Mechanisms of functional specificity among plasma-membrane syntaxins in Arabidopsis.

Abstract

Syntaxins and interacting SNARE proteins enable membrane fusion in diverse trafficking pathways. The Arabidopsis SYP1 family of plasma membrane-localized syntaxins comprises nine members, of which KNOLLE and PEN1 play specific roles in cytokinesis and innate immunity, respectively. To identify mechanisms conferring specificity of action, we examined one member of each subfamily-KNOLLE/SYP111, PEN1/SYP121 and SYP132-in regard to subcellular localization, dynamic behavior and complementation of knolle and pen1 mutants when expressed from the same promoters. Our results suggest that cytokinesis-specific syntaxin requires high-level accumulation during cell-plate formation, which necessitates de novo synthesis rather than endocytosis of pre-made protein from the plasma membrane. In contrast, syntaxin in innate immunity does not need upregulation of expression but instead requires pathogen-induced and endocytosis-dependent retargeting to the infection site. This feature of PEN1 is not afforded by SYP132. Additionally, PEN1 could not substitute for KNOLLE because of SNARE domain differences, as revealed by protein chimeras. In contrast, SYP132 was able to rescue knolle as did KNOLLE-SYP132 chimeras. Unlike KNOLLE and PEN1, which appear to have evolved to perform specialized functions, SYP132 stably localized at the plasma membrane and thus might play a role in constitutive membrane fusion.

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Traffic 2011; 12: 1269–1280
© 2011 John Wiley & Sons A/S
doi:10.1111/j.1600-0854.2011.01222.x
Mechanisms of Functional Specificity Among
Plasma-Membrane Syntaxins in Arabidopsis
Ilka Reichardt1,2, Daniel Slane1, Farid El Kasmi1,
Christian Kn¨ oll1, Rene Fuchs3,4, Ulrike Mayer1,
Volker Lipka3,4and Gerd J¨ urgens1,∗
1ZMBP, Entwicklungsgenetik, Universit¨ at T¨ ubingen, Auf
der Morgenstelle 3, 72076 T¨ ubingen, Germany
2Current address: Institute of Molecular Biotechnology of
the Austrian Academy of Sciences (IMBA), Dr. Bohr
Gasse 3, 1030 Vienna, Austria
3Sainsbury Laboratory, John Innes Centre, Norwich
Research Park, Norwich NR4 7UH, UK
4Current address: Albrecht-von-Haller-Institute for Plant
Sciences, Georg-August-Universit¨ at G¨ ottingen, Untere
Karsp¨ ule 2, 37073 G¨ ottingen, Germany
*Corresponding author: Gerd J¨ urgens, gerd.juergens@
zmbp.uni-tuebingen.de
Syntaxins and interacting SNARE proteins enable mem-
brane fusion in diverse trafficking pathways. The Ara-
bidopsis SYP1 family of plasma membrane-localized
syntaxins comprises nine members, of which KNOLLE
and PEN1 play specific roles in cytokinesis and innate
immunity, respectively. To identify mechanisms confer-
ring specificity of action, we examined one member
of each subfamily – KNOLLE/SYP111, PEN1/SYP121 and
SYP132 – in regard to subcellular localization, dynamic
behavior and complementation of knolle and pen1
mutants when expressed from the same promoters.
Our results suggest that cytokinesis-specific syntaxin
requires high-level accumulation during cell-plate forma-
tion, which necessitates de novo synthesis rather than
endocytosis of pre-made protein from the plasma mem-
brane. In contrast, syntaxin in innate immunity does not
need upregulation of expression but instead requires
pathogen-induced and endocytosis-dependent retarget-
ing to the infection site. This feature of PEN1 is not
afforded by SYP132. Additionally, PEN1 could not substi-
tute for KNOLLE because of SNARE domain differences,
as revealed by protein chimeras. In contrast, SYP132 was
able to rescue knolle as did KNOLLE-SYP132 chimeras.
Unlike KNOLLE and PEN1, which appear to have evolved
to perform specialized functions, SYP132 stably localized
at the plasma membrane and thus might play a role in
constitutive membrane fusion.
Key words: Arabidopsis, cell cycle, cytokinesis, innate
immunity, plasma membrane, protein dynamics, recy-
cling, syntaxin
Received 20 April 2011, revised and accepted for
publication 1 June 2011, published online 28 June 2011
SNARE proteins constitute a family of membrane-
anchored proteins that play key roles in membrane fusion
events of intracellular trafficking pathways by forming
SNARE complexes that dock membranes to be fused.
Their main characteristic feature is an evolutionarily
conserved domain of 60–70 amino acids arranged
in heptad repeats, which has been designated the
SNARE domain (1). Based on the conserved amino-acid
residue at the center of the SNARE domain, SNARE
proteins have been classified into R- (arginine) and Q-
(glutamine) SNAREs. The Q-SNARE family is further
divided into four subfamilies (Qa-, Qb-, Qc- and Qb,c-
SNAREs) based on differences in the structure of the
SNARE domain (2). Each SNARE complex is formed by
association of four interacting SNARE domains, one each
from VAMP/R-SNARE on the donor membrane and three
from Q-SNAREs on the acceptor membrane: one from
syntaxin/Qa-SNARE and two from either SNAP25/Qb,c-
SNARE or one each from two t-SNARE light chains/Qb-
and Qc-SNAREs (1).
Syntaxins/Qa-SNAREsare conserved among alleukaryotic
organisms, a fact that emphasizes a universal role
for syntaxins in membrane trafficking (3). Compared
to other organisms like yeast and mammals, which
have two and four genes encoding plasma membrane-
localized syntaxins, respectively, plant genomes harbor an
increased number of genes for syntaxins involved in the
late secretory pathway (4–6). The Arabidopsis genome
encodes 18 putative syntaxins representing 5 different
Syntaxin of Plant (SYP) families, of which the 9 members
of the SYP1 family have been localized to the plasma
membrane (5–7). Although, in principle, redundancy
would explain the occurrence of the numerous SYP1
syntaxins in Arabidopsis, there is also evidence for
functional diversification.
SYP1 syntaxins show different spatio-temporal expres-
sion profiles (8). For instance, SYP132 is expressed
ubiquitously in all tissues throughout plant develop-
ment, whereas SYP124, SYP125 and SYP131 are only
expressed in pollen, and SYP123 appears to be exclu-
sively expressed in root hair cells during root develop-
ment (8). The SYP1 family also includes the functionally
well-characterized syntaxins KNOLLE/SYP111 (9,10) and
PEN1/SYP121/SYR1 (11–14). KNOLLE is a specialized
SYP1 syntaxin of flowering plants that seems to be exclu-
sively required for cytokinesis and has no ortholog in lower
plants or non-plant organisms, suggesting that other SYP1
syntaxins played a comparable role in plant cytokinesis
before KNOLLE evolved (6). KNOLLE gene expression
is confined to late G2 and M phases of the cell cycle,
which is mediated by mitosis-specific activator (MSA) pro-
moter elements that bind R1R2R3-Myb transcription fac-
tors (9,15). In addition, KNOLLE protein only accumulates
during mitosis, localizing to the forming cell plate that
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eventually separates the daughter cells, and is degraded
immediately after completion of cytokinesis (10,16). Like
KNOLLE, the plasma membrane-localized syntaxin PEN1
also arose late in plant evolution (6). PEN1 is involved
in non-host penetration resistance against the powdery
mildew Blumeria graminis f. sp. hordei (B. g. hordei)
and mediates vesicle fusion at B. g. hordei – Arabidopsis
interaction sites (12,13). PEN1/SYP121 also known as
SYR1 was originally identified in tobacco for its involve-
ment in potassium and chloride channel response to the
plant hormone ABA in guard cells (11). SYR1/SYP121 has
been shown to affect KAT1 potassium channel activity,
KAT1 trafficking to the plasma membrane, and directly
interacts with the channel subunit KC1 via an FxRF
motif (14,17,18). PEN1/SYP121 has also been subjected
to structure–function analysis. However, that study did
not address the problem of syntaxin specificity (19). A
third member of the SYP1 family, SYP132, is ubiquitously
expressed in Arabidopsis (5) and has been related to the
evolutionarily mostancient branchof SYP1 proteins (6).So
far no function for SYP132 has been reported in Arabidop-
sis. The tobacco ortholog NbSYP132 contributes to resis-
tance against bacterial pathogens, mediating secretion of
pathogenesis-related protein 1 (20), whereas Medicago
MtSYP132 has been localized to the plasma membrane
surrounding Rhizobium infection threads and to the sym-
biosome membrane (21).
We have previously analyzed mechanisms of KNOLLE
specificity in cytokinesis and identified the mitosis-specific
expression of KNOLLE as a major determinant (22). The
closest paralog of KNOLLE named SYP112 was func-
tionally equivalent to KNOLLE when expressed from the
KNOLLE promoter. In contrast, PEN1 was not able to
substitute for KNOLLE in cytokinesis when expressed
like KNOLLE. The results suggested functional diver-
gence of the two SYP1 proteins but did not offer any
mechanistic explanation. To analyze mechanisms that
ensure the specific biological activity of KNOLLE and
PEN1, we expressed these specialized SYP1 syntaxins
and SYP132 from the same set of promoters in sta-
bly transformed Arabidopsis plants. The transgenically
made proteins were analyzed for their ability to substi-
tute for KNOLLE or PEN1 in the respective mutant as
well as their subcellular localization and dynamics. We
also tested chimeric proteins that were generated by
exchanging the SNARE domain between KNOLLE and
PEN1 or SYP132. We show that KNOLLE is completely
functional during cytokinesis when carrying the SNARE
motif of SYP132 but fails to fulfill proper cell-plate for-
mation when carrying the SNARE motif of PEN1. Our
results suggest that functional specificity of the spe-
cialized SYP1 syntaxins KNOLLE and PEN1 diverged
from the presumably ancient SYP132. KNOLLE function
appears to require high-level expression during the mitosis
immediately preceding cytokinesis, whereas PEN1 func-
tion displays striking subcellular protein dynamics that
seems to enable rapid retargeting to infection sites via
endocytosis.
Results
Unlike KNOLLE, PEN1 and SYP132 syntaxins
are stable proteins
In dividing cells of the Arabidopsis seedling root, KNOLLE
accumulates at the trans-Golgi network (TGN) in early
mitosis, localizes to the cell plate during cytokinesis and
takes the degradation route via multivesicular bodies
(MVBs) to the lytic vacuole shortly after completion of
the newly made plasma membrane (10,16,23). To analyze
the subcellular localization and protein behavior of PEN1
and SYP132 in comparison to KNOLLE, we generated
transgenic lines stably expressing Myc-tagged PEN1
(Myc-PEN1) or SYP132 (Myc-SYP132) under control of
the KNOLLE cis-regulatory sequences (22). Colocalization
analyses indicated that both Myc-PEN1 and Myc-SYP132
accumulated at KNOLLE-positive compartments: at the
TGN in early mitosis and at the cell plate during
cytokinesis, although the SYP132 signal was weaker
than the PEN1 signal at the cell plate (Figure 1A–F;
22). After formation of the cell plate, neither Myc-PEN1
nor Myc-SYP132 colocalized at KNOLLE-labeled MVBs
(Figure 1G–L). Both Myc-PEN1 and Myc-SYP132 proteins
were more stable than endogenous KNOLLE as well as
transgenically made Myc-KNOLLE and still detectable in
interphase cells, when the KNOLLE promoter is not active
(Figure 1N,O;comparewithFigure 1M).Intriguingly,PEN1
accumulated much more strongly at the cell plate than
at the plasma membrane during cytokinesis, whereas
SYP132 accumulated evenly at both the cell plate and
the plasma membrane (Figure S1). Thus, both PEN1 and
SYP132 localize at the division plane in dividing cells and
at the plasma membrane in interphase, indicating that
they do not take the KNOLLE degradation pathway after
cell-plate formation.
SYP132 but not PEN1 can substitute for KNOLLE
function
Previously we have shown that PEN1 cannot substitute
for KNOLLE function; however, the reason for this
has not been clarified so far (22). To characterize this
inability in more detail we performed a comparative
analysis of the knolle complementation competence
of PEN1, SYP132 and KNOLLE when expressed from
the KNOLLE promoter. Progeny from five KN:Myc-
KNOLLE, nine KN:Myc-PEN1 and five KN:Myc-SYP132
transgenic lines that were also knolle heterozygous
were phenotypically analyzed. All five KN:Myc-KNOLLE
transgenic lines completely rescued the knolle mutant
phenotype. Two out of five KN:Myc-SYP132 transgenic
lines rescued the knolle mutant phenotype completely,
which was not the case for any of the nine KN:Myc-
PEN1 transgenic lines. Examples of high-level and low-
level expression lines for each transgene are shown in
Figure 2 and Table S1. We observed phenotypic variation
between different transgenic lines ranging from severe
knolle seedlings(norescue)tonormalseedlings(complete
rescue) (Figure 2A/1). Partially rescued seedlings initially
developed like wild type but were later arrested in
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Arabidopsis SYP1 Syntaxin Specificity
ABC
FED
GHI
LJ
MN
K
O
Figure 1: Subcellular localization of SYP1 syntaxins in
seedling root-tip cells. A–F) MYC-PEN1 (red; A–C) and MYC-
SYP132(red;D–F)colocalizewithKNOLLE(green)atthecellplate
(asterisks in B, E) in dividing cells. Blue, DAPI. G–L) MYC-PEN1
(G–I) and MYC-SYP132 (J–L) colocalize with KNOLLE (green) at
the TGN in early mitotic cells but not at MVBs (arrowheads in I,
L). M–O) KNOLLE (M) only labels mitotic cells, whereas MYC-
PEN1 (N) and MYC-SYP132 (O) are more stable and also label the
plasma membrane in interphase cells. Scale bars, 5 μm.
growth and eventually died (Figure 2A/2,3). To address
why different Myc-PEN1 and Myc-SYP132 transgenic
lines rescued knolle mutants to different degrees, we
analyzed their expression levels (Figure 2B,C). Whereas
PEN1 transgenic lines only rescued knolle partially at both
lowandhigh levels ofexpression,SYP132 transgeniclines
rescued knolle partially or completely, depending on the
level of expression (Figure 2A,B). Surprisingly, the protein
levels of the low expression lines were still much higher
than the Myc-KNOLLE protein level required for rescuing
the knolle mutant completely (Figure 2B). As PEN1 and
SYP132 are more stable than KNOLLE, the protein
level we detected by immuno-blotting analysis does not
represent their expression levels in mitotic cells. KNOLLE
mRNA and protein are only present in dividing cells (9,10)
and thus, the transcript level of each SYP1 transgene
should represent the respective de novo synthesized
protein amount in dividing cells. Therefore, we compared
levels of transcript accumulation between the different
SYP1 transgenes. Intriguingly, although all Myc-KNOLLE
transgenic lines rescued the knolle mutant, some lines
displayed a lower level of transcript accumulation than did
Myc-PEN1 or Myc-SYP132 transgenic lines (Figure 2C).
Thus, the rescue ability appeared not to depend on mere
expression quantity but rather might reflect a qualitative
difference between the SYP1 syntaxins in regard to their
ability to promote cytokinesis.
SYP132 cannot replace PEN1 in pathogen
penetration resistance
PEN1 was shown to play a role in non-host resis-
tance to fungal pathogens by contributing to localized
cell wall deposition (formation of papillae), which pre-
vents barley powdery mildew Blumeria graminis f. sp.
hordei (B. g. hordei) spores from invading Arabidopsis
leaves (12). Although pen1 mutants display no obvious
phenotype in the absence of pathogen attack, papilla
formation upon attempted fungal ingress is delayed
and penetration resistance significantly reduced (12,24).
To analyze whether SYP1 syntaxins are able to sub-
stitute for PEN1 in penetration resistance, transgenic
plant lines constitutively expressing SYP1 syntaxins from
the UBIQUITIN 10 (UBQ10) promoter (25) were gener-
ated and these plants were crossed with pen1-1 mutant
plants (12). pen1-1 mutant plants expressing UBQ10:RFP-
PEN1 or UBQ10:RFP-SYP132 were inoculated with
powdery mildew B. g. hordei. B. g. hordei–Arabidopsis
interaction sites were visualized by established coomassie
blueandcallosestainingprotocolsthatallowquantification
of fungal invasion rates (Figure 3C; 12). Only about 20% of
the fungal penetration attempts were successful in wild-
type control plants, whereas the success rate increased
to about 90% in the pen1-1 mutant (Figure 3C). This was
also reflected in significantly different levels of fluores-
centepidermalcellsshowinghypersensitive-likecelldeath
response (Figure 3D,E) because of activation of post-
invasion defense mechanisms (26). UBQ10:RFP-PEN1
fully restored wild-type invasion resistance, whereas
UBQ10:RFP-SYP132 complemented pen1-1 only partially,
allowing an intermediate 60% of fungal sporelings to
invade epidermal leaf cells (Figure 3C). We did not
recover any UBQ10:RFP-KNOLLE transgenic plants that
expressed KNOLLE at detectable levels. To analyze how
thisfunctionaldifferencebetween thetwoSYP1 syntaxins
PEN1 and SYP132 in pathogen resistance comes about,
we investigated their behavior at the subcellular level.
Quantitative live-cell imaging revealed that RFP-PEN1
expressed from the UBQ10 promoter strongly accu-
mulated at the Arabidopsis–B. g. hordei interaction
sites 16 h after inoculation (Figure 4A–C). At sites of
attempted fungal penetration, RFP-PEN1 signal inten-
sity was about 4.5 times higher than at other plasma
membrane regions of the same cell (Figures 4C and
S2), indicative of the recently described active translo-
cation to and accumulation in a pathogen-induced plasma-
membrane microdomain (24,27). In contrast, RFP-SYP132
expressed from the UBQ10 promoter showed only about
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A
B
anti-MYC
anti-KN
C
KN::MYC-KN #7
Col-0
KN::MYC-PEN1 #6
KN::MYC-PEN1 #9
1
23
*
*
*
*
35 kDa
35 kDa
KN::MYC-KN #1
KN::MYC-KN #2
KN::MYC-KN #4
KN::MYC-KN #6
genomic DNA control
H2Ocontrol
KN::MYC-KN #1
Col-0 cDNA
Myc-SYP1
Actin2
KN::MYC-SYP132 #8
KN::MYC-SYP132 #5
Rubisco
65 kDa
*
ab
KN::MYC-KN #2
KN::MYC-KN #4
KN::MYC-KN #6
KN::MYC-KN #7
KN::MYC-PEN1 #6
KN::MYC-PEN1 #9
KN::MYC-SYP132 #8
KN::MYC-SYP132 #5
kn
*
cd
Figure 2: knolle rescue ability of transgenic PEN1 and SYP132. A) Improved morphology (asterisks) of partially rescued transgenic
seedlings (1) which, however, die on soil as growth-retarded plants (2,3). Partially rescued transgenic seedlings from each line analyzed
were genotyped by PCR after transfer to soil. Lines analyzed: (1a) KN::MYC-PEN1 #6, (1b) KN::MYC-PEN1 #9, (1c) KN::MYC-SYP132
#8 and (1d) KN::MYC-SYP132 #5. Arrowheads indicate wild-type seedlings; kn, knolle. Scale bars, 2 mm. B) Transgenic proteins from
seedling extracts of the same transgenic lines detected with anti-Myc monoclonal antibody and anti-KNOLLE antiserum. Rubisco loading
control stained with Ponceau S. C) Analysis of transcript levels (MYC-SYP1) from the same transgenic SYP1 lines as in (B). Actin2,
loading control.
1.4timeshighersignalintensityatplant–pathogeninterac-
tion sites than elsewhere at the same plasma membrane
(Figures 4D–F and S2). In summary, the poor ability of
UBQ10:RFP-SYP132 to prevent pathogen entry correlated
well with its comparatively inefficient recruitment to fun-
gal invasion sites rather than with any conceivable activity
differences to PEN1 at the penetration site.
PEN1 but not SYP132 constitutively cycles between
the plasma membrane and endosomes
To address why PEN1 and SYP132 differed in their ability
to substitute for knocked-out SYP1 family members,
we analyzed their subcellular localization and dynamics
in seedling root cells. Both Myc-PEN1 and Myc-
SYP132 expressed from the KNOLLE promoter localize
at the plasma membrane
Additionally, Myc-PEN1 stained some endomembrane
compartments in interphase cells, unlike SYP132 (Figure
S3). To identify these compartments we did double-
labeling experiments with specific subcellular markers
(Figure 5A–H). Myc-PEN1-positive puncta were distinct
from the Golgi stacks that were labeled by the γCOP
subunit of the coat protein I (COPI) complex mediating
retrograde transport from the cis-Golgi to the endoplasmic
reticulum (ER) (Figure 5A; 28). Additionally, there was
also no colocalization with the trans-Golgi labeled by
andatthe cell plate.
the yellow fluorescent protein (YFP)-tagged rat sialyl
transferase, N-ST-YFP (Figure 5E; 29). In contrast, the
vesicle formation-initiating GTPase ARF1 that localizes to
the Golgi/TGN/early endosome (16,23) labeled some Myc-
PEN1-positive punctate structures (Figure 5B–D). ARF1
was localized mainly to the TGN and also to the Golgi
stacks by immunogold labeling (23). Thus, we would
expect some ARF1-positive compartments not to be
labeled by endocytosed RFP-PEN1. In addition, live-cell
imaging revealed complete colocalization of GFP-PEN1-
positive puncta with the endocytic tracer FM4-64 within
10 minofincubation(Figure 5F–H;12).Thus,ininterphase
cells, PEN1 localizes to the plasma membrane and to the
TGN, which functionally corresponds to early endosomes
in plants (30,31).
The fungal toxin brefeldin A (BFA) reversibly inhibits
vesicle trafficking by blocking the activity of sensitive ARF
guanine-nucleotide exchange factors (ARF-GEFs) (32,33).
BFA treatment of Arabidopsis seedling roots traps
cycling plasma-membrane proteins in endosomal BFA
compartments by inhibiting ARF-GEFs required for
recycling (31,32). In contrast, secretory traffic from the
ER to the plasma membrane is not inhibited by BFA
in Arabidopsis so that newly synthesized proteins are
not trapped in BFA compartments (16,31,34). When
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Arabidopsis SYP1 Syntaxin Specificity
35 kDa
35 kDa
anti-KNOLLE
35 kDa
50 kDa
anti-Myc anti-Myc
anti-Tubulin
Protein extract from leaves
Protein extract from flowers
AB
C
KN::MYC-KNOLLE
Col-0
KN::MYC-PEN1
KN::MYC-SYP132
KN::MYC-KNOLLE
Col-0
KN::MYC-PEN1
KN::MYC-SYP132
Col-0
pen1
KN::Myc-PEN1 in pen1 KN::Myc-SYP132 in pen1

KN::Myc-SYP132 in Col-0
DE
FGH
Col-0
pen1
UBQ10::RFP-PEN1
in pen1
UBQ10::RFP-SYP132
in pen1
KN::MYC-SYP132
in pen1
KN::MYC-PEN1
in pen1
KN::MYC-SYP132
in Col-0
**
100
90
80
70
60
50
40
30
20
10
0
(%)
n
o i t a r t e
n
e
p f o y
c
n
e
u
q
e r f
Figure 3: Incomplete rescue of pen1 mutants by transgenic
SYP132. A and B) SYP1 syntaxins expressed from the KNOLLE
promoter in flowers (A) and leaves (B). Note the absence of
Myc-tagged KNOLLE in leaf extracts. Vertical bars indicate
junction sites in the same gel edited with imaging software.
In brief, bands of no interest for the experiment were cut
out and the band corresponding to myc-SYP132 transgenic
protein moved to the right of the junction site. C) Frequency
of cell death reflecting successful penetration events at B.
g. hordei–Arabidopsis interaction sites in leaves of transgenic
plants. n = 6; error bars indicate standard deviation (∗∗p < 0.01,
t-test). D–H) Aniline-blue staining of B. g. hordei–Arabidopsis
interaction sites in infected leaves. Leaf cells respond with papilla
formation in wild type (D), in KN:MYC-PEN1 transgenic pen1
lines (F) and in KN:MYC-SYP132 transgenic lines (H). B. g. hordei
successfully penetrated leaf cells, leading to cell wall deposition,
in pen1 (E) and in KN:MYC-SYP132 transgenic pen1 lines (G).
Col-0, wild-type control. Scale bars, 100 μm.
KN:Myc-SYP1 transgenic root tips were treated with BFA,
all three transgenically made SYP1 proteins accumulated
inlargeBFAcompartmentsinmitoticcells(FigureS4A–C).
UBQ10::RFP-PEN1 UBQ10::RFP-SYP132
PM
site
UBQ10::RFP-PEN1 UBQ10::RFP-SYP132
AD
BE
FC
PM
Figure 4: Live imaging of RFP-SYP1 proteins in infected leaf
cells. A and B) UBQ10:RFP-PEN1 and (D, E) UBQ10:RFP-
SYP132 label the plasma membrane (PM) and the B. g.
hordei–Arabidopsis interaction sites (PS); (A and D) fluorescence,
(B and E) fluorescence superimposed on bright-field images.
Fungal appressoria (FA) and scan lines (between arrowheads)
are outlined in (A, D). C and F) Quantitative scans of RFP-SYP1
protein accumulation from the plasma membrane (PM) across
the cell to the penetration site (PS; arrowheads in A, D). Scale
bars, 5 μm.
In non-dividing cells, however, Myc-PEN1 localized to BFA
compartments, whereas Myc-SYP132 still labeled the
plasma membrane (Figure S4B,C) and KNOLLE protein
was not detected because of its specific degradation
shortly after completion of cytokinesis (Figure S4A).
Because the transgenically made SYP1 proteins were
expressed from the KNOLLE promoter, and therefore
not synthesized during interphase, only labeled SYP1
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J
N-ST-YFP
GNL1-BFAsens-YFP
*
I
KN::MYC-PEN1
gnl1
*
M
gnl1
gnl1
+BFA
gnl1
+BFA
K
KN::MYC-PEN1
KN::MYC-SYP132
QS
TV
L
NOP
R
U
KN::MYC-SYP132
H4::RFP-PEN1
KNOLLE
H4::RFP-SYP132
KNOLLE
A
E
B
KN::MYC-PEN1
ARF1
35S::GFP-PEN1
FM4-64
D
H
C
FG
KN::MYC-PEN1
γCOP
KN::MYC-PEN1
N-ST-YFP
Figure 5: PEN1 but not SYP132 cycles constitutively in
seedling root cells. A–E) MYC-PEN1 (red) does not colocalize
with the Golgi-markers (green) γCOP (A) or N-ST-YFP (E) but
colocalizes with the TGN/endosomal marker ARF1 (green, B–D).
F–H) Live imaging: GFP-PEN1 completely colocalizes with FM4-
64 after 10 min of treatment. I–P) Immunostaining of gnl1 mutant
without (I, M) or with 25 μM BFA treatment for 1 h (J–L, N–P).
Blue, DAPI. I, M) MYC-PEN1 (I) and MYC-SYP132 (M) localize
at the cell plate in gnl1 mutant cells (asterisks). J–L) After
BFA treatment, MYC-PEN1 (red) accumulates at the ER (open
arrowhead, K), as does N-ST-YFP, and also in endosomal BFA
compartments (closed arrowhead, K). N–P) After BFA treatment,
MYC-SYP132 (red) only accumulates at the ER (open arrowhead,
O) as does BFA-sensitive GNL1-YFP. Q–V) Immuno-localization
of RFP-PEN1 (Q–S) and RFP-SYP132 (T–V) expressed from the
HISTONE 4 (H4) promoter. Q–S) RFP-PEN1 (red) colocalizes with
KNOLLE (green) at the cell plate, although it is not expressed in
dividing cells. T–V) RFP-SYP132 (red) does not colocalize with
KNOLLE (green) at the cell plate in dividing cells but labels the
plasma membrane. Blue, DAPI. Scale bars, 5 μm.
protein that was endocytosed from the plasma membrane
accumulatedinBFAcompartments.These resultsindicate
that PEN1, but not SYP132, cycles constitutively between
the plasma membrane and endosomal compartment(s) in
interphase cells.
In mitotic cells, both PEN1 and SYP132 could be detected
in BFA compartments. To determine whether PEN1 and
SYP132 are newly synthesized or endocytosed before
accumulating in BFA compartments during mitosis, we
blocked the secretory pathway by BFA treatment of gnl1
mutant seedlings expressing KN:Myc-PEN1 and KN:Myc-
SYP132 transgenes. GNL1 is a BFA-resistant ARF-GEF
that mediates retrograde transport from the Golgi stacks
totheER (34,35).Treatinggnl1mutantseedlingswithBFA
leads to the inhibition of ER–Golgi traffic, as shown by
the ER accumulation of KNOLLE and the Golgi marker
N-ST-YFP as well as the fusion of Golgi stacks with
the ER (16,35). Without BFA treatment, both Myc-PEN1
and Myc-SYP132 accumulated at the cell plate in mitotic
cells of gnl1 mutant seedlings (Figure 5I,M). After BFA
treatment, however, the newly synthesized Myc-SYP1
syntaxins were trapped in the ER and, thus, did not
reach the plane of cell division (Figure 5J–L,N–P, open
arrowheads). Intriguingly, Myc-PEN1 also accumulated in
BFA compartments, whereas Myc-SYP132 did not but
rather labeled the plasma membrane (Figure 5K; closed
arrowhead, compare with Figure 5O). This indicates that
PEN1 is both secreted and endocytosed during mitosis,
whereas SYP132 is not endocytosed and only the de novo
synthesized protein is transported through the secretory
pathway to the cell plate. It should be noted that genomic
GFP fusions of SYP132 also label the cell plate in dividing
cells, in addition to labeling the plasma membrane in
all cells (8). Considering our results, this observation
suggests that the endogenous promoter of SYP132 is
also active during M phase such that newly synthesized
SYP132 is targeted to the cell plate.
Although BFA is commonly believed to be a highly specific
inhibitor of membrane trafficking with known molecular
targets,there is always a remote possibility of non-specific
side effects (36). To examine in a different way whether
SYP132 is indeed not endocytosed during cytokinesis,
we expressed SYP132 exclusively during interphase and
investigated its ability to reach the cell plate. PEN1 was
used as a control and both SYP1 proteins were expressed
from the cis-regulatory elements of the HISTONE 4 (H4)
gene because H4 mRNA appears in S-phase and is
completely degraded before the onset of mitosis (37,38).
Both RFP-PEN1 and RFP-SYP132 were present at the
plasma membrane in interphase cells as they were
when expressed from the KNOLLE promoter (Figure
S4D–F,H–J, compare with Fig. 1H,I). Additionally, RFP-
PEN1 but not RFP-SYP132 colocalized with ARF1-labeled
endosomes and accumulated in BFA compartments upon
BFA treatment (Figure S4G,K). In mitotic cells, we
observed RFP-PEN1 fluorescence at the division plane,
colocalizing with KNOLLE at the cell plate (Figure 5Q–S).
In contrast, RFP-SYP132 did not label the developing cell
plate at all, indicating that the protein is not internalized
during mitosis (Figure 5T–V). Taken together, our results
demonstrate that SYP1 syntaxins behave differently
at the subcellular level: PEN1 cycles constitutively
betweentheplasmamembraneandendosomes,whereas
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SYP132, once delivered, remains statically at the plasma
membrane. This difference in localization dynamics might
also explain two observations reported above. First, PEN1
but not SYP132 accumulates more strongly at the cell
plate than at the plasma membrane in dividing cells
(Figure S1). In the case of cycling PEN1, both de novo
synthesized and endocytosed proteins are targeted to the
cell plate, whereas in the case of SYP132, only de novo
synthesized protein accumulates at the cell plate. Second,
SYP132 when expressed from the UBQ10 promoter
was not able to completely rescue the pen1 mutant
during pathogen attack because only newly synthesized
protein was targeted to the infection site. To perform
a more rigorous test of this idea, we crossed KN:Myc-
SYP1 transgenic plants with pen1-1 mutant plants and
analyzed their homozygous pen1-1 mutant progeny that
also expressed the transgene. In accordance with the
exclusive activity of the KNOLLE promoter in proliferating
tissues (10), all transgenically made SYP1 proteins were
detected in flowers (Figure 3A). However, Myc-PEN1 and
Myc-SYP132 also accumulated in leaves, again indicating
that these proteins were more stable than KNOLLE
(Figure 3B). We tested their ability to restrict pathogen
entry in pen1-1 mutants (Figure 3C–H; 12). Myc-PEN1
completely rescued pen1-1, reducing fungal entry rates to
the wild-type level (Figure 3C,F). In contrast, Myc-SYP132
entirely failed to rescue pen1-1 (Figure 3C,G). In addition,
Myc-SYP132 expressed in the wild-type background
did not interfere with endogenous PEN1 function
(Figure 3C,H). Thus, when expressed from the KNOLLE
promoter, SYP132 cannot take over PEN1 function in
pathogenresistance.Thus,theabilityofsyntaxintorescue
pen1 requires dynamic localization behavior. However,
this cannot explain the ability of SYP132 to rescue knolle
in cytokinesis. As SYP132 but not PEN1 can substitute
for KNOLLE function (Figure 2), differences in amino-acid
sequence rather than protein abundance at the cell plate
account for their different rescuing ability.
Significance of the SNARE domain for KNOLLE
function
Although PEN1 localized at the cell plate, it did not
substitute for KNOLLE during cell-plate formation. A
possible reason for its failure might be an insufficient
interaction with other SNARE partners of the cytokinesis
SNARE complex. The SNARE domain was identified as
the main determinant of specificity between interacting
SNARE proteins (39–43). Although the SNARE domain
of SYP1 syntaxins is very conserved by amino-acid
sequence, we cannot rule out that subtle differences
account for the efficiency of interaction. To test if the
SNARE domain contributes to functional specificity of
SYP1 syntaxins, we generated chimeric proteins by
swapping the SNARE domains between KNOLLE and
PEN1 or SYP132, and expressed these chimeric proteins
from the KNOLLE promoter (Figure 6A). Both KNOLLE
carrying the SNARE domain of PEN1 (KNOLLE-PEN1SND)
and PEN1 carrying the SNARE domain of KNOLLE (PEN1-
KNOLLESND) localized at the cell plate and the plasma
membrane in dividing cells (Figure 6B,C). In interphase
cells both chimeric proteins were detected at the plasma
membrane, indicating that they were stable and not
degraded after cytokinesis. The same was observed
for the KNOLLE-SYP132 chimeras as both ‘KNOLLE-
SYP132SND’ and ‘SYP132-KNOLLESND’ localized at the
cell plate as well as the plasma membrane in dividing cells
and at the plasma membrane in non-dividing cells, further
demonstrating high protein stability (Figure 6D,E). Thus,
replacingtheSNAREdomainofKNOLLEwiththatofPEN1
or SYP132 leads to increased protein stability, suggesting
that the SNARE domain of KNOLLE in conjunction with
the remainder of the KNOLLE protein promotes its rapid
turnover. All chimeric constructs were tested for their
abilitytorescuetheknollemutant.Remarkably,allproteins
harboring the SNARE domain of KNOLLE were able to
completely rescue the knolle mutant phenotype (Table
S1). In addition, chimeric KNOLLE protein harboring the
SNARE domain of SYP132 was also able to rescue the
knolle mutant phenotype, consistent with the rescue
ability of SYP132 (Table S1). On the contrary, chimeric
KNOLLE protein carrying the SNARE domain of PEN1
did not rescue the knolle phenotype, and thus behaved
like the wild-type PEN1 protein (Table S1). These results
suggest that the SNARE domain contributes to syntaxin
function in cytokinesis.
knolle mutant rescue by PEN1-KNSNDis limited
to expression during mitosis
Replacing the SNARE domain of PEN1 with that of
KNOLLE rendered PEN1 competent to rescue the knolle
mutant when expressed during mitosis. Considering that
PEN1 is endocytosed and reaches the plane of cell
division during cytokinesis, we addressed the possibility
that chimeric PEN1-KNOLLESND protein can rescue the
knolle mutant if expressed before mitosis. To this end
we generated plants expressing RFP-PEN1-KNOLLESNDor
the reciprocal construct RFP-KNOLLE-PEN1SNDunder the
control of the S-phase-specific HISTONE 4 promoter. The
chimeric proteins accumulated at the plasma membrane
in non-dividing cells and at the cell plate during mitosis
(Figure 6F,G). H4:RFP-KNOLLE-PEN1SNDwas not able to
rescue the knolle mutant phenotype as might have been
expected because KN:RFP-KNOLLE-PEN1SND also did
not rescue knolle. Surprisingly, H4:RFP-PEN1-KNOLLESND
also did not rescue the knolle mutant phenotype
in contrast to KN:RFP-PEN1-KNOLLESND (Table S1),
although expression levels of the RFP-PEN1-KNOLLESND
protein were comparable for either promoter (Figure S5).
These results clearly demonstrate that KNOLLE function
depends on (i) high-level de novo protein synthesis during
mitosis and (ii) its specific SNARE domain.
Discussion
Our
redundancy among members of the SYP1 syntaxin
family, focusing on one representative from each of
study addressed functionaldivergenceversus
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KN::KNOLLE-PEN1SND
KN::PEN1-KNOLLESND
KN::KNOLLE-SYP132SND
KN::SYP132-KNOLLESND
H4::KNOLLE-PEN1SND
H4::PEN1-KNOLLESND
A
SYP111/KNOLLE
SYP121/PEN1
N-terminusHa HbHc SNARE domainTM
KNOLLE-PEN1SND
PEN1-KNOLLESND
KNOLLE-SYP132SND
SYP132-KNOLLESND
SYP132
B
C
D
E
F
G
Figure 6: Subcellular localization of chimeric SYP1 proteins. A) Diagram of chimeric proteins of KNOLLE (blue), PEN1 (red) and
SYP132 (green). B–I) Live imaging of RFP-tagged syntaxins with swapped SNARE-domains. B–E) When expressed from the KNOLLE
promoter, KNOLLE-PEN1SND(B), PEN1-KNOLLESND(C), KNOLLE-SYP132SND(D) and SYP132-KNOLLESND(E) localize at the cell plate
(asterisks) in dividing cells and show high protein stability. F–G) When expressed from the HISTONE 4 promoter, KNOLLE-PEN1SND
(F) and PEN1-KNOLLESND(G) localize at the cell plate (asterisks) in dividing cells and show high protein stability. Scale bars, 5 μm.
the three subgroups, KNOLLE/SYP111, PEN1/SYP121
and SYP132. KNOLLE and PEN1 have their specific
biological roles in cytokinesis and non-host pathogen
defense, respectively (9,12). To identify mechanisms
defining specificity of protein function, we expressed
SYP1 proteins from a specific set of promoters, which
eliminated differences in gene expression conferred by
the endogenous promoters (8), and we swapped protein
domains.
KNOLLE plays a unique role in somatic cytokinesis, which
cannot easily be substituted for by other syntaxins (22). Its
closest paralog SYP112 is essentially functionally equiva-
lent, including rapid degradation at the end of cytokinesis,
but lacks the strong expression of KNOLLE during mito-
sis preceding cytokinesis (22). The same study revealed
the inability of PEN1 to substitute for KNOLLE when
expressed from the KNOLLE cis-regulatorysequences but
did not identify a plausible molecular mechanism for this
failure. As PEN1 when expressed like KNOLLE accumu-
lated at the cell plate there seemed to be some functional
difference between the two proteins, although the lev-
els of protein accumulation had not been compared. Our
present study revealed that PEN1 was indeed expressed
during M phase at least as strongly as KNOLLE but failed
to rescue the knolle mutant. By contrast, SYP132 when
expressed like KNOLLE rescued the knolle mutant com-
pletely, indicating a clear functional difference between
PEN1 on one hand and KNOLLE and SYP132 on the other.
SNARE domain swaps between KNOLLE and PEN1 or
SYP132 yielded chimeric proteins of which PEN1 protein
with the SNARE domain of KNOLLE was able to rescue
the knolle mutant completely. Thus, the SNARE domain
appears to be a critical determinant of KNOLLE protein
functional specificity. This is a surprising result, consider-
ing the earlier observation that both KNOLLE and PEN1
interact with the same Qb,c-SNARE SNAP33 (13,44).
Although the same interacting Qb,c-SNARE is involved
in cytokinesis and in non-host pathogen defense, the
R-SNARE might be different between the two SNARE
complexes. Alternatively, the rate of assembly or disas-
sembly of the two SNARE complexes might be different.
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KNOLLE expression outside mitosis appears to be
detrimental, although 35S promoter-driven expression
resulted in KNOLLE accumulation near the apical plasma
membrane in growing root hairs (45). However, using the
S-phase-specificH4 promoter,KNOLLEaccumulationwas
severely impaired, whereas both PEN1 and SYP132 were
expressed and accumulated at the plasma membrane. As
PEN1-KNOLLESNDprotein was able to rescue the knolle
mutant completely when expressed from the KNOLLE
promoter, i.e. during M phase immediately preceding
cytokinesis, and this chimeric protein was stable and not
deleterious, we expressed the same protein from the S-
phase-specific H4 promoter. Surprisingly, early expression
of the PEN1-KNOLLESNDfollowed by its transient storage
at the plasma membrane and subsequent endocytosis
during cytokinesis enabled its accumulation in the plane
of cell division but did not rescue the knolle mutant,
although the protein level was comparable to that of the
same protein made from the KNOLLE promoter. This
strongly suggests that the cytokinesis-specific syntaxin
needs to be synthesized immediately before cell-plate
formation, whereas the same protein when endocytosed
is ineffective. This is in line with the observation that
BFA-induced inhibition of ER–Golgi traffic in gnl1 mutant
seedlings impairs cytokinesis (16). There is no obvious
reason for this difference in efficacy between newly
made and endocytosed syntaxin in cytokinesis, especially
because the TGN acts as a sorting station that directs
both secretory and endocytosed proteins to the plane of
cell division in cytokinesis (31).
The comparative analysis of PEN1 and SYP132 revealed
an important difference in the dynamic behavior of the
two proteins. Whereas PEN1 cycled continually between
the plasma membrane and endosomal compartments,
SYP132 appeared to associate stably with the plasma
membrane. This was observed in the root cells in
which ER–Golgi traffic was inhibited by BFA treatment
of gnl1 mutant seedlings: when expressed from the
KNOLLE promoter, PEN1 was trapped in the ER but still
accumulated in endosomal BFA compartments, whereas
SYP132 was only detected in the ER and at the plasma
membrane. The same difference was observed when the
two proteins were expressed from the S-phase-specific
H4 promoter: only PEN1 accumulated at the plane of
division (cell plate) during cytokinesis, whereas SYP132
stayed at the plasma membrane. Thus, PEN1 appears to
be a highly dynamic protein, whereas SYP132 once made
appears to be firmly anchored at the plasma membrane.
PEN1 plays an important role in Arabidopsis non-host
resistancetofungalpathogens (12,24).Endogenous PEN1
appears to be moderately up-regulated in response to
pathogen attack and accumulates rapidly at the site of
infection. We used two different promoters to analyze
the relevance of syntaxin retargeting for mounting a
successful defense against non-adapted powdery mildew
fungi. The UBQ10 promoter is constitutively active
and thus provides, during pathogen attack, both newly
synthesized syntaxin and syntaxin made earlier and then
stored at the plasma membrane. In contrast, the KNOLLE
promoter is only active in proliferating cells but not in
mature leaf cells. Thus, in the latter case, only syntaxin
made earlier and then stored at the plasma membrane is
available during pathogen attack. Our data suggest that
PEN1 is highly dynamic such that endocytic retargeting
of plasma membrane-localized syntaxin is sufficient and
no newly made syntaxin is necessary for mounting a
successful defense during pathogen attack. In contrast,
SYP132 only partially rescued the compromised pathogen
defense of pen1 leaves when expressed from the UBQ10
promoter but had no effect when expressed from the
KNOLLE promoter.Thus,evennewlysynthesizedSYP132
might not be efficiently targeted to the site of infection.
One possible explanation for this difference in subcellular
dynamics between PEN1 and SYP132 might be that the
strong accumulation of syntaxin at the infection site does
not result from directional secretion but rather requires
endosomal retargeting. Similar observations were made
in polar targeting of PIN proteins in Arabidopsis (46,47).
Compared to the two specialized SYP1 syntaxins KNOLLE
and PEN1, SYP132 might represent a rather general syn-
taxin function at the plasma membrane, possibly involved
in constitutive fusion of secretory vesicles. No knockout
mutantsof SYP132 areknown. However, SYP132 appears
to be broadly if not ubiquitously expressed during develop-
ment (8). Furthermore, sequence comparisons with SYP1
syntaxins from lower plants suggest that SYP132 might
play an ancient role in secretory traffic to the plasma
membrane (6).
In an evolutionary scenario that associates SYP132 with
plasma-membrane syntaxins of primitive land plants,
KNOLLE and PEN1 appear to have evolved divergently to
serve their respective highly specialized function. KNOLLE
has adopted an exclusive role in membrane fusion during
cytokinesis, which for yet unknown reasons requires
high-level expression immediately before cytokinesis.
In addition to, and possibly as a consequence of, the
dramatic change in gene regulation leading to high-level
protein accumulation, KNOLLE protein has become highly
unstable, being targeted to the vacuole for degradation
at the end of cytokinesis. Interestingly, during KNOLLE
evolution, there seems to have been no substantial
functional change from the presumably ancient SNARE
domain of SYP132, unlike the SNARE domain of PEN1.
In contrast, PEN1 displays dramatic subcellular protein
dynamics, as evidenced by its continual cycling in non-
infected cells and the retargeting to the plane of cell
division during cytokinesis. This dynamic behavior also
enables PEN1 to act in plant innate immunity, facilitating
its rapid relocation from the plasma membrane to
fungal infection sites via endocytosis and retargeting. It
remains to be determined how the attacked plant cell
reorganizes its membrane trafficking to fend off fungal
intruders.
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Methods
Plant growth, transformation and selection
Arabidopsis thaliana plants were grown on half-strength Murashige and
Skoog (MS) medium (+1% sucrose for microscopy) or on soil at 18–23◦C,
with cyclesof16 h lightand8 h dark.Landsberg/Niederzenz(Ler/Nd)plants
heterozygous for the knolle mutation X37-2 (9) or Columbia (Col) plants
homozygous for the pen1-1 mutation (12) were transformed with Agrobac-
terium tumefaciens, using the floral-dip method (48). T1 plants from
bulk-harvested seeds were selected for transformants either grown on soil
bysprayingtwicewitha1:1000dilutionofBasta®(45)(183 g/LGlufosinate-
ammonium,Bayer)orgrownonhalf-strengthMSmediumcontaining50 μM
kanamycin. BASTA-resistant plants were genotyped for knolle X37-2 (22)
and kanamycin-resistant plants for pen1-1 (12) as described.
Molecular biology
Transgenic constructs for subcellular localization and for knolle rescue
were expressed from the mitosis-specific KNOLLE promoter. KN and
PEN1 coding sequences were cloned into the KNOLLE cassette as
described (22). SYP132 CDS was amplified from a flower and silique
cDNA library of the Landsberg ecotype (49) by polymerase chain reaction
(PCR) according to standard procedures using Taq DNA Polymerase (Peq
Lab Biotechnologie GmbH). SYP132 CDS was directionally cloned into the
KNOLLE cassette in the pBluescript vector via restriction sites SmaI and
EcoRI (22). The KN:Myc-SYP132 insert was introduced into the pBAR-B
vector via the restriction sites HpaI and SpeI.
Constructs for SNARE domain swaps were generated by primer extension
PCRaccordingtostandardproceduresusingTaqDNAPolymerase(PeqLab
Biotechnologie GmbH). Constructs were cloned into the multiple cloning
site (MCS) of the pGreenIIB containing the KNOLLE cassette carrying an
N-terminal RFP-tag.
AHISTONE4 (H4)expressioncassettewasgeneratedbyamplifying543bp
upstreamand177bpdownstreamoftheH4 gene.The5?and3?sequences
were directionallycloned into the binary pGreenIIB-vector using the restric-
tion sites SacI/XbaI and EcoRI/KpnI, respectively, surrounding an MCS
containing XbaI, SmaI and EcoRI. An N-terminal RFP-tag was introducedby
XbaI/SmaI. SYP1 coding sequences were introduced via SmaI/EcoRI sites.
The UBQ10 promoter (25) constructsfor pen1-1 rescue were generatedby
directionally cloning Arabidopsis syntaxin coding sequences into the binary
pGreenIIB-vector downstream of the UBQ10 promoter via the restriction
sites SmaI and SpeI.
The constructs were checked by restriction digest and sequencing
using the ABI PRISM Big Dye Terminator Cycle Sequencing Kit and
the ABI-Sequencer 310 (Applied Biosystems) or using GATC (Konstanz)
service before transformation into A. tumefaciens strain GV3101. Standard
protocols were used for molecular biology(50). Restriction enzymes were
purchased from MBI Fermentas and synthetic oligonucleotides from ARK
(Sigma-Aldrich).
For RT-PCR analysis, total RNA was isolated from 100 mg Arabidopsis
seedlings, using the ’Trizol-method’(51) or the RNAeasy plant mini kit
(Qiagen). After removal of contaminating DNA (DNase I, Fermentas),
first strand cDNA was synthesized with Superscript II RNaseH-Reverse
Transcriptase (Invitrogen), using the dT-anchor-random II primer. As a
control, we used ACTIN2. All primer sequences are listed in Table S2.
Western blot analysis
Preparation of protein extracts and Western blots was performed as
described (10). For protein extraction we used one inflorescence, five
rosette leaves or 50 mg seedlings. Rabbit anti-KNOLLE antiserum was
used at 1:5000 dilution (10), mouse anti-α-tubulin monoclonal antibody
at 1:4000 (Sigma-Aldrich), rat anti-RFP monoclonal antibody at 1:1500
(chromotek), sheep anti-rabbit IgPOD polyclonal antibody at 1:1000
(Boehringer), goat anti-mouse IgPOD polyclonal antibody at 1:10 000
(Boehringer), goat anti-rat IgPOD at 1:1000 (Sigma-Aldrich) and mouse
anti-Myc-POD monoclonal antibody at 1:1000 (Roche). Independent T1
knolle heterozygouslines for each transgene (KNOLLE, PEN1 and SYP132)
were investigated for expression level. For phenotypic and transcript level
analyses, we used the strongest and weakest expression line of each
transgenic construct.
Inhibitor treatment and FM 4-64 staining
Three- to five-day-old seedlings were incubated in 1 mL of liquid medium
(half-strength MS medium) containing 50 μM BFA. FM 4-64 dissolved in
water was used at 4 μM final concentration. Seedlings were incubated
with inhibitors and dye at room temperature for the indicated times
followed by fixation with 4% paraformaldehyde in microtuble stabilizing
buffer (MTSB) (50 mM PIPES, 5 mM EGTA, 5 mM MgSO4, adjust pH with
KOH).Thefollowingstocksolutionswereused:50 mMBFA(Sigma-Aldrich)
in dimethylsulphoxide(DMSO):ethanol(1:1), and 2 mM FM 4-64 (Molecular
Probes) in water. Control treatments were performed with equal amounts
of the respective solvents.
Antibody staining and confocal laser-scanning
microscopy
Whole-mount immunofluorescence was performed as described (10).
Antibodies and dilutions were as follows: rabbit anti-KNOLLE anti-
serum (1:2000) (10), mouse anti-Myc monoclonal antibody 9E10 (1:600;
Santa Cruz Biotechnology), rabbit anti-SEC21/γCOP polyclonal antibody
(1:1000) (16), rabbit anti-ARF1 polyclonal antibody (1:5000) (52), fluores-
cein isothiocyanate (FITC)-conjugated secondary goat anti-rabbit antibody
(1:600, Dianova), Cy3-conjugated secondary goat anti-mouse antibody
(1:600, Dianova). DAPI (4’,6-diamidino-2-phenylindole) staining was per-
formed as described (45). Immunofluorescence and live-cell microscopy
were done with a Leica TCS-SP2/SP5 confocal laser-scanning microscope.
All confocal laser-scanning microscopy (CLSM) images were obtained
using the LEICA CONFOCAL software and a 63× water-immersion objective.
Images were processed using ADOBE PHOTOSHOP CS3.
B. g. hordei inoculation and quantification of pen1
mutant phenotype
Four-week-old Arabidopsis plants were inoculated with powdery mildew
Blumeria graminis hordei from barley. Live-cell imaging of single leaves
was performed at 12–16 h after inoculation. Penetration rescue analyses
were performed at 72 h after inoculation.
Individual B. g. hordei–Arabidopsis interaction sites were characterized
microscopically for failed and successful invasion (efficient papilla forma-
tion versus haustorium formation and hypersensitive-response-like cell
death) using aniline blue and coomassie blue as recently described (12).
The experiment was repeated three times, and 100 interaction sites per
genotype were scored each time.
Acknowledgments
We thank Stefan Driessen, Ulrike Hiller and Alexandra Matei for
technical assistance. This work was supported by the Deutsche
Forschungsgemeinschaft through an AFGN grant to G. J.
Supporting Information
Additional Supporting Information may be found in the online version of
this article:
Figure S1: Subcellular localization of PEN1 and SYP132 during
cytokinesis. A–F) Like KNOLLE (A, D), MYC-PEN1 (B) and MYC-SYP132
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(E) accumulate at the cell plate (arrows). MYC-PEN1 accumulates less
at the plasma membrane, whereas MYC-SYP132 shows no difference
(arrowheads). C and F) Overlay counterstained with DAPI (DNA, blue). G
and H) Quantitative scans of MYC-PEN1 (G) and MYC-SYP132 (H) protein
accumulation from the plasma membrane (PM) across the cell including
the cell plate (CP). Scale bars, 5 μm.
Figure S2: Quantification of RFP-SYP1 protein accumulation at
infected leaf cells. A–C, upper panels) UBQ:RFP-PEN1 and (D–F, upper
panels) UBQ:RFP-SYP132 label the plasma membrane and the B. g.
hordei–Arabidopsisinteraction sites. Scale bars, 5 mm. A–F, lower panels)
Quantitative scans of RFP-SYP1 protein accumulation from the plasma
membrane across the cell to the penetration site. G) Quantification of
signal intensity of the RFP-SYP1 proteins at the penetration site compared
to the plasma membrane. n = 5; error bars indicate standard deviation.
Figure S3:Co-labelingof GFP-PEN1 andRFP-SYP132. A–C)Subcellular
localization of GFP-PEN1 (A) and RFP-SYP132 (B) in Arabidopsis root-tip
cells. Both SYP1 proteins label the plasma membrane (arrows), whereas
only PEN1 labels some endosomes (arrowheads); (C) merged image. D–F)
After BFA treatment PEN1 (D) but not SYP132 (E) accumulates in BFA
compartments; (F) merged image. Scale bars, 5 μm.
Figure S4: Subcellular behavior of SYP1 syntaxins. A–C) Like KNOLLE
(A), MYC-PEN1 (B, red) and MYC-SYP132 (C, red; arrowhead) accumulate
in BFA compartments in mitotic cells. Only PEN1 accumulates in BFA
compartments in interphase cells (B). D–K) Immuno-localization of RFP-
PEN1 (D–G) and RFP-SYP132 (H–K) expressed from the HISTONE 4 (H4)
promoter. D–F) RFP-PEN1 (red) localizes at the plasma membrane and
colocalizes with the TGN/endosomal marker ARF1 (green). G) RFP-PEN1
accumulatesin BFA compartments.H–J) RFP-SYP132(red) localizesat the
plasma membrane but does not colocalize with ARF1 (green). K) KNOLLE
(green) but not RFP-SYP132 (red) accumulates in BFA compartments
(arrowhead). Scale bars, 5 μm.
Figure S5: Protein levels of PEN1-KNOLLESND. Protein expression of
transgenic PEN1-KNOLLESNDfrom several independent transgenic lines
was analyzed with anti-RFP antibody. Note that protein levels are nearly
equal when expressed from the KNOLLE (KN) promoter or from the
HISTONE 4 (H4) promoter. KN:RFP-PEN1-KNOLLESNDtransgenic lines
#2 and #4 were shown to rescue knolle mutants like wild type (Table S1).
Table S1:
plant lines were grown on agar plates to seedling stage and phenotypically
analyzed. knolle and partial rescue phenotypes were counted.
Rescue analysis of SYP1 syntaxins. Progeny of transgenic
Table S2: Oligonucleotide sequences.
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