Biochemical and Biophysical Research Communications 524 (2020) 977e982

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

CCOAOMT1, a candidate cargo secreted via VAMP721/722 secretory

vesicles in Arabidopsis
Hyeokjin Kwon a, 1, Da Jeong Cho b, 1, Horim Lee c, Myung Hee Nam d, Chian Kwon b,
Hye Sup Yun a, *
a
Department of Biological Sciences, Konkuk University, Seoul, 05029, South Korea
b
Department of Molecular Biology, Dankook University, Cheonan, 31116, South Korea
c
Department of Biotechnology, Duksung Women’s University, Seoul, 01369, South Korea
d
Environmental Risk and Welfare Research Team, Korea Basic Science Institute (KBSI), Seoul, 02855, South Korea

a r t i c l e i n f o a b s t r a c t

Article history: We previously found that VAMP721/722 SNARE proteins guide secretory vesicles to pathogen-attacking
Received 2 February 2020 sites during immune responses in Arabidopsis, which suggests that these vesicles should deliver immune
Accepted 5 February 2020 molecules. However, the lethality of vamp721 vamp722 double null mutant makes it difficult to under-
Available online 12 February 2020
stand the nature of cargo transported via VAMP721/722 vesicles. Since VAMP721/722-depleted
(VAMP721þ/ VAMP722/ and VAMP721/ VAMP722þ/) plants show compromised resistance to
Keywords:
extracellular pathogens, we assume that an immune protein secreted through the VAMP721/722-
Arabidopsis
engaged exocytosis would be remained more in VAMP721/722-depleted plants than WT. By
CCOAOMT1
P. syringae DC3000
comparing intracellular proteins between WT and VAMP721/722-depleted plants, we found caffeoyl-CoA
Secretion O-methyltransferase 1 (CCOAOMT1) involved in the lignin biosynthesis was more abundantly detected in
VAMP721/722 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.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction mitochondrion-localized PEN2 atypical myrosinase and the

Plant immune responses to extracellular pathogens are initiated
by the perception of pathogen-derived nonself molecules by sur-
face receptors, and finalized by the secretion of immune molecules
to terminate pathogenesis [1e4]. In the dicot model plant, Arabi-
dopsis thaliana, two prominent immune secretory pathways have
been identified [5]. One is the transporter-mediated extrusion of
pathogen-toxic compounds that engages the peroxisome/
Abbreviations: CCR1, cinnamoyl-CoA reductase 1; CCOAOMT1, caffeoyl-CoA O-
methyltransferase 1; CLPP5, ATP-dependent Clp protease proteolytic subunit 5; CSE,
caffeoyl shikimate esterase; GDH3, glycine cleavage system H protein 3; PAL1,
phenylalanine ammonialyase 1; SNARE, soluble N-ethylmaleimide sensitive factor
attachment protein receptor; TCA, tricholoroacetic acid; VAMP721/722, vesicle-
associated membrane protein 721/722; WT, wild-type.
* Corresponding author.
E-mail address: hsyun@konkuk.ac.kr (H.S. Yun).
1
These authors contributed equally to this work.
plasma membrane (PM)-residing PEN3 ATP-binding cassette (ABC)
transporter [6e8]. The other is the vesicle-mediated exocytosis that
involves the PM-localized PEN1 (SYP121) and SYP132 syntaxins, the
PM-attached SNAP33 adaptor, and the functionally redundant
vesicle-associated membrane protein (VAMP) 721/722 soluble N-
ethylmaleimide sensitive factor attachment protein receptor
(SNARE) proteins [9e11]. Orthologous SNARE proteins found in the
monocot barley (HvROR2, HvSNAP34 and HvVAMP721 to the Ara-
bidopsis PEN1, SNAP33 and VAMP721/722, respectively) [9,11]
indicate that the latter secretory pathway is an ancient immune
exocytosis before the divergence between monocot and dicot
plants.
It was recently found that PEN3 in the former export pathway
secretes antimicrobial camalexins [12]. However, little is known for
the nature of cargo transported and secreted by VAMP721/722
vesicles in the latter exocytic pathway. Delayed deposition of cal-
lose, a component of pathogen-induced secondary cell wall called

https://doi.org/10.1016/j.bbrc.2020.02.029
0006-291X/© 2020 Elsevier Inc. All rights reserved.
978 H. Kwon et al. / Biochemical and Biophysical Research Communications 524 (2020) 977e982
papilla, in pen1 and VAMP721/722-silenced plants [11,13] rather
suggests cell wall materials and/or cell wall-modifying enzymes as
cargos for VAMP721/722 vesicles. Indeed, recent proteomic ana-
lyses revealed that PEN1 and VAMP721 are important for the
secretion of some cell wall-related proteins [14,15]. We previously
found that flg22, a part of bacterial flagellin that induces plant
immune responses, elevated VAMP721/722 abundance [16].
Therefore, to understand an immune protein that is transported via
VAMP721/722 vesicles, we in this study compared intracellular
proteins between wild-type (WT) and VAMP721/722-depleted
(VAMP721þ/ VAMP722/ and VAMP721/ VAMP722þ/) plants
treated with flg22, because vamp721 vamp722 double mutant is
lethal. By mass spectrometry, we found that caffeoyl-CoA O-
methyltransferase 1 (CCOAOMT1), an enzyme in the lignin
biosynthetic pathway, is significantly more accumulated in
VAMP721/722-depleted plants than WT. Extracellular detection of
CCOAOMT1 in protoplasts and compromised defense against
Pseudomonas syringae DC3000 in ccoaomt1 plants suggest that
plants secrete cell wall-reinforcing proteins including CCOAOMT1
as a part of immune responses.
2. Materials and methods
2.1. Plant materials
Plants used for experiments were Arabidopsis thaliana Col-
0 grown at 22  C with 10 h light/14 h dark photoperiod. To isolate
ccoaomt1-1 and ccoaomt1-2, GABI_007F02 and SALK_151507 were
obtained from ABRC and GABI-Kat, and analyzed by genomic DNA
PCR and immunoblot with anti-CCOAOMT1 antibody. Since the
haplo-insufficient VAMP721þ/ VAMP722/ and VAMP721/
VAMP722þ/ lines are segregating, to isolate intracellular proteins,
plants were first grown in solid MS medium containing 1% sucrose
for 10 d, and genotyped by genomic DNA PCR with detached leaves
as previously described [17]. Selected plants were then transferred
to liquid MS medium containing 1% sucrose, further grown for 2 d,
and treated with 1 mM flg22 for 8 h. To analyze CCOAOMT1
expression or secretion and bacterial growth, plants were grown in
liquid MS medium containing 1% sucrose for 10 d, and untreated or
treated with 1 mM flg22 for the indicated time. For protoplast
isolation, plants were grown in soil for 4e5 weeks.
2.2. Protein identification by mass spectrometry
To obtain intracellular proteins, apoplastic fluid was removed
from flg22-treated WT, VAMP721þ/ VAMP722/ and VAMP721/
VAMP722þ/ plants as previously described. Then total proteins
were extracted, separated on 2-D gels, and stained with Coomassie
as previously described [18]. The intensity of protein spots from 3
biological repetitions were analyzed by the SameSpots Software
(TotalLab). Protein spots, whose intensity was higher in both
VAMP721þ/ VAMP722/ and VAMP721/ VAMP722þ/ plants than
WT plants by more than 1.2-fold, were selected and subject to mass
spectrometry for protein identification, as previously described
[18].
2.3. Expression analysis
To analyze transcript levels, total RNA was extracted from flg22-
treated WT plants and subject to RT-qPCR with LightCycler® Nano
System (Roche). Primers used are; 50 -GAGGAGTGATTGGCTACGACA
and 50 -TCCGACGGCAGATAGTGATTC for CCOAOMT1, and 50 -ATG-
GAAGCTTCTGGAATCCAC and 50 -TTTGCTCATACCTTCAGCGAT for
Actin2. Relative transcript amounts of CCOAOMT1 against Actin2
were calculated by LightCycler® Nano Software (Roche).
To analyze protein levels, proteins were extracted in 1 x PBS (pH
8.0) containing 1% Triton X-100 from WT, ccoaomt1-1 and
ccoaomt1-2 plants either untreated or treated with 1 mM flg22 for
the indicated time. Proteins were then separated on a poly-
acrylamide gel and blotted with anti-CCOAOMT1 antibody. To
visualize equal loading, separated proteins were stained with
Ponceau S. Anti-CCOAOMT1 antibody was generated in a rabbit
with the full-length CCOAOMT1 that was purified as previously
described [16].
2.4. Secretion analysis
Protoplasts were isolated from soil-grown plants as previously
described [19]. Protoplasts in WI solution (0.5 M mannitol, 20 mM
KCl, and 4 mM MES, pH 5.7) were treated with 1 mM flg22 for the
indicated time. After removal of protoplasts by centrifugation,
secreted proteins in the protoplast-free medium were precipitated
with 20% tricholoroacetic acid (TCA). Precipitated proteins were
then resuspended in 1 x PBS (pH 8.0) containing 1% Triton X-100,
and subject to immunoblot with anti-CCOAOMT1 antibody.
2.5. Pathogenicity test
Liquid MS medium-grown plants were inoculated with
P. syringae DC3000 (1  105 cfu) by adding bacteria to the medium
as previously described [27]. At 2 d post inoculation, the number of
in planta bacteria was counted and normalized against plant fresh
weight.
3. Results and discussion
Functional redundancy between VAMP721 and VAMP722, and
the lethality of vamp721 vamp722 double null mutant [11] lead to
difficulty in understanding what are secreted via VAMP721/722
vesicles. However, compromised disease resistance in the haplo-
insufficient VAMP721þ/ VAMP722/ and VAMP721/ VAMP722þ/

plants, compared to WT and either single mutant plants [11],
suggests that the lower gene dosage accompanied by reduced
protein levels in those plants may result in impaired secretion of
immune molecules. This additionally suggests that at least a part of
immune molecules might be more accumulated in VAMP721þ/
VAMP722/ and VAMP721/ VAMP722þ/ plants than WT plants.
Therefore, we tried to isolate a candidate cargo protein which is
transported via VAMP721/722 vesicles by comparing intracellular
proteins among WT, VAMP721þ/ VAMP722/ and VAMP721/
VAMP722þ/ plants.
We previously found that VAMP721/722 abundance is elevated
by the bacterial flg22 MAMP treatment in Arabidopsis [16]. This
implies that at least a portion of defense-related proteins whose
production is induced by flg22 would be secreted via VAMP721/722
vesicles. Therefore, we treated WT, VAMP721þ/ VAMP722/ and
VAMP721/ VAMP722þ/ plants with 1 mM flg22 for 8 h, followed
by extracting intracellular proteins from those plants. After sepa-
rating extracted proteins on 2-dimensional (D) gels, we analyzed
gels from three independent biological replicates of each genotype
(Supplementary Fig. S1A-C) with the SameSpots software. Since
one non-mutated allele of either VAMP721 or VAMP722 is still
present in VAMP721þ/ VAMP722/ or VAMP721/ VAMP722þ/
plants, we expected that the difference in intracellularly accumu-
lated protein levels, if any, would be not such big between WT and
VAMP721þ/ VAMP722/ or VAMP721/ VAMP722þ/ plants. With
this, we found in total 8 protein spots whose average volumes are
higher by more than 1.2-fold both in VAMP721þ/ VAMP722/ and
VAMP721/ VAMP722þ/ plants than in WT plants (Supplementary
Fig. S2 and Supplementary Table S1). Among these, we by LC-MS/
 H. Kwon et al. / Biochemical and Biophysical Research Communications 524 (2020) 977e982 979

MS identified 3 proteins, caffeoyl-CoA O-methyltransferase 1
(CCOAOMT1), ATP-dependent Clp protease proteolytic subunit 5
(CLPP5) and glycine cleavage system H protein 3 (GDH3)
(Supplementary Table S2). However, since spot volumes of CLPP5
and GDH3 were not significantly different between WT and both
VAMP721þ/ VAMP722/ and VAMP721/ VAMP722þ/ plants
(Supplementary Fig. S2B), we in this study focused on CCOAOMT1
whose spot volumes were significantly more accumulated both in
VAMP721þ/ VAMP722/ and VAMP721/ VAMP722þ/ than in WT
plants (Fig. 1A and B; Supplementary Fig. S2B).
CCOAOMT1 is a key enzyme to convert caffeoyl-CoA to feruloyl-
CoA in the lignin biosynthetic pathway in plants [20e22]. It was
recently reported that CCOAOMT1 was identified as a co-
precipitated protein with the PM-residing SYP132 in Arabidopsis
[23]. Since SYP132 is required for defense against bacterial
pathogens and specifically interacts with VAMP721/722 [10,16],
this suggests that CCOAOMT1 might be delivered via VAMP721/722
vesicles to bacteria-attempting sites likely to form the secondary
cell walls. Therefore, we first examined its expression in response
to flg22 by treating WT plants with 1 mM flg22. flg22 rapidly
induced CCOAOMT1 transcription, and maintained its elevated
transcription levels until 24 h (Fig. 2A). By immunoblot with anti-
CCOAOMT1 antibody that was raised in a rabbit and specifically
detects CCOAOMT1 (Fig. 3A), we also found that its increased
transcription levels were accompanied by elevated abundance of
CCOAOMT1 protein levels (Fig. 2A).
Since we isolated and identified CCOAOMT1 as a more accu-
mulated protein within the exocytosis-impaired VAMP721/722-
depleted plant cells (Fig. 1), we next tested whether CCOAOMT1
is secreted from plant cells or not. For this, we isolated protoplasts

Fig. 1. Identification of CCOAOMT1 as an intracellularly more accumulated protein in VAMP721/722-depleted plants. (A) CCOAOMT1-corresponding spots (red circles) on two
dimensionally separated intracellular proteins from the indicated WT, VAMP721þ/ VAMP722/ and VAMP721/ VAMP722þ/ plants treated with 1 mM flg22 for 8 h. Parts of 2-D
gels stained with Coomassie from three biologically replications are shown. (B) Significant elevation of CCOAOMT1 amounts in both VAMP721þ/ VAMP722/ and VAMP721/
VAMP722þ/ plants. Spot volumes corresponding to CCOAOMT1 in (A) were analyzed by the SameSpots Software. Bar, mean ± SD. *, P < 0.05. (C) Identification of CCOAOMT1 by LC-
MS/MS. A protein spot corresponding to CCOAOMT1 was subject to LC-MS/MS analysis. The identified peptides were shown in red and underlined. (For interpretation of the
references to colour in this figure legend, the reader is referred to the Web version of this article.)
980 H. Kwon et al. / Biochemical and Biophysical Research Communications 524 (2020) 977e982

Fig. 2. Secretion of CCOAOMT1. (A) Induction of CCOAOMT1 by flg22. WT seedlings grown in liquid MS medium were treated with 1 mM flg22 for the indicated time. RNA extracts
were subject to realtime RT-PCR. Relative CCOAOMT1 transcript levels were normalized against Actin2. Protein extracts were subject to immunoblot with anti-CCOAOMT1 antibody.
Equal loading was shown by Ponceau S-stained Rubisco. Bar, mean ± SE (three biological replicates). *, P < 0.05; **, P < 0.01. (B) Extracellular detection of CCOAOMT1 in protoplasts.
Leaf-driven protoplasts were incubated with or without 1 mM flg22 for the indicated time. Protein extracts from protoplasts (intracellular) and precipitated proteins from protoplast-
incubated medium (extracellular) were subject to immunoblot with anti-CCOAOMT1 antibody. Equal loading was shown by Ponceau S staining.

from WT leaf tissues, treated them with 1 mM flg22, and precipi-
tated proteins in the protoplast-free medium. We then examined
the presence of CCOAOMT1 in the medium-precipitated proteins by
immunoblot with anti-CCOAOMT1 antibody. Unexpectedly, we
detected CCOAOMT1 in the medium even in the absence of flg22
(Fig. 2B). Since protein bands were barely detectable in precipitated
proteins from the medium (Fig. 2B), this implies that a part of
CCOAOMT1 might be constitutively secreted from plant cells.
Interestingly, we found that its levels in the medium were tran-
siently elevated at 4 h and diminished at 8 h post protoplast in-
cubation (hpi) in the absence of flg22 (Fig. 2B). However,
intracellular CCOAOMT1 levels were gradually increased during
protoplast incubation (Fig. 2B). Since protoplasts are highly stressed
during their isolation especially by removing the cell walls, and
since stresses are known to induce the secretion of proteases [24],
the reduction of CCOAOMT1 levels in the medium at 8 hpi might be
due to its proteolytic degradation. Interestingly, we found that
CCOAOMT1 level in the medium was more elevated in the presence
of flg22 at 4 hpi (Fig. 2B), suggesting that flg22 induces its secretion
from protoplasts.
Downregulation of CCOAOMT1 was reported to result in
compromised defense against powdery mildew fungi in wheat [25].
In addition, it was recently reported that the guaiacyl (G) lignin
biosynthetic pathway including CCOAOMT1 is governed by the
MYB15 transcription factor and is required for defense against
Pseudomonas syringae DC3000 bacterium in Arabidopsis [26]. Since
flg22 induces CCOAOMT1 elevation and secretion (Fig. 2), we then
examined its function in immunity to P. syringae DC3000. For this,
we isolated two independent T-DNA insertion mutant lines,
ccoaomt1-1 (GABI_007F02) and ccoaomt1-2 (SALK_151507), in
which T-DNA is inserted in an intron in the former, but in an exon in
the latter (Fig. 3A). By immunoblot with anti-CCOAOMT1, we found
that CCOAOMT1 is barely detectable in both ccoaomt1 plants
(Fig. 3A). We then liquid-inoculated WT and ccoaomt1 plants with
P. syringae DC3000 and counted the number of in-plant bacteria at
2 d post inoculation (dpi). As previously reported [27], the in-plant
bacterial growth in this experiment was dependent on the presence
of the flagellin-recognizing FLS2 receptor (Fig. 3B). Interestingly, we
found that bacterial growth at 2 dpi in ccoaomt1 plants was
elevated compared to WT plants in the absence of flg22 treatment
(Fig. 3B), indicating that CCOAOMT1 is required for defense against
P. syringae DC3000. However, in flg22 pretreatment experiments,
bacterial growth in ccoaomt1 plants was comparable to that in WT
plants (Fig. 3B). This suggests that CCOAOMT1 is required for basal
but not induced immunity to P. syringae DC3000. The lack of
CCOAOMT1 might be compensated by flg22-induced expression of
 H. Kwon et al. / Biochemical and Biophysical Research Communications 524 (2020) 977e982
Fig. 3. Compromised defense against P. syringae DC3000 in ccoaomt1 plants. (A)
Isolation of two independent ccoaomt1 T-DNA insertion lines. (A) A schematic repre-
sentation of CCOAOMT1 gene. Box, exon; line, intron; triangle, T-DNA insertion site.
CCOAOMT1 is not detected in isolated two independent ccoaomt1-1 and ccoaomt1-2
mutant plants. Protein extracts from the indicated genotype plants were subject to
immunoblot with anti-CCOAOMT1 antibody. Equal loading was shown by Ponceau S-
stained Rubisco. (B) Elevated growth of P. syringae DC3000 in ccoaomt1 plants
compared to WT. The indicated genotype plants grown in liquid MS medium were
liquid-inoculated with P. syringae DC3000 (1  105 cfu) following treatment with 1 mM
flg22 (black) or not (gray) for 1 d. The in-planta bacterial growth was measured at 2 d
post inoculation. Bar, mean ± SD (three biological replicates). Different letters repre-
sent significant differences analyzed by one-way ANOVA with Turkey’s post-hoc test
(P < 0.05).
other lignin biosynthetic genes and/or by CCOAOMT1-like genes
(CCOAOMT2 e 7) [28] in flg22-pretreated ccoaomt1 plants.
In this study, we identified CCOAOMT1, one of lignin biosyn-
thetic enzymes, as a candidate cargo protein to be secreted by
VAMP721/722 vesicles by comparing flg22-accumulated intracel-
lular proteins between WT and VAMP721/722-deficient
(VAMP721þ/ VAMP722/ and VAMP721/ VAMP722þ/) plants
(Fig. 1). Detection of elevated CCOAOMT1 levels in the protoplast-
removed medium after flg22 treatment (Fig. 2) indicates that
CCOAOMT1 is secreted from plant cells and its secretion is stimu-
lated by flg22. It was recently reported that CCOAOMT6
(At1g67980) was found in WT seedling-incubated medium [14].
Since CCOAOMT6 is homologous to CCOAOMT1, this supports our
results of CCOAOMT1 secretion. Interestingly, CCOAOMT6 was not
detected in syp121 seedling-grown medium [14], suggesting that
CCOAOMT6 and likely CCOAOMT1 may be secreted by the SYP121-
associated exocytosis. The identification of CCOAOMT1 in the GFP-
SYP132 immunoprecipitates from GFP-SYP132-expressing trans-
genic plants [23] additionally suggests a possibility that CCOAOMT1
is extracellularly discharged by the SYP132-dependent exocytosis.
These PM-residing SYP121 and SYP132 syntaxins intriguingly form
the in planta SNARE complex with VAMP721/722 to drive the im-
mune exocytosis [11,16], additionally supporting the secretion of
CCOAOMT1 via VAMP721/722 vesicles.
During immune responses, plant cells are known to deposit the
secondary cell wall that is called papilla containing callose and
lignin [5], which is regarded as a physical immune barrier to per-
turb pathogenesis. Indeed, it was found that deletion of lignin
biosynthetic genes such as phenylalanine ammonialyase 1 (PAL1),
caffeoyl shikimate esterase (CSE) and cinnamoyl-CoA reductase 1
(CCR1), whose transcription is controlled by MYB15, resulted in
impaired resistance to P. syringae DC3000 in Arabidopsis [26].
981
Similarly, we found the elevated P. syringae DC3000 growth in
ccoaomt1 plants compared to WT plants (Fig. 3B). We previously
found that this papilla formation is dependent on VAMP721/722,
because callose deposition underneath fungal penetration sites is
delayed in VAMP721/722-deficient plants [11]. Recent secretome
analysis studies revealed that a variety of cell wall-associated
proteins are secreted from plants, and that the secretion of a part
of those proteins is dependent on VAMP721 [14,15]. Therefore, this
and the directional movement of GFP-VAMP722 to fungal entry
sites [11] strongly suggest that the VAMP721/722-engaged immune
exocytosis is responsible for the secondary cell wall formation at
pathogen-contacting sites in plants, likely by transporting
demanded cell wall materials and/or related enzymes at least
including CCOAOMT1. Interestingly, the majority of identified pro-
teins in the above-mentioned secretome studies are lacking of
signal peptides [14,15], indicating that those proteins are uncon-
ventionally (not via the ER/Golgi route) secreted from plant cells.
Likewise, CCOAOMT1 that we identified in this study lacks a signal
peptide. This and the observation of GFP-VAMP722 in the extra-
cellular space in infected plants suggest that VAMP721/722 might
extracellularly discharge CCOAOMT1 partly in an exosome-driven
unconventional secretory pathway [29].
Declaration of competing interest
There is no conflict of interest.
Acknowledgements
We thank Dr. Kun Cho at Korea Basic Science Institute for
technical assistance in mass spectrometry. This work was sup-
ported by grants (2016R1D1A1B02007322 to CK and
2017R1D1A1B03029802 to HSY) from National Research Founda-
tion, Korea and a grant (PJ01477001 to CK) from Rural Development
Administration, Korea.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2020.02.029.
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