Salicylic acid-mediated plasmodesmal closure via
Remorin-dependent lipid organization
Dingquan Huanga,b,1, Yanbiao Suna,b,1, Zhiming Mac,1, Meiyu Kea,b, Yong Cuid, Zichen Chenb, Chaofan Chena,b,
Changyang Jid, Tuan Minh Tranc, Liang Yangc,e, Sin Man Lamf, Yanhong Hanb, Guanghou Shuf, Ji rí Frimlg,
Yansong Miaoc,h, Liwen Jiangd,i, and Xu Chenb,2
aCollege of Life Science and Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Fujian Agriculture and Forestry University, Fuzhou,
Fujian 350002, China; bFujian Agriculture and Forestry University–University of California, Riverside, Joint Center for Horticultural Biology and
Metabolomics, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, China; cSchool of Biological Sciences,
Nanyang Technological University, 637551, Singapore; dSchool of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of
Agrobiotechnology, The Chinese University of Hong Kong, 999077, Hong Kong; eSingapore Centre for Environmental Life Sciences Engineering, Nanyang
Technological University, 637551 Singapore; fState Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology,
Chinese Academy of Sciences, Beijing 100101, China; gInstitute of Science and Technology Austria (IST Austria), 3400 Klosterneuburg, Austria; hSchool of
Chemical and Biomedical Engineering, Nanyang Technological University, 637459, Singapore; and iThe Chinese University of Hong Kong Shenzhen Research
Institute, Shenzhen 518057, China
Edited by Natasha V. Raikhel, Center for Plant Cell Biology, Riverside, CA, and approved September 12, 2019 (received for review July 12, 2019)
Plasmodesmata (PD) are plant-specific membrane-lined channelsDownloaded by guest on January 7, 2020 of the SAR-generated chemical signals, salicylic acid (SA),
that create cytoplasmic and membrane continuities between adjacent contributes to the regulation of PD permeability, and exogenous
cells, thereby facilitating cell–cell communication and virus move- PLANT BIOLOGYapplication of SA causes PD closure via regulation of callose
ment. Plant cells have evolved diverse mechanisms to regulate PD deposition (9). Apparently, PD establish a battleground for plant
plasticity in response to numerous environmental stimuli. In particu- defenses against pathogen attacks.
lar, during defense against plant pathogens, the defense hormone,
salicylic acid (SA), plays a crucial role in the regulation of PD perme- PD membranes are enriched in sterols and sphingolipids with
ability in a callose-dependent manner. Here, we uncover a mechanism very long chain saturated fatty acids (10), constituting crucial
by which plants restrict the spreading of virus and PD cargoes using components of membrane lipid raft nanodomains (11, 12).
SA signaling by increasing lipid order and closure of PD. We showed Remorin (REM) represents one of the best-characterized mem-
that exogenous SA application triggered the compartmentalization brane lipid nanodomain-localized proteins; its assembly pattern is
of lipid raft nanodomains through a modulation of the lipid raft- critical for determining the formation of lipid nanodomains (13).
regulatory protein, Remorin (REM). Genetic studies, superresolution In Arabidopsis, the REM family comprises 16 members (14). In-
imaging, and transmission electron microscopy observation together dividual REM proteins are associated with distinct membrane
demonstrated that Arabidopsis REM1.2 and REM1.3 are crucial for nanodomains, providing a platform for specific interactions be-
plasma membrane nanodomain assembly to control PD aperture tween membrane lipids and membrane-resident proteins (15–17).
and functionality. In addition, we also found that a 14-3-3 epsilon
protein modulates REM clustering and membrane nanodomain com- In this study, we adopted REM as a well-characterized nano-
partmentalization through its direct interaction with REM proteins. domain marker to investigate the correlation between membrane
This study unveils a molecular mechanism by which the key plant
defense hormone, SA, triggers membrane lipid nanodomain reorga- Significance
nization, thereby regulating PD closure to impede virus spreading.
Plasmodesmata (PD) create cytoplasmic and membrane conti-
| | |Remorin plasmodesmata closure SA-controlled lipid order membrane nuities between adjacent cells to facilitate cell–cell communi-
cation and virus movement. Plant cells have evolved diverse
nanodomain compartmentalization mechanisms to regulate PD plasticity against plant pathogens,
including the accumulation of the defense hormone, salicylic
Plasmodesmata (PD) are highly plastic nanosized membrane- acid (SA). However, the mechanism of how this occurs is not
lined channels that serve as gatekeepers for cell-to-cell well understood. Here, we uncover a mechanism by which SA
transportation and communication in plants (1). Previous ultra- triggers Remorin-dependent membrane lipid nanodomain as-
structural analysis by transmission electron microscopy (TEM) sembly, leading to enhancement of the liquid-ordered phase.
have described PD as tunnels connecting neighboring cells, con- The higher-ordered lipids, which are particularly enriched at PD
taining inner components of desmotubule, cytoplasmic sleeve, and membrane, decreased PD membrane plasticity, and thus re-
deposited callose (2–4). Although these structural features of PD stricted PD opening and impeded virus spreading. Our findings
have been broadly described and the functions are essential, the address a knowledge gap in plant defense mechanisms at the
mechanisms underlying PD permeability regulation are still poorly membrane level that rely on SA-controlled lipid order and
understood. Therefore, elucidating the regulatory mechanism of PD closure.
PD plasticity and its role in fine-tuning cell-to-cell communication
are critical for understanding the functions of PD in plant devel- Author contributions: D.H., J.F., Y.M., L.J., and X.C. designed research; D.H., Y.S., Z.M.,
opment and responses to environmental stimuli. M.K., Y.C., Z.C., C.C., C.J., T.M.T., L.Y., and S.M.L. performed research; Y.H. and G.S.
contributed new reagents/analytic tools; X.C. analyzed data; and X.C. wrote the
The plant systemic acquired resistance (SAR) depends on the paper.
spreading of defense signals between cells and requires PD-
mediated transportation (5). As channels connecting the sym- The authors declare no competing interest.
plastic cell-to-cell network, PD can also be hijacked by pathogens
such as viruses to facilitate the trafficking of viral particles (6). This article is a PNAS Direct Submission.
On one hand, viruses spread from one cell to adjacent cells by
manipulating the PD architecture (6). On the other hand, upon This open access article is distributed under Creative Commons Attribution-NonCommercial-
pathogen infection, plants induce SAR involving the local ac- NoDerivatives License 4.0 (CC BY-NC-ND).
cumulation of defense signals in the infected tissues (7, 8). One
1D.H., Y.S., and Z.M. contributed equally to this work.
2To whom correspondence may be addressed. Email: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1911892116 PNAS Latest Articles | 1 of 11
lipid raft compartmentalization and PD plasticity. We demon- data support the conclusion that SA caused PD closure and disrupted
strated that SA signaling has a direct impact on membrane PD conductivity.
nanodomain formation and PD closure. Based on the partition
of membrane liquid-ordered/disordered phases, SA triggers A previous study had demonstrated that SA-mediated PD closure
recompartmentalization of lipid nanodomains and promotes relies on the deposited callose (9). We therefore tracked callose
ordered phase formation. PD membranes that are enriched with abundance by aniline blue staining and simultaneously used the PD-
lipid components are greatly affected by SA, and PD perme- callose binding protein 1 (PDCB1), which binds callose (20), as
ability is attenuated by SA application. Remorin proteins serve another indicator to assay for callose location. SA significantly in-
as key regulators to coordinate the events of SA signaling, duced aniline blue-stained callose on both apical/basal and lateral
nanodomain organization, and PD structural plasticity. Alto- cell sides (SI Appendix, Fig. S1 A and B). Similar to callose de-
gether, our study underscores the fundamental role of SA in position, SA induced a PDCB1 signal on the lateral side, but
membrane lipid raft organization, uncovering the regulation of PDCB1 signal was diminished on apical/basal sides (SI Appendix,
PD closure during defense responses. Fig. S1 A–D). PDCB1 binds callose at PD and preferentially
anchors to the sterol and sphingolipid-enriched membrane raft
Results via glycosylphosphatidylinositol (GPI) motif (20–22). There-
fore, the accumulation of PDCB1 to the lateral sides might be due
SA Causes PD Closure. To investigate the mechanism by which SA to the overproduction of callose or to a rearrangement in the
regulates PD permeability, we initially examined the ultrastruc- membrane system. To test these hypotheses, we used 2-deoxy-
tural changes of root meristematic cells in SA-treated wild-type D-glucose (DDG), a callose synthesis inhibitor (23) (SI Ap-
(WT) plants. Using TEM, we observed that SA significantly pendix, Fig. S1 E and F), applied with SA-treated PDCB1-YFP
impaired PD opening, as exhibited by long, narrow, and straight (yellow fluorescent protein). DDG significantly disrupted the SA-
PD channels with 31- to 33-nm aperture, compared with the induced PDCB1 signal on the lateral sides, whereas it did not fully
typical sandglass-shaped PD with a dilated neck and a 42- to inhibit the SA effect (SI Appendix, Fig. S1 A–D), indicating the
44-nm aperture in untreated plants (mock) (Fig. 1 A and B). PD existence of an additional, callose-independent mechanism of PD
density in the cell wall along the cell division plane (apical/basal regulation downstream of SA signaling.
sides) or along root growth axis (lateral sides) was not influenced
by SA (Fig. 1C), indicating that PD structure is controlled locally Lipid Rafts Are Essential for SA-Induced PD Closure. Compared with
by SA. To determine whether PD functionality is influenced by the surrounding plasma membrane (PM), PD membranes con-
SA, we designed a dye-loading assay employing carboxy-fluorescein tain strikingly abundant lipid raft constituents that are homo-
(CF) diacetate (CFDA) to track symplastic transportation (18, 19). geneously distributed and fundamental for PD functionality (10,
CFDA assay showed that PD permeability was significantly blocked 24, 25). We thus hypothesized that SA might influence PD ap-
by SA (Fig. 1 D and E and Movies S1 and S2). Altogether, these erture by modulating lipid rafts. Methyl-β-cyclodextrin (mßcd) is
Downloaded by guest on January 7, 2020 Fig. 1. SA causes PD closure. (A–C) PD structure was visualized by TEM, in root meristematic zone of mock, SA-treated (50 μM, 24 h), mßcd-treated (10 mM,
24 h), and SA-plus-mßcd–treated WT. PD aperture and density (number per micrometer) were observed and quantified on the apical/basal and lateral sides
(B: n = 98, 134, 121, 112, 203, 145, 174, and 171; C: n = 115, 92, 92, 91, 115, 92, 92, and 91). (D and E) PD permeability was detected by CFDA assay in WT
(100 μM SA, 10 mM mßcd, SA plus mßcd for 24 h), and PD permeability was quantified as CF signal in the root (E) (n = 16, 21, 19, and 20). (Scale bars: A,
100 nm; D, 10 μm.) Error bars represent SD. P values were determined by 2-tailed Student’s t test (*P < 0.05, **P < 0.01, ****P < 0.0001; ns, not significant).
2 of 11 | www.pnas.org/cgi/doi/10.1073/pnas.1911892116 Huang et al.
an efficient raft-disrupting agent and operates by depleting sterol Lipid rafts are defined as transient, relatively liquid-ordered
from the membrane (11). To examine the possible relationship membrane nanoscale domains (<200 nm), enriched in various
between SA and lipid raft on the control of PD aperture and sterols and sphingolipids (25). We stained WT roots using the
conductivity, we treated WT with SA and mßcd separately or in widely used plant fluorescent probe, di-4-ANEPPDHQ, which is
combination. Although mßcd was not able to change callose level a styryl dye that senses the dipole potential changes of lipid bi-
(SI Appendix, Fig. S1 A and B), mßcd and SA cotreatment effi- layer (26). When di-4-ANEPPDHQ molecules detect the lipid
ciently impaired the SA effect on PD closure, as shown by a 39-nm domains with different dipole potential in the cell membrane,
PD aperture compared to a 33-nm PD after sole SA treatment there is a large shift in the peak emission wavelength of the dye
(Fig. 1 A and B). PD density was not influenced by mßcd (Fig. from 630 nm in liquid-disordered phase (nonnanodomain) to
1C). This result showed that removal of sterols significantly offset 570 nm in liquid-ordered phase (nanodomain) (26, 27) (SI Ap-
the SA effect on PD closure, indicating that an appropriate order pendix, Fig. S2A). Compared with the nontreated plants, di-4-
of the membrane lipid raft is essential for SA-mediated PD gating. ANEPPDHQ displayed a significantly higher generalized po-
larization (GP) value (28) in SA-treated WT, suggesting that SA
We next examined whether lipid rafts influence PD conductivity enhances the formation of an ordered membrane phase in the
using CFDA assay. SA and mßcd cotreatment partially restored the apical/basal or lateral PM (Fig. 2 A and B and SI Appendix, Fig.
inhibitory effect of SA on CF unloading (Fig. 1 D and E). In- S2B). To validate this SA effect, we examined lipid order in the
terestingly, mßcd also partially restored the SA effect on PDCB1 presence of mßcd, which depletes sterols, reducing the ordered
lateral accumulation (SI Appendix, Fig. S1 C and D). All of these data phase. As expected, the GP value was significantly reduced by
unequivocally support the notion that SA locally influences PD ap- mßcd. Interestingly, mßcd also strongly attenuated the SA effect
erture and permeability through a lipid raft-dependent mechanism. on shifting lipids toward the higher-ordered phase (Fig. 2 A and
B). We then asked whether the pronounced effect of SA was
SA Increases the Proportion of Ordered Lipid Phase and Regulates the dependent on SA signaling. Thus, we quantified the effect of SA-
Compartmentalization of Membrane Nanodomains. We next in- mediated lipid order in 2 mutants, the nonexpresser of PR gene 1
vestigated whether SA affects lipid raft organization in the PM.
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Fig. 2. SA triggers ordered lipid formation and induces REM assembly on the PM. (A and B) PM lipid order visualization in root meristematic cells. WT, npr1,
and npr3/4 seedlings were treated with mock (DMSO), SA (100 μM, 24 h), in the absence or presence of 10 mM mßcd (30 min), and then stained by di-4-
ANEPPDHQ. The radiometric color-coded GP images were generated in HSB pictures. The white triangles indicate the membrane regions used for GP
quantification (A) (B: n = 82, 78, 75, 73, 78, 85, 71, and 84). (C) pREM1.2::GFP:REM1.2 seedlings were treated with SA (100 μM, 24 h), mßcd (10 mM, 30 min), or
SA-plus-mßcd cotreatment, and GFP signal was observed by 2D-SIM. The Inset images display the 2× enlarged views of boxed areas in the original images.
(D–F) Quantitative analysis of the individual GFP:REM1.2-marked nanodomains, with respect to diameter (D), density distribution (E), and intensity (F). n =
500 for each column in D and F from at least 10 images. The density graph (E) was generated by counting the nanodomain number in the selected regions of
interest (n = 30, 24, 31, and 25). (G and H) REM1.2 distribution pattern was visualized by pREM1.2::GFP: REM1.2 in npr1 and npr3 npr4 in the absence or
presence of SA (100 μM, 24 h). (H) Chart represents the histogram of signal distribution frequency of REM1.2 signal (n = 6,282 [from 40 cells, 20 roots], 5,750
[38 cells, 16 roots], 6,437 [45 cells,13 roots], 8,205 [50 cells, 18 roots], 7,756 [51 cells, 16 roots], and 7,299 [43 cells, 16 roots]). (Scale bars: A and G, 5 μm; C, 2 μm.)
Error bars represent SD. P values were determined by 2-tailed Student’s t test (*P < 0.05; ****P < 0.0001; ns, not significant).
Huang et al. PNAS Latest Articles | 3 of 11
Downloaded by guest on January 7, 2020 (npr1) and npr3 npr4, which are defective in SA perception (29, exhibited much smaller cotyledons than WT and single mutants
30). Both npr mutants showed a strong resistance to lipid order (Fig. 3 A and B).
upon SA stimulation (Fig. 2 A and B), indicating that the NPR
receptor-mediated SA signaling regulates lipid order in the PM. We also generated plant lines that conditionally overexpressed
REM1.2 and REM1.3 lines using the β-estradiol–inducible sys-
To provide insights into SA-dependent regulation on lipid raft tem (XVE) (35), termed XVE:REM lines. We obtained several
organization at nanometer resolution, we employed superresolution independent XVE:REM1.2 and XVE:REM1.3 lines (SI Appendix,
structured illumination microscopy (SR-SIM) to observe the Fig. S3 K and L), and all of them consistently displayed severely
lipid nanodomains using a well-studied nanodomain marker, dwarf plants, short and agravitropic roots when continuously
REM. REM proteins are highly concentrated in sterol-enriched growing on estradiol-supplemented medium (Fig. 3 C and D, SI
lipid environments and are required for the assembly of raft Appendix, Fig. S3O, and Movies S3 and S4). Validation by
nanodomains (31). We constructed the green fluorescent protein Western blotting showed that the quantity of REM proteins was
(GFP) fused with coding sequences under the control of their native gradually elevated in a time course manner of estradiol induction
promoters plasmid and established stable transgenic Arabidopsis and reached maximal levels at 24 h postinduction (SI Appendix,
plants of pREM1.2::GFP:REM1.2 and pREM1.3::GFP:REM1.3. Fig. S3 M and N).
Compared with a homogeneous distribution pattern of REMs in
the PM of untreated plants, SA induced pronounced clusters of To further access the role of REM1.2/1.3 in lipid raft forma-
REM1.2 and REM1.3 proteins (SI Appendix, Fig. S2C). We then tion, we stained roots of overexpressed REM1.2 line and rem1.2
examined the GFP-REM1.2 signals by SR-SIM and used them as 1.3c by di-4-ANEPPDHQ to examine the lateral segregation of
bona fide raft markers to quantitatively determine the compart- lipid species into liquid-ordered or liquid-disordered phases. The
mentalization of individual raft nanodomain. SA greatly enhanced transient and robust induction of REM1.2 (XVE:REM1.2) gen-
the size of higher-signal intensity REM1.2-marked nanodomains erated significantly higher GP values, indicating an increased
but decreased nanodomain density (Fig. 2 C–F). The changes of level of ordered lipid domains on the PM (Fig. 3 E and F). A
nanodomain organization reported above are consistent with the coincubation with mßcd entirely abolished the lipid order change
overall increase in lipid order by SA. Owing to the importance of in XVE:REM1.2 (Fig. 3 E and F). The rem1.2 1.3c mutant did not
sterols for the ordered lipid phase formation (32), all SA-triggered show obvious changes on lipid order (Fig. 3 G and H). We then
changes in REM1.2-marked nanodomains were largely abolished examined the sensitivity of rem1.2 1.3c mutant to SA-triggered
by mßcd (Fig. 2 C–F). higher ordered lipid formation using a serial concentration of
SA. We observed a clear difference in lipid order between rem1.2
We then tested the assembly pattern of REMs in npr1 and npr3 1.3c and WT under the treatments using 25 and 50 μM SA, re-
npr4 mutants by expressing pREM::GFP:REM in these mutants. In spectively. Thus, rem1.2 1.3c showed significantly less sensitivity
npr1 and npr3 npr4, the SA impacts on REM1.2/1.3 clustering to lipid order change upon SA elicitation compared with WT
were largely abolished (Fig. 2 G and H and SI Appendix, Fig. S2 D (Fig. 3 G and H).
and E). This provides evidence that NPR-dependent SA signaling
organizes lipid raft nanodomains. We next asked whether the increase in lipid order and nano-
domain compartmentalization by SA or REM overproduction is
Arabidopsis inoculated with the plant pathogenic virus, cucum- due to REM clustering or the change of lipid species. We per-
ber mosaic virus (CMV), primes the induction of SA biosynthesis formed a lipidomics analysis to compare lipid components upon
(33), which simulates the status of elevated SA level in vivo. To SA treatment or in REM mutants and overexpressed plants. We
examine whether CMV-triggered endogenous SA accumulation did not observe strong lipid imbalance in SA-treated WT, rem1.2
causes a comparable assembly of REM-labeled nanodomains, rem1.3c mutant, or XVE:REM1.2 line, except for slight changes
we inoculated pREM1.2::GFP:REM1.2 with CMV. As expected, in several phosphatidylinositol, phosphatidylglycerol, phosphati-
CMV-infected roots also displayed obvious REM1.2 clusters, dylcholine, phosphatidic acid, and phosphatidylserine species (SI
which were similar to the effects of exogenous SA application (SI Appendix, Fig. S4 and Datasets S1–S3). It is worthy to note that
Appendix, Fig. S2 F–H). Together, these data consistently neither SA treatment nor REM transgenic plants were able to
showed that elevation of SA enhanced REM-labeled lipid raft change the levels of sphingolipids and sterols, which are the
nanodomain compartmentalization. typical nanodomain lipid components (SI Appendix, Fig. S4 A
and B and Datasets S1 and S2). Taken together, these data in-
Remorins Are Crucial Regulators of Lipid Raft Formation and Plant dicate that, without significantly changing the abundance of
Development. Lipid raft nanodomains are indispensable for sterol and sphingolipid components, a modulation of REMs
controlling plant development, under physiological conditions as clustering is sufficient to regulate lipid order on the PM as well as
well as during pathogenic attack (34). Although recent bio- the compartmentalization of raft nanodomains. The lipidomics
chemical studies have provided insights into the structural data suggest a primary role of REM clustering in fine-tuning SA-
property of REM proteins and their potential functions in lipid mediated nanodomain assembly.
raft organization (13, 16), the genetic analysis of REM has
remained limited owing to the functional redundancy of these Remorins Regulate Plasmodesmal Aperture and Functionality. Pre-
proteins. To examine the expression profiles of 16 Arabidopsis vious studies have shown that particular lipid components are
REM homologs (15), we performed qRT-PCR to search for enriched in PD membranes rather than in the surrounding PM
root-abundant REMs. Our results showed that the REM1 sub- (10), suggesting that SA regulates lipid order to change PD
family, especially REM1.2, REM1.3, and REM1.4 were ubiqui- functionality. To examine PD functionality, we first detected PD
tously expressed in most tissues (SI Appendix, Fig. S3 A–E). Of permeability in rem mutants and overexpressors by CFDA assay.
all REMs, the sequences of REM1.2 and REM1.3 were mostly CF signals in the root tip showed that PD permeability was en-
closely matched (14), and therefore most likely to be redundant. hanced in rem mutants, whereas it declined in REM overexpressors
We also identified null alleles of rem1.2 and rem1.3 single mu- (SI Appendix, Fig. S5 A–C). Moreover, using a functional comple-
tants (SI Appendix, Fig. S3 F–H), and neither of them displayed mentation test by introducing pREM::GFP:REM in rem1.2 or
significant developmental defects. We then generated 2 in- rem1.3, respectively (called rem1.2-Comp or rem1.3-Comp), we
dependent rem1.2 1.3 double mutants by designing a CRISPR- showed that PD permeability in the complementation lines was
Cas9–mediated REM1.3 knockout in rem1.2 mutant, termed restored to almost a WT level (SI Appendix, Fig. S5 A and C),
rem1.2 1.3c. We validated the mutation of REM1.2 and REM1.3 confirming that REMs are key components involved in PD-
knockouts by Western blotting (SI Appendix, Fig. S3 I and J). mediated symplastic communication.
Further phenotypic analysis revealed that rem1.2 1.3c mutants
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Fig. 3. Remorins are crucial regulators for proper lipid order formation. (A and B) Phenotypes of 5-d-old rem1.2, rem1.3 single mutants, and 2 independent
rem1.2 1.3c double mutants (L15 and L27). Cotyledon areas were quantified (B: n = 50, 50, 54, 55, and 52). (C and D) WT, XVE:REM1.2, and XVE:REM1.3 were
germinated and continuously grown on 5 μM estradiol-supplemented medium for 8 d. Enlarged pictures of XVE:REM1.2 and XVE:REM1.3 are shown in the
Inset boxes, and the arrows highlight the primary roots (C). Primary root length was quantified (D: n = 40, 40, and 42). (E and F) Lipid order visualization in
root cells of WT and XVE:REM1.2 (±10 mM mßcd, 30 min), which were stained by di-4-ANEPPDHQ. GP value was quantified (F: n = 76, 77, and 67). (G and H)
Lipid order was tested in WT and rem1.21.3c, which were treated with a serial concentration of SA for 24 h, followed by di-4-ANEPPDHQ staining. GP value
was quantified (H: n = 80, 78, 73, 118, 93, 88, 85, 78, 74, and 102). The percentages indicate the increase ratios of GP value induced by SA treatments
comparing with each mock treatment. (Scale bars: C, 2 mm; E and G, 5 μm.) Error bars represent SD. P values were determined by 2-tailed Student’s t test (*P <
0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant).
To clarify the regulatory mechanism by which REM regulates similar to SA-treated WT plants (Figs. 1 A and B and 4 B and C).
PD permeability, we examined the subcellular distributions of Consistent with the PD phenotypes on apical/basal sides, rem
REM1.2 and REM1.3 by immunogold labeling on pREM::GFP:REM mutants promoted and XVE:REM1.2 impaired PD opening also
seedlings. Compared with the negative control of the antibody on the lateral sides (SI Appendix, Fig. S6B). Thus, REM pro-
added to WT cells or no antibody in pREM1.3::GFP:REM1.3 cells teins function as negative regulators involved in the modulation
(SI Appendix, Fig. S6A), gold particles against GFP in GFP:REM of PD gating. Similar to the effects of SA, rem mutant and
cells showed a clear localization of REM1.2 and REM1.3 in the overexpressors were not able to alter PD density (SI Appendix,
PM as well as at PD, supporting the functionality of REMs at PD Fig. S6C), further supporting a specific role for REM in control
and PM (Fig. 4A). of PD aperture.
We next analyzed the ultrastructural structure of PD in rem To examine PD structure in-depth, we reconstructed ultra-
mutants and overexpressors by TEM. Since there is a differential structural PD morphology by 3D electronic tomography. Rep-
expression pattern of REM1.2 and REM1.3 in the cortex and resentative 3D-PD structures of rem mutants and overexpressors
endodermis of root cell layers (SI Appendix, Fig. S5A), we ana- revealed that rem1.2 and rem1.2 1.3c mutants had wider PD
lyzed the PD structure of rem mutants in cortex and endodermis, aperture and apparent cytoplasmic sleeves between the desmo-
respectively. Although the canonical architecture of PD was in- tubule and the PM, whereas cytoplasmic sleeves disappeared in
tact, PD aperture was significantly modified in rem mutants and XVE:REM1.2 (Fig. 4B, SI Appendix, Fig. S5D, and Movies S5–S8).
overexpressors. On the apical/basal sides of WT cells, we saw a Thus, 2D and 3D TEM observations unequivocally demonstrated
∼40-nm aperture of PD channels, compared with a wider ∼43-nm that REMs are crucial regulators for determining the diameter of
PD aperture in rem1.2 and rem1.3 mutants. Moreover, rem1.2 1.3c PD channels. We then used pSUC2-GFP as an additional in-
had an even wider PD aperture of ∼48 nm (Fig. 4 B and C). In dicator of PD conductivity (36). CFDA assay or expressing
contrast, XVE:REM1.2 and XVE:REM1.3 exhibited severely im- pSUC2-GFP in XVE:REMs both showed that PD conductivity was
paired PD openings and exhibited narrow PD channels (∼36 nm),
Huang et al. PNAS Latest Articles | 5 of 11
Fig. 4. Rem mutants increase and overexpressed REMs decrease PD aperture. (A) REM1.2 and REM1.3 proteins were detectable at PM and PD by immunogold
labeling with GFP antibody. The red boxes indicate PD and the black boxes indicate PM, and the arrows highlight the gold-labeled REMs. The Inset images
display the 2× enlarged views of boxed areas in the original images. (B and C) PD structure was visualized by TEM in WT, rem mutants, and XVE:REM at the
cell layers of cortex and endodermis, respectively. PD aperture was quantified on the apical/basal sides (C: n = 120, 228, 179, 126, 150, 125, 182, and 126).
Tomographic slices and 3D models show dimensional PD structures. ER (green) structure and PM (blue) are differently color-coded (B, Lower). (D and E) WT
and XVE:REM1.2/1.3 seedlings were treated with 10 mM mßcd for 24 h, compared with nontreated samples. PD permeability was measured by CFDA
assay (E: n = 19, 20, 22, 17, 20, and 19). (Scale bars: A and B, 100 nm; D, 10 μm.) Error bars represent SD. P values were determined by 2-tailed Student’s t test
(*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
Downloaded by guest on January 7, 2020 severely blocked in XVE:REMs (Fig. 4 D and E and SI Appendix, motivated us to test whether REM would also functionally reg-
Fig. S6 D–F). ulate lipid rafts downstream of SA-mediated signaling pathways.
Transcript abundance of REMs was slightly elevated by SA;
We next asked whether such REM-mediated PD closure requires meanwhile, protein levels of REM1.2 and REM1.3 were also ele-
key nanodomain components, such as the plant phytosterols. To vated 1.4- to 1.7-fold upon SA stimulation (Fig. 5A and SI Ap-
test this, mßcd, which depletes membrane sterols, was applied to pendix, Fig. S7 A and B).
XVE:REMs. mßcd significantly restored the weak unloading of
CFDA or aberrant expression of the GFP signal in XVE:REM To prove the functionality of REMs in SA-mediated PD clo-
lines (Fig. 4 D and E and SI Appendix, Fig. S6 E and F), further sure, we examined the sensitivity of rem1.2 1.3c mutant to SA
supporting the importance of lipid raft in REM-mediated PD using CFDA assay. CFDA-indicated PD permeability of SA-
closure. To further examine whether REM-stimulated callose over- treated WT was decreased to 5% upon 100 μM SA treatment,
production resulted in PD closure (37), we also applied DDG in compared with nontreated WT (Fig. 5 B and C and SI Appendix,
XVE:REM1.2 SUC2-GFP. The decreased callose level as a result of Fig. S7 C and D). In contrast, SA was less effective in rem1.2 1.3c
DDG treatment was not able to rescue the impaired SUC2-GFP double mutant, compared with WT (Fig. 5 B and C and SI Ap-
signal in XVE:REM1.2 line (SI Appendix, Fig. S6 E and G). There- pendix, Fig. S7 C and D). To further dissect the functional cor-
fore, we conclude that REM-dependent lipid raft organization is relation between REMs and the SA signaling receptors NPRs,
necessary to maintain appropriate PD aperture and permeability. we overexpressed REM1.2 and REM1.3 in npr1 and npr3 npr4
mutants. Compared with the background plants from which
Remorins-Mediated PD Closure Is Downstream of SA Signaling. The the XVE:REMs or npr mutants were derived, the leaf and root
discovered SA effect on the assembly of REM1.2/1.3 pro- morphology of XVE:REMs npr1 and XVE:REMs npr3 npr4 all
teins and the compartmentalization of membrane nanodomains
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Fig. 5. REMs are crucial regulators involved in SA signaling pathway. (A) Protein level of REM1.2 and REM1.3 (labeled as arrows) was detected in WT
seedlings by Western blotting (with REM1.2/1.3 antibody) with or without SA treatment (100 μM, 24 h). (B–E) PD permeability was detected by CFDA assay.
The 50 or 100 μM SA was applied for 24 h in WT and rem1.2 1.3c (n = 17, 16, 16, 17, 16, and 16) (B and C). rem mutants, npr mutants, and XVE:REM npr
seedlings were treated with or without SA (50 μM, 24 h) treatment (n = 22, 18, 19, 19, 18, 20, 22, 17, 18, 18, 18, 18, 19, 18, 19, 18, 18, and 18) (D and E). The
percentage indicates the signal ratio to compare SA-treated group with each mock, respectively. (Scale bars: B and D, 10 μm.) Error bars represent SD. P values
were determined by 2-tailed Student’s t test (**P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant).
resembled XVE:REMs (SI Appendix, Fig. S7E). CFDA assay in the (SI Appendix, Fig. S8A). When sprayed with luciferin to detect
above lines consistently showed that PD permeability in XVE:REMs maximum Luc-complementation activity for both REM1.2-
npr1 and XVE:REMs npr3 npr4 was severely compromised, similar GRFs and REM1.3-GRFs, only pairs of REM1.2-GRF10 and
to XVE:REMs (Fig. 5 D and E). Thus, REMs are epistatic to NPRs REM1.3-GRF10 showed a strong reconstituted Luc-signal,
involved in SA signaling. compared with the negative controls (SI Appendix, Fig. S8 B
and C). These results demonstrated that REM1.2 and REM1.3
14-3-3 Protein Acts as an Adaptor to Maintain the Assembly Pattern interact with GRF10 protein in plants. We then performed a
of REM and Membrane Nanodomains. To address how SA regulates yeast 2-hybrid assay to test the existence of a physical interaction
REM proteins and lipid raft assembly, we used pREM1.2::GFP:REM1.2 between REM1.2/1.3 and GRF10. Compared with the negative
stable transgenic seedlings to screen for candidate proteins inter- controls, the positive colonies of GRF10-REM1.2/1.3 pairs on
acting with REM1.2 by immunoprecipitation coupled to mass the selective yeast medium (SD/-LTHA) confirmed that GRF10
spectrometry (IP-MS) assay. Analysis of IP-MS data revealed that directly interacted with REM proteins (SI Appendix, Fig. S8D).
several isoforms of 14-3-3 protein, including 14-3-3 epsilon (general
regulatory factor 10 [GRF10]), chi (GRF1), mu (GRF9), omega The 14-3-3 protein has a wide range of roles in cell signaling
(GRF2), nu (GRF7), and phi (GRF4) were obtained among pro- pathways within the nucleus, cytoplasm, and PM (39–43). To
teins that coimmunoprecipitated (co-IP) with REM1.2 (Dataset understand the location for REM1.2/1.3 and GRF10 interaction,
S4). We further performed co-IP to confirm these protein–protein we utilized the BiFC-YFP system to coexpress GRF-n/cYFP and
interactions in vivo using overexpressed GFP-tagged REM1.2 n/cYFP-REM in N. benthamiana leaves. Compared with the
transgenic Arabidopsis plants (GFP-REM1.2). Interestingly, when negative controls, GRF10-REM pairs showed strong recon-
REM1.2 proteins were pulled down by GFP magnetic beads, the stituted YFP signal on the PM, which was colocalized with FM4-
proteins at the molecular weight of 14-3-3 monomers and dimers 64–labeled membrane (Fig. 6B and SI Appendix, Fig. S8 E and
were both detectable in GFP-REM1.2 line, compared with only the F), indicating that GRF10 directly binds to REM proteins on the
monomer 14-3-3 bands in WT (Fig. 6A), implying that 14-3-3 might PM. Interestingly, detection of GRF10 protein level by sepa-
work together with REM through its dimeric form. rately extracting total proteins or membrane-associated proteins
showed that SA significantly enhanced membrane-associated of
In an attempt to examine whether REMs and 14-3-3 interac- GRF10, whereas the total protein abundance of GRF10 was not
tion exists under in vivo physiological condition in plant, we changed (Fig. 6C). These data imply that GRF10 might act as a
applied the bimolecular fluorescence complementation (BiFC)- stabilization factor to mediate the oligomerization of REM on
luciferase (Luc) reconstitute imaging assay (38) by coexpressing the PM.
GRF-nLUC and cLUC-REM in Nicotiana benthamiana leaves
Huang et al. PNAS Latest Articles | 7 of 11
Fig. 6. 14-3-3 proteins interact with REMs and are required for REM oligomerization and assembly. (A) Co-IP showed the interaction between REM1.2 and
14-3-3. GFP-tagged REM1.2 were immunoprecipitated by GFP magnetic beads. The coimmunoprecipitated 14-3-3 proteins were detected by the endogenous
14-3-3 antibody. Monomeric and oligomeric 14-3-3 proteins are marked according to molecular weight. The corresponding blot detected by the GFP antibody
was used as the positive control. Input indicates the flow-through samples before the incubation of GFP beads. (B) BiFc-YFP system shows the interactions of
REMs with GRF10. REMs and GRFs were individually fused with nYFP or cYFP. BiFc pairs of other unrelated n/cYFP with n/cYFP-REM are shown as negative
controls. The pictures with increased laser setting of weak signal are shown in the Bottom Right corner. Lower panels show the colocalization between FM4-
64–labeled PM (red) and REM1.2–GRF10 interaction (yellow). Colocalization signal profile chart was generated based on the white dot line. (C) Total protein
and membrane protein levels of GRF10 were detected in 35S:GFP or 35S::GRF10:GFP seedlings by Western blotting (with GFP antibody) under mock or SA
treatment (100 μM, 24 h). GRF10-GFP and GFP proteins are individually labeled as arrows according to molecular weight. (D and E) pREM1.2::GFP:REM1.2
signal was visualized in WT or GRF-amiR-L1 background (with estradiol induction) with mock or SA (100 μM, 24 h) (D). (E) Chart represents the relative signal
profile of REM1.2 signal intensity in different genotypes along the PM upon mock or SA treatment (n = 11,642 [from 58 cells, 18 roots], 8,315 [42 cells, 15
roots], 11,522 [51 cells, 17 roots], and 11,814 [55 cells, 13 roots]). (F and G) VAEM image of pREM1.2::GFP:REM1.2 in WT or GRF-amiR-L1 roots by mock and SA
(100 μM) treatment for 24 h (F). Stepwise photobleaching counting experiments of GFP:REM1.2 (n = 105, 100, 111, and 125) in G. (Scale bars: B, 50 μm; D, 2 μm;
and F, 1 μm.) Error bars represent SD. P values were determined by 2-tailed Student’s t test (**P < 0.01, ****P < 0.0001).
Downloaded by guest on January 7, 2020 Increasing evidence has shown that 14-3-3 serves as primary induced the formation of the higher-order protein oligomeriza-
pathogen targets in plant immunity (44, 45), and a 14-3-3 protein tion of GFP-REM1.2; however, this oligomerization disappeared
was significantly enriched in detergent-resistant membranes in GRF-amiR-L1 (Fig. 6G and SI Appendix, Fig. S8I), suggesting
upon cryptogein treatment (46). To examine the contribution of a GRF10-mediated REM clustering and protein oligomerization
GRF10 in mediating REM oligomerization, we firstly generated on the PM under SA exposure. Correspondingly, PD perme-
a GRF10-deficient mutant to silence the 14-3-3 epsilon sub- ability analysis by CFDA assay showed that GRF-amiR-L1 was
family, GRF9 and GRF10 simultaneously (called GRF-amiR insensitive to the SA effect on PD closure (SI Appendix, Fig.
lines). These lines displayed growth defects with a higher in- S9D). Therefore, we conclude that 14-3-3 protein acts as an
cidence of root agravitropism (SI Appendix, Fig. S9 A and B), adaptor to maintain the organizational pattern of REM and
consistent with the prior publication (47). Furthermore, we in- membrane nanodomains.
troduced pREM1.2::GFP:REM1.2 into GRF-amiR-L1 to in-
vestigate the assembly pattern of REM1.2 proteins on the PM. Remorins Are Important Regulatory Components of Plant Defense to
Strikingly, GFP:REM1.2 assemblies were no longer enhanced by Virus. Through the dynamic control of PD aperture by SA sig-
SA in GRF-amiR-L1 seedlings, compared with SA effects on naling, plants may establish an unelucidated defense system
GFP-REM1.2 in WT (Fig. 6 D and E and SI Appendix, Fig. S8 G against virus infection. In order to verify this hypothesis, we in-
and H). Using the variable-angle epifluorescence microscopy oculated N. benthamiana leaves with agrobacterial strains that
(VAEM) approach in GRF-amiR-L1, the brighter punctuates harbored 35S:RFP-REM1.2/1.3 or 35S:RFP (control), together
of GFP-REM1.2 that were previously induced by SA in WT with a modified tobacco rattle virus (TRV)-GFP (48) to assess
plants were no longer clustered on PM upon SA treatment (Fig. the impact of the altered cell-to-cell movement of virus. We used
6F). A fluorescent bleaching assay demonstrated that SA directly fluorescent microscopy to analyze the size of TRV infection foci
8 of 11 | www.pnas.org/cgi/doi/10.1073/pnas.1911892116 Huang et al.
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Fig. 7. Model of SA-mediated plant defense response by REM-dependent lipid raft organization. (A and B) TRV–GFP. N. benthamiana leaves were coin-
filtrated with agrobacterial strains containing diluted TRV1 and TRV-GFP as well as 35S:RFP-REM1.2/1.3. Infection efficiency of TRV-GFP was quantified.
Coinfiltration of TRV/TRV1-GFP and 35S:RFP was used as a control. The 100 μM SA was sprayed, and GFP spread areas were observed and quantified at 5 dpi
(n = 33, 32, 35, 35, 30, and 31). (C) Speculated models: Membrane system shows tighter packed liquid-ordered phase (nanodomain) and less packed liquid-
disordered phase (nonnanodomain). The ordered lipid phase requires the integration of sterols. Under low-SA conditions, the ordered and disordered phases
of lipids are homogeneously distributed and well organized by monomer/oligomer states of REM proteins, maintaining PD membrane plasticity. Under high-
SA conditions, REM oligomers are recruited by 14-3-3 dimers on the PM; REM clusters cause nanodomain compartmentalization and higher-ordered lipid
phase, thereby reducing membrane plasticity to block PD opening. The red arrows with dotted lines represent the possible efficiency of PD cargoes movement
during low- or high-SA circumstance. The Right panel shows the possible signaling transduction described in this study. (Scale bars: A, 200 μm.) Error bars
represent SD. P values were determined by 2-tailed Student’s t test (****P < 0.0001; ns, not significant).
at 5 d after inoculation to reveal the efficiency of virus particle contrast with the unsaturated lipids that are assembled into a less
movement. In RFP-inoculated leaves, exogenous SA significantly tightly packed liquid-disordered (Ld) phase (52, 53). Tilsner
decreased TRV-GFP spreading area to one-third of that of the et al. (54) also proposed that a key attribute of PD architecture is
nontreated sample (Fig. 7 A and B and SI Appendix, Fig. S10). In its high degree of membrane curvature, involved in a plastic PD
contrast, overexpressed RFP-REMs pronouncedly restricted membrane system. REM proteins that were known as the foci of
TRV-GFP movement, in line with our conclusion that overex- ordered nanodomains (55), are required for structuring of lipid
pressed REMs conferred PD closure (Fig. 7 A and B and SI ordered domains. Small Lo domains of 10 to 20 nm form
Appendix, Fig. S10). Collectively, these results provide robust spontaneously with a very short lift-time unless they are stabi-
evidence for the involvement of REM-dependent lipid raft or- lized by membrane-anchored proteins (such as REM proteins),
ganization in SA-triggered plant defense pathway. which assemble at nanodomains reaching 100 to 200 nm in size
(53). Although REM is detectable on PD (Fig. 4A), it is not a
Discussion specific PD-located protein. The PD location of REMs is caused
by the enrichment of ordered lipids on the membrane sur-
Accumulating evidence indicates that membrane lipid rafts act as rounding the PD (10, 25). When REM proteins are incorporated
platforms to mediate spatiotemporal organization of protein between lipid molecules, they modify the mean size of the or-
complexes, thereby influencing downstream cellular cascades dered domains and increase membrane tension (56). Perraki
(49–51). In particular, during pathogen infection, membrane et al. (37) have demonstrated that potato virus X (PVX) in-
lipid rafts are essential to establish the appropriate pathogen fection increased REM1.3 mobility and stimulated bigger
recognition sites, providing a large surface area for pathogen nanodomain formation, which is consistent with the phenome-
colonization. In our study, we have revealed that high SA pro- non in our observation that high SA or CMV infection promotes
motes the assembly of lipid nanodomains, leading to the en- REM-labeled nanodomain compartmentalization. REM showed
hancement of the liquid-ordered phase. The higher-ordered a dynamic and tunable range for protein clustering upon dif-
lipids, which are particularly enriched at the PD membrane, ferent treated conditions that are suggested by the different steps
might decrease PD membrane plasticity and thus restrict PD of bleaching and different intensity from our SIM data. The
opening against virus spreading (Fig. 7C). range of lipid order change could depend on the range of the
changing in REM clustering. Therefore, once the overexpressed
Similar to lipid raft components on PM, the PD membrane REM or SA treatment results in REM clustering to a much
also contains a variety of abundant lipids (10), which are segre-
gated into a more tightly packed, liquid-ordered (Lo) phase, in
Huang et al. PNAS Latest Articles | 9 of 11
higher level, a tough and rigid membrane system is formed to PDCB1 signal only on the lateral cell sides (SI Appendix, Fig. S1 A–
constrict the membrane curvature at the PD neck region. Thus, D), which is similar with but also different from SA-induced callose
funnel-shaped PD appear during SA treatment or when REM is deposition (SI Appendix, Fig. S1 A and B). Even though PDCB1
overexpressed. mßcd strongly reverts SA or overexpressed REM localizes at PD due to its callose binding activity (20), PDCB1 protein
on lipid order change, and this process requires the control and abundance is not linearly related to callose abundance. Our study
buffering by REM protein. showed that PDCB1 and callose begin to appear on the lateral
sides under SA treatment; PD were not formed de novo due to
Due to the presence of a rigid cell wall, viruses have to exploit the missing unknown factors for PD biogenesis (Fig. 1C).
existing channels to facilitate their movement into plants. PD are Addressing these interesting questions in the near future would
ideal means by which virus can get through the plant cell and deliver more comprehensive understanding of the biogenesis
serve as gateways for systemic virus movement (57). PD func- of PD.
tionality requires the regulations of several reported factors, such
as callose, callose binding proteins (PDCBs), and PD localizing Methods
proteins (PDLPs) (4, 20), etc. Wang et al. (9) have demonstrated
that SA-mediated PD closure is dependent on callose accumu- Confocal Microscopy Observation. Images were taken by either Zeiss LSM 880
lation, which requires the action of PDLP5 protein. Consistently, (with Airyscan) or Leica SP8 confocal microscopes, or 2D-SIM. The settings of
our study also showed that callose levels are increased on both excitation and detection were as follows: GFP, 488 nm, 505 to 550 nm; YFP,
apical/basal and lateral walls of SA-treated root cells (SI Ap- 488 nm, 495 to 550 nm; aniline blue, 405 nm, 420 to 480 nm; FM4-64, 561 nm,
pendix, Fig. S1 A and B), implying that SA utilizes multiple 590 to 760 nm. All images in a single experiment were captured with the same
regulatory components including callose and lipid order to me- setting. Root meristematic zone of 4-d-old seedlings was consistently used for
diated PD gating. However, PDLP5 cannot directly close PD in confocal microscopy observation.
the absence of SA signaling components (9), and thus a potential
unknown regulator is present downstream of PDLP5 to manip- The rest of the protocols used for plant growth, phenotype analysis,
ulate PD aperture. We identified that REM protein in our study cloning, data quantification, etc., are described in SI Appendix.
fits well with the properties of this unknown regulator, which
directly modifies PD structure and is downstream of SA signaling ACKNOWLEDGMENTS. This work was supported by the National Key
receptors. Research and Development Program of China (2016YFD0100705 and
2017YFA0506100) and National Science Foundation (Grants 31701168 and
PDCB1 is another type of PD-associated protein, which binds 31870170) (to X.C.); Nanyang Technological University (NTU) startup grant
callose at PD and preferentially anchors to the sterol and (M4081533) and NIM/01/2016 (NTU, Singapore) (to Y.M.); and the Research
sphingolipid-enriched membrane raft via its GPI motif (20). Grants Council of Hong Kong (AoE/M-05/12 and C4012-16E) (to L.J.). We
Thus, the PDCB1 distribution pattern is influenced by callose thank Shouwei Ding and Zhenbiao Yang for manuscript comments; Justice
homeostasis, lipid raft organization, and potentially other un- Norvienyeku for manuscript revision; Shi Xiao for phospholipids detection;
known factors. Interestingly, we occasionally found that SA increased Shunping Yan for donation of npr1 and npr3 npr4 mutants; Zhongxin Guo
and Huishan Guo for donation of TRV-GFP constructs; Wenfei Wang for
donation of BiFc-YFP constructs; and Lei Shi and Zhongquan Lin for microscopy
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