Qa-SNAREs localized to the trans-Golgi network regulate multiple transport pathways and extracellular disease resistance in plants.
ABSTRACT In all eukaryotic cells, a membrane-trafficking system connects the post-Golgi organelles, such as the trans-Golgi network (TGN), endosomes, vacuoles, and the plasma membrane. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. Unlike its roles in animal and yeast cells, the TGN has also been reported to function like early endosomal compartments in plant cells. However, the physiological roles of the TGN functions in plants are not understood. Here, we report a study of the SYP4 group (SYP41, SYP42, and SYP43), which represents the plant orthologs of the Tlg2/syntaxin16 Qa-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) that localizes on the TGN in yeast and animal cells. The SYP4 group regulates the secretory and vacuolar transport pathways in the post-Golgi network and maintains the morphology of the Golgi apparatus and TGN. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance responses to a fungal pathogen. We also reveal a plant cell-specific higher-order role of the SYP4 group in the protection of chloroplasts from salicylic acid-dependent biotic stress.
[show abstract] [hide abstract]
ABSTRACT: A subset of intracellular transmembrane proteins such as acid-hydrolase receptors, processing peptidases and SNAREs, as well as extracellular protein toxins such as Shiga toxin and ricin, undergoes 'retrograde' transport from endosomes to the trans-Golgi network. Here, we discuss recent studies that have begun to unravel the molecular machinery that is involved in this process. We also propose a central role for a 'tubular endosomal network' in sorting to recycling pathways that lead not only to the trans-Golgi network but also to different plasma-membrane domains and to specialized storage vesicles.Nature Reviews Molecular Cell Biology 09/2006; 7(8):568-79. · 39.12 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Now that whole genome sequences are available for many eukaryotic organisms from yeast to man, we can form broad hypotheses on the basis of the relative expansion of protein families. To investigate the molecular mechanisms responsible for the organization of membrane compartments, we identified members of the SNARE, coat complex, Rab and Sec1 protein families in four eukaryotic genomes. Of these families only the Rab family expanded from the unicellular yeast to the multicellular fly and worm. All families were expanded in humans, where we find 35 SNAREs, 60 Rabs and 53 coat complex subunits. In addition, we were able to resolve the SNARE class of proteins into four distinct subfamilies.Nature 03/2001; 409(6822):839-41. · 36.28 Impact Factor
Article: SNAREs--engines for membrane fusion.[show abstract] [hide abstract]
ABSTRACT: Since the discovery of SNARE proteins in the late 1980s, SNAREs have been recognized as key components of protein complexes that drive membrane fusion. Despite considerable sequence divergence among SNARE proteins, their mechanism seems to be conserved and is adaptable for fusion reactions as diverse as those involved in cell growth, membrane repair, cytokinesis and synaptic transmission. A fascinating picture of these robust nanomachines is emerging.Nature Reviews Molecular Cell Biology 10/2006; 7(9):631-43. · 39.12 Impact Factor
Qa-SNAREs localized to the trans-Golgi network
regulate multiple transport pathways and
extracellular disease resistance in plants
Tomohiro Uemuraa,1, Hyeran Kimb, Chieko Saitoc, Kazuo Ebinea,2, Takashi Uedaa, Paul Schulze-Lefertb,
and Akihiko Nakanoa,c
aDepartment of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan;bDepartment of Plant Microbe
Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany; andcMolecular Membrane Biology Laboratory, RIKEN Advanced
Science Institute, Wako, Saitama 351-0198, Japan
Edited by Jeffery L. Dangl, University of North Carolina, Chapel Hill, NC, and approved December 19, 2011 (received for review September 14, 2011)
In all eukaryotic cells, a membrane-trafficking system connects the
somes, vacuoles, and the plasma membrane. This complex network
plays critical roles in several higher-order functions in multicellular
organisms. The TGN, one of the important organelles for protein
transport in the post-Golgi network, functions as a sorting station,
where cargo proteins are directed to the appropriate post-Golgi
compartments. Unlike its roles in animal and yeast cells, the TGN
has also been reported to function like early endosomal compart-
ments in plant cells. However, the physiological roles of the TGN
functions in plants are not understood. Here, we report a study of
the SYP4 group (SYP41, SYP42, and SYP43), which represents the
plant orthologs of the Tlg2/syntaxin16 Qa-SNARE (soluble N-ethyl-
maleimide sensitive factor attachment protein receptor) that local-
izes on the TGN in yeast and animal cells. The SYP4 group regulates
the secretory and vacuolar transport pathways in the post-Golgi
network and maintains the morphology of the Golgi apparatus
and TGN. Consistent with a secretory role, SYP4 proteins are re-
quired for extracellular resistance responses to a fungal pathogen.
We also reveal a plant cell-specific higher-order role of the SYP4
group in the protection of chloroplasts from salicylic acid-depen-
dent biotic stress.
Arabidopsis|membrane traffic|membrane fusion
fusion occurs with the concerted functions of several molecules,
including SNAREs (soluble N-ethylmaleimide sensitive factor
attachment protein receptors), Rab GTPases, tethering factors,
and Sec1p/Munc18 (SM) proteins. SNAREs are membrane-an-
chored proteins that contain α-helical heptad repeats and a
characteristic central amino acid within the SNARE motif. The
SNARE complex comprises four SNAREs, including three with a
central glutamine residue in SNARE motif (Q-SNAREs: Qa, Qb,
and Qc) and one with a central arginine (R-SNARE) in SNARE
motif. The Q-SNAREs reside on the target membrane, and the
R-SNARE resides on the transport vesicle. This complex first
forms a bridge between the target organelle membrane and the
vesicle, and then compresses to bring the two membranes close
enough to mediate specific membrane fusion. After fusion is
complete, the SNARE complex is dissociated by a NSF (N-eth-
ylmaleimide-sensitive factor), and the SNAREs are recycled. To
execute the correct membrane fusion, SNAREs must be localized
on the membrane of specific organelles or transport vesicles. Qa-
SNAREs also serve as organelle markers (3–5), by virtue of their
specific localization on the membrane of target organelles.
The TGN was first defined as a special organelle on the trans-
side of the Golgi stack that is responsible for protein sorting to the
plasma membrane or lysosomes (6). The TGN contains multiple
In addition, the TGN is the interface between the Golgi apparatus
n eukaryotic cells, membrane fusion is an essential process in
protein secretion and endocytosis (1, 2). Selective membrane
and endocytic pathways (7). Syntaxin 16, a TGN-localized Qa-
SNARE in animals, functions in the retrograde transport from
early endosomes or recycling endosomes to the TGN (8–10). Tlg2,
the yeast ortholog of syntaxin 16, plays a role in transport in the
endocytic pathway, but not the secretory pathway. Mutants with a
disrupted Tlg2 gene grow slowly, but remain viable (11–13). In
plants, theTGN was alsoreportedtofunctionasthesortingstation
for secretory and endocytosed cargos (14). In addition, recent ev-
idence has indicated that the plant TGN also functions as the early
endosome, the first compartment in the endocytic pathway (15).
However, the physiological function of the plant orthologs of the
TGN-localized Qa-SNAREs (the SYP4 group) is not fully un-
derstood. In this study,weaimedtoelucidate thephysiological role
of the SYP4 group (SYP41, SYP42, and SYP43) in Arabidopsis.
Results and Discussion
Arabidopsis SYP4 Group Possesses Redundant Functions. In Arabi-
dopsis, the SYP4 group represents the plant orthologs of Tlg2/
syntaxin16 and comprises three proteins, SYP41, SYP42, and
SYP43 (16, 17). Previous studies reported that SYP41 and SYP42
localized to different subdomains of the TGN and had non-
redundant functions because individual syp41 and syp42 knockout
mutants were gametophytic lethal (18–20). However, in the
present study, we found that individual, single homozygous
mutants (syp41, syp42, and syp43) were fully viable in the Col-
0 accession. We found that the root length of the syp42 mutant
was slightly shorter than that of the wild-type plant, and the syp41
and syp43 mutants exhibited no visible abnormalities. The double
mutants, syp41−/−syp42−/−and syp41−/−syp43−/−, also showed
shorter and normal roots like their parents, respectively. How-
number of lateral roots, semidwarfism, and early senescence (Fig. 1
A and B and Fig. S1a). Further genetic analyses among the syp4
mutants revealed that syp41+/−syp42−/−syp43−/−mutants (heterozy-
gous for syp41 and homozygous for syp42 and syp43) were seedling
grew almost normally except for short roots like the syp42 single
mutant. syp41−/−syp42−/−syp43+/−mutants were not lethal but
showed phenotypes of short roots, small and white cotyledons, and
Author contributions: T. Uemura, T. Ueda, P.S.-L., and A.N. designed research; T. Uemura,
H.K., C.S., and K.E. performed research; T. Uemura, H.K., C.S., T. Ueda, P.S.-L., and A.N.
analyzed data; and T. Uemura, T. Ueda, P.S.-L., and A.N. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
2Present address: Department of Parasitology, National Institute of Infectious Diseases,
1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 31, 2012
| vol. 109
| no. 5www.pnas.org/cgi/doi/10.1073/pnas.1115146109
normal rosette leaves (Fig. 1A and Fig. S1a). We obtained no triple
homozygous mutants (syp41−/−syp42−/−syp43−/−) in progeny derived
from self-pollinated syp41−/−syp42+/−syp43−/−or syp41−/−syp42−/−
triple mutant was impaired in fertilization competence (Tables S1
and S2). These results indicated that SYP41, SYP42, and SYP43
possessed partly overlapping functions and that SYP42 and SYP43
played major roles in the Col-0 accession.
Members of the SYP4 Group Localize on the Same TGN Compartment.
To determine the subcellular localizations of the SYP4 group
members, we generated transgenic plants expressing SYP4 pro-
teins with fluorescent [green fluorescent protein (GFP), Venus,
and monomeric red fluorescent protein (mRFP)] tags, under the
control of their respective, native, 5′ regulatory sequences. The
expression of GFP-tagged SYP42 and SYP43 proteins com-
plemented the abnormalities of the syp42syp43 mutant, indicating
that these GFP-tagged proteins are functional (Fig. S1b). In-
terestingly, overexpression of GFP-SYP41 (wild-type copies plus
externally added GFP-SYP41) also rescued the defects in the
syp42syp43 mutant. This result further supported the notion that
the proteins of the SYP4 group had partly overlapping functions.
Coexpression of Venus-SYP42 and GFP-SYP43 revealed that
they were completely colocalized in root cells (Fig. 1C). In ad-
dition, expression of individual, GFP-tagged SYP4 proteins
(GFP-SYP41, GFP-SYP42, and GFP-SYP43) demonstrated that
they were colocalized with the TGN marker Venus-SYP61 (Fig.
1D and Fig. S2). Further observation with other organelle
markers indicated that GFP-SYP43 colocalized with VHAa1-
mRFP (vacuolar ATPase a1 subunit; a TGN marker) (Fig. 1E)
of sialyl transferase; a trans-Golgi marker) (Fig. 1F). VAMP722,
an Arabidopsis R-SNARE, was reported to be localized on mo-
bile, punctate structures; moreover, VAMP722 was shown to
function in the secretory pathways that regulate the plant immune
response (21). We found that mRFP-VAMP722 partially colo-
calized with SYP43 but not with ST-Venus or ARA6-GFP (an
endosomal marker) (Fig. 1G and Fig. S2 b and c). Furthermore,
mRFP-VAMP722 generated aggregations after treatment with
Brefeldin A (BFA) (Fig. S2c). BFA blocks the function of BFA-
sensitive ARF-GEFs, and this leads to the aggregation of the
TGN and endosomes in Arabidopsis root and cotyledon cells,
whereas BFA blocks the retrograde membrane traffic between
the Golgi and the endoplasmic reticulum (ER), leading to a block
of the early secretory pathway in mature leaves (22). Plants that
coexpressed GFP-SYP43, ST-Venus, and mRFP-VAMP722
showed that GFP-SYP43 appeared to localize between the trans-
Golgi cisternae labeled with ST-Venus and the compartments
labeled with mRFP-VAMP722 (Fig. 1H). This observation sug-
gested that mRFP-VAMP722 might localize to the more trans
side of the SYP43-labeled compartment for secretion. Taken
together, these results indicated that SYP41, SYP42, and SYP43
localized on the same TGN compartment and had partly over-
SYP4 Group Regulates Secretory and Vacuolar Transport. To de-
termine the physiological significance of the SYP4 group in
plants, we further investigated the nonlethal syp42syp43 mutant
the seedling stage. Wild-type (Wt) and mutant plants are 12 d old. (B) Phenotypes of the aerial parts of Wt and syp42syp43 plants at 35 d. (C–G) GFP-SYP43
colocalized with Venus-SYP42, Venus-SYP61, and VHAa1-mRFP and partially overlapped with ST-mRFP and mRFP-VAMP722. (H) Fluorescent images of root
epidermal cells from transgenic plants that expressed GFP-SYP43, ST-Venus, and mRFP-VAMP722. The inset represents the magnified image of a dot-like
structure with triple labeling. [Scale bars in C–H: 10 μm.)
Members of the SYP4 group have redundant functions. (A) Phenotypes of each syp4 single mutant and all combinations of SYP4 multiple mutants at
Uemura et al.PNAS
| January 31, 2012
| vol. 109
| no. 5
that exhibited several abnormalities. First, we evaluated SYP4-
mediated transport in wild type and syp42syp43 mutant with
a tracer of the endocytic pathway, FM4-64. In wild type, 15 min
after adding FM4-64, the dye appears on the part of the TGN
labeled by VHAa1-GFP, then, after 2 h, it appears on the vac-
uolar membrane. Notably, in the syp42syp43 mutant, after 15
min, we observed weak FM4-64 staining on the vacuolar mem-
brane (Fig. 2A). This early arrival of FM4-64 to the vacuolar
membrane may have been attributable to impaired recycling
from the TGN to the plasma membrane or bypass transport from
the plasma membrane to late endosomes. These results indicated
that internalization from the plasma membrane to the endocytic
pathway was not impaired in the syp42syp43 mutant.
Next, we used secGFP, a signal peptide added to a variant of
GFP, to analyze the secretory pathway. In wild type, secGFP is
synthesized in the ER, traverses the secretory pathway and is fi-
nally secreted to the apoplast. In the syp42syp43 mutant cells, we
clearly observed secGFP accumulation (Fig. 2B, compare with
the wild type), which suggested that secGFP was not secreted.
Furthermore, secGFP florescence did not accumulate inside
the vacuoles after dark treatment (Fig. S3), which suggested
that secGFP was not misdirected to the vacuole. These results
indicate that the secretory pathway was defective in the
We also investigated the vacuolar transport pathway by moni-
toring the processing of a seed storage protein, 12S globulin, in
dry seeds (23). The precursors of 12S globulin did not accumulate
in any of the single mutants or in the double mutants, syp41syp42
or syp41syp43. In contrast, 12S globulin precursor accumulation
was substantial in the syp42syp43 mutant (Fig. 2C). This accu-
mulation of precursors disappeared in the mutant complemented
with GFP-tagged SYP4s (Fig. 2C). One possible explanation for
thisoutcomemight bethatthe syp42syp43mutant wasimpaired in
the recycling of vacuolar sorting receptors from the late endo-
somes/prevacuolar compartment (LE/PVC) to the TGN. These
findings clearly indicated that the plant SYP4 group members
were involved in multiple transport pathways, including the
secretory pathway, the vacuolar transport pathway, and perhaps
the retrieval pathway from the LE/PVC to the TGN.
In addition, we investigated the subcellular localization of the
PIN proteins (auxin efflux carriers), which are localized on the
plasma membrane with a characteristic polarity. As shown in Fig.
3 A and B, the expression of PIN1-GFP (24) and PIN2-GFP (24)
in syp42syp43 mutant was a little restricted compared with the
wild type, but the polar localization of these proteins was not
disturbed. These results could imply that, although the SYP4
groupplayed a substantial role in the secretory pathway (asshown
by the secGFP results), it is not necessarily required for all plasma
membrane-localized cargos. The PIN2 protein is constitutively
recycled and transported to the vacuole for degradation (25).
Indeed, we observed that the fluorescent signal from PIN2-GFP
was observed in the vacuole under dark conditions in wild type
(Fig. 3B, arrowheads). However, in the syp42syp43 mutant, PIN2-
GFP did not accumulate in the vacuole (Fig. 3B). These results
implied that the vacuolar transport of PIN2-GFP was impaired.
Consequently, we reasoned that this might result in disturbed
auxin distribution in the syp42syp43 mutant. Thus, we monitored
auxin distribution by expressing a reporter gene that carried the
synthetic auxin response element DR5 fused to the GFP gene
(26). Activation by auxin induces proportional expression of the
GFP reporter, and thus, reflects the auxin distribution. Until
5 days after germination (DAG), the pattern of fluorescence
signals from DR5::GFP in the syp42syp43 mutant was not altered
compared with the wild type. However, from 7 DAG, the fluo-
rescent pattern of the syp42syp43 mutant began to show clear
difference from the wild type (Fig. S4a). The GFP fluorescence
became more restricted in the quiescent center of the syp42syp43
mutant (Fig. S4b). Consistent with these results, the syp42syp43
mutant showed a weak defect in root gravitropism (Fig. 3C). In
addition, this disturbed auxin distribution might be responsible
for developmental abnormalities, like the short roots and the
large number of lateral roots observed in the syp42syp43 mutant.
type (wt) (Left) and syp42syp43 mutant plants (Right). FM4-64 is transported to the part of the TGN labeled with VHAa1-GFP (15 min) and to the vacuoles
(2 h). (Scale bars: 10 μm.) (B) secGFP accumulated inside syp42syp43 cells. Arrowheads show the strong signal from secGFP accumulation within the cell. (Scale
bars: 10 μm.) (C) Immunoblot of seed proteins probed with the anti-12S globulin antibody. Arrowhead indicates unprocessed precursors.
SYP4 group regulates secretory and vacuolar transport and maintains Golgi/TGN morphology. (A) Uptake of FM4-64 by endocytosis is normal in wild-
| www.pnas.org/cgi/doi/10.1073/pnas.1115146109Uemura et al.
SYP4 Proteins Are Required to Maintain the Morphology of the Golgi/
TGN. To address whether the syp42syp43 mutant had altered
TGN properties, we treated the plants with BFA and then an-
alyzed the TGN. In wild-type cells, BFA treatment (25 μM; 30
min) caused aggregation of the TGN compartment labeled with
VHAa1-GFP. In the syp42syp43 mutant, the aggregation was
markedly delayed in cells from roots and leaves (cotyledons)
(Fig. S5 a and b). In contrast, there was no difference between
wild type and mutants by treatment with wortmannin (Wm) (a
PI3-kinase inhibitor) (Fig. S5a).
We also observed the morphology of the TGN at the ultra-
structural level in the syp42syp43 mutant by transmission electron
microscopy. The morphology of the trans side of the Golgi appa-
ratus, including the trans-Golgi cisternae and the TGN, was al-
tered in the syp42syp43 mutant. The curved trans cisternae of the
Golgi and TGN were clearly observed in the syp42syp43 mutant
compared with the wild type (Fig. 4A). Quantitative analysis
measuring the angles of the last cisternae of the trans side of the
Golgi apparatus, we found that the Golgi or TGN of the syp42-
syp43 mutants had a larger number of curved trans cisternae than
of cisternae within individual stacks were not altered compared
with the wild type (Fig. 4 B–D). In the syp41+/−syp42−/−syp43−/−
triple mutant, which exhibited a severe phenotype (Fig.1A, inset),
we observed several unusual structures in the Golgi/TGN mor-
phology, including a horseshoe-like shape and a small number of
cisternae (Fig. 4E). These results implied that the SYP4 group
members were required to maintain the morphology of the Golgi/
TGN, probably by regulating post-Golgi trafficking.
SYP4 Group Plays Plant Cell-Specific, Higher-Order Roles. To reveal
the physiological roles of the SYP4 group, we focused on stress
responses and investigated the response of the syp42syp43 mu-
tant to biotic or abiotic stress. Through these experiments, we
found prominent phenotypes after inoculation with pathogenic
powdery mildew fungi. We noted a striking leaf chlorosis of
syp42syp43 plants 7 d after pathogen challenge with con-
idiospores of the host-adapted virulent powdery fungus Golovi-
nomyces orontii (27) (Fig. 5A). This chlorosis is dependent on an
intact pathogen-inducible salicylic acid (SA) biosynthesis path-
way because leaves of syp42syp43sid2 triple mutants, lacking
additionally the chloroplast-targeted SID2 isochorismate syn-
thase (28, 29), showed a wild-type-like green leaf coloration (Fig.
5A). These findings point to a potential chloroplast dysfunction
in syp42syp43 plants during SA-dependent biotic stress. Despite
the leaf chlorosis and semidwarfism, G. orontii pathogenesis on
syp42syp43 plants was indistinguishable compared with the wild
type, as evidenced by the development of a profuse fungal my-
celium on the leaf surface in time-series experiments and mi-
croscopic inspection of leaf specimen (Fig. S6). The syp42syp43
mutants also retain the ability to accumulate high SA levels in
response to powdery mildew challenge albeit SA levels in non-
inoculated syp42syp43 plants are moderately elevated in the ab-
sence of pathogen (Fig. 5B). Consistent with an intact SA-
dependent defense signaling pathway in syp42syp43 plants, we
observed a fungus-induced activation of the defense response
marker gene PR-1 but not in sid2 or syp42syp43sid2 genotypes
(Fig. 5C). Thus, the biotic stress-induced and SA-dependent
chloroplast dysfunction of syp42syp43 plants occurs in the pres-
ence of an otherwise intact pathogen-induced and SA-dependent
defense signaling. Together, this reveals an unexpected link be-
tween the TGN and chloroplasts and points to a plant-specific
higher-order role of the TGN-resident SYP4 group in protecting
chloroplasts from SA-dependent biotic stress.
Next, we inoculated syp42syp43 plants with the nonadapted
powdery mildew fungus Erysiphe pisi, the pathogenesis of which
postinvasive resistance responses (27, 30). In comparison with
wild type, syp42syp43 plants fail to effectively restrict hyphal
Because this step in fungal pathogenesis requires entry of fungal
a function in secretion-dependent disease resistance responses.
Possible Mechanism for the Higher-Order Role of the SYP4 Group. In
this study, we demonstrated that the SYP4 group maintained the
morphology of the Golgi apparatus and TGN. Although the SYP4
group was localized on the TGN, the morphology of the Golgi
apparatus inthe syp42syp43 mutant wasabnormal. The mechanism
of transport between the trans-Golgi cisternae and the TGN
remains unknown, even in animal and yeast cells. Thus, it was not
clear why the Golgi apparatus morphology was affected in the
syp42syp43 mutant. One possibility is that the SYP4 group might
mediate membrane fusion between the transport vesicles budding
from the trans-Golgi cisterna and the TGN. In that case, in the
syp42syp43 mutant, the abnormal morphology of Golgi apparatus
might result from the accumulation of transport vesicles that left
possibility is that the trans-Golgi cisternae might not mature suffi-
ciently to form the TGN in the syp42syp43 mutant because of the
defective TGN function, given that the SYP4 group mediated
vacuole. (A) Expression and subcellular localization of PIN1-GFP in wild type
(wt) and the syp42syp43 mutant. Mislocalization of PIN1-GFP was not ob-
served in the syp42syp43 mutant. Plants were grown in continuous light for
7 d. (B) Expression and subcellular localization of PIN2-GFP in wt and the
syp42syp43 mutant. Accumulation of PIN2-GFP in the vacuole (arrowheads)
was not detected in the syp42syp43 mutant. Plants were grown in continuous
light for 5 d and transferred to dark conditions. (Scale bars: 10 μm.) (C) Phe-
notype of root gravitropism in the syp42syp43 mutant. Plants were grown in
continuous light for 5 d and transferred to dark conditions. Plants were
grown in dark conditions for 1 d vertically and turned counter clockwise
through 90°. After 24 h incubation, the angles of the roots were measured.
syp42syp43 mutants exhibit defective PIN2-GFP transport to the
Uemura et al. PNAS
| January 31, 2012
| vol. 109
| no. 5
several post-Golgi transport pathways and maintained TGN func-
tion. Cargo molecules destined for post-Golgi organelles might not
be selected correctly at the abnormal TGN, resulting in the traf-
ficking defects of FM4-64, secGFP, PIN2, and defense molecules.
Our results also indicated that the SYP4 group regulated
multiple transport pathways, including pathways clearly linked to
higher-order physiological functions. The secretory pathway
regulated by the SYP4 group appears to play an important role in
is altered in syp4 mutants. (A) Transmission
electron micrographs of Golgi/TGN struc-
curved structures that represent the cister-
nae of the trans-Golgi and/or TGN. (Scale
bars: 500 nm.) (B) Quantitative analysis of
the curved cisternae of the trans-Golgi and/
or TGN. The angles of the curved cisternae
were measured, and those with angles un-
der 150° were counted. (C) Quantitative
analysis of the total number of cisternae in
the Golgi stacks of wild type (wt) and
analysis of the lengths of the longest cis-
ternae of the Golgi stacks in wt and
syp42syp43 mutants. (E) Transmission elec-
tron micrographs of Golgi/TGN structures
showing abnormal structures of the Golgi
stacks. (Scale bars: 500 nm.)
The morphology of the Golgi/TGN
chloroplasts from SA-dependent biotic stress and for ex-
tracellular resistance responses to a powdery mildew
fungus. (A) Macroscopic images of infection phenotypes
of virulent G. orontii inoculated on the indicated plant
genotypes. Images were taken at 7 d after conidiospore
inoculation. (Scale bars: 1 cm.) Note the pathogen-in-
duced leaf chlorosis phenotype of syp42 syp43 plants. (B)
Total salicylic acid levels of the indicated genotypes of
noninoculated and G. orontii-inoculated plants at 3 d af-
ter pathogen challenge. (C) Quantitative RT-PCR gene
expression of the defense marker PR1 in noninoculated
and G. orontii-inoculated plants of the indicated plant
genotypes. Gene expression levels were normalized by
actin2 expression. (D) Secondary hyphae formation of
E. pisi sporelings (%) on leaves of the indicated geno-
types at 72 h after inoculation.
SYP4 proteins are required for the protection of
| www.pnas.org/cgi/doi/10.1073/pnas.1115146109Uemura et al.
extracellular defense responses: a defense cargo in the TGN
might be sorted by the SYP4 group and delivered via VAMP721/
722-containing vesicles to the plasma membrane for subsequent
PEN1(penetration1)-dependent exocytosisin asecretory defense
pathway (21, 32). The current findings assign to the TGN com-
partment a role as an entry portal to post-Golgi transport path-
ways for several higher-order plant functions. Interestingly,
despite the leaf chlorosis of infected syp42syp43 plants, pathogen-
inducible SA accumulation, PR-1 expression, and pathogenesis of
host-adapted G. orontii remain largely unaffected in the double
mutant. Thus, the SYP4 group is not essential, per se, for the
responsiveness of the plants to biotic stress. Our data, instead,
show that SYP4 regulates a pathogen-inducible and SA-de-
pendent pathway required for chloroplast function during biotic
stress. This points to an unexpected functional link between the
TGN and chloroplasts, although it remains to be seen whether
this link is direct. For example, SYP4 proteins in the TGN might
be essential for sorting nucleus-encoded proteins targeted to
chloroplasts during SA-dependent defense responses. Another
possibility is that the TGN directly contacts chloroplasts to ex-
change materials. Taken together, our study reveals the engage-
ment of the SYP4 group in multiple intracellular transport
pathways, thereby highlighting functional links between the post-
Golgi network and higher-order processes in plants.
Materials and Methods
Electron microscopy, immunoblot analysis, root gravitropism assay, pathogen
assays, and SA measurements were performed as described in SI Materials
Plant Materials and Plasmids. The syp41 (Salk_060613), syp42 (Salk_116966),
and syp43 (Salk_144268) Arabidopsis thaliana mutants were obtained from
the Arabidopsis Biological Resource Center. Details regarding plant materi-
als and plasmids are described in SI Materials and Methods.
Confocal Laser-Scanning Microscopy. For single-color imaging, GFP and mRFP
with a confocal scanner unit (CSU10; Yokogawa Electric) and a cooled CCD
camera (ORCA-AG; Hamamatsu Photonics). Multicolor observations were
carried out with a LSM710 confocal microscope (Carl Zeiss). Details regarding
drug treatment analysis are available in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Drs. F. Ausubel, K. Schumacher, J. Friml,
B. Scheres, and I. Hara-Nishimura for sharing materials; The Salk Institute and
theArabidopsisBiological Resource Center for providing A.thaliana mutants;
and Dr. Chian Kwon for the suggestion to generate syp42 syp43 sid2 triple
mutants. This work was supported by a Grant-in-Aid for Specially Promoted
Research, Grants-in-Aid for Scientific Research, and the Targeted Proteins
Research Program from the Ministry of Education, Culture, Sports, Science
and Technology of Japan; in part, by the Bioarchitect and the Cellular Systems
Biology Projects of RIKEN; and, in part, by Deutsche Forschungsgemeinschaft
Research Grant SPP1212 (to H.K. and P.S.-L.).
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Uemura et al.PNAS
| January 31, 2012
| vol. 109
| no. 5