- Email: [email protected]
Bacterial Effectors Induce Oligomerization of Immune Receptor ZAR1 In Vivo
Journal Pre-proof Bacterial effectors induce oligomerization of immune receptor ZAR1 in vivo Meijuan Hu, Jinfeng Qi, Guozhi Bi, Jian-Min Zhou
PII: DOI: Reference:
S1674-2052(20)30066-6 https://doi.org/10.1016/j.molp.2020.03.004 MOLP 907
To appear in: MOLECULAR PLANT Accepted Date: 11 March 2020
Please cite this article as: Hu M., Qi J., Bi G., and Zhou J.-M. (2020). Bacterial effectors induce oligomerization of immune receptor ZAR1 in vivo. Mol. Plant. doi: https://doi.org/10.1016/ j.molp.2020.03.004. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs. © 2020 The Author
1
Bacterial effectors induce oligomerization of immune receptor ZAR1 in vivo
2
Meijuan Hu1,2,3, Jinfeng Qi1,2,3, Guozhi Bi1,*, and Jian-Min Zhou1,2,*
3 4
1
5
Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences,
6
Beijing 100101, P. R. China
7
2
8
Sciences, Beijing 100049, P. R. China
9
3
10 11
State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental
CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of
These authors contributed equally to this article
*Correspondence: Guozhi ([email protected])
Bi
([email protected]),
Jian-Min
Zhou
12 13 14
Short Summary
15
The bacterial pathogen effectors AvrAC and HopZ1a induce oligomerization of the
16
Arabidopsis NLR protein ZAR1 in protoplasts. Structural requirements for ZAR1
17
resistosome assembly in vitro are also essential for HopZ1a-induced ZAR1
18
oligomerization in vivo and disease resistance in plants, providing evidence that the
19
ZAR1 resistosome forms in vivo during immune activation.
20
21
ABSTRACT
22
Plants utilize nucleotide-binding (NB), leucine-rich repeat (LRR) receptors (NLRs) to
23
detect pathogen effectors, leading to effector-triggered immunity. The NLR ZAR1
24
indirectly recognizes the Xanthomonas campestris pv. campestris effector AvrAC and
25
Pseudomonas syingae effector HopZ1a, by associating with closely related
26
receptor-like cytoplasmic kinase subfamily XII-2 (RLCK XII-2) members RKS1 and
27
ZED1,
28
AvrAC-modified decoy PBL2UMP form a pentameric resistosome in vitro, and the
29
ability of resistosome formation is required for AvrAC-triggered cell death and
30
disease resistance. However, it remains unknown whether the effectors induce ZAR1
31
oligomerization in the plant cell. Here, we show that both AvrAC and HopZ1a can
32
induce oligomerization of ZAR1 in Arabidopsis protoplasts. Residues mediating
33
ZAR1-ZED1
34
oligomerization in vivo and disease resistance. In addition, ZAR1 residues required
35
for the assembly of ZAR1 resistosome in vitro are also essential for HopZ1a-induced
36
ZAR1 oligomerization in vivo and disease resistance. Our study provides evidence
37
that pathogen effectors induce ZAR1 resistosome formation in the plant cell and that
38
the resistosome formation triggers disease resistance.
respectively.
Recently,
interaction
are
we
showed
that
indispensable
for
ZAR1,
RKS1,
HopZ1a-induced
and
the
ZAR1
39
INTRODUCTION
40
Plants deploy cell surface receptors and intracellular nucleotide-binding (NB),
41
leucine-rich repeat (LRR) receptors (NLRs) for pathogen perception (Dodds and
42
Rathjen, 2010; Maekawa et al., 2011; Monaghan and Zipfel, 2012). Cell surface
43
pattern recognition receptors (PRRs) recognize microbe- and host-derived molecular
44
patterns, and activate immunity (Tang et al., 2017). However, pathogenic microbes
45
often deliver effector proteins into the plant cell where they suppress PRR signaling
46
and promote microbial virulence (Feng and Zhou, 2012). In turn, plants have evolved
47
NLRs to monitor effector proteins and trigger robust immune responses, which often
48
results in localized programmed cell death called hypersensitive response (HR) and
49
accumulation of defense hormone salicylic acid (SA) (Jones and Dangl, 2006;
50
Maekawa et al., 2011; Fu and Dong, 2013; Cui et al., 2015).
51
Plant NLRs detect pathogen effectors either directly or indirectly (Jones and Dangl,
52
2006; Cui et al., 2015; Kourelis and van der Hoorn, 2018). While direct recognition
53
follows a receptor-ligand model in which an NLR physically interacts with an effector
54
(Dangl and McDowell, 2006; Dodds et al., 2006; Krasileva et al., 2010; Ravensdale et
55
al., 2012), more often an NLR forms a complex with another host protein that is
56
modified by pathogen effectors (Chung et al., 2011; Wang et al., 2015). The modified
57
host protein is either an effector virulence target or a molecular mimic of a virulence
58
target, which are called “guardee” and “decoy”, respectively (Zhou and Chai, 2008;
59
van der Hoorn and Kamoun, 2008).
60
Arabidopsis HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) was first identified as a
61
NLR protein that is responsible for the recognition of Pseudomonas syringae effector
62
protein HopZ1a, an acetyl transferase belonging to the YopJ/HopZ superfamily
63
(Lewis et al., 2008, 2010). ZAR1 interacts with HOPZ-ETI-DEFICIENT 1 (ZED1), a
64
pseudokinase from receptor-like cytoplasmic kinase subfamily XII-2 (RLCK XII-2)
65
required for HopZ1a recognition (Lewis et al., 2013). Interestingly, ZAR1 also
66
associates with other RLCK XII-2 proteins, enabling a single ZAR1 to recognize
67
multiple effectors (Wang et al., 2015; Khan et al., 2016). Thus, the association of
68
Arabidopsis ZAR1 with RESISTANCE RELATED KINASE 1 (RKS1) and
69
ZED1-RELATED KINASE 3 (ZRK3) confers resistance to Xanthomonas campestris
70
pv. campestris carrying AvrAC (an uridylyl transferase) and P. syringae carrying
71
HopF2 (a ribosyltransferase), respectively. Wang et al., 2015; Seto et al., 2017). A
72
recent study indicates that ZAR1 also confers resistance to P. syringae carrying three
73
additional effectors, HopBA1, HopO1, and HopX1 (Laflamme et al., 2020). ZAR1
74
also exists in Nicotiana benthamiana (NbZAR1) and it interacts with the RLCK XII-2
75
member XOPJ4 IMMUNITY 2 (JIM2) to confer resistance to Xanthomonas perforans
76
carrying XopJ4, another YopJ/HopZ superfamily acetyl transferase (Schultink et al.,
77
2019). AvrAC uridylylates multiple RLCK VII members and inhibits PTI responses
78
(Feng et al., 2012). Among these, PBL2 is a decoy (Guys et al., 2013; Wang et al.,
79
2015), and the uridylylated PBL2 (PBL2UMP) is recruited to the ZAR1-RKS1 complex,
80
to activate immunity (Wang et al., 2015). Although HopZ1a can acetylate ZED1, it is
81
not clear whether this modification is required for HopZ1a-triggered disease
82
resistance (Lewis et al., 2013). Recent studies showed that HopZ1a promotes
83
interaction between ZED1 and several RLCK VII members (Bastedo et al., 2019), and
84
two closely related RLCKs, SUPPRESSOR OF ZED1-D1 (SZE1) and SZE2, are
85
required for HopZ1a-induced disease resistance (Liu et al., 2019), suggesting that
86
these RLCK members may act as decoy or guardee for HopZ1a recognition.
87
How NLRs initiate immune signaling is a fundamental question of immunology in
88
plants and animals. Animal NLR apoptosis inhibitory protein 2 (NAIP2) directly
89
binds bacterial T3SS rod protein PrgJ, and then catalyzes its helper NLR NLRC4
90
polymerization to form oligomeric inflammasome, which is mainly mediated by NB
91
and oligomerization domain (NOD) (Hu et al., 2015). In addition, NAIP1 and
92
NAIP5/6 form oligomeric inflammasomes with NLRC4 in response to T3SS needle
93
protein and bacterial flagellin, respectively (Kofoed and Vance, 2011; Yang et al.,
94
2013). Plant and animal NLRs share similar structural domains including a C-terminal
95
LRR domain, a variable N-terminal domain and a conserved central NOD (a NACHT
96
domain in animals and an NB-ARC domain in plants) (Jones et al., 2016). Full-length
97
NLR proteins, like RPM1, MLA, Sr33, Sr50, RPS5, Rx, self-associate before
98
activation (Ade et al., 2007; Cesari et al., 2016; Gutierrez et al., 2010; El Kasmi et al.,
99
2017), whereas the tobacco NLR protein N interacts with itself only in the presence of
100
the TMV P50 elicitor (Mestre and Baulcombe, 2006), suggesting self-association
101
plays a role in NLR-mediated defense signaling. However, whether effectors have the
102
ability to induce oligomerization of NLRs in the plant cell remains unknown, as
103
detection of NLR protein oligomerization in vivo remains technically challenging.
104
ZAR1 is a canonical CC-NB-LRR which contains a C-terminal LRR domain, an N
105
terminal CC domain, and NOD (Lewis et al., 2010; Baudin et al., 2017). We recently
106
solved by cryo-EM three structures of ZAR1 protein complexes, including an inactive
107
ZAR1-RKS1 complex, an intermediate ZAR1-RKS1-PBL2UMP complex, and an
108
active ZAR1-RKS1-PBL2UMP pentameric complex (Wang et al., 2019a, 2019b). The
109
LRR domain of ZAR1 (ZAR1LRR) interacts with the N terminus of RKS1 in the
110
preformed complex. PBL2UMP interacts with RKS1, resulting in conformation
111
changes of a RKS1 segment, which then sterically clashes with the ZAR1 NB domain
112
(ZAR1NBD) to dislodge ADP. In vitro, the ADP-depleted ZAR1-RKS1-PBL2UMP
113
complex binds ATP to trigger drastic conformational changes in ZAR1 to expose
114
surfaces required for inter-molecular interactions between neighboring ZAR1, leading
115
to the assembly of the active pentamer called resistosome. The structural features
116
required for resistosome assembly are correlated with disease resistance and cell death
117
function triggered by AvrAC. Furthermore, a segment of the CC domain is organized
118
into a barrel-like structure on plasma membrane (PM), and this is also required for
119
AvrAC-triggered disease resistance and cell death. However, whether ZAR1
120
oligomerizes in the plant cell remains to be investigated. Furthermore, whether
121
resistosome formation is similarly required for immune activation by additional
122
effector proteins such as HopZ1a remains unknown.
123
Here, we show that Blue Native polyacrylamide gel electrophoresis (BN-PAGE) and
124
gel filtration assays can be successfully applied to detect NLR oligomerization in vivo
125
and that both AvrAC and HopZ1a can induce ZAR1 oligomerization in Arabidopsis
126
protoplasts. ZED1 and ZAR1 residues required for ZAR1-ZED1 interaction are
127
essential for ZAR1 oligomerization. In addition, ZAR1 residues required for in vitro
128
assembly of ZAR1-RKS1-PBL2UMP resistosome are essential for HopZ1a-induced
129
ZAR1 oligomerization and disease resistance. Furthermore, N-terminal α helix of
130
ZAR1 is indispensable for HopZ1a-induced disease resistance. These results indicate
131
that bacterial effectors induce ZAR1 oligomerization in vivo, confirming resistosome
132
formation observed in vitro.
133 134
RESULTS AND DISCUSSION
135
AvrAC and HopZ1a induce oligomerization of ZAR1 in Arabidopsis protoplasts
136
A major technical difficulty in the investigation of NLR protein activation in plants is
137
the rapid cell death associated with NLR activation, which hampers protein detection.
138
The cryo-EM structures of the ZAR1 resistosome reveal that the very N-terminal
139
amphipathic helices are released and form a funnel-shaped structure, which promotes
140
ZAR1 association with plasma membrane (Wang et al., 2019b). The inner surface of
141
the funnel structure contains several negatively charged residues. Mutations of two of
142
these residues, Glu11 and Glu18, impair ZAR1-mediated cell death activity without
143
affecting oligomerization and PM-association. We sought to take advantage of these
144
mutations and asked whether effectors induce oligmerization of ZAR1E11A/E18A in vivo
145
by using a transient expression system in protoplasts. We transfected the
146
ZAR1E11A/E18A construct into zar1 protoplasts along with RKS1, PBL2, and AvrAC or
147
the catalytic-deficient variant AvrACH469A, and subjected total protein to BN-PAGE
148
assay. In the absence of AvrAC (resting state), the ZAR1E11A/E18A protein existed in a
149
small molecular mass complex (Figure 1A), which probably contains unidentified
150
components. When co-expressed with AvrACH469A, the majority of ZAR1E11A/E18A
151
protein remained in the low molecular mass, and a small amount of ZAR1E11A/E18A
152
shifted to a large complex of ~900 kDa. In contrast, co-expression of AvrAC resulted
153
in all ZAR1E11A/E18A protein shifted to the large complex of about 900 kDa, which is
154
similar to that of ZAR1 resistosome in vitro (Wang et al., 2019b). The reason that a
155
small amount of ZAR1E11A/E18A was present in the ~900 kDa complex is not
156
understood. AvrACH469A displayed no activity in uridylylation of RIPK and PBL2 in
157
vitro (Feng et al., 2012; Wang et al., 2015), but a partial reduction of flg22-induced
158
FRK1 expression in protoplasts has been observed previously (Feng et al., 2012),
159
suggesting that AvrACH469A retains residual activity in vivo.
160
We next sought to verify whether the observed oligomerization can be observed with
161
a wild-type ZAR1. To prevent cell death and harvest sufficient protein for analysis,
162
we treated Arabidopsis protoplasts with LaCl3, a channel blocker that is known to
163
inhibit AvrRpm1-induced cell death (El Kasmi et al., 2017). We found that LaCl3 can
164
indeed inhibit HR in Col-0 leaves infiltrated with a high concentration of P. syringae
165
carrying hopZ1a (Supplemental Figure 1A). In Col-0 protoplasts co-transfected with
166
ZAR1, RKS1, PBL2, and AvrAC, no protein was detected because of complete cell
167
death. However, the same protoplasts treated with LaCl3 allowed accumulation of
168
ZAR1, RKS1, and PBL2 proteins (Supplemental Figure 1B). Still, the protein levels
169
were much less compared to protoplasts transfected with ZAR1, RKS1, PBL2 in the
170
absence of AvrAC, suggesting that LaCl3 only partially blocked deleterious effects
171
during ZAR1 activation. Because the BN-PAGE assay allows only a small volume of
172
sample in each well (10 µL or less), it was not suitable for the analysis of the protein
173
harvested after LaCl3 treatment in our study. Note that proteins in BN-PAGE typically
174
appear in smearing patterns, which further hampers the detection. To circumvent the
175
problem, we adopted gel filtration assays which allowed us to scale up the amounts of
176
protoplasts and protein. The wild-type ZAR1 was co-transfected along with RKS1,
177
PBL2, and AvrAC or AvrACH469A into Col-0 protoplasts. Consistent with the
178
BN-PAGE data, when co-expressed with AvrACH469A, the majority of ZAR1 and
179
RKS1 co-migrated in a small molecular mass complex indicative of an inactive
180
pre-formed complex and only a small amount of ZAR1 and RKS1 migrated to a large
181
molecular mass complex (Figure 1B). When co-expressed with AvrAC, almost all
182
ZAR1 and RKS1 migrated to the large complex (Figure 1B). These experiments
183
validated the results observed in BN-PAGE assay using the ZAR1E11A/E18A variant.
184
Together,
185
ZAR1-RKS1-PBL2UMP observed in vitro also exists in plant protoplasts.
186
These results described above indicated that both BN-PAGE and gel filtration can be
187
applied to detect ZAR1 oligomerization in vivo. Because gel filtration required large
188
amounts of materials and long handling of samples, we decided to use ZAR1 variants
189
that are defective in triggering cell death and BN-PAGE assays for ZAR1
190
oligomerization in the rest of the study.
191
We next investigated whether HopZ1a can similarly induce the oligomerization of
192
ZAR1. The enzymatic dead variant HopZ1aC216A, which does not trigger HR, failed to
193
induce ZAR1E11A/E18A oligomerization, whereas HopZ1a induced an oligomeric
194
complex of ZAR1E11A/E18A with a molecular mass of about 900 kDa (Figure 1C).
195
These results indicate that HopZ1a, in addition to AvrAC, also induced the formation
196
of ZAR1 resistosome in plant protoplasts.
197
ZED1-ZAR1 interaction is critical for HopZ1a triggered immunity
198
We next sought to determine whether the ZAR1 oligomerization observed in
199
protoplasts has similar structural requirements as the ZAR1 ressitosome assembled in
200
vitro. The interaction of ZAR1-RKS1 mainly results from the hydrophobic contacts
201
mediated by N terminal α helix of RKS1 and ZAR1LRR (Wang et al., 2019a).
202
Sequence alignment showed that the residues of RKS1 responsible for association
203
with ZAR1 are highly conserved in Arabidopsis RLCK XII-2 subfamily. To evaluate
204
the effect of these residues on ZAR1-ZED1 interaction, we generated two ZED1
205
mutations, I24E (ZED1I24E) and G29E (ZED1G29E), and transfected these constructs
206
into Arabidopsis protoplasts. Both mutations of ZED1 severely diminished the
these
observations
suggest
that
the
oligomeric
complex
of
207
interaction with ZAR1 (Figure 2A), further explaining the association between ZAR1
208
and diverse proteins from RLCK XII-2 subfamily. We next tested whether the
209
RKS1-interacting residues of ZAR1 are required for ZED1 association in Arabidopsis
210
protoplasts. The ZAR1V544E, ZAR1H597E and ZAR1W825A/F839A variants, which are
211
impaired in the interaction with RKS1, displayed a weaker interaction with ZED1
212
compared to wild-type ZAR1 (Figure 2B). The ZAR1I600E mutation had negligible
213
effect on ZAR1-RKS1 and ZAR1-ZED1 interactions (Wang et al., 2019a; Figure 2B).
214
We next tested how these mutations affect oligomerization of ZAR1 in Arabidopsis
215
protoplasts. The mutation ZED1I24E impaired the shift of ZAR1E11A/E18A mobility
216
induced by HopZ1a (Figure 2C). Furthermore, the ZAR1W825A/F839A mutation
217
completely abolished oligomerization of ZAR1 when co-expressed with HopZ1a and
218
ZED1 (Figure 2D). These results indicated that ZAR1-ZED1 interaction is
219
indispensable for the formation of ZAR1-ZED1 oligomeric complex in protoplasts.
220
We then tested the impact of these mutations on HopZ1a-induced cell death in
221
protoplasts by using the Cell Titer-Glo Luminescent Cell Viability Assay, which
222
measures cellular ATP. The ZED1 mutations, ZED1I24E and ZED1G29E, greatly
223
reduced ZAR1 mediated cell death compared to wild-type ZED1 (Figure 2E). In
224
addition, ZAR1 mutations, ZAR1V544E, ZAR1H597E and ZAR1W825A/F839A, markedly
225
reduced HopZ1a-induced cell death (Figure 2F). To further evaluate the role of these
226
mutations on HopZ1a-induced disease resistance, we introduced ZED1 variants with
227
their native promoters into the zed1 mutant. T1 transgenic plants were challenged
228
with wild-type P. syringae hopZ1a. As expected, plants carrying wild type ZED1
229
transgene restored resistance to P. syringae hopZ1a, whereas plants expressing the
230
ZED1I24E and ZED1G29E variants were fully susceptible compared to the plants
231
complemented with wild-type ZED1 (Figure 2G). All constructs accumulated
232
ZED1-HA protein (Supplemental Figure 2), suggesting that the lack of resistance in
233
ZED1I24E and ZED1G29E plants was not because of a lack of protein. We further
234
verified these results by testing two independent T2 lines for each construct, and the
235
results are completely consistent with those observed in T1 plants (Supplemental
236
Figure 3). We further tested representative transgenic lines (zar1 background)
237
carrying ZAR1V544E, ZAR1H597E and ZAR1W825A/F839A variants for resistance to P.
238
syringae hopZ1a. These lines accumulate similar amounts of ZAR1 protein and have
239
been shown to be compromised in resistance to X. campestris campestris avrAC
240
(Wang et al., 2019a). As expected, the wild-type ZAR1, but not mutant variants,
241
restored disease resistance (Figure 2H). Among the three independent experiments,
242
the ZAR1H597E line showed partial resistance compared to controls, but this was not
243
repeated in the other two experiments (Supplemental Figure 4). Taken together, our
244
results support the idea that ZAR1 interacts with ZED1 in a similar manner shown by
245
the structure of ZAR1-RKS1 complex, and this interaction is required for
246
oligomerization in vivo, cell death and disease resistance (Wang et al., 2019a).
247
Oligomerization of ZAR1 is critical for HopZ1a induced immunity
248
We further compared structural requirements for ZAR1 oligomerization in vivo and
249
ZAR1 resistosome assembly in vitro. In the ZAR1-RKS1-PBL2UMP resistosome, the
250
interaction of two adjacent ZAR1 proteins is mediated by all the structural domains of
251
ZAR1 including LRR domain, NB domain, helical domain 1 (HD1), winged-helix
252
domain (WHD) and CC domain (Wang et al., 2019b). In addition, ATP binding is
253
also essential for oligomerization of ZAR1-RKS1-PBL2UMP, as phosphate group of
254
the bound dATP forms a hydrogen bond with Ser403 which results in further
255
stabilizing the active conformation of ZAR1. We selected three mutations, including
256
ZAR1W150A and ZAR1S152E in the ZAR1NBD- ZAR1NBD interface, and ZAR1R194A/R297A
257
in residues specifically interacting with dATP but not ADP. When co-expressed with
258
HopZ1a and ZED1, the mutations ZAR1W150A and ZAR1S152E completely abolished
259
HopZ1a-induced oligomerization of ZAR1E11A/E18A in BN-PAGE assay (Figure 3A),
260
indicating
261
ZAR1-RKS1-PBL2UMP in vitro are also essential for HopZ1a-induced oligomerization
262
of ZAR1 in protoplasts. The introduction of the ZAR1R194A/R297A mutation led to a
that
these
residues
required
for
the
oligomeric
complex
of
263
smaller oligomeric complexes with a molecular mass of ~700 kDa irrespective of
264
HopZ1a or HopZ1aC216A (Figure 3A), suggesting that the mutation ZAR1R194A/R297A
265
resulted in aberrant complex formation in protoplasts that no longer respond to the
266
effector.
267
Oligomerization of ZAR1 play crucial roles in AvrAC-induced cell death and disease
268
resistance (Wang et al., 2019b). To further evaluate the impact of these mutations on
269
HopZ1a-induced cell death, we co-expressed indicated constructs in Arabidopsis
270
protoplasts. ZAR1W150A and ZAR1S152E mutant proteins showed a reduction of
271
HopZ1a-induced cell death compared to wild-type ZAR1, and ZAR1R194A/R297A
272
completely lost cell death triggering activity (Figure 3B). To determine the role of
273
these mutations on HopZ1a-induced disease resistance, wild-type, zar1 and
274
representative transgenic lines complemented with wild-type ZAR1, ZAR1W150A,
275
ZAR1S152E and ZAR1R194A/R297A were challenged with P. syringae hopZ1a. These
276
transgenic lines were selected because they have been fully characterized and tested
277
for resistance to X. campestris campestris avrAC (Wang et al., 2019b). The
278
ZAR1W150A, ZAR1S152E and ZAR1R194A/R297A lines were significantly more susceptible
279
to P. syringae hopZ1a compared to wild-type lines (Figure 3C), indicating the
280
HopZ1a-induced normal oligomerization activity of ZAR1 is necessary for
281
ZAR1-mediated disease resistance. The lack of cell death activity and disease
282
resistance function for ZAR1R194A/R297A further confirm that the aberrant aggregation
283
at about 700 kDa is non-functional.
284
N-terminal α helix of ZAR1 is critical for HopZ1a induced immunity
285
In the ZAR1 resistosome, the very N-terminal α1 helix of ZAR1 forms a
286
funnel-shaped structure that is required for AvrAC-induced cell death and
287
PM-association of ZAR1. However, the α1 helix does not appear to be necessary for
288
ZAR1 oligomerization in vitro (Wang et al., 2019b). We tested various α1 helix
289
variants for ZAR1 oligomerization in vivo by using BN-PAGE. Arabidopsis
290
protoplasts of the zar1 background were transfected with ZAR1F9A/L10A/L14A, which
291
carried mutations in residues located at the outer surface of the funnel-shaped
292
structure, and ZAR1∆10, which lacked the first 10 residues of the α helix. Both
293
ZAR1F9A/L10A/L14A and ZAR1∆10 retained the ability to form oligomeric complex as
294
indicated by BN-PAGE assay, although the amount is less compared to ZAR1E11A/E18A
295
(Figure 4A). Thus the α1 helix did not appear to be required for ZAR1
296
oligomerization in vivo, which is consistent with our previous in vitro study (Wang et
297
al., 2019b).
298
We next asked whether the α1 helix mutations affect HopZ1a-induced cell death as
299
they did to the AvrAC-induced cell death (Wang et al., 2019b). zar1 protoplasts
300
transfected with ZAR1F9A/L10A/L14A, ZAR1∆10 , or ZAR1E11A/E18A along with HopZ1a
301
showed much less cell death compared to those expressing wild-type ZAR1 and
302
HopZ1a (Figure 4B). To further examine the effect of these mutations on
303
HopZ1a-induced
304
complemented with wild-type ZAR1, ZAR1F9A/L10A/L14A, ZAR1∆10, or ZAR1E11A/E18A
305
were challenged with P. syringae hopZ1a. These lines accumulate similar amounts of
306
ZAR1 protein and have been tested for resistance to X. campestris campestris avrAC
307
(Wang et al., 2019b). The ZAR1F9A/L10A/L14A, ZAR1∆10, and ZAR1E11A/E18A lines were
308
significantly more susceptible compared to wild-type ZAR1 line (Figure 4C),
309
indicating that the α1 helix plays a critical role not only AvrAC-specified disease
310
resistance, but also HopZ1a-specified resistance.
311
Together, the results described above indicate that both AvrAC and HopZ1a induce
312
oligomerization of ZAR1 in vivo, which can be detected by using BN-PAGE or gel
313
filtration. These assays are probably also suitable for analyses of other NLR proteins
314
in vivo. Indeed, an independent study showed that BN-PAGE can be used to detect the
315
oligomerization NLR protein RPP7 when co-expressed with an immune activating
316
allele of RPW8 protein (Li et al., 2020). Our results also support that structural
317
requirements for HopZ1a-induced ZAR1 oligomerization and immunity are highly
318
consistent with ZAR1-RKS1-PBL2UMP resistosome assembly in vitro. Thus ZAR1
disease
resistance,
Transgenic
lines
of
zar1
background
319
resistosome formation in vivo is important for HopZ1a- and AvrAC-triggered
320
immunity.
321
METHODS
322
Plant Materials and Growth Conditions
323
Arabidopsis thaliana plants used in this study include Col-0, zed1, zar1-1 and zar1
324
transgenic lines complemented with various mutants (Lewis et al., 2010; Wang et al.,
325
2019a; Wang et al., 2019b). The plants used for protoplasts transfection and pathogen
326
inoculation were grown in soil with a photoperiod of 10 h of white light and 14 h
327
darkness at 23°C for 4-5 weeks. The intensity of white light was 90 µE m−2 s−1
328
provided with white fluorescent bulbs.
329
Constructs, Transgenic Plants and Protoplast Transformation
330
To generate ProZED1:ZED1-HA transgenic plants with mutant variants, the
331
full-length genomic DNA fragments containing promoter and coding sequence of
332
ZED1 were PCR amplified from Col-0 genomic DNA and cloned into
333
pCAMBIA1300 vector. The constructs of ZED1 mutations were generated by
334
site-directed mutagenesis. These constructs were introduced into zed1 mutant plants
335
by Agrobacterium tumefaciens-mediated transformation. Transgenic plants of T2
336
generation were identified for transgene expression by anti-HA immunoblot.
337
ProZAR1:ZAR1-HA and
338
Constructs of AvrAC, PBL2, RKS1, ZED1, HopZ1a and ZAR1 were under control of
339
the 35S promoter and have been reported previously (Wang et al., 2015; Wang et al.,
340
2019a; Wang et al., 2019b). New ZED1 mutant constructs were generated by
341
site-directed mutagenesis, and all these genes were cloned to pUC19-35S-HA-RBS or
342
pUC19-35S-FLAG-RBS for protoplasts transfection as previously described (He et al.,
343
2007)(He et al., 2007).
344
Blue Native PAGE assay
345
To determine ZAR1 oligomerization in protoplasts, Blue Native polyacrylamide gel
346
electrophoresis (BN-PAGE) was performed using the Bis-Tris Native PAGE system
347
(Invitrogen) according to the manufacturer’s instructions. Briefly, protoplasts
348
expressing indicated plasmids were incubated for 12 h, and total protein was extracted
349
with 1×Native PAGE Sample Buffer (Invitrogen) containing 1% digitonin and
350
protease inhibitor cocktail. Protein samples containing 0.25% Coomassie G-250 was
351
loaded and run on a Native PAGE 3-12% Bis-Tris gel. The proteins were then
352
transferred to PVDF membranes using NuPAGE Transfer Buffer, followed by
353
immunoblot analysis with the desired antibodies.
354
Gel filtration assay
355
For gel filtration assay, Arabidopsis protoplasts were transfected with the indicated
356
plasmids and incubated with 1 mM laCl3 for 12 h. Total protein was then isolated with
357
protein extraction buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.3%
358
Trition-X100, 1 mM DTT, protease inhibitor cocktail). Protein samples were filtered
359
through a 0.22 µm low-protein binding filter (Millipore) and analyzed by gel filtration.
360
AKTA Purifier system (GE Healthcare) was used to perform these experiments and
361
Superdex 200 Increase 10/300 GL column (GE Healthcare) was used at a flow rate of
362
0.4 mL/min. The buffer used in elution containing 50 mM HEPES [pH 7.5], 150 mM
363
NaCl, 1 mM EDTA, 1 mM DTT. The eluted fractions were analyzed by SDS-PAGE
364
and detected by anti-HA immunoblot.
365
Co-immunoprecipitation assay
366
For co-immunoprecipitation assay, protoplasts were transfected with the indicated
367
plasmids and incubated for 12 h. Total protein was extracted with protein extraction
368
buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 1 mM EDTA, 0.5% Trition-X100, 1
369
mM DTT, protease inhibitor cocktail). 50 µL anti-FLAG M2 agarose (Sigma) were
370
incubated with total protein for 2 h at 4°C, washed six times with protein extraction
371
buffer, and eluted with 60 µL of 0.5 mg/mL 3 × FLAG peptide (Sigma) for 1 h at 4°C.
372
Immunoprecipitates were separated on a 10% SDS PAGE gel and detected by the
373
desired antibodies.
374
Protoplasts viability assay
375
For protoplast viability assay, the zed1 or zar1 protoplasts transfected with the
376
indicated plasmids were incubated for 12 h as previously described (Wang et al., 2019;
377
Wang et al., 2019b). Cell viability was determined by the Cell Titer-Glo Luminescent
378
Cell Viability Assay according to the manufacturer’s instructions (Promega, G7570).
379
ATP-based Luminescence intensity were measured by the EnSpire Multimode plate
380
Reader (Perkin Elmer). The experimental treatment cells were normalized against the
381
control, assigned as 100 percent, to calculate the percentage of cell survival.
382
Pathogen Strains and Inoculations
383
The bacterial strain P. syringae DC3000 carrying hopZ1a, which was originally
384
isolated from P. syringae pv. syringae A2 (Lewis et al., 2008), was used in this work.
385
For bacterial growth assay, 4-week-old Arabidopsis plants were infiltrated with
386
bacteria at 1 × 106 colony-forming units/mL by a needleless syringe. The bacterial
387
number in leaves was determined at 3 d after inoculation.
388
Accession Numbers
389
Sequences of genes described in this work can be found in The Arabidopsis
390
Information Resource using the following accession numbers: ZAR1 (AT3G50950)
391
and ZED1 (AT3G57750).
392
SUPPLEMENTAL INFORMATION
393
Supplemental Information is available at Molecular Plant Online.
394
AUTHOR CONTRIBUTIONS
395
J.-M.Z. designed the research. M.H. and J.Q. performed the experiments. J.-M.Z. and
396
G.B. wrote the manuscript.
397
ACKNOWLEDGMENTS
398
The work was supported by grants from Ministry of Science and Technology of China
399
(2016YFD0100601), the Chinese Academy of Sciences international cooperation key
400
project grant GJHZ1311, and the State Key Laboratory of Plant Genomics
401
(SKLPG2016B-2) to J.-M.Z..
402
REFERENCES
403
Ade, J., DeYoung, B.J., Golstein, C., and Innes, R.W. (2007). Indirect activation of
404
a plant nucleotide binding site–leucine-rich repeat protein by a bacterial protease.
405
Proc. Natl. Acad. Sci. USA 104: 2531-2536.
406
Bastedo, D.P., Khan, M., Martel, A., Seto, D., Kireeva, I., Zhang, J., Masud, W.,
407
Millar, D., Lee, J.Y., Lee, A.H., et al. (2019). Perturbations of the ZED1
408
pseudokinase activate plant immunity. PLoS Pathog. 15: e1007900.
409
Baudin, M., Hassan, J.A., Schreiber, K.J., and Lewis, J.D. (2017). Analysis of the
410
ZAR1 immune complex reveals determinants for immunity and molecular
411
interactions. Plant Physiol. 174:2038-2053.
412
Cesari, S., Moore, J., Chen, C., Webb, D., Periyannan, S., Mago, R., Bernoux, M.,
413
Lagudah, E.S., and Dodds, P.N. (2016). Cytosolic activation of cell death and stem
414
rust resistance by cereal MLA-family CC–NLR proteins. Proc. Natl. Acad. Sci. USA
415
113: 10204-10209.
416
Chung, E.H., da Cunha, L., Wu, A.J., Gao, Z., Cherkis, K., Afzal, A.J., Mackey,
417
D., and Dangl, J.L. (2011). Specific threonine phosphorylation of a host target by
418
two unrelated type III effectors activates a host innate immune receptor in plants. Cell
419
Host Microbe 9:125-136.
420
Cui, H., Tsuda, K., and Parker, J.E. (2015). Effector-triggered immunity: from
421
pathogen perception to robust defense. Annu. Rev. Plant Biol. 66:487-511.
422
Dangl, J.L., and McDowell, J.M. (2006). Two modes of pathogen recognition by
423
plants. Proc. Natl. Acad. Sci. USA 103:8575-8576.
424
Dodds, P.N., Lawrence, G.J., Catanzariti, A.M., Teh, T., Wang, C.I., Ayliffe, M.A.,
425
Kobe, B., and Ellis, J.G. (2006). Direct protein interaction underlies gene-for-gene
426
specificity and coevolution of the flax resistance genes and flax rust avirulence genes.
427
Proc. Natl. Acad. Sci. USA 103:8888-8893.
428
Dodds, P.N., and Rathjen, J.P. (2010). Plant immunity: towards an integrated view
429
of plant-pathogen interactions. Nat. Rev. Genet. 11:539-548.
430
El Kasmi, F., Chung, E.H., Anderson, R.G., Li, J., Wan, L., Eitas, T.K., Gao, Z.,
431
and Dangl, J.L. (2017). Signaling from the plasma-membrane localized plant
432
immune receptor RPM1 requires self-association of the full-length protein. Proc. Natl.
433
Acad. Sci. USA 114: E7385-E7394.
434
Feng, F., and Zhou, J.M. (2012). Plant-bacterial pathogen interactions mediated by
435
type III effectors. Curr. Opin. Plant Biol. 15:469-476.
436
Feng, F., Yang, F., Rong, W., Wu, X., Zhang, J., Chen, S., He, C., and Zhou, J.-M.
437
(2012). A Xanthomonas uridine 5’-monophosphate transferase inhibits plant immune
438
kinases. Nature 485:114–118.
439
Fu, Z.Q., and Dong, X. (2013). Systemic acquired resistance: turning local infection
440
into global defense. Annu. Rev. Plant Biol. 64:839-863.
441
Gutierrez, J.R., Balmuth, A.L., Ntoukakis, V., Mucyn, T.S., Gimenez‐ ‐Ibanez, S.,
442
Jones, A.M., and Rathjen, J.P. (2010). Prf immune complexes of tomato are
443
oligomeric and contain multiple Pto-like kinases that diversify effector recognition.
444
Plant J. 61: 507-518.
445
He, P., Shan, L., and Sheen, J. (2007). The use of protoplasts to study innate
446
immune responses. In Plant-Pathogen Interactions (Springer), pp. 1-9.
447
Hu, Z., Zhou, Q., Zhang, C., Fan, S., Cheng, W., Zhao, Y., Shao, F., Wang, H.-W.,
448
Sui, S.-F., and Chai, J. (2015). Structural and biochemical basis for induced
449
self-propagation of NLRC4. Science 350: 399-404.
450
Jones, J.D., and Dangl, J.L. (2006). The plant immune system. Nature 444:323–329.
451
Jones, J.D., Vance, R.E., and Dangl, J.L. (2016). Intracellular innate immune
452
surveillance devices in plants and animals. Science 354: aaf6395.
453
Khan, M., Subramaniam, R., and Desveaux, D. (2016). Of guards, decoys, baits
454
and traps: pathogen perception in plants by type III effector sensors. Curr. Opin.
455
Microbiol. 29: 49-55
456
Kofoed, E.M., and Vance, R.E. (2011). Innate immune recognition of bacterial
457
ligands by NAIPs determines inflammasome specificity. Nature 477: 592.
458
Kourelis, J., and van der Hoorn, R.A.L. (2018). Defended to the nines: 25 Years of
459
resistance gene cloning identifies nine mechanisms for R protein function. Plant Cell
460
30:285-299.
461
Krasileva, K.V., Dahlbeck, D., and Staskawicz, B.J. (2010). Activation of an
462
Arabidopsis resistance protein is specified by the in planta association of its
463
leucine-rich repeat domain with the cognate oomycete effector. Plant Cell
464
22:2444-2458.
465
Laflamme, B., Dillon, M.M., Martel, A., Almeida, R.N., Desveaux, D., and
466
Guttman, D.S. (2020). The pan-genome effector-triggered immunity landscape of a
467
host-pathogen interaction. Science 367: 763-768.
468
Lewis, J.D., Abada, W., Ma, W., Guttman, D.S., and Desveaux, D. (2008). The
469
HopZ family of Pseudomonas syringae type III effectors require myristoylation for
470
virulence and
471
190:2880-2891.
472
Lewis, J.D., Wu, R., Guttman, D.S., and Desveaux, D. (2010). Allele-specific
473
virulence attenuation of the Pseudomonas syringae HopZ1a type III effector via the
474
Arabidopsis ZAR1 resistance protein. PLoS Genet. 6:e1000894.
475
Lewis, J.D., Lee, A.H.-Y., Hassan, J.A., Wan, J., Hurley, B., Jhingree, J.R., Wang,
476
P.W., Lo, T., Youn, J.-Y., Guttman, D.S., and Desveaux, D. (2013). The Arabidopsis
477
ZED1 pseudokinase is required for ZAR1-mediated immunity induced by the
478
Pseudomonas syringae type III effector HopZ1a. Proc. Natl. Acad. Sci. USA
479
110:18722–18727.
480
Li, L., Habring, A., Wang, K., and Weigel, D. (2020). Atypical resistance protein
481
RPW8/HR triggers oligomerization of the NLR immune receptor RPP7 and
avirulence functions
in
Arabidopsis
thaliana.
J. Bacteriol.
482
autoimmunity. Cell Host Microbe.
483
Liu, C., Cui, D., Zhao, J., Liu, N., Wang, B., Liu, J., Xu, E., Hu, Z., Ren, D., Tang,
484
D., and Hu, Y. (2019). Two Arabidopsis receptor-like cytoplasmic kinases SZE1 and
485
SZE2 associate with the ZAR1-ZED1 complex and are required for effector-triggered
486
immunity. Mol. Plant 12: 967-983.
487
Maekawa, T., Kufer, T.A., and Schulze-Lefert, P. (2011). NLR functions in plant
488
and animal immune systems: so far and yet so close. Nat. Immunol. 12:817-826.
489
Mestre, P., and Baulcombe, D.C. (2006). Elicitor-mediated oligomerization of the
490
tobacco N disease resistance protein. Plant Cell 18: 491-501.
491
Monaghan, J., and Zipfel, C. (2012). Plant pattern recognition receptor complexes at
492
the plasma membrane. Curr. Opin. Plant Biol. 15:349-357.
493
Schultink, A., Qi, T., Bally, J., and Staskawicz, B. (2019). Using forward genetics in
494
Nicotiana benthamiana to uncover the immune signaling pathway mediating
495
recognition
496
221:1001-1009.
497
Seto, D., Koulena, N., Lo, T., Menna, A., Guttman, D.S., and Desveaux, D. (2017).
498
Expanded type III effector recognition by the ZAR1 NLR protein using ZED1-related
499
kinases. Nat. Plants 3:17027.
500
Tang, D., Wang, G., and Zhou, J.M. (2017). Receptor kinases in plant-pathogen
501
Interactions: more than pattern recognition. Plant Cell 29:618-637.
502
van der Hoorn, R.A., and Kamoun, S. (2008). From guard to decoy: a new model
503
for perception of plant pathogen effectors. Plant Cell 20:2009-2017.
504
Wang, G., Roux, B., Feng, F., Guy, E., Li, L., Li, N., Zhang, X., Lautier, M.,
505
Jardinaud, M.F., Chabannes, M., et al. (2015). The decoy substrate of a pathogen
506
effector and a pseudokinase specify pathogen-induced modified-self recognition and
507
immunity in plants. Cell Host Microbe 18:285-295.
of the Xanthomonas
perforans
effector XopJ4.
New Phytol.
508
Wang, J., Wang, J., Hu, M., Wu, S., Qi, J., Wang, G., Han, Z., Qi, Y., Gao, N.,
509
Wang, H.W., et al. (2019a). Ligand-triggered allosteric ADP release primes a plant
510
NLR complex. Science 364:eaav5868.
511
Wang, J., Hu, M., Wang, J., Qi, J., Han, Z., Wang, G., Qi, Y., Wang, H.W., Zhou,
512
J.M., and Chai, J. (2019b). Reconstitution and structure of a plant NLR resistosome
513
conferring immunity. Science 364:eaav5870.
514
Ravensdale, M., Bernoux, M., Ve, T., Kobe, B., Thrall, P.H., Ellis, J.G., and
515
Dodds, P.N. (2012). Intramolecular interaction influences binding of the flax L5 and
516
L6 resistance proteins to their AvrL567 ligands. PLoS Pathog. 8:e1003004.
517
Yang, J., Zhao, Y., Shi, J., and Shao, F. (2013). Human NAIP and mouse NAIP1
518
recognize bacterial type III secretion needle protein for inflammasome activation.
519
Proc. Natl. Acad. Sci. USA 110: 14408-14413.
520
Zhou, J.M., and Chai, J. (2008). Plant pathogenic bacterial type III effectors subdue
521
host responses. Curr. Opin. Microbiol. 11: 179-185.
522
Figure legends
523
Figure 1. AvrAC and HopZ1a induce oligomerization of ZAR1 in Arabidopsis
524
protoplasts.
525
(A) BN-PAGE assay for AvrAC-induced oligomerization of ZAR1 in Arabidopsis
526
protoplasts. The indicated constructs were transfected into zar1 protoplasts. Total
527
protein was subjected to BN-PAGE and detected by immunoblotting with anti-HA
528
and anti-FLAG antibodies. All assays were performed three times, and a
529
representative photograph is shown.
530
(B) Gel filtration assay for AvrAC-induced oligomerization of ZAR1-RKS1-PBL2UMP
531
in Arabidopsis protoplasts. ZAR1-HA, RKS1-HA and PBL2-HA were coexpressed
532
with AvrACH469A (upper panel) or AvrAC (bottom panel) in Col-0 protoplasts,
533
incubated with 1 mM LaCl3, and total protein was subjected to gel filtration. The
534
eluted fractions were analyzed by immunoblotting with anti-HA antibody. Relative
535
grayscales (right panel) indicate the arbitrary densitometry units of different proteins
536
shown by immunoblots (left panel). Dashed lines indicate positions of standard
537
molecular masses. All assays were performed three times, and a representative
538
photograph is shown.
539
(C) BN-PAGE assay for HopZ1a-induced oligomerization of ZAR1 in Arabidopsis
540
protoplasts. The indicated constructs were transfected into zar1 protoplasts, and total
541
protein was subjected to BN-PAGE and immunoblot analysis. All assays were
542
performed three times, and a representative photograph is shown.
543
Figure 2. ZED1-ZAR1 interaction is critical for HopZ1a-induced immunity
544
(A and B) ZED1 (A) or ZAR1 (B) mutations reduce or abolish ZAR1-ZED1
545
interaction in protoplasts. The indicated constructs were transfected into zed1 and
546
zar1 protoplasts, respectively. Total protein was subjected to co-IP assays. All assays
547
were performed three times, and a representative photograph is shown.
548
(C and D) ZED1 (C) or ZAR1 (D) mutations abolish HopZ1a-induced
549
oligomerization of ZAR1 in protoplasts. The indicated constructs were transfected
550
into zed1 and zar1 protoplasts, respectively. Total protein was subjected to BN-PAGE.
551
All assays were performed three times, and a representative photograph is shown.
552
(E and F) The ZAR1-ZED1 interaction is required for HopZ1a-induced cell death in
553
protoplasts. ZED1 mutants (E) were co-expressed with HopZ1a and ZAR1 in zed1
554
protoplasts, and ZAR1 mutants (F) were co-expressed with HopZ1a and ZED1 in
555
zar1 protoplasts. The protoplasts were incubated for 12 h and cell viability was
556
measured by the Cell Titer-Glo Luminescent Cell Viability Assay. Data are presented
557
as mean ± SE. Different letters indicate significant difference at P < 0.05. (n =3,
558
one-way ANOVA, Tukey post-test, three independent experiments).
559
(G and H) Compromising the ZAR1-ZED1 interaction impairs HopZ1a-induced
560
antibacterial immunity. (G) Col-0, zed1, and T1 transgenic plants (zed1 background)
561
carrying the indicated ZED1 variants (G) and T2 transgenic lines (zar1 background)
562
carrying the indicated ZAR1 variants (H) were inoculated with P. syringae hopZ1a,
563
and bacterial population in the leaf was determined 3 d after inoculation. Boxplots
564
represent 16 and 24 data points from two (G) or three (H) independent experiments,
565
each of which contains eight plants. Colors indicate independent experiments.
566
Different letters indicate significant difference at P < 0.05. (one-way ANOVA, Tukey
567
post-test).
568
Figure 3. Oligomerization of ZAR1 is critical for HopZ1a-induced immunity
569
(A) ZAR1 residues required for resistosome assembly in vitro are essential for
570
HopZ1a-induced oligomerization in vivo. zar1 protoplasts expressing indicated
571
proteins were incubated for 12 h, and total protein was subjected to BN-PAGE. All
572
assays were performed three times, and a representative photograph is shown.
573
(B) Oligomerization of ZAR1 is required for HopZ1a-induced cell death in
574
protoplasts. ZAR1 mutants were co-expressed with HopZ1a and ZED1 in zar1
575
protoplasts, and cell viability was measured by the Cell Titer-Glo Luminescent Cell
576
Viability Assay. Data are presented as mean ± SE. Different letters indicate significant
577
difference at P < 0.05. (n =3, one-way ANOVA, Tukey post-test, three independent
578
experiments).
579
(C) Compromising oligomerization of ZAR1 impairs HopZ1a-induced antibacterial
580
immunity. Transgenic lines were inoculated with P. syringae hopZ1a, and bacterial
581
population in the leaf was determined 3 d after inoculation. Boxplots represent 24 data
582
points from three biological replicates, each of which contains eight technical
583
replicates. Colors indicate biological replicates. Different letters indicate significant
584
difference at P < 0.05. (one-way ANOVA, Tukey post-test).
585
Figuer 4. The N-terminal α1 helix of ZAR1 is critical for HopZ1a-induced
586
immunity
587
(A) Functional analysis of the N-terminal α1 helix of ZAR1 in HopZ1a-induced
588
ZAR1 oligomerization. zar1 protoplasts expressing indicated proteins were incubated
589
for 12 h, and total protein was subjected to BN-PAGE. All assays were performed
590
three times, and a representative photograph is shown.
591
(B) The α1 helix of ZAR1 is essential for HopZ1a-induced cell death in protoplasts.
592
ZAR1 mutants were coexpressed with HopZ1a and ZED1 in zar1 protoplasts, and cell
593
viability was measured by the Cell Titer-Glo Luminescent Cell Viability Assay. Data
594
are presented as mean ± SE. Different letters indicate significant difference at P <
595
0.05. (n =3, one-way ANOVA, Tukey post-test, three independent experiments).
596
(C) The α1 helix of ZAR1 is required for HopZ1a-induced bacterial resistance.
597
Transgenic lines were inoculated with P. syringae hopZ1a, and bacterial population in
598
the leaf was determined 3 d after inoculation. Boxplots represent 24 data points from
599
three biological replicates, each of which contains eight technical replicates. Colors
600
indicate biological replicates. Different letters indicate significant difference at P <
601
0.05. (one-way ANOVA, Tukey post-test).
602
Supplemental Figure 1.
603
affects AvrAC-induced protein degradation.
604
(A) LaCl3 blocks HopZ1a-induced cell death. Col-0 and zar1 plants were infiltrated
605
with P. syringae DC3000 hopZ1a (1 × 108 cfu/mL) and indicated concerntration of
606
LaCl3, and Macroscopic HR in leaves was recorded 5 h after inoculation.
607
(B) LaCl3 inhibits AvrAC-induced protein degradation. Protoplasts expressing
608
indicated proteins were incubated with LaCl3 for 12 h and total protein was subjected
609
to SDS-PAGE and detected by immunoblotting with anti-FLAG antibody.
610
Supplemental Figure 2. Accumulation of ZED1, ZEDI24E and ZED1G29E proteins
611
in T1 transgenic plants.
612
Positive T1 plants carrying ZED1 variant transgenes were pooled, and total protein
613
was isolated from the indicated transgenic lines and anti-HA immunoblot was done to
614
detect the accumulation of ZED1-HA protein. Upper and lower bands are non-specific
615
cross-reacting proteins. Ponceau staining of Rubisco indicates loading of protein.
616
Supplemental Figure 3. ZED1 mutants impaired in ZAR1-interaction fail to
617
confer antibacterial resistance.
618
Col-0, zed1, and T2 transgenic lines (zed1 background) carrying the indicated
619
transgenes were inoculated with P. syringae DC3000 hopZ1a, and bacterial growth
620
assay was performed as in Fig. 2G. Boxplots represent 8 data points from a single
621
experiment containing eight plants/line. Different letters indicate significant
622
difference at P < 0.05. (one-way ANOVA, Tukey post-test).
623
Supplemental Figure 4. ZAR1 mutants impaired in ZED1-interaction fail to
LaCl3 blocks HopZ1a-induced cell death and partially
624
confer antibacterial resistance (related to Figure 2H).
625
Col-0, zar1, and T2 transgenic lines (zar1 background) carrying the indicated
626
transgenes were inoculated with P. syringae DC3000 hopZ1a, and bacterial
627
population in the leaf was determined 3 d after inoculation. Boxplots represent 8 data
628
points from one replicate of this experiment shown in Figure 2H with green dots.
629
Different letters indicate significant difference at P < 0.05. (one-way ANOVA, Tukey
630
post-test).
C + + + +
+ + + + -
ZAR1E11A/E18A-HA ZED1-FLAG HopZ1a-FLAG HopZ1aC216A-FLAG
kDa 1048
Oligomer 720 α-HA
480 242
+ + -
+ + +
+ + + -
kDa 1048
Oligomer 720 α-HA
480
100 70
AvrAC-FLAG
55
PBL2-FLAG RKS1-FLAG
40
Rubisco
10% SDS-PAGE
242 α-HA
α-HA
100
55
HopZ1a-FLAG ZED1-FLAG
40
Rubisco
3-12% native PAGE
+ + -
3-12% native PAGE
ZAR1E11A/E18A-HA PBL2-FLAG RKS1-FLAG AvrAC-FLAG AvrACH469A-FLAG
10% SDS-PAGE
A
B
Input 7
8
9
10
11
12
13 mL
kDa 100
ZAR1 AvrACH469A
70
PBL2 RKS1
55 40
Relative grayscale
669 kDa 440 kDa 400000 400000 300000 300000 200000 200000
AvrAC PBL2 RKS1
10
11
12
13 mL kDa 100 70 55 40
Relative grayscale
ZAR1
9
440 kDa
8
8.5 8.5
9 9
9.5 9.5
10 10
10.5 11 11 11.5 11.5 12 12 12.5 12.5 13 13 10.5
Elution volume (mL)
669 kDa 440 kDa 8
669 kDa
100000 100000 00
Input 7
AvrACH469A
ZAR1 PBL2 RKS1
AvrAC
500000 500000
ZAR1 PBL2 RKS1
400000 400000
300000 300000
669 kDa
200000 200000
440 kDa
100000 100000 00 7.5 7.5
8
8.5 8.5
99
9.5 10 10 10.5 11 11.5 11.5 12 12 12.5 12.5 13 13 9.5 10.5 11
Elution volume (mL)
B -
40
ZAR1-FLAG
ZAR1-HA
E
C + + + -
+ + +
+ + + -
kDa 1048 720
α-FLAG
480 242
α-FALG
100 55
α-HA
40 Rubisco
10% SDS-PAGE 3-12% native PAGE
ZED1-HA ZED1I24E-HA HopZ1a-HA HopZ1aC216A-HA
+ + +
+ + + -
+ + +
+ + + -
G
kDa
1048 α-HA
720 480 242
α-HA α-FLAG Rubisco
100 55 40
10% SDS-PAGE 3-12% native PAGE
+ + +
α-FLAG IP
40
120 100 80 60 40 20 0
120 100 80 60 40 20 0
a c d
b
F
D ZAR1E11A/E18A-HA ZAR1W825A/F839A-HA ZED1-FLAG HopZ1a-FLAG HopZ1aC216A-FLAG
100
ZED1-FLAG
100
ZAR1E11A/E18A-FLAG
40
Cell viability (%)
ZED1-HA
ZED1-FLAG
Cell viability (%)
100
100
H
Bacteria [log10 (CFU cm-2)]
ZAR1-FLAG
kDa
ZAR1-HA
Bacteria [log10 (CFU cm-2)]
40
Input
ZED1-HA
α-FLAG IP
kDa
Input
A
a
c
c
c b
7
b
b
b
6 a
5
a
4 3 2
6 5 4 3 2
b
b
7 a
a
b
b
B
ZAR1E11A/E18A-HA ZAR1W150A-HA ZAR1S152E-HA ZAR1R149A/R297A-HA ZED1-FLAG HopZ1a-FLAG HopZ1aC216A-FLAG
+ + +
+ + + -
+ + +
+ + + -
+ + +
+ + + -
+ + +
Cell viability (%)
A + + + -
120
α-HA
100 55
α-FLAG 40 Rubisco
d
60
c
40
b
20
C Bacteria [log10 (CFU cm-2)]
242
3-12% native PAGE
480
10% SDS-PAGE
α-HA
720
80
0
kDa 1048
a
a
100
b
7
b
6 a 5
4 3 2
1
a
b
b
B + + + -
+ + +
+ + + -
+ + +
+ + + -
kDa 1048
Oligomer 720 α-HA
480
α-HA α-FLAG
100 55 40
Rubisco
10% SDS-PAGE
242
120 100 80 60 40 20 0
a
ac
c d b
C Bacteria [log10 (CFU cm-2)]
+ + +
3-12% native PAGE
ZAR1E11A/E18A-HA ZAR1F9A/L10A/L14A-HA ZAR1Δ10-HA ZED1-FLAG HopZ1a-FLAG HopZ1aC216A-FLAG
Cell viability (%)
A
7 6 5 4 3 2
1
b a
bd c
d
bd
PII: DOI: Reference:
S1674-2052(20)30066-6 https://doi.org/10.1016/j.molp.2020.03.004 MOLP 907
To appear in: MOLECULAR PLANT Accepted Date: 11 March 2020
Please cite this article as: Hu M., Qi J., Bi G., and Zhou J.-M. (2020). Bacterial effectors induce oligomerization of immune receptor ZAR1 in vivo. Mol. Plant. doi: https://doi.org/10.1016/ j.molp.2020.03.004. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs. © 2020 The Author
1
Bacterial effectors induce oligomerization of immune receptor ZAR1 in vivo
2
Meijuan Hu1,2,3, Jinfeng Qi1,2,3, Guozhi Bi1,*, and Jian-Min Zhou1,2,*
3 4
1
5
Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences,
6
Beijing 100101, P. R. China
7
2
8
Sciences, Beijing 100049, P. R. China
9
3
10 11
State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental
CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of
These authors contributed equally to this article
*Correspondence: Guozhi ([email protected])
Bi
([email protected]),
Jian-Min
Zhou
12 13 14
Short Summary
15
The bacterial pathogen effectors AvrAC and HopZ1a induce oligomerization of the
16
Arabidopsis NLR protein ZAR1 in protoplasts. Structural requirements for ZAR1
17
resistosome assembly in vitro are also essential for HopZ1a-induced ZAR1
18
oligomerization in vivo and disease resistance in plants, providing evidence that the
19
ZAR1 resistosome forms in vivo during immune activation.
20
21
ABSTRACT
22
Plants utilize nucleotide-binding (NB), leucine-rich repeat (LRR) receptors (NLRs) to
23
detect pathogen effectors, leading to effector-triggered immunity. The NLR ZAR1
24
indirectly recognizes the Xanthomonas campestris pv. campestris effector AvrAC and
25
Pseudomonas syingae effector HopZ1a, by associating with closely related
26
receptor-like cytoplasmic kinase subfamily XII-2 (RLCK XII-2) members RKS1 and
27
ZED1,
28
AvrAC-modified decoy PBL2UMP form a pentameric resistosome in vitro, and the
29
ability of resistosome formation is required for AvrAC-triggered cell death and
30
disease resistance. However, it remains unknown whether the effectors induce ZAR1
31
oligomerization in the plant cell. Here, we show that both AvrAC and HopZ1a can
32
induce oligomerization of ZAR1 in Arabidopsis protoplasts. Residues mediating
33
ZAR1-ZED1
34
oligomerization in vivo and disease resistance. In addition, ZAR1 residues required
35
for the assembly of ZAR1 resistosome in vitro are also essential for HopZ1a-induced
36
ZAR1 oligomerization in vivo and disease resistance. Our study provides evidence
37
that pathogen effectors induce ZAR1 resistosome formation in the plant cell and that
38
the resistosome formation triggers disease resistance.
respectively.
Recently,
interaction
are
we
showed
that
indispensable
for
ZAR1,
RKS1,
HopZ1a-induced
and
the
ZAR1
39
INTRODUCTION
40
Plants deploy cell surface receptors and intracellular nucleotide-binding (NB),
41
leucine-rich repeat (LRR) receptors (NLRs) for pathogen perception (Dodds and
42
Rathjen, 2010; Maekawa et al., 2011; Monaghan and Zipfel, 2012). Cell surface
43
pattern recognition receptors (PRRs) recognize microbe- and host-derived molecular
44
patterns, and activate immunity (Tang et al., 2017). However, pathogenic microbes
45
often deliver effector proteins into the plant cell where they suppress PRR signaling
46
and promote microbial virulence (Feng and Zhou, 2012). In turn, plants have evolved
47
NLRs to monitor effector proteins and trigger robust immune responses, which often
48
results in localized programmed cell death called hypersensitive response (HR) and
49
accumulation of defense hormone salicylic acid (SA) (Jones and Dangl, 2006;
50
Maekawa et al., 2011; Fu and Dong, 2013; Cui et al., 2015).
51
Plant NLRs detect pathogen effectors either directly or indirectly (Jones and Dangl,
52
2006; Cui et al., 2015; Kourelis and van der Hoorn, 2018). While direct recognition
53
follows a receptor-ligand model in which an NLR physically interacts with an effector
54
(Dangl and McDowell, 2006; Dodds et al., 2006; Krasileva et al., 2010; Ravensdale et
55
al., 2012), more often an NLR forms a complex with another host protein that is
56
modified by pathogen effectors (Chung et al., 2011; Wang et al., 2015). The modified
57
host protein is either an effector virulence target or a molecular mimic of a virulence
58
target, which are called “guardee” and “decoy”, respectively (Zhou and Chai, 2008;
59
van der Hoorn and Kamoun, 2008).
60
Arabidopsis HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) was first identified as a
61
NLR protein that is responsible for the recognition of Pseudomonas syringae effector
62
protein HopZ1a, an acetyl transferase belonging to the YopJ/HopZ superfamily
63
(Lewis et al., 2008, 2010). ZAR1 interacts with HOPZ-ETI-DEFICIENT 1 (ZED1), a
64
pseudokinase from receptor-like cytoplasmic kinase subfamily XII-2 (RLCK XII-2)
65
required for HopZ1a recognition (Lewis et al., 2013). Interestingly, ZAR1 also
66
associates with other RLCK XII-2 proteins, enabling a single ZAR1 to recognize
67
multiple effectors (Wang et al., 2015; Khan et al., 2016). Thus, the association of
68
Arabidopsis ZAR1 with RESISTANCE RELATED KINASE 1 (RKS1) and
69
ZED1-RELATED KINASE 3 (ZRK3) confers resistance to Xanthomonas campestris
70
pv. campestris carrying AvrAC (an uridylyl transferase) and P. syringae carrying
71
HopF2 (a ribosyltransferase), respectively. Wang et al., 2015; Seto et al., 2017). A
72
recent study indicates that ZAR1 also confers resistance to P. syringae carrying three
73
additional effectors, HopBA1, HopO1, and HopX1 (Laflamme et al., 2020). ZAR1
74
also exists in Nicotiana benthamiana (NbZAR1) and it interacts with the RLCK XII-2
75
member XOPJ4 IMMUNITY 2 (JIM2) to confer resistance to Xanthomonas perforans
76
carrying XopJ4, another YopJ/HopZ superfamily acetyl transferase (Schultink et al.,
77
2019). AvrAC uridylylates multiple RLCK VII members and inhibits PTI responses
78
(Feng et al., 2012). Among these, PBL2 is a decoy (Guys et al., 2013; Wang et al.,
79
2015), and the uridylylated PBL2 (PBL2UMP) is recruited to the ZAR1-RKS1 complex,
80
to activate immunity (Wang et al., 2015). Although HopZ1a can acetylate ZED1, it is
81
not clear whether this modification is required for HopZ1a-triggered disease
82
resistance (Lewis et al., 2013). Recent studies showed that HopZ1a promotes
83
interaction between ZED1 and several RLCK VII members (Bastedo et al., 2019), and
84
two closely related RLCKs, SUPPRESSOR OF ZED1-D1 (SZE1) and SZE2, are
85
required for HopZ1a-induced disease resistance (Liu et al., 2019), suggesting that
86
these RLCK members may act as decoy or guardee for HopZ1a recognition.
87
How NLRs initiate immune signaling is a fundamental question of immunology in
88
plants and animals. Animal NLR apoptosis inhibitory protein 2 (NAIP2) directly
89
binds bacterial T3SS rod protein PrgJ, and then catalyzes its helper NLR NLRC4
90
polymerization to form oligomeric inflammasome, which is mainly mediated by NB
91
and oligomerization domain (NOD) (Hu et al., 2015). In addition, NAIP1 and
92
NAIP5/6 form oligomeric inflammasomes with NLRC4 in response to T3SS needle
93
protein and bacterial flagellin, respectively (Kofoed and Vance, 2011; Yang et al.,
94
2013). Plant and animal NLRs share similar structural domains including a C-terminal
95
LRR domain, a variable N-terminal domain and a conserved central NOD (a NACHT
96
domain in animals and an NB-ARC domain in plants) (Jones et al., 2016). Full-length
97
NLR proteins, like RPM1, MLA, Sr33, Sr50, RPS5, Rx, self-associate before
98
activation (Ade et al., 2007; Cesari et al., 2016; Gutierrez et al., 2010; El Kasmi et al.,
99
2017), whereas the tobacco NLR protein N interacts with itself only in the presence of
100
the TMV P50 elicitor (Mestre and Baulcombe, 2006), suggesting self-association
101
plays a role in NLR-mediated defense signaling. However, whether effectors have the
102
ability to induce oligomerization of NLRs in the plant cell remains unknown, as
103
detection of NLR protein oligomerization in vivo remains technically challenging.
104
ZAR1 is a canonical CC-NB-LRR which contains a C-terminal LRR domain, an N
105
terminal CC domain, and NOD (Lewis et al., 2010; Baudin et al., 2017). We recently
106
solved by cryo-EM three structures of ZAR1 protein complexes, including an inactive
107
ZAR1-RKS1 complex, an intermediate ZAR1-RKS1-PBL2UMP complex, and an
108
active ZAR1-RKS1-PBL2UMP pentameric complex (Wang et al., 2019a, 2019b). The
109
LRR domain of ZAR1 (ZAR1LRR) interacts with the N terminus of RKS1 in the
110
preformed complex. PBL2UMP interacts with RKS1, resulting in conformation
111
changes of a RKS1 segment, which then sterically clashes with the ZAR1 NB domain
112
(ZAR1NBD) to dislodge ADP. In vitro, the ADP-depleted ZAR1-RKS1-PBL2UMP
113
complex binds ATP to trigger drastic conformational changes in ZAR1 to expose
114
surfaces required for inter-molecular interactions between neighboring ZAR1, leading
115
to the assembly of the active pentamer called resistosome. The structural features
116
required for resistosome assembly are correlated with disease resistance and cell death
117
function triggered by AvrAC. Furthermore, a segment of the CC domain is organized
118
into a barrel-like structure on plasma membrane (PM), and this is also required for
119
AvrAC-triggered disease resistance and cell death. However, whether ZAR1
120
oligomerizes in the plant cell remains to be investigated. Furthermore, whether
121
resistosome formation is similarly required for immune activation by additional
122
effector proteins such as HopZ1a remains unknown.
123
Here, we show that Blue Native polyacrylamide gel electrophoresis (BN-PAGE) and
124
gel filtration assays can be successfully applied to detect NLR oligomerization in vivo
125
and that both AvrAC and HopZ1a can induce ZAR1 oligomerization in Arabidopsis
126
protoplasts. ZED1 and ZAR1 residues required for ZAR1-ZED1 interaction are
127
essential for ZAR1 oligomerization. In addition, ZAR1 residues required for in vitro
128
assembly of ZAR1-RKS1-PBL2UMP resistosome are essential for HopZ1a-induced
129
ZAR1 oligomerization and disease resistance. Furthermore, N-terminal α helix of
130
ZAR1 is indispensable for HopZ1a-induced disease resistance. These results indicate
131
that bacterial effectors induce ZAR1 oligomerization in vivo, confirming resistosome
132
formation observed in vitro.
133 134
RESULTS AND DISCUSSION
135
AvrAC and HopZ1a induce oligomerization of ZAR1 in Arabidopsis protoplasts
136
A major technical difficulty in the investigation of NLR protein activation in plants is
137
the rapid cell death associated with NLR activation, which hampers protein detection.
138
The cryo-EM structures of the ZAR1 resistosome reveal that the very N-terminal
139
amphipathic helices are released and form a funnel-shaped structure, which promotes
140
ZAR1 association with plasma membrane (Wang et al., 2019b). The inner surface of
141
the funnel structure contains several negatively charged residues. Mutations of two of
142
these residues, Glu11 and Glu18, impair ZAR1-mediated cell death activity without
143
affecting oligomerization and PM-association. We sought to take advantage of these
144
mutations and asked whether effectors induce oligmerization of ZAR1E11A/E18A in vivo
145
by using a transient expression system in protoplasts. We transfected the
146
ZAR1E11A/E18A construct into zar1 protoplasts along with RKS1, PBL2, and AvrAC or
147
the catalytic-deficient variant AvrACH469A, and subjected total protein to BN-PAGE
148
assay. In the absence of AvrAC (resting state), the ZAR1E11A/E18A protein existed in a
149
small molecular mass complex (Figure 1A), which probably contains unidentified
150
components. When co-expressed with AvrACH469A, the majority of ZAR1E11A/E18A
151
protein remained in the low molecular mass, and a small amount of ZAR1E11A/E18A
152
shifted to a large complex of ~900 kDa. In contrast, co-expression of AvrAC resulted
153
in all ZAR1E11A/E18A protein shifted to the large complex of about 900 kDa, which is
154
similar to that of ZAR1 resistosome in vitro (Wang et al., 2019b). The reason that a
155
small amount of ZAR1E11A/E18A was present in the ~900 kDa complex is not
156
understood. AvrACH469A displayed no activity in uridylylation of RIPK and PBL2 in
157
vitro (Feng et al., 2012; Wang et al., 2015), but a partial reduction of flg22-induced
158
FRK1 expression in protoplasts has been observed previously (Feng et al., 2012),
159
suggesting that AvrACH469A retains residual activity in vivo.
160
We next sought to verify whether the observed oligomerization can be observed with
161
a wild-type ZAR1. To prevent cell death and harvest sufficient protein for analysis,
162
we treated Arabidopsis protoplasts with LaCl3, a channel blocker that is known to
163
inhibit AvrRpm1-induced cell death (El Kasmi et al., 2017). We found that LaCl3 can
164
indeed inhibit HR in Col-0 leaves infiltrated with a high concentration of P. syringae
165
carrying hopZ1a (Supplemental Figure 1A). In Col-0 protoplasts co-transfected with
166
ZAR1, RKS1, PBL2, and AvrAC, no protein was detected because of complete cell
167
death. However, the same protoplasts treated with LaCl3 allowed accumulation of
168
ZAR1, RKS1, and PBL2 proteins (Supplemental Figure 1B). Still, the protein levels
169
were much less compared to protoplasts transfected with ZAR1, RKS1, PBL2 in the
170
absence of AvrAC, suggesting that LaCl3 only partially blocked deleterious effects
171
during ZAR1 activation. Because the BN-PAGE assay allows only a small volume of
172
sample in each well (10 µL or less), it was not suitable for the analysis of the protein
173
harvested after LaCl3 treatment in our study. Note that proteins in BN-PAGE typically
174
appear in smearing patterns, which further hampers the detection. To circumvent the
175
problem, we adopted gel filtration assays which allowed us to scale up the amounts of
176
protoplasts and protein. The wild-type ZAR1 was co-transfected along with RKS1,
177
PBL2, and AvrAC or AvrACH469A into Col-0 protoplasts. Consistent with the
178
BN-PAGE data, when co-expressed with AvrACH469A, the majority of ZAR1 and
179
RKS1 co-migrated in a small molecular mass complex indicative of an inactive
180
pre-formed complex and only a small amount of ZAR1 and RKS1 migrated to a large
181
molecular mass complex (Figure 1B). When co-expressed with AvrAC, almost all
182
ZAR1 and RKS1 migrated to the large complex (Figure 1B). These experiments
183
validated the results observed in BN-PAGE assay using the ZAR1E11A/E18A variant.
184
Together,
185
ZAR1-RKS1-PBL2UMP observed in vitro also exists in plant protoplasts.
186
These results described above indicated that both BN-PAGE and gel filtration can be
187
applied to detect ZAR1 oligomerization in vivo. Because gel filtration required large
188
amounts of materials and long handling of samples, we decided to use ZAR1 variants
189
that are defective in triggering cell death and BN-PAGE assays for ZAR1
190
oligomerization in the rest of the study.
191
We next investigated whether HopZ1a can similarly induce the oligomerization of
192
ZAR1. The enzymatic dead variant HopZ1aC216A, which does not trigger HR, failed to
193
induce ZAR1E11A/E18A oligomerization, whereas HopZ1a induced an oligomeric
194
complex of ZAR1E11A/E18A with a molecular mass of about 900 kDa (Figure 1C).
195
These results indicate that HopZ1a, in addition to AvrAC, also induced the formation
196
of ZAR1 resistosome in plant protoplasts.
197
ZED1-ZAR1 interaction is critical for HopZ1a triggered immunity
198
We next sought to determine whether the ZAR1 oligomerization observed in
199
protoplasts has similar structural requirements as the ZAR1 ressitosome assembled in
200
vitro. The interaction of ZAR1-RKS1 mainly results from the hydrophobic contacts
201
mediated by N terminal α helix of RKS1 and ZAR1LRR (Wang et al., 2019a).
202
Sequence alignment showed that the residues of RKS1 responsible for association
203
with ZAR1 are highly conserved in Arabidopsis RLCK XII-2 subfamily. To evaluate
204
the effect of these residues on ZAR1-ZED1 interaction, we generated two ZED1
205
mutations, I24E (ZED1I24E) and G29E (ZED1G29E), and transfected these constructs
206
into Arabidopsis protoplasts. Both mutations of ZED1 severely diminished the
these
observations
suggest
that
the
oligomeric
complex
of
207
interaction with ZAR1 (Figure 2A), further explaining the association between ZAR1
208
and diverse proteins from RLCK XII-2 subfamily. We next tested whether the
209
RKS1-interacting residues of ZAR1 are required for ZED1 association in Arabidopsis
210
protoplasts. The ZAR1V544E, ZAR1H597E and ZAR1W825A/F839A variants, which are
211
impaired in the interaction with RKS1, displayed a weaker interaction with ZED1
212
compared to wild-type ZAR1 (Figure 2B). The ZAR1I600E mutation had negligible
213
effect on ZAR1-RKS1 and ZAR1-ZED1 interactions (Wang et al., 2019a; Figure 2B).
214
We next tested how these mutations affect oligomerization of ZAR1 in Arabidopsis
215
protoplasts. The mutation ZED1I24E impaired the shift of ZAR1E11A/E18A mobility
216
induced by HopZ1a (Figure 2C). Furthermore, the ZAR1W825A/F839A mutation
217
completely abolished oligomerization of ZAR1 when co-expressed with HopZ1a and
218
ZED1 (Figure 2D). These results indicated that ZAR1-ZED1 interaction is
219
indispensable for the formation of ZAR1-ZED1 oligomeric complex in protoplasts.
220
We then tested the impact of these mutations on HopZ1a-induced cell death in
221
protoplasts by using the Cell Titer-Glo Luminescent Cell Viability Assay, which
222
measures cellular ATP. The ZED1 mutations, ZED1I24E and ZED1G29E, greatly
223
reduced ZAR1 mediated cell death compared to wild-type ZED1 (Figure 2E). In
224
addition, ZAR1 mutations, ZAR1V544E, ZAR1H597E and ZAR1W825A/F839A, markedly
225
reduced HopZ1a-induced cell death (Figure 2F). To further evaluate the role of these
226
mutations on HopZ1a-induced disease resistance, we introduced ZED1 variants with
227
their native promoters into the zed1 mutant. T1 transgenic plants were challenged
228
with wild-type P. syringae hopZ1a. As expected, plants carrying wild type ZED1
229
transgene restored resistance to P. syringae hopZ1a, whereas plants expressing the
230
ZED1I24E and ZED1G29E variants were fully susceptible compared to the plants
231
complemented with wild-type ZED1 (Figure 2G). All constructs accumulated
232
ZED1-HA protein (Supplemental Figure 2), suggesting that the lack of resistance in
233
ZED1I24E and ZED1G29E plants was not because of a lack of protein. We further
234
verified these results by testing two independent T2 lines for each construct, and the
235
results are completely consistent with those observed in T1 plants (Supplemental
236
Figure 3). We further tested representative transgenic lines (zar1 background)
237
carrying ZAR1V544E, ZAR1H597E and ZAR1W825A/F839A variants for resistance to P.
238
syringae hopZ1a. These lines accumulate similar amounts of ZAR1 protein and have
239
been shown to be compromised in resistance to X. campestris campestris avrAC
240
(Wang et al., 2019a). As expected, the wild-type ZAR1, but not mutant variants,
241
restored disease resistance (Figure 2H). Among the three independent experiments,
242
the ZAR1H597E line showed partial resistance compared to controls, but this was not
243
repeated in the other two experiments (Supplemental Figure 4). Taken together, our
244
results support the idea that ZAR1 interacts with ZED1 in a similar manner shown by
245
the structure of ZAR1-RKS1 complex, and this interaction is required for
246
oligomerization in vivo, cell death and disease resistance (Wang et al., 2019a).
247
Oligomerization of ZAR1 is critical for HopZ1a induced immunity
248
We further compared structural requirements for ZAR1 oligomerization in vivo and
249
ZAR1 resistosome assembly in vitro. In the ZAR1-RKS1-PBL2UMP resistosome, the
250
interaction of two adjacent ZAR1 proteins is mediated by all the structural domains of
251
ZAR1 including LRR domain, NB domain, helical domain 1 (HD1), winged-helix
252
domain (WHD) and CC domain (Wang et al., 2019b). In addition, ATP binding is
253
also essential for oligomerization of ZAR1-RKS1-PBL2UMP, as phosphate group of
254
the bound dATP forms a hydrogen bond with Ser403 which results in further
255
stabilizing the active conformation of ZAR1. We selected three mutations, including
256
ZAR1W150A and ZAR1S152E in the ZAR1NBD- ZAR1NBD interface, and ZAR1R194A/R297A
257
in residues specifically interacting with dATP but not ADP. When co-expressed with
258
HopZ1a and ZED1, the mutations ZAR1W150A and ZAR1S152E completely abolished
259
HopZ1a-induced oligomerization of ZAR1E11A/E18A in BN-PAGE assay (Figure 3A),
260
indicating
261
ZAR1-RKS1-PBL2UMP in vitro are also essential for HopZ1a-induced oligomerization
262
of ZAR1 in protoplasts. The introduction of the ZAR1R194A/R297A mutation led to a
that
these
residues
required
for
the
oligomeric
complex
of
263
smaller oligomeric complexes with a molecular mass of ~700 kDa irrespective of
264
HopZ1a or HopZ1aC216A (Figure 3A), suggesting that the mutation ZAR1R194A/R297A
265
resulted in aberrant complex formation in protoplasts that no longer respond to the
266
effector.
267
Oligomerization of ZAR1 play crucial roles in AvrAC-induced cell death and disease
268
resistance (Wang et al., 2019b). To further evaluate the impact of these mutations on
269
HopZ1a-induced cell death, we co-expressed indicated constructs in Arabidopsis
270
protoplasts. ZAR1W150A and ZAR1S152E mutant proteins showed a reduction of
271
HopZ1a-induced cell death compared to wild-type ZAR1, and ZAR1R194A/R297A
272
completely lost cell death triggering activity (Figure 3B). To determine the role of
273
these mutations on HopZ1a-induced disease resistance, wild-type, zar1 and
274
representative transgenic lines complemented with wild-type ZAR1, ZAR1W150A,
275
ZAR1S152E and ZAR1R194A/R297A were challenged with P. syringae hopZ1a. These
276
transgenic lines were selected because they have been fully characterized and tested
277
for resistance to X. campestris campestris avrAC (Wang et al., 2019b). The
278
ZAR1W150A, ZAR1S152E and ZAR1R194A/R297A lines were significantly more susceptible
279
to P. syringae hopZ1a compared to wild-type lines (Figure 3C), indicating the
280
HopZ1a-induced normal oligomerization activity of ZAR1 is necessary for
281
ZAR1-mediated disease resistance. The lack of cell death activity and disease
282
resistance function for ZAR1R194A/R297A further confirm that the aberrant aggregation
283
at about 700 kDa is non-functional.
284
N-terminal α helix of ZAR1 is critical for HopZ1a induced immunity
285
In the ZAR1 resistosome, the very N-terminal α1 helix of ZAR1 forms a
286
funnel-shaped structure that is required for AvrAC-induced cell death and
287
PM-association of ZAR1. However, the α1 helix does not appear to be necessary for
288
ZAR1 oligomerization in vitro (Wang et al., 2019b). We tested various α1 helix
289
variants for ZAR1 oligomerization in vivo by using BN-PAGE. Arabidopsis
290
protoplasts of the zar1 background were transfected with ZAR1F9A/L10A/L14A, which
291
carried mutations in residues located at the outer surface of the funnel-shaped
292
structure, and ZAR1∆10, which lacked the first 10 residues of the α helix. Both
293
ZAR1F9A/L10A/L14A and ZAR1∆10 retained the ability to form oligomeric complex as
294
indicated by BN-PAGE assay, although the amount is less compared to ZAR1E11A/E18A
295
(Figure 4A). Thus the α1 helix did not appear to be required for ZAR1
296
oligomerization in vivo, which is consistent with our previous in vitro study (Wang et
297
al., 2019b).
298
We next asked whether the α1 helix mutations affect HopZ1a-induced cell death as
299
they did to the AvrAC-induced cell death (Wang et al., 2019b). zar1 protoplasts
300
transfected with ZAR1F9A/L10A/L14A, ZAR1∆10 , or ZAR1E11A/E18A along with HopZ1a
301
showed much less cell death compared to those expressing wild-type ZAR1 and
302
HopZ1a (Figure 4B). To further examine the effect of these mutations on
303
HopZ1a-induced
304
complemented with wild-type ZAR1, ZAR1F9A/L10A/L14A, ZAR1∆10, or ZAR1E11A/E18A
305
were challenged with P. syringae hopZ1a. These lines accumulate similar amounts of
306
ZAR1 protein and have been tested for resistance to X. campestris campestris avrAC
307
(Wang et al., 2019b). The ZAR1F9A/L10A/L14A, ZAR1∆10, and ZAR1E11A/E18A lines were
308
significantly more susceptible compared to wild-type ZAR1 line (Figure 4C),
309
indicating that the α1 helix plays a critical role not only AvrAC-specified disease
310
resistance, but also HopZ1a-specified resistance.
311
Together, the results described above indicate that both AvrAC and HopZ1a induce
312
oligomerization of ZAR1 in vivo, which can be detected by using BN-PAGE or gel
313
filtration. These assays are probably also suitable for analyses of other NLR proteins
314
in vivo. Indeed, an independent study showed that BN-PAGE can be used to detect the
315
oligomerization NLR protein RPP7 when co-expressed with an immune activating
316
allele of RPW8 protein (Li et al., 2020). Our results also support that structural
317
requirements for HopZ1a-induced ZAR1 oligomerization and immunity are highly
318
consistent with ZAR1-RKS1-PBL2UMP resistosome assembly in vitro. Thus ZAR1
disease
resistance,
Transgenic
lines
of
zar1
background
319
resistosome formation in vivo is important for HopZ1a- and AvrAC-triggered
320
immunity.
321
METHODS
322
Plant Materials and Growth Conditions
323
Arabidopsis thaliana plants used in this study include Col-0, zed1, zar1-1 and zar1
324
transgenic lines complemented with various mutants (Lewis et al., 2010; Wang et al.,
325
2019a; Wang et al., 2019b). The plants used for protoplasts transfection and pathogen
326
inoculation were grown in soil with a photoperiod of 10 h of white light and 14 h
327
darkness at 23°C for 4-5 weeks. The intensity of white light was 90 µE m−2 s−1
328
provided with white fluorescent bulbs.
329
Constructs, Transgenic Plants and Protoplast Transformation
330
To generate ProZED1:ZED1-HA transgenic plants with mutant variants, the
331
full-length genomic DNA fragments containing promoter and coding sequence of
332
ZED1 were PCR amplified from Col-0 genomic DNA and cloned into
333
pCAMBIA1300 vector. The constructs of ZED1 mutations were generated by
334
site-directed mutagenesis. These constructs were introduced into zed1 mutant plants
335
by Agrobacterium tumefaciens-mediated transformation. Transgenic plants of T2
336
generation were identified for transgene expression by anti-HA immunoblot.
337
ProZAR1:ZAR1-HA and
338
Constructs of AvrAC, PBL2, RKS1, ZED1, HopZ1a and ZAR1 were under control of
339
the 35S promoter and have been reported previously (Wang et al., 2015; Wang et al.,
340
2019a; Wang et al., 2019b). New ZED1 mutant constructs were generated by
341
site-directed mutagenesis, and all these genes were cloned to pUC19-35S-HA-RBS or
342
pUC19-35S-FLAG-RBS for protoplasts transfection as previously described (He et al.,
343
2007)(He et al., 2007).
344
Blue Native PAGE assay
345
To determine ZAR1 oligomerization in protoplasts, Blue Native polyacrylamide gel
346
electrophoresis (BN-PAGE) was performed using the Bis-Tris Native PAGE system
347
(Invitrogen) according to the manufacturer’s instructions. Briefly, protoplasts
348
expressing indicated plasmids were incubated for 12 h, and total protein was extracted
349
with 1×Native PAGE Sample Buffer (Invitrogen) containing 1% digitonin and
350
protease inhibitor cocktail. Protein samples containing 0.25% Coomassie G-250 was
351
loaded and run on a Native PAGE 3-12% Bis-Tris gel. The proteins were then
352
transferred to PVDF membranes using NuPAGE Transfer Buffer, followed by
353
immunoblot analysis with the desired antibodies.
354
Gel filtration assay
355
For gel filtration assay, Arabidopsis protoplasts were transfected with the indicated
356
plasmids and incubated with 1 mM laCl3 for 12 h. Total protein was then isolated with
357
protein extraction buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.3%
358
Trition-X100, 1 mM DTT, protease inhibitor cocktail). Protein samples were filtered
359
through a 0.22 µm low-protein binding filter (Millipore) and analyzed by gel filtration.
360
AKTA Purifier system (GE Healthcare) was used to perform these experiments and
361
Superdex 200 Increase 10/300 GL column (GE Healthcare) was used at a flow rate of
362
0.4 mL/min. The buffer used in elution containing 50 mM HEPES [pH 7.5], 150 mM
363
NaCl, 1 mM EDTA, 1 mM DTT. The eluted fractions were analyzed by SDS-PAGE
364
and detected by anti-HA immunoblot.
365
Co-immunoprecipitation assay
366
For co-immunoprecipitation assay, protoplasts were transfected with the indicated
367
plasmids and incubated for 12 h. Total protein was extracted with protein extraction
368
buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 1 mM EDTA, 0.5% Trition-X100, 1
369
mM DTT, protease inhibitor cocktail). 50 µL anti-FLAG M2 agarose (Sigma) were
370
incubated with total protein for 2 h at 4°C, washed six times with protein extraction
371
buffer, and eluted with 60 µL of 0.5 mg/mL 3 × FLAG peptide (Sigma) for 1 h at 4°C.
372
Immunoprecipitates were separated on a 10% SDS PAGE gel and detected by the
373
desired antibodies.
374
Protoplasts viability assay
375
For protoplast viability assay, the zed1 or zar1 protoplasts transfected with the
376
indicated plasmids were incubated for 12 h as previously described (Wang et al., 2019;
377
Wang et al., 2019b). Cell viability was determined by the Cell Titer-Glo Luminescent
378
Cell Viability Assay according to the manufacturer’s instructions (Promega, G7570).
379
ATP-based Luminescence intensity were measured by the EnSpire Multimode plate
380
Reader (Perkin Elmer). The experimental treatment cells were normalized against the
381
control, assigned as 100 percent, to calculate the percentage of cell survival.
382
Pathogen Strains and Inoculations
383
The bacterial strain P. syringae DC3000 carrying hopZ1a, which was originally
384
isolated from P. syringae pv. syringae A2 (Lewis et al., 2008), was used in this work.
385
For bacterial growth assay, 4-week-old Arabidopsis plants were infiltrated with
386
bacteria at 1 × 106 colony-forming units/mL by a needleless syringe. The bacterial
387
number in leaves was determined at 3 d after inoculation.
388
Accession Numbers
389
Sequences of genes described in this work can be found in The Arabidopsis
390
Information Resource using the following accession numbers: ZAR1 (AT3G50950)
391
and ZED1 (AT3G57750).
392
SUPPLEMENTAL INFORMATION
393
Supplemental Information is available at Molecular Plant Online.
394
AUTHOR CONTRIBUTIONS
395
J.-M.Z. designed the research. M.H. and J.Q. performed the experiments. J.-M.Z. and
396
G.B. wrote the manuscript.
397
ACKNOWLEDGMENTS
398
The work was supported by grants from Ministry of Science and Technology of China
399
(2016YFD0100601), the Chinese Academy of Sciences international cooperation key
400
project grant GJHZ1311, and the State Key Laboratory of Plant Genomics
401
(SKLPG2016B-2) to J.-M.Z..
402
REFERENCES
403
Ade, J., DeYoung, B.J., Golstein, C., and Innes, R.W. (2007). Indirect activation of
404
a plant nucleotide binding site–leucine-rich repeat protein by a bacterial protease.
405
Proc. Natl. Acad. Sci. USA 104: 2531-2536.
406
Bastedo, D.P., Khan, M., Martel, A., Seto, D., Kireeva, I., Zhang, J., Masud, W.,
407
Millar, D., Lee, J.Y., Lee, A.H., et al. (2019). Perturbations of the ZED1
408
pseudokinase activate plant immunity. PLoS Pathog. 15: e1007900.
409
Baudin, M., Hassan, J.A., Schreiber, K.J., and Lewis, J.D. (2017). Analysis of the
410
ZAR1 immune complex reveals determinants for immunity and molecular
411
interactions. Plant Physiol. 174:2038-2053.
412
Cesari, S., Moore, J., Chen, C., Webb, D., Periyannan, S., Mago, R., Bernoux, M.,
413
Lagudah, E.S., and Dodds, P.N. (2016). Cytosolic activation of cell death and stem
414
rust resistance by cereal MLA-family CC–NLR proteins. Proc. Natl. Acad. Sci. USA
415
113: 10204-10209.
416
Chung, E.H., da Cunha, L., Wu, A.J., Gao, Z., Cherkis, K., Afzal, A.J., Mackey,
417
D., and Dangl, J.L. (2011). Specific threonine phosphorylation of a host target by
418
two unrelated type III effectors activates a host innate immune receptor in plants. Cell
419
Host Microbe 9:125-136.
420
Cui, H., Tsuda, K., and Parker, J.E. (2015). Effector-triggered immunity: from
421
pathogen perception to robust defense. Annu. Rev. Plant Biol. 66:487-511.
422
Dangl, J.L., and McDowell, J.M. (2006). Two modes of pathogen recognition by
423
plants. Proc. Natl. Acad. Sci. USA 103:8575-8576.
424
Dodds, P.N., Lawrence, G.J., Catanzariti, A.M., Teh, T., Wang, C.I., Ayliffe, M.A.,
425
Kobe, B., and Ellis, J.G. (2006). Direct protein interaction underlies gene-for-gene
426
specificity and coevolution of the flax resistance genes and flax rust avirulence genes.
427
Proc. Natl. Acad. Sci. USA 103:8888-8893.
428
Dodds, P.N., and Rathjen, J.P. (2010). Plant immunity: towards an integrated view
429
of plant-pathogen interactions. Nat. Rev. Genet. 11:539-548.
430
El Kasmi, F., Chung, E.H., Anderson, R.G., Li, J., Wan, L., Eitas, T.K., Gao, Z.,
431
and Dangl, J.L. (2017). Signaling from the plasma-membrane localized plant
432
immune receptor RPM1 requires self-association of the full-length protein. Proc. Natl.
433
Acad. Sci. USA 114: E7385-E7394.
434
Feng, F., and Zhou, J.M. (2012). Plant-bacterial pathogen interactions mediated by
435
type III effectors. Curr. Opin. Plant Biol. 15:469-476.
436
Feng, F., Yang, F., Rong, W., Wu, X., Zhang, J., Chen, S., He, C., and Zhou, J.-M.
437
(2012). A Xanthomonas uridine 5’-monophosphate transferase inhibits plant immune
438
kinases. Nature 485:114–118.
439
Fu, Z.Q., and Dong, X. (2013). Systemic acquired resistance: turning local infection
440
into global defense. Annu. Rev. Plant Biol. 64:839-863.
441
Gutierrez, J.R., Balmuth, A.L., Ntoukakis, V., Mucyn, T.S., Gimenez‐ ‐Ibanez, S.,
442
Jones, A.M., and Rathjen, J.P. (2010). Prf immune complexes of tomato are
443
oligomeric and contain multiple Pto-like kinases that diversify effector recognition.
444
Plant J. 61: 507-518.
445
He, P., Shan, L., and Sheen, J. (2007). The use of protoplasts to study innate
446
immune responses. In Plant-Pathogen Interactions (Springer), pp. 1-9.
447
Hu, Z., Zhou, Q., Zhang, C., Fan, S., Cheng, W., Zhao, Y., Shao, F., Wang, H.-W.,
448
Sui, S.-F., and Chai, J. (2015). Structural and biochemical basis for induced
449
self-propagation of NLRC4. Science 350: 399-404.
450
Jones, J.D., and Dangl, J.L. (2006). The plant immune system. Nature 444:323–329.
451
Jones, J.D., Vance, R.E., and Dangl, J.L. (2016). Intracellular innate immune
452
surveillance devices in plants and animals. Science 354: aaf6395.
453
Khan, M., Subramaniam, R., and Desveaux, D. (2016). Of guards, decoys, baits
454
and traps: pathogen perception in plants by type III effector sensors. Curr. Opin.
455
Microbiol. 29: 49-55
456
Kofoed, E.M., and Vance, R.E. (2011). Innate immune recognition of bacterial
457
ligands by NAIPs determines inflammasome specificity. Nature 477: 592.
458
Kourelis, J., and van der Hoorn, R.A.L. (2018). Defended to the nines: 25 Years of
459
resistance gene cloning identifies nine mechanisms for R protein function. Plant Cell
460
30:285-299.
461
Krasileva, K.V., Dahlbeck, D., and Staskawicz, B.J. (2010). Activation of an
462
Arabidopsis resistance protein is specified by the in planta association of its
463
leucine-rich repeat domain with the cognate oomycete effector. Plant Cell
464
22:2444-2458.
465
Laflamme, B., Dillon, M.M., Martel, A., Almeida, R.N., Desveaux, D., and
466
Guttman, D.S. (2020). The pan-genome effector-triggered immunity landscape of a
467
host-pathogen interaction. Science 367: 763-768.
468
Lewis, J.D., Abada, W., Ma, W., Guttman, D.S., and Desveaux, D. (2008). The
469
HopZ family of Pseudomonas syringae type III effectors require myristoylation for
470
virulence and
471
190:2880-2891.
472
Lewis, J.D., Wu, R., Guttman, D.S., and Desveaux, D. (2010). Allele-specific
473
virulence attenuation of the Pseudomonas syringae HopZ1a type III effector via the
474
Arabidopsis ZAR1 resistance protein. PLoS Genet. 6:e1000894.
475
Lewis, J.D., Lee, A.H.-Y., Hassan, J.A., Wan, J., Hurley, B., Jhingree, J.R., Wang,
476
P.W., Lo, T., Youn, J.-Y., Guttman, D.S., and Desveaux, D. (2013). The Arabidopsis
477
ZED1 pseudokinase is required for ZAR1-mediated immunity induced by the
478
Pseudomonas syringae type III effector HopZ1a. Proc. Natl. Acad. Sci. USA
479
110:18722–18727.
480
Li, L., Habring, A., Wang, K., and Weigel, D. (2020). Atypical resistance protein
481
RPW8/HR triggers oligomerization of the NLR immune receptor RPP7 and
avirulence functions
in
Arabidopsis
thaliana.
J. Bacteriol.
482
autoimmunity. Cell Host Microbe.
483
Liu, C., Cui, D., Zhao, J., Liu, N., Wang, B., Liu, J., Xu, E., Hu, Z., Ren, D., Tang,
484
D., and Hu, Y. (2019). Two Arabidopsis receptor-like cytoplasmic kinases SZE1 and
485
SZE2 associate with the ZAR1-ZED1 complex and are required for effector-triggered
486
immunity. Mol. Plant 12: 967-983.
487
Maekawa, T., Kufer, T.A., and Schulze-Lefert, P. (2011). NLR functions in plant
488
and animal immune systems: so far and yet so close. Nat. Immunol. 12:817-826.
489
Mestre, P., and Baulcombe, D.C. (2006). Elicitor-mediated oligomerization of the
490
tobacco N disease resistance protein. Plant Cell 18: 491-501.
491
Monaghan, J., and Zipfel, C. (2012). Plant pattern recognition receptor complexes at
492
the plasma membrane. Curr. Opin. Plant Biol. 15:349-357.
493
Schultink, A., Qi, T., Bally, J., and Staskawicz, B. (2019). Using forward genetics in
494
Nicotiana benthamiana to uncover the immune signaling pathway mediating
495
recognition
496
221:1001-1009.
497
Seto, D., Koulena, N., Lo, T., Menna, A., Guttman, D.S., and Desveaux, D. (2017).
498
Expanded type III effector recognition by the ZAR1 NLR protein using ZED1-related
499
kinases. Nat. Plants 3:17027.
500
Tang, D., Wang, G., and Zhou, J.M. (2017). Receptor kinases in plant-pathogen
501
Interactions: more than pattern recognition. Plant Cell 29:618-637.
502
van der Hoorn, R.A., and Kamoun, S. (2008). From guard to decoy: a new model
503
for perception of plant pathogen effectors. Plant Cell 20:2009-2017.
504
Wang, G., Roux, B., Feng, F., Guy, E., Li, L., Li, N., Zhang, X., Lautier, M.,
505
Jardinaud, M.F., Chabannes, M., et al. (2015). The decoy substrate of a pathogen
506
effector and a pseudokinase specify pathogen-induced modified-self recognition and
507
immunity in plants. Cell Host Microbe 18:285-295.
of the Xanthomonas
perforans
effector XopJ4.
New Phytol.
508
Wang, J., Wang, J., Hu, M., Wu, S., Qi, J., Wang, G., Han, Z., Qi, Y., Gao, N.,
509
Wang, H.W., et al. (2019a). Ligand-triggered allosteric ADP release primes a plant
510
NLR complex. Science 364:eaav5868.
511
Wang, J., Hu, M., Wang, J., Qi, J., Han, Z., Wang, G., Qi, Y., Wang, H.W., Zhou,
512
J.M., and Chai, J. (2019b). Reconstitution and structure of a plant NLR resistosome
513
conferring immunity. Science 364:eaav5870.
514
Ravensdale, M., Bernoux, M., Ve, T., Kobe, B., Thrall, P.H., Ellis, J.G., and
515
Dodds, P.N. (2012). Intramolecular interaction influences binding of the flax L5 and
516
L6 resistance proteins to their AvrL567 ligands. PLoS Pathog. 8:e1003004.
517
Yang, J., Zhao, Y., Shi, J., and Shao, F. (2013). Human NAIP and mouse NAIP1
518
recognize bacterial type III secretion needle protein for inflammasome activation.
519
Proc. Natl. Acad. Sci. USA 110: 14408-14413.
520
Zhou, J.M., and Chai, J. (2008). Plant pathogenic bacterial type III effectors subdue
521
host responses. Curr. Opin. Microbiol. 11: 179-185.
522
Figure legends
523
Figure 1. AvrAC and HopZ1a induce oligomerization of ZAR1 in Arabidopsis
524
protoplasts.
525
(A) BN-PAGE assay for AvrAC-induced oligomerization of ZAR1 in Arabidopsis
526
protoplasts. The indicated constructs were transfected into zar1 protoplasts. Total
527
protein was subjected to BN-PAGE and detected by immunoblotting with anti-HA
528
and anti-FLAG antibodies. All assays were performed three times, and a
529
representative photograph is shown.
530
(B) Gel filtration assay for AvrAC-induced oligomerization of ZAR1-RKS1-PBL2UMP
531
in Arabidopsis protoplasts. ZAR1-HA, RKS1-HA and PBL2-HA were coexpressed
532
with AvrACH469A (upper panel) or AvrAC (bottom panel) in Col-0 protoplasts,
533
incubated with 1 mM LaCl3, and total protein was subjected to gel filtration. The
534
eluted fractions were analyzed by immunoblotting with anti-HA antibody. Relative
535
grayscales (right panel) indicate the arbitrary densitometry units of different proteins
536
shown by immunoblots (left panel). Dashed lines indicate positions of standard
537
molecular masses. All assays were performed three times, and a representative
538
photograph is shown.
539
(C) BN-PAGE assay for HopZ1a-induced oligomerization of ZAR1 in Arabidopsis
540
protoplasts. The indicated constructs were transfected into zar1 protoplasts, and total
541
protein was subjected to BN-PAGE and immunoblot analysis. All assays were
542
performed three times, and a representative photograph is shown.
543
Figure 2. ZED1-ZAR1 interaction is critical for HopZ1a-induced immunity
544
(A and B) ZED1 (A) or ZAR1 (B) mutations reduce or abolish ZAR1-ZED1
545
interaction in protoplasts. The indicated constructs were transfected into zed1 and
546
zar1 protoplasts, respectively. Total protein was subjected to co-IP assays. All assays
547
were performed three times, and a representative photograph is shown.
548
(C and D) ZED1 (C) or ZAR1 (D) mutations abolish HopZ1a-induced
549
oligomerization of ZAR1 in protoplasts. The indicated constructs were transfected
550
into zed1 and zar1 protoplasts, respectively. Total protein was subjected to BN-PAGE.
551
All assays were performed three times, and a representative photograph is shown.
552
(E and F) The ZAR1-ZED1 interaction is required for HopZ1a-induced cell death in
553
protoplasts. ZED1 mutants (E) were co-expressed with HopZ1a and ZAR1 in zed1
554
protoplasts, and ZAR1 mutants (F) were co-expressed with HopZ1a and ZED1 in
555
zar1 protoplasts. The protoplasts were incubated for 12 h and cell viability was
556
measured by the Cell Titer-Glo Luminescent Cell Viability Assay. Data are presented
557
as mean ± SE. Different letters indicate significant difference at P < 0.05. (n =3,
558
one-way ANOVA, Tukey post-test, three independent experiments).
559
(G and H) Compromising the ZAR1-ZED1 interaction impairs HopZ1a-induced
560
antibacterial immunity. (G) Col-0, zed1, and T1 transgenic plants (zed1 background)
561
carrying the indicated ZED1 variants (G) and T2 transgenic lines (zar1 background)
562
carrying the indicated ZAR1 variants (H) were inoculated with P. syringae hopZ1a,
563
and bacterial population in the leaf was determined 3 d after inoculation. Boxplots
564
represent 16 and 24 data points from two (G) or three (H) independent experiments,
565
each of which contains eight plants. Colors indicate independent experiments.
566
Different letters indicate significant difference at P < 0.05. (one-way ANOVA, Tukey
567
post-test).
568
Figure 3. Oligomerization of ZAR1 is critical for HopZ1a-induced immunity
569
(A) ZAR1 residues required for resistosome assembly in vitro are essential for
570
HopZ1a-induced oligomerization in vivo. zar1 protoplasts expressing indicated
571
proteins were incubated for 12 h, and total protein was subjected to BN-PAGE. All
572
assays were performed three times, and a representative photograph is shown.
573
(B) Oligomerization of ZAR1 is required for HopZ1a-induced cell death in
574
protoplasts. ZAR1 mutants were co-expressed with HopZ1a and ZED1 in zar1
575
protoplasts, and cell viability was measured by the Cell Titer-Glo Luminescent Cell
576
Viability Assay. Data are presented as mean ± SE. Different letters indicate significant
577
difference at P < 0.05. (n =3, one-way ANOVA, Tukey post-test, three independent
578
experiments).
579
(C) Compromising oligomerization of ZAR1 impairs HopZ1a-induced antibacterial
580
immunity. Transgenic lines were inoculated with P. syringae hopZ1a, and bacterial
581
population in the leaf was determined 3 d after inoculation. Boxplots represent 24 data
582
points from three biological replicates, each of which contains eight technical
583
replicates. Colors indicate biological replicates. Different letters indicate significant
584
difference at P < 0.05. (one-way ANOVA, Tukey post-test).
585
Figuer 4. The N-terminal α1 helix of ZAR1 is critical for HopZ1a-induced
586
immunity
587
(A) Functional analysis of the N-terminal α1 helix of ZAR1 in HopZ1a-induced
588
ZAR1 oligomerization. zar1 protoplasts expressing indicated proteins were incubated
589
for 12 h, and total protein was subjected to BN-PAGE. All assays were performed
590
three times, and a representative photograph is shown.
591
(B) The α1 helix of ZAR1 is essential for HopZ1a-induced cell death in protoplasts.
592
ZAR1 mutants were coexpressed with HopZ1a and ZED1 in zar1 protoplasts, and cell
593
viability was measured by the Cell Titer-Glo Luminescent Cell Viability Assay. Data
594
are presented as mean ± SE. Different letters indicate significant difference at P <
595
0.05. (n =3, one-way ANOVA, Tukey post-test, three independent experiments).
596
(C) The α1 helix of ZAR1 is required for HopZ1a-induced bacterial resistance.
597
Transgenic lines were inoculated with P. syringae hopZ1a, and bacterial population in
598
the leaf was determined 3 d after inoculation. Boxplots represent 24 data points from
599
three biological replicates, each of which contains eight technical replicates. Colors
600
indicate biological replicates. Different letters indicate significant difference at P <
601
0.05. (one-way ANOVA, Tukey post-test).
602
Supplemental Figure 1.
603
affects AvrAC-induced protein degradation.
604
(A) LaCl3 blocks HopZ1a-induced cell death. Col-0 and zar1 plants were infiltrated
605
with P. syringae DC3000 hopZ1a (1 × 108 cfu/mL) and indicated concerntration of
606
LaCl3, and Macroscopic HR in leaves was recorded 5 h after inoculation.
607
(B) LaCl3 inhibits AvrAC-induced protein degradation. Protoplasts expressing
608
indicated proteins were incubated with LaCl3 for 12 h and total protein was subjected
609
to SDS-PAGE and detected by immunoblotting with anti-FLAG antibody.
610
Supplemental Figure 2. Accumulation of ZED1, ZEDI24E and ZED1G29E proteins
611
in T1 transgenic plants.
612
Positive T1 plants carrying ZED1 variant transgenes were pooled, and total protein
613
was isolated from the indicated transgenic lines and anti-HA immunoblot was done to
614
detect the accumulation of ZED1-HA protein. Upper and lower bands are non-specific
615
cross-reacting proteins. Ponceau staining of Rubisco indicates loading of protein.
616
Supplemental Figure 3. ZED1 mutants impaired in ZAR1-interaction fail to
617
confer antibacterial resistance.
618
Col-0, zed1, and T2 transgenic lines (zed1 background) carrying the indicated
619
transgenes were inoculated with P. syringae DC3000 hopZ1a, and bacterial growth
620
assay was performed as in Fig. 2G. Boxplots represent 8 data points from a single
621
experiment containing eight plants/line. Different letters indicate significant
622
difference at P < 0.05. (one-way ANOVA, Tukey post-test).
623
Supplemental Figure 4. ZAR1 mutants impaired in ZED1-interaction fail to
LaCl3 blocks HopZ1a-induced cell death and partially
624
confer antibacterial resistance (related to Figure 2H).
625
Col-0, zar1, and T2 transgenic lines (zar1 background) carrying the indicated
626
transgenes were inoculated with P. syringae DC3000 hopZ1a, and bacterial
627
population in the leaf was determined 3 d after inoculation. Boxplots represent 8 data
628
points from one replicate of this experiment shown in Figure 2H with green dots.
629
Different letters indicate significant difference at P < 0.05. (one-way ANOVA, Tukey
630
post-test).
C + + + +
+ + + + -
ZAR1E11A/E18A-HA ZED1-FLAG HopZ1a-FLAG HopZ1aC216A-FLAG
kDa 1048
Oligomer 720 α-HA
480 242
+ + -
+ + +
+ + + -
kDa 1048
Oligomer 720 α-HA
480
100 70
AvrAC-FLAG
55
PBL2-FLAG RKS1-FLAG
40
Rubisco
10% SDS-PAGE
242 α-HA
α-HA
100
55
HopZ1a-FLAG ZED1-FLAG
40
Rubisco
3-12% native PAGE
+ + -
3-12% native PAGE
ZAR1E11A/E18A-HA PBL2-FLAG RKS1-FLAG AvrAC-FLAG AvrACH469A-FLAG
10% SDS-PAGE
A
B
Input 7
8
9
10
11
12
13 mL
kDa 100
ZAR1 AvrACH469A
70
PBL2 RKS1
55 40
Relative grayscale
669 kDa 440 kDa 400000 400000 300000 300000 200000 200000
AvrAC PBL2 RKS1
10
11
12
13 mL kDa 100 70 55 40
Relative grayscale
ZAR1
9
440 kDa
8
8.5 8.5
9 9
9.5 9.5
10 10
10.5 11 11 11.5 11.5 12 12 12.5 12.5 13 13 10.5
Elution volume (mL)
669 kDa 440 kDa 8
669 kDa
100000 100000 00
Input 7
AvrACH469A
ZAR1 PBL2 RKS1
AvrAC
500000 500000
ZAR1 PBL2 RKS1
400000 400000
300000 300000
669 kDa
200000 200000
440 kDa
100000 100000 00 7.5 7.5
8
8.5 8.5
99
9.5 10 10 10.5 11 11.5 11.5 12 12 12.5 12.5 13 13 9.5 10.5 11
Elution volume (mL)
B -
40
ZAR1-FLAG
ZAR1-HA
E
C + + + -
+ + +
+ + + -
kDa 1048 720
α-FLAG
480 242
α-FALG
100 55
α-HA
40 Rubisco
10% SDS-PAGE 3-12% native PAGE
ZED1-HA ZED1I24E-HA HopZ1a-HA HopZ1aC216A-HA
+ + +
+ + + -
+ + +
+ + + -
G
kDa
1048 α-HA
720 480 242
α-HA α-FLAG Rubisco
100 55 40
10% SDS-PAGE 3-12% native PAGE
+ + +
α-FLAG IP
40
120 100 80 60 40 20 0
120 100 80 60 40 20 0
a c d
b
F
D ZAR1E11A/E18A-HA ZAR1W825A/F839A-HA ZED1-FLAG HopZ1a-FLAG HopZ1aC216A-FLAG
100
ZED1-FLAG
100
ZAR1E11A/E18A-FLAG
40
Cell viability (%)
ZED1-HA
ZED1-FLAG
Cell viability (%)
100
100
H
Bacteria [log10 (CFU cm-2)]
ZAR1-FLAG
kDa
ZAR1-HA
Bacteria [log10 (CFU cm-2)]
40
Input
ZED1-HA
α-FLAG IP
kDa
Input
A
a
c
c
c b
7
b
b
b
6 a
5
a
4 3 2
6 5 4 3 2
b
b
7 a
a
b
b
B
ZAR1E11A/E18A-HA ZAR1W150A-HA ZAR1S152E-HA ZAR1R149A/R297A-HA ZED1-FLAG HopZ1a-FLAG HopZ1aC216A-FLAG
+ + +
+ + + -
+ + +
+ + + -
+ + +
+ + + -
+ + +
Cell viability (%)
A + + + -
120
α-HA
100 55
α-FLAG 40 Rubisco
d
60
c
40
b
20
C Bacteria [log10 (CFU cm-2)]
242
3-12% native PAGE
480
10% SDS-PAGE
α-HA
720
80
0
kDa 1048
a
a
100
b
7
b
6 a 5
4 3 2
1
a
b
b
B + + + -
+ + +
+ + + -
+ + +
+ + + -
kDa 1048
Oligomer 720 α-HA
480
α-HA α-FLAG
100 55 40
Rubisco
10% SDS-PAGE
242
120 100 80 60 40 20 0
a
ac
c d b
C Bacteria [log10 (CFU cm-2)]
+ + +
3-12% native PAGE
ZAR1E11A/E18A-HA ZAR1F9A/L10A/L14A-HA ZAR1Δ10-HA ZED1-FLAG HopZ1a-FLAG HopZ1aC216A-FLAG
Cell viability (%)
A
7 6 5 4 3 2
1
b a
bd c
d
bd