP33 antiserum for showing steady state levels of SYT5, SYP121, SYP132, VAMP721/722, or SNAP33, respectively.
Plants were grown in soil for 4-5 weeks to be inoculated with
For immunostaining, 7-day-old transgenic Arabidopsis seedlings expressing either GFP-SYP132 or mRFP-VAMP722 were fixed in MTSB buffer (50 mM PIPES, 5 mM EGTA and 5 mM MgSO4, pH 7.0 adjusted with KOH) containing 4% paraformaldehyde for 1 h at room temperature under vacuum infiltration (Sauer et al., 2006). Fixed seedlings were placed onto poly-L-Lys-coated glass slides, and washed with MTSB buffer containing 0.1% Triton X-100 and with deionized water. Fixed seedlings were then incubated with 2% driselase in MTSB buffer for 40 min to digest their cell wall, and incubated with PBS containing 20% DMSO and 3% NP40 for 1 h. Following washing with MTSB buffer containing 0.1% Triton and with deionized water, fixed seedlings were pre-incubated with a blocking buffer (PBS containing 5% BSA) at 37°C for 1 h and incubated with anti-SYT5 antibody in the blocking buffer at 4°C overnight. After washing with PBS containing 0.1% Triton X-100, fixed seedlings were incubated with either Alexa 488- or Alexa 546-conjugated rabbit igG antibody (Invitrogen, USA) in the blocking buffer at room temperature for 3 h. Following washing with PBS containing 0.1% Triton X-100, these seedlings were finally transferred into a mounting medium (100 mM Tris [pH 8.5] containing 25% glycerol) containing Mowiol 4-88 (Calbiochem, USA). All fluorescent images were taken by LSM780 confocal microscope (Zeiss, Germany) equipped with a 40× objective (C-Apochromat 40×/1.1 W) and processed by Zen 2011 software (Zeiss) and Adobe Photoshop CS5 (Adobe, USA). GFP and Alexa 488 were excited with an argon laser at 488 nm, and mRFP and Alexa 546 were at 561 nm. For two-color imaging, multitracking was configured to avoid cross-talk between fluorescence channels.
It was reported that transiently expressed SYT5-GFP is localized to endomembrane compartments in Arabidopsis protoplasts (Yamazaki et al., 2010). SYT5-GFP was also reported to be localized to the ER/PM contact sites by transient expression in
We previously reported that SYT1 interacts with the PM-residing SYP121 syntaxin to modulate plant immune responses by controlling SYP121 level in plants (Kim et al., 2016). We therefore tested whether SYT5 is also able to interact with a PM syntaxin. It is known that SYP121 is required for defense against powdery mildew fungi, whereas SYP132 is for resistance to bacteria (Kalde et al., 2007; Kwon et al., 2008). We found that SYP121 levels in both
We next tested whether SYT5 and SYP132 also interact in plants. For this, we generated anti-SYT5 and anti-SYP132 antibodies in a rabbit and a chicken, respectively. The anti-SYT5 antibody detects endogenous SYT5 in WT but not in
SYP132 specifically interacts with VAMP721/722 that reside in the trans-Golgi network (TGN) and secretory vesicles (Ichikawa et al., 2014; Yun et al., 2013). Since SYT5 interacts with SYP132 (Fig. 2A, Supplementary Fig. S2A), we then tested whether SYT5 is able to regulate SYP132-VAMP721/722 interaction. We first examined the effect of SYT5 on SYP132-VAMP722 interaction
Since all reported
We previously reported that VAMP721/722 are indispensable for plant resistance to
We next examined subcellular localization of endogenous SYT5 in plants. We immuno-fluorescently stained endogenous SYT5 with two distinct secondary antibodies (Alexa 546 with red fluorescence and Alexa 488 with green one) in roots of transgenic plants expressing either the PM-localized GFP-SYP132 or the TGN/vesicle-located mRFP-VAMP722 (Ichikawa et al., 2014). Due to the difficulty in immunostaining root elongating cells leading to smear images (data not shown), we microscopically observed meristematic root cells (Supplementary Fig. S3, Fig. 4). As previously reported (Lee et al., 2020), we detected immuno-fluorescently marked SYT5 in perinuclear area (Figs. 4B and 4E). Distinct localization patterns of SYT5 from GFP-SYP132 (Figs. 4A-4C) and mRFP-VAMP722 (Figs. 4D-4F) indicate that SYT5 is largely localized to the ER. In a dividing root meristematic cell, we interestingly found that SYT5 is not detected in the developing cell plate (Supplementary Fig. S3D-S3F), where SYT1 was reported to be localized (Yamazaki et al., 2010). Since SYP121 level is elevated in
Together with SYT1, SYT5 was recently reported to be engaged in stress responses to rare earth elements and viral pathogens by regulating the rearrangement and intactness of ER-PM contact sites (Ishikawa et al., 2020; Lee et al., 2020). We previously found that SYT1 has an additional immune activity to its ER-PM contact-controlling function (Kim et al., 2016). Elevated SYP121 abundance in
VAMP721/722 are the major exocytosis-associated R-SNAREs in Arabidopsis. They participate in a number of physiological processes such as cell division, growth, and biotic/abiotic stress responses even including symbiosis in plants (El Kasmi et al., 2013; Ichikawa et al., 2014; Ivanov et al., 2012; Kim et al., 2019; Kwon et al., 2008; Sogawa et al., 2019; Yi et al., 2013; Yun et al., 2013). In contrast to VAMP721/722, a plant PM syntaxin is involved rather in a specific biological process. Although how VAMP721/722 can be engaged in such diverse processes is largely unknown, their interactions with distinct PM syntaxins such as SYP111, SYP121, SYP122, SYP123, and SYP132 (El Kasmi et al., 2013; Ichikawa et al., 2014; Kwon et al., 2008; Pajonk et al., 2008; Yun et al., 2013) implicate that VAMP721/722 may work for a cellular activity by interacting with a respective PM syntaxin. It is regarded that SYP121 immune function is restricted to fungal pathogens, whereas SYP132 defense activity is limited to resistance to bacterial pathogens (Kalde et al., 2007; Kwon et al., 2008). Based on elevated bacterial growth in
Unlike typical SYTs, all known plant SYTs contain an additional SYT-like mitochondrial lipid-binding (SMP) domain (Saheki and De Camilli, 2017). In animals, these SYTs called extended-SYTs (E-SYTs) are responsible for tethering the ER to the PM to generate the ER-PM contact sites at which lipids are transported through the SMP domain between two distinct membranes (Saheki and De Camilli, 2017). Likewise, plant SYTs, especially SYT1, SYT5, and SYT7 (also called Ca2+- and lipid-binding protein 1 [CLB1]) were found to play a critical role in maintaining the ER-PM contact sites for responses to various biotic/abiotic stresses (Ishikawa et al., 2020; Lee et al., 2020; Levy et al., 2015; Lewis and Lazarowitz, 2010; Perez-Sancho et al., 2015; Schapire et al., 2008; Yamazaki et al., 2008). Interestingly, it was recently reported that a part of endocytosis-associated autophagy pathway is initiated at the ER-PM contact sites (Wang et al., 2019), suggesting that the ER-PM contact sites may control vesicle trafficking in plants. Indeed, SYT2 is suggested to play a role in secretion in Arabidopsis (Wang et al., 2015; Zhang et al., 2011). In addition, E-SYTs were recently reported to regulate membrane trafficking even in animals (El Kasmi et al., 2018; Kikuma et al., 2017). In response to pathogen attack, plant cells reorganize all subcellular compartments near to the pathogen-attempted area (Bestwick et al., 1997; Koh et al., 2005; Takemoto et al., 2003), likely for rapid and efficient production and secretion of immune molecules including secondary metabolites (Khare et al., 2020). In this scenario, SYT5 may contribute to plant immunity to bacteria by promoting the SYP132/VAMP721/722-associated exocytosis at the ER-PM contact sites. The ‘kiss-and-run’ exocytosis may explain the observed distinct localization of SYT5, SYP132, and VAMP722 in plant cells (Fig. 4, Supplementary Fig. S3), in spite of their interactions (Fig. 2, Supplementary Fig. S2).
Although we previously reported that VAMP721/722 are dispensable for plant immune responses to
Although VAMP721/722 are required for immune responses in plants, any other direct immune molecules but RPW8.2 and phospholipase Dδ (PLDδ) were not identified so far to be transported by VAMP721/722 vesicles (Kim et al., 2014; Xing et al., 2019). Proteomic approaches using VAMP721/722-depleted plants found that mostly cell wall-associated proteins are likely to be delivered by VAMP721/722 vesicles in plants (Kwon et al., 2020b; Uemura et al., 2019). Enhanced resistance to fungal pathogens but not to
This work was supported by a 2019 Research Grant (520190026 to H.K.) from Kangwon National University, Korea, a grant (PJ01477001 to C.K.) from Rural Development Administration, Korea, a grant (P0016045 to C.K.) from Korea Institute for Advancement of Technology, Korea, and grants (2016R1D1A1B02007322 to C.K.; 2021R1F1A1063111 to H.S.Y.) from National Research Foundation, Korea.
We thank Hae Ri Kwon and Yunjin Choi for technical assistance.
All authors (S.K., H.K., K.P., D.J.C., M.K.K., C.K., and H.S.Y.)conceived and performed experiments. H.K., C.K., and H.S.Y. wrote the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2021; 44(9): 670-679
Published online September 30, 2021 https://doi.org/10.14348/molcells.2021.0100
Copyright © The Korean Society for Molecular and Cellular Biology.
Soohong Kim1,4 , Hyeran Kim2,4
, Keunchun Park1,4
, Da Jeong Cho1
, Mi Kyung Kim1
, Chian Kwon1
, and Hye Sup Yun3,*
1Department of Molecular Biology, Dankook University, Cheonan 31116, Korea, 2Department of Biological Sciences, Kangwon National University, Chuncheon 24341, Korea, 3Department of Biological Sciences, Konkuk University, Seoul 05029, Korea, 4These authors contributed equally to this work.
Correspondence to:hsyun@konkuk.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
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.
Keywords: plant immunity, Pst DC3000, SYP132, SYT5, VAMP721/722
Soluble
The first identified SNARE components to form a biologically relevant SNARE complex in plants are the plasma membrane (PM)-localized SYP121 (syntaxin of plant 121, also called PEN1) Qa-SNARE, the PM-attached SNAP33 Qbc-SNARE and the vesicle-residing VAMP721/722 (vesicle-associated membrane proteins 721 and 722) R-SNAREs (Collins et al., 2003; Kwon et al., 2008). The SYP121-SNAP33-VAMP721/722 ternary SNARE complex drives an immune exocytosis. Interestingly, while SYP121 function is limited to defense against fungal pathogens, VAMP721/722 are additionally required for resistance to oomycete pathogens (Kwon et al., 2008). VAMP721/722 are also required for growth, cell division and abiotic stress responses (El Kasmi et al., 2013; Ichikawa et al., 2014; Kim et al., 2019; Kwon et al., 2008; Yi et al., 2013; Yun et al., 2013). Promiscuous SNARE complex formation of VAMP721/722 with distinct Qa-SNAREs such as SYP121, SYP111 (KNOLLE), SYP123 and SYP132 (El Kasmi et al., 2013; Ichikawa et al., 2014; Kwon et al., 2008; Yun et al., 2013) strongly suggests that at least one regulatory protein should control the SNARE complex formation of VAMP721/722 with a specific Qa-SNARE in a particular biological process in plants. This can be additionally supported by promiscuous SNARE complex formation of SYP121 with members in the VAMP72 R-SNARE group (Kwon et al., 2008).
In plants, three groups of regulatory proteins such as Sec1/Munc18 (SM), small GTPase and synaptotagmin (SYT) have been studied for modulating the trafficking functionality of SNAREs. The KEULE (also called SEC11) SM protein controls the SNARE complex formation of VAMP721/722 with KNOLLE for cytokinesis (Karnahl et al., 2018; Park et al., 2012), but with SYP121 for potassium uptake and growth (Karnik et al., 2013; 2015; Waghmare et al., 2019; Zhang et al., 2019) by binding to the N-terminus of respective Qa-SNARE. Recently, a KEULE paralog, SEC1B, was found to be predominantly engaged in regulating the SYP132-dependent secretion during pollen growth (Karnahl et al., 2018). The plant-specific ARA6 GTPase is regarded to switch the interaction of SYP121 between VAMP721/722 and VAMP727 (Ebine et al., 2011). SYP121 is known to continuously cycle between the PM and endosomes (Reichardt et al., 2011). Focal accumulation of endocytosed SYP121 or its barley ortholog, ROR2, to fungal entry sites was found to require the GNOM ADP-ribosylation factor-guanine nucleotide exchange factor (ARF-GEF) in Arabidopsis or ARFA1b/c GTPases in barely, respectively (Bohlenius et al., 2010; Nielsen et al., 2012). SYT1 preferentially residing in the endoplasmic reticulum (ER) (Levy et al., 2015; Perez-Sancho et al., 2015) was originally found to repair damaged PM by abiotic stresses and control endocytosis often resulting in promoting cell-to-cell movement of viral movement proteins (Lewis and Lazarowitz, 2010; Schapire et al., 2008; Yamazaki et al., 2008). SYT1 together with SYT5 was recently found to regulate the intactness and rearrangement of ER-PM contact sites for stress responses to rare earth elements and viral pathogens in plants (Ishikawa et al., 2020; Lee et al., 2020).
We previously found that SYT1 additionally down-regulates SYP121 abundance possibly via endocytosis to control plant disease resistance to fungal pathogens (Kim et al., 2016). We therefore examined whether SYT5 also functions in plant immunity. Based on specific impairment of resistance to
Plants used for experiments were grown at 22°C with 10-h light/14-h dark photoperiod. To isolate T-DNA-inserted
To express recombinant proteins, cDNAs corresponding to
To test the interaction between SYP132 and SYT5, equimolar recombinant HA-SYP132 and GST-SYT5∆TM were incubated in the absence or presence of 1 mM CaCl2. To assess the effect of Ca2+ on SYT5-SYP132 interaction, 1 mM EDTA was added during incubation. To test the interaction between SYP132 and VAMP722, equimolar recombinant HA-SYP132 and GST-VAMP722 were mixed together with GST-free SYT5∆TM in the absence or presence of 1 mM CaCl2. To analyze whether the Ca2+-promoted SYT5∆TM-SYP132 interaction affects SYP132-VAMP722 interaction, 1 mM EDTA was added during incubation. Interacted proteins were then precipitated with glutathione-Sepharose 4B and the precipitates were analyzed by immunoblot with anti-HA antibody to detect HA-SYP132 in the precipitates.
Proteins were extracted from the indicated genotype plants by suspending ground plant materials in 1× phosphate-buffered saline (PBS) containing 1% Triton X-100. Protein amounts were measured by the Bio-Rad protein assay (Bio-Rad, USA). Protein extracts were first pre-cleared with Protein A/G-agarose beads (Santa Cruz Biotechnology, USA) and incubated with anti-SYT5 or anti-VAMP721/722 antibody. Anti-SYT5 antibody-bound or anti-VAMP721/722 antibody-bound proteins were then retrieved by precipitation with Protein A/G-agarose beads. The immunoprecipitates were finally analyzed by immunoblot with anti-SYP132 antibody. A part (3%) of protein extracts used for immunoprecipitation were subject to immunoblot with anti-SYT5, anti-SYP121, anti-SYP132, anti-VAMP721/722 antibody, or anti-SNAP33 antiserum for showing steady state levels of SYT5, SYP121, SYP132, VAMP721/722, or SNAP33, respectively.
Plants were grown in soil for 4-5 weeks to be inoculated with
For immunostaining, 7-day-old transgenic Arabidopsis seedlings expressing either GFP-SYP132 or mRFP-VAMP722 were fixed in MTSB buffer (50 mM PIPES, 5 mM EGTA and 5 mM MgSO4, pH 7.0 adjusted with KOH) containing 4% paraformaldehyde for 1 h at room temperature under vacuum infiltration (Sauer et al., 2006). Fixed seedlings were placed onto poly-L-Lys-coated glass slides, and washed with MTSB buffer containing 0.1% Triton X-100 and with deionized water. Fixed seedlings were then incubated with 2% driselase in MTSB buffer for 40 min to digest their cell wall, and incubated with PBS containing 20% DMSO and 3% NP40 for 1 h. Following washing with MTSB buffer containing 0.1% Triton and with deionized water, fixed seedlings were pre-incubated with a blocking buffer (PBS containing 5% BSA) at 37°C for 1 h and incubated with anti-SYT5 antibody in the blocking buffer at 4°C overnight. After washing with PBS containing 0.1% Triton X-100, fixed seedlings were incubated with either Alexa 488- or Alexa 546-conjugated rabbit igG antibody (Invitrogen, USA) in the blocking buffer at room temperature for 3 h. Following washing with PBS containing 0.1% Triton X-100, these seedlings were finally transferred into a mounting medium (100 mM Tris [pH 8.5] containing 25% glycerol) containing Mowiol 4-88 (Calbiochem, USA). All fluorescent images were taken by LSM780 confocal microscope (Zeiss, Germany) equipped with a 40× objective (C-Apochromat 40×/1.1 W) and processed by Zen 2011 software (Zeiss) and Adobe Photoshop CS5 (Adobe, USA). GFP and Alexa 488 were excited with an argon laser at 488 nm, and mRFP and Alexa 546 were at 561 nm. For two-color imaging, multitracking was configured to avoid cross-talk between fluorescence channels.
It was reported that transiently expressed SYT5-GFP is localized to endomembrane compartments in Arabidopsis protoplasts (Yamazaki et al., 2010). SYT5-GFP was also reported to be localized to the ER/PM contact sites by transient expression in
We previously reported that SYT1 interacts with the PM-residing SYP121 syntaxin to modulate plant immune responses by controlling SYP121 level in plants (Kim et al., 2016). We therefore tested whether SYT5 is also able to interact with a PM syntaxin. It is known that SYP121 is required for defense against powdery mildew fungi, whereas SYP132 is for resistance to bacteria (Kalde et al., 2007; Kwon et al., 2008). We found that SYP121 levels in both
We next tested whether SYT5 and SYP132 also interact in plants. For this, we generated anti-SYT5 and anti-SYP132 antibodies in a rabbit and a chicken, respectively. The anti-SYT5 antibody detects endogenous SYT5 in WT but not in
SYP132 specifically interacts with VAMP721/722 that reside in the trans-Golgi network (TGN) and secretory vesicles (Ichikawa et al., 2014; Yun et al., 2013). Since SYT5 interacts with SYP132 (Fig. 2A, Supplementary Fig. S2A), we then tested whether SYT5 is able to regulate SYP132-VAMP721/722 interaction. We first examined the effect of SYT5 on SYP132-VAMP722 interaction
Since all reported
We previously reported that VAMP721/722 are indispensable for plant resistance to
We next examined subcellular localization of endogenous SYT5 in plants. We immuno-fluorescently stained endogenous SYT5 with two distinct secondary antibodies (Alexa 546 with red fluorescence and Alexa 488 with green one) in roots of transgenic plants expressing either the PM-localized GFP-SYP132 or the TGN/vesicle-located mRFP-VAMP722 (Ichikawa et al., 2014). Due to the difficulty in immunostaining root elongating cells leading to smear images (data not shown), we microscopically observed meristematic root cells (Supplementary Fig. S3, Fig. 4). As previously reported (Lee et al., 2020), we detected immuno-fluorescently marked SYT5 in perinuclear area (Figs. 4B and 4E). Distinct localization patterns of SYT5 from GFP-SYP132 (Figs. 4A-4C) and mRFP-VAMP722 (Figs. 4D-4F) indicate that SYT5 is largely localized to the ER. In a dividing root meristematic cell, we interestingly found that SYT5 is not detected in the developing cell plate (Supplementary Fig. S3D-S3F), where SYT1 was reported to be localized (Yamazaki et al., 2010). Since SYP121 level is elevated in
Together with SYT1, SYT5 was recently reported to be engaged in stress responses to rare earth elements and viral pathogens by regulating the rearrangement and intactness of ER-PM contact sites (Ishikawa et al., 2020; Lee et al., 2020). We previously found that SYT1 has an additional immune activity to its ER-PM contact-controlling function (Kim et al., 2016). Elevated SYP121 abundance in
VAMP721/722 are the major exocytosis-associated R-SNAREs in Arabidopsis. They participate in a number of physiological processes such as cell division, growth, and biotic/abiotic stress responses even including symbiosis in plants (El Kasmi et al., 2013; Ichikawa et al., 2014; Ivanov et al., 2012; Kim et al., 2019; Kwon et al., 2008; Sogawa et al., 2019; Yi et al., 2013; Yun et al., 2013). In contrast to VAMP721/722, a plant PM syntaxin is involved rather in a specific biological process. Although how VAMP721/722 can be engaged in such diverse processes is largely unknown, their interactions with distinct PM syntaxins such as SYP111, SYP121, SYP122, SYP123, and SYP132 (El Kasmi et al., 2013; Ichikawa et al., 2014; Kwon et al., 2008; Pajonk et al., 2008; Yun et al., 2013) implicate that VAMP721/722 may work for a cellular activity by interacting with a respective PM syntaxin. It is regarded that SYP121 immune function is restricted to fungal pathogens, whereas SYP132 defense activity is limited to resistance to bacterial pathogens (Kalde et al., 2007; Kwon et al., 2008). Based on elevated bacterial growth in
Unlike typical SYTs, all known plant SYTs contain an additional SYT-like mitochondrial lipid-binding (SMP) domain (Saheki and De Camilli, 2017). In animals, these SYTs called extended-SYTs (E-SYTs) are responsible for tethering the ER to the PM to generate the ER-PM contact sites at which lipids are transported through the SMP domain between two distinct membranes (Saheki and De Camilli, 2017). Likewise, plant SYTs, especially SYT1, SYT5, and SYT7 (also called Ca2+- and lipid-binding protein 1 [CLB1]) were found to play a critical role in maintaining the ER-PM contact sites for responses to various biotic/abiotic stresses (Ishikawa et al., 2020; Lee et al., 2020; Levy et al., 2015; Lewis and Lazarowitz, 2010; Perez-Sancho et al., 2015; Schapire et al., 2008; Yamazaki et al., 2008). Interestingly, it was recently reported that a part of endocytosis-associated autophagy pathway is initiated at the ER-PM contact sites (Wang et al., 2019), suggesting that the ER-PM contact sites may control vesicle trafficking in plants. Indeed, SYT2 is suggested to play a role in secretion in Arabidopsis (Wang et al., 2015; Zhang et al., 2011). In addition, E-SYTs were recently reported to regulate membrane trafficking even in animals (El Kasmi et al., 2018; Kikuma et al., 2017). In response to pathogen attack, plant cells reorganize all subcellular compartments near to the pathogen-attempted area (Bestwick et al., 1997; Koh et al., 2005; Takemoto et al., 2003), likely for rapid and efficient production and secretion of immune molecules including secondary metabolites (Khare et al., 2020). In this scenario, SYT5 may contribute to plant immunity to bacteria by promoting the SYP132/VAMP721/722-associated exocytosis at the ER-PM contact sites. The ‘kiss-and-run’ exocytosis may explain the observed distinct localization of SYT5, SYP132, and VAMP722 in plant cells (Fig. 4, Supplementary Fig. S3), in spite of their interactions (Fig. 2, Supplementary Fig. S2).
Although we previously reported that VAMP721/722 are dispensable for plant immune responses to
Although VAMP721/722 are required for immune responses in plants, any other direct immune molecules but RPW8.2 and phospholipase Dδ (PLDδ) were not identified so far to be transported by VAMP721/722 vesicles (Kim et al., 2014; Xing et al., 2019). Proteomic approaches using VAMP721/722-depleted plants found that mostly cell wall-associated proteins are likely to be delivered by VAMP721/722 vesicles in plants (Kwon et al., 2020b; Uemura et al., 2019). Enhanced resistance to fungal pathogens but not to
This work was supported by a 2019 Research Grant (520190026 to H.K.) from Kangwon National University, Korea, a grant (PJ01477001 to C.K.) from Rural Development Administration, Korea, a grant (P0016045 to C.K.) from Korea Institute for Advancement of Technology, Korea, and grants (2016R1D1A1B02007322 to C.K.; 2021R1F1A1063111 to H.S.Y.) from National Research Foundation, Korea.
We thank Hae Ri Kwon and Yunjin Choi for technical assistance.
All authors (S.K., H.K., K.P., D.J.C., M.K.K., C.K., and H.S.Y.)conceived and performed experiments. H.K., C.K., and H.S.Y. wrote the manuscript.
The authors have no potential conflicts of interest to disclose.
Geun Cheol Song, and Choong-Min Ryu
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