Bacterial Effectors Induce Oligomerization of Immune ...Bacterial Effectors Induce Oligomerization...

Molecular PlantResearch Report

Bacterial Effectors Induce Oligomerization ofImmune Receptor ZAR1 In VivoMeijuan Hu1,2,3, Jinfeng Qi1,2,3, Guozhi Bi1,* and Jian-Min Zhou1,2,*1State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of

Sciences, Beijing 100101, P. R. China

2CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing 100049, P. R. China

3These authors contributed equally to this article.

*Correspondence: Guozhi Bi ([email protected]), Jian-Min Zhou ([email protected])

https://doi.org/10.1016/j.molp.2020.03.004

ABSTRACT

Plants utilize nucleotide-binding, leucine-rich repeat receptors (NLRs) to detect pathogen effectors, lead-

ing to effector-triggered immunity. The NLR ZAR1 indirectly recognizes the Xanthomonas campestris pv.

campestris effector AvrAC and Pseudomonas syringae effector HopZ1a by associating with closely

related receptor-like cytoplasmic kinase subfamily XII-2 (RLCK XII-2) members RKS1 and ZED1,

respectively. ZAR1, RKS1, and the AvrAC-modified decoy PBL2UMP form a pentameric resistosome

in vitro, and the ability of resistosome formation is required for AvrAC-triggered cell death and disease

resistance. However, it remains unknown whether the effectors induce ZAR1 oligomerization in the plant

cell. In this study, we show that both AvrAC and HopZ1a can induce oligomerization of ZAR1 in Arabidopsis

protoplasts. Residuesmediating ZAR1–ZED1 interaction are indispensable for HopZ1a-induced ZAR1 olig-

omerization in vivo and disease resistance. In addition, ZAR1 residues required for the assembly of ZAR1

resistosome in vitro are also essential for HopZ1a-induced ZAR1 oligomerization in vivo and disease resis-

tance. Our study provides evidence that pathogen effectors induce ZAR1 resistosome formation in the

plant cell and that the resistosome formation triggers disease resistance.

Key words: plant immunity, NLR, ZAR1 resistosome, HopZ1a, oligomerization

Hu M., Qi J., Bi G., and Zhou J.-M. (2020). Bacterial Effectors Induce Oligomerization of Immune Receptor ZAR1In Vivo. Mol. Plant. 13, 793–801.

Published by the Molecular Plant Shanghai Editorial Office in association with

Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, CAS.

INTRODUCTION

Plants deploy cell-surface receptors and intracellular nucleotide-

binding (NB), leucine-rich repeat (LRR) receptors (NLRs) for path-

ogen perception (Dodds and Rathjen, 2010; Maekawa et al.,

2011; Monaghan and Zipfel, 2012). Cell-surface pattern

recognition receptors (PRRs) recognize microbe- and host-

derived molecular patterns and activate immunity (Tang et al.,

2017; Wang et al., 2019c). However, pathogenic microbes often

deliver effector proteins into the plant cell where they suppress

PRR signaling and promote microbial virulence (Feng and

Zhou, 2012). In turn, plants have evolved NLRs to monitor

effector proteins and trigger robust immune responses, which

often results in localized programmed cell death called

hypersensitive response (HR) and accumulation of defense

hormone salicylic acid (Jones and Dangl, 2006; Maekawa et al.,

2011; Fu and Dong, 2013; Cui et al., 2015).

Plant NLRs detect pathogen effectors either directly or indirectly

(Jones and Dangl, 2006; Cui et al., 2015; Kourelis and van der

Hoorn, 2018). While direct recognition follows a receptor–ligand

model in which an NLR physically interacts with an effector

(Dangl and McDowell, 2006; Dodds et al., 2006; Krasileva et al.,

2010; Ravensdale et al., 2012), more often an NLR forms a

complex with another host protein that is modified by pathogen

effectors (Chung et al., 2011; Wang et al., 2015). The modified

host protein is either an effector virulence target or a molecular

mimic of a virulence target, which are called ‘‘guardee’’ and

‘‘decoy,’’ respectively (van der Hoorn and Kamoun, 2008; Zhou

and Chai, 2008).

Arabidopsis HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) was first

identified as an NLR protein that is responsible for the recognition

of Pseudomonas syringae effector protein HopZ1a, an acetyl-

transferase belonging to the YopJ/HopZ superfamily (Lewis

et al., 2008, 2010). ZAR1 interacts with HOPZ-ETI-DEFICIENT 1

(ZED1), a pseudokinase from receptor-like cytoplasmic kinase

subfamily XII-2 (RLCK XII-2) required for HopZ1a recognition

(Lewis et al., 2013). Interestingly, ZAR1 also associates with

other RLCK XII-2 proteins, enabling a single ZAR1 to recognize

Molecular Plant 13, 793–801, May 4 2020 ª The Author 2020. 793

mailto:[email protected]


https://doi.org/10.1016/j.molp.2020.03.004

Molecular Plant Oligomerization of Immune Receptor ZAR1 In Vivo

multiple effectors (Wang et al., 2015; Khan et al., 2016). Thus, the

association of Arabidopsis ZAR1 with RESISTANCE-RELATED

KINASE 1 (RKS1) and ZED1-RELATED KINASE 3 (ZRK3) confers

resistance to Xanthomonas campestris pv. campestris carrying

AvrAC (an uridylyl transferase) and P. syringae carrying HopF2

(a ribosyltransferase), respectively (Wang et al., 2015; Seto

et al., 2017). A recent study indicates that ZAR1 also confers

resistance to P. syringae carrying three additional effectors,

HopBA1, HopO1, and HopX1 (Laflamme et al., 2020). ZAR1

also exists in Nicotiana benthamiana (NbZAR1) and interacts

with the RLCKXII-2member XOPJ4 IMMUNITY 2 (JIM2) to confer

resistance to Xanthomonas perforans carrying XopJ4, another

YopJ/HopZ superfamily acetyltransferase (Schultink et al.,

2019). AvrAC uridylylates multiple RLCK VII members and

inhibits PTI responses (Feng et al., 2012). Among these, PBL2

is a decoy (Guy et al., 2013; Wang et al., 2015), and the

uridylylated PBL2 (PBL2UMP) is recruited to the ZAR1–RKS1

complex to activate immunity (Wang et al., 2015). Although

HopZ1a can acetylate ZED1, it is not clear whether this

modification is required for HopZ1a-triggered disease resistance

(Lewis et al., 2013). Recent studies showed that HopZ1a

promotes interaction between ZED1 and several RLCK VII

members (Bastedo et al., 2019), and two closely related

RLCKs, SUPPRESSOR OF ZED1-D1 (SZE1) and SZE2, are

required for HopZ1a-induced disease resistance (Liu et al.,

2019), suggesting that these RLCK members may act as decoy

or guardee for HopZ1a recognition.

How NLRs initiate immune signaling is a fundamental question of

immunology in plants and animals. Animal NLR apoptosis inhib-

itory protein 2 (NAIP2) directly binds bacterial T3SS rod protein

PrgJ and then catalyzes its helper NLR NLRC4 polymerization

to form oligomeric inflammasome, which is mainly mediated by

NB and oligomerization domain (NOD) (Hu et al., 2015). In

addition, NAIP1 and NAIP5/6 form oligomeric inflammasomes

with NLRC4 in response to T3SS needle protein and bacterial

flagellin, respectively (Kofoed and Vance, 2011; Yang et al.,

2013). Plant and animal NLRs share similar structural domains

including a C-terminal LRR domain, a variable N-terminal

domain, and a conserved central NOD (an NACHT domain in

animals and an NB-ARC domain in plants) (Jones et al., 2016).

Full-length NLR proteins, such as RPM1, MLA, Sr33, Sr50,

RPS5, and Rx, self-associate before activation (Ade et al.,

2007; Gutierrez et al., 2010; Cesari et al., 2016; El Kasmi et al.,

2017), whereas the tobacco NLR protein N interacts with itself

only in the presence of the TMV P50 elicitor (Mestre and

Baulcombe, 2006), suggesting that self-association plays a role

in NLR-mediated defense signaling. However, whether effectors

have the ability to induce oligomerization of NLRs in the plant cell

remains unknown, as detection of NLR protein oligomerization

in vivo remains technically challenging.

ZAR1 is a canonical CC-NB-LRR that contains a C-terminal LRR

domain, an N-terminal coiled- coil (CC) domain, and NOD (Lewis

et al., 2010; Baudin et al., 2017). We recently solved by

cryoelectron microscopy (cryo-EM) three structures of ZAR1

protein complexes, including an inactive ZAR1–RKS1 complex,

an intermediate ZAR1–RKS1–PBL2UMP complex, and an active

ZAR1–RKS1–PBL2UMP pentameric complex (Wang et al.,

2019a, 2019b). The LRR domain of ZAR1 (ZAR1LRR) interacts

with the N terminus of RKS1 in the preformed complex.

794 Molecular Plant 13, 793–801, May 4 2020 ª The Author 2020.

PBL2UMP interacts with RKS1, resulting in conformation

changes of a RKS1 segment, which then sterically clashes with

the ZAR1 NB domain (ZAR1NBD) to dislodge ADP. In vitro, the

ADP-depleted ZAR1–RKS1–PBL2UMP complex binds dATP to

trigger drastic conformational changes in ZAR1 to expose sur-

faces required for intermolecular interactions between neigh-

boring ZAR1, leading to the assembly of the active pentamer

called resistosome. The structural features required for resisto-

some assembly are correlated with disease resistance and cell-

death function triggered by AvrAC. Furthermore, a segment of

the CC domain is organized into a barrel-like structure on plasma

membrane (PM), and this is also required for AvrAC-triggered dis-

ease resistance and cell death. However, whether ZAR1 oligo-

merizes in the plant cell remains to be investigated. Furthermore,

whether resistosome formation is similarly required for immune

activation by additional effector proteins such as HopZ1a re-

mains unknown.

Here, we show that Blue Native polyacrylamide gel electropho-

resis (BN-PAGE) and gel-filtration assays can be successfully

applied to detect NLR oligomerization in vivo and that both AvrAC

and HopZ1a can induce ZAR1 oligomerization in Arabidopsis

protoplasts. ZED1 and ZAR1 residues required for ZAR1–ZED1

interaction are essential for ZAR1 oligomerization. In addition,

ZAR1 residues required for in vitro assembly of ZAR1–RKS1–

PBL2UMP resistosome are essential for HopZ1a-induced ZAR1

oligomerization and disease resistance. Furthermore, N-terminal

a helix of ZAR1 is indispensable for HopZ1a-induced disease

resistance. These results indicate that bacterial effectors induce

ZAR1 oligomerization in vivo, confirming resistosome formation

observed in vitro.

RESULTS AND DISCUSSION

AvrAC and HopZ1a Induce Oligomerization of ZAR1 inArabidopsis Protoplasts

Amajor technical difficulty in the investigation of NLR protein acti-

vation in plants is the rapid cell death associated with NLR activa-

tion, which hampers protein detection. The cryo-EM structures of

the ZAR1 resistosome reveal that the very N-terminal amphi-

pathic helices are released and form a funnel-shaped structure,

which promotes ZAR1 association with PM (Wang et al.,

2019b). The inner surface of the funnel structure contains

several negatively charged residues. Mutations of two of these

residues, Glu11 and Glu18, impair ZAR1-mediated cell-death ac-

tivity without affecting oligomerization and PM association. We

sought to take advantage of these mutations and asked whether

effectors induce oligomerization of ZAR1E11A/E18A in vivo by using

a transient expression system in protoplasts. We transfected the

ZAR1E11A/E18A construct into zar1 protoplasts along with RKS1,

PBL2, and AvrAC or the catalytic-deficient variant AvrACH469A,

and subjected the total protein to BN-PAGE assay. In the

absence of AvrAC (resting state), the ZAR1E11A/E18A protein ex-

isted in a small molecular mass complex (Figure 1A), which

probably contains unidentified components. When co-

expressed with AvrACH469A, themajority of ZAR1E11A/E18A protein

remained in the low molecular mass, and a small amount of

ZAR1E11A/E18A shifted to a large complex of ~900 kDa. In

contrast, co-expression of AvrAC resulted in all ZAR1E11A/E18A

protein shifted to the large complex of about 900 kDa, which is

A C

B

Figure 1. AvrAC and HopZ1a Induce Oligomerization of ZAR1 in Arabidopsis Protoplasts.(A) BN-PAGE assay for AvrAC-induced oligomerization of ZAR1 in Arabidopsis protoplasts. The indicated constructs were transfected into zar1 pro-

toplasts. Total protein was subjected to BN-PAGE and detected by immunoblotting with anti-HA and anti-FLAG antibodies. All assays were performed

three times, and a representative photograph is shown.

(B)Gel-filtration assay for AvrAC-induced oligomerization of ZAR1-RKS1-PBL2UMP in Arabidopsis protoplasts. ZAR1-HA, RKS1-HA, and PBL2-HA were

co-expressed with AvrACH469A (upper panel) or AvrAC (bottom panel) in Col-0 protoplasts and incubated with 1 mM LaCl3, and total protein was sub-

jected to gel filtration. The eluted fractions were analyzed by immunoblotting with anti-HA antibody. Relative gray scales (right panel) indicate the arbitrary

densitometry units of different proteins shown by immunoblots (left panel). Closed triangles indicate positions of standard molecular masses. All assays

were performed three times, and a representative photograph is shown.

(C) BN-PAGE assay for HopZ1a-induced oligomerization of ZAR1 in Arabidopsis protoplasts. The indicated constructs were transfected into zar1

protoplasts, and total protein was subjected to BN-PAGE and immunoblot analysis. All assays were performed three times, and a representative

photograph is shown.

Oligomerization of Immune Receptor ZAR1 In Vivo Molecular Plant

similar to that of ZAR1 resistosome in vitro (Wang et al., 2019b).

The reason that a small amount of ZAR1E11A/E18A was present

in the ~900 kDa complex is not understood. AvrACH469A

displayed no activity in uridylylation of RIPK and PBL2 in vitro

(Feng et al., 2012; Wang et al., 2015), but a partial reduction of

flg22-induced FRK1 expression in protoplasts has been

observed previously (Feng et al., 2012), suggesting that

AvrACH469A retains residual activity in vivo.



We next sought to verify whether the observed oligomerization

can be observed with a wild-type ZAR1. To prevent cell death

and harvest sufficient protein for analysis, we treated Arabidop-

sis protoplasts with LaCl3, a channel blocker that is known to

inhibit AvrRpm1-induced cell death (El Kasmi et al., 2017). We

found that LaCl3 can indeed inhibit HR in Columbia-0 (Col-0)

leaves infiltrated with a high concentration of P. syringae

carrying hopZ1a (Supplemental Figure 1A). In Col-0 protoplasts

co-transfected with ZAR1, RKS1, PBL2, and AvrAC, no protein

was detected because of complete cell death. However, the

same protoplasts treated with LaCl3 allowed accumulation of

ZAR1, RKS1, and PBL2 proteins (Supplemental Figure 1B).

Still, the protein levels were much less compared with

protoplasts transfected with ZAR1, RKS1, and PBL2 in the

absence of AvrAC, suggesting that LaCl3 only partially

blocked deleterious effects during ZAR1 activation. Because

the BN-PAGE assay allows only a small volume of sample in

each well (15 ml or less), it was not suitable for the analysis of

the protein harvested after LaCl3 treatment in our study. Note

that proteins in BN-PAGE typically appear in smearing patterns,

which further hampers the detection. To circumvent the

problem we adopted gel-filtration assays, which allowed us to

scale up the amounts of protoplasts and protein. The wild-

type ZAR1 was co-transfected along with RKS1, PBL2, and

AvrAC or AvrACH469A into Col-0 protoplasts. Consistent with

the BN-PAGE data, when co-expressed with AvrACH469A, the

majority of ZAR1 and RKS1 co-migrated in a small molecular

mass complex indicative of an inactive preformed complex,

and only a small amount of ZAR1 and RKS1 migrated to a large

molecular mass complex (Figure 1B). When co-expressed with

AvrAC, almost all ZAR1 and RKS1 migrated to the large com-

plex (Figure 1B). These experiments validated the results

observed in the BN-PAGE assay using the ZAR1E11A/E18A

variant. Together, these observations suggest that the

oligomeric complex of ZAR1–RKS1–PBL2UMP observed in vitro

also exists in plant protoplasts. The results described above

also indicate that both BN-PAGE and gel filtration can be

applied to detect ZAR1 oligomerization in vivo. Because gel

filtration required large amounts of materials and long handling

of samples, for the rest of the study we decided to use ZAR1

variants that are defective in triggering cell death and BN-

PAGE assays for ZAR1 oligomerization.

We next investigated whether HopZ1a can similarly induce the

oligomerization of ZAR1. The enzymatic dead variant Hop-

Z1aC216A, which does not trigger HR, failed to induce ZAR1E11A/

E18A oligomerization, whereas HopZ1a induced an oligomeric

complex of ZAR1E11A/E18A with a molecular mass of about

900 kDa (Figure 1C). These results indicate that HopZ1a, in

addition to AvrAC, also induced the formation of ZAR1

resistosome in plant protoplasts.

ZED1–ZAR1 Interaction Is Critical for HopZ1a-TriggeredImmunity

We next sought to determine whether the ZAR1 oligomerization

observed in protoplasts has structural requirements similar to

those of the ZAR1 resistosome assembled in vitro. The interaction

of ZAR1–RKS1 mainly results from the hydrophobic contacts

mediated by N-terminal a helix of RKS1 and ZAR1LRR (Wang

et al., 2019a). Sequence alignment showed that the residues of


RKS1 responsible for association with ZAR1 are highly

conserved in the Arabidopsis RLCK XII-2 subfamily. To evaluate

the effect of these residues on ZAR1–ZED1 interaction, we gener-

ated two ZED1 mutations, I24E (ZED1I24E) and G29E (ZED1G29E),

and transfected these constructs into Arabidopsis protoplasts.

Both mutations of ZED1 severely diminished the interaction with

ZAR1 (Figure 2A), further explaining the association between

ZAR1 and diverse proteins from the RLCK XII-2 subfamily. We

next tested whether the RKS1-interacting residues of ZAR1 are

required for ZED1 association in Arabidopsis protoplasts. The

ZAR1V544E, ZAR1H597E, and ZAR1W825A/F839A variants, which are

impaired in the interaction with RKS1, displayed a weaker interac-

tion with ZED1 compared with wild-type ZAR1 (Figure 2B). The

ZAR1I600E mutation had a negligible effect on ZAR1–RKS1 and

ZAR1–ZED1 interactions (Wang et al., 2019a; Figure 2B).

We next tested how these mutations affect oligomerization of

ZAR1 in Arabidopsis protoplasts. The mutation ZED1I24E

impaired the shift of ZAR1E11A/E18A mobility induced by HopZ1a

(Figure 2C). Furthermore, the ZAR1W825A/F839A mutation

completely abolished oligomerization of ZAR1 when co-

expressed with HopZ1a and ZED1 (Figure 2D). These results

indicated that ZAR1–ZED1 interaction is indispensable for the

formation of ZAR1–ZED1 oligomeric complex in protoplasts.

We then tested the impact of thesemutationsonHopZ1a-induced

cell death in protoplasts by using the Cell Titer-Glo Luminescent

Cell Viability Assay, which measures cellular ATP. The ZED1 mu-

tations, ZED1I24E and ZED1G29E, greatly reduced ZAR1-mediated

cell death compared with wild-type ZED1 (Figure 2E). In addition,

ZAR1 mutations, ZAR1V544E, ZAR1H597E, and ZAR1W825A/F839A,

markedly reduced HopZ1a-induced cell death (Figure 2F). To

further evaluate the role of these mutations on HopZ1a-induced

disease resistance, we introduced ZED1 variants with their native

promoters into the zed1 mutant. T1 transgenic plants were chal-

lenged with wild-type P. syringae hopZ1a. As expected, plants

carryingwild-typeZED1 transgene restored resistance toP. syrin-

gae hopZ1a, whereas plants expressing the ZED1I24E and

ZED1G29E variants were fully susceptible compared with the

plants complemented with wild-type ZED1 (Figure 2G). All

constructs accumulated ZED1-HA protein (Supplemental

Figure 2), suggesting that the lack of resistance in ZED1I24E and

ZED1G29E plants was not because of a lack of protein. We

further verified these results by testing two independent T2 lines

for each construct, and these results are completely consistent

with those observed in T1 plants (Supplemental Figure 3). We

further tested representative transgenic lines (zar1 background)

carrying ZAR1V544E, ZAR1H597E, and ZAR1W825A/F839A variants

for resistance to P. syringae hopZ1a. These lines accumulate

similar amounts of ZAR1 protein and have been shown to be

compromised in resistance to X. campestris campestris avrAC

(Wang et al., 2019a). As expected, the wild-type ZAR1, but not

mutant variants, restored disease resistance (Figure 2H). Among

the three independent experiments, the ZAR1H597E line showed

partial resistance compared with controls, but this was not

repeated in the other two experiments (Supplemental Figure 4).

Taken together, our results support the idea that ZAR1 interacts

with ZED1 in a similar manner shown by the structure of

ZAR1–RKS1 complex, and this interaction is required for

oligomerization in vivo, cell death, and disease resistance (Wang

et al., 2019a).

A B

C E

D

F

G

H

Figure 2. ZED1–ZAR1 Interaction Is Critical for HopZ1a-Induced Immunity.(A and B) ZED1 (A) or ZAR1 (B) mutations reduce or abolish ZAR1–ZED1

interaction in protoplasts. The indicated constructs were transfected into

zed1 and zar1 protoplasts, respectively. Total protein was subjected to

co-immunoprecipitation assays. All assays were performed three times,

and a representative photograph is shown.

(C and D) ZED1 (C) or ZAR1 (D) mutations abolish HopZ1a-induced

oligomerization of ZAR1 in protoplasts. The indicated constructs were

transfected into zed1 and zar1 protoplasts, respectively. Total protein was

subjected to BN-PAGE. All assays were performed three times, and a

representative photograph is shown.

(E and F) The ZAR1–ZED1 interaction is required for HopZ1a-induced cell

death in protoplasts. ZED1 mutants (E) were co-expressed with HopZ1a

and ZAR1 in zed1 protoplasts, and ZAR1 mutants (F) were co-expressed

with HopZ1a and ZED1 in zar1 protoplasts. The protoplasts were incu-

bated for 12 h and cell viability was measured by the Cell Titer-Glo

Luminescent Cell Viability Assay. Data are presented as mean ± SE.

Different letters indicate significant difference at P < 0.05 (n = 3, one-way

ANOVA, Tukey’s post-test, three independent experiments).

(G and H) Compromising the ZAR1–ZED1 interaction impairs HopZ1a-

induced antibacterial immunity. Col-0, zed1, and T1 transgenic plants


Oligomerization of ZAR1 Is Critical for HopZ1a-InducedImmunity

We further compared structural requirements for ZAR1 oligo-

merization in vivo and ZAR1 resistosome assembly in vitro. In

the ZAR1–RKS1–PBL2UMP resistosome, the interaction of two

adjacent ZAR1 proteins is mediated by all the structural domains

of ZAR1 including the LRR domain, NB domain, helical domain 1

(HD1), winged-helix domain, and CC domain (Wang et al.,

2019b). In addition, ATP binding is essential for oligomerization

of ZAR1–RKS1–PBL2UMP, as the phosphate group of the

bound dATP forms a hydrogen bond with Ser403, which results

in further stabilizing of the active conformation of ZAR1. We

selected three mutations, including ZAR1W150A and ZAR1S152E

in the ZAR1NBD–ZAR1NBD interface, and ZAR1R149A/R297A in

residues specifically interacting with dATP but not ADP. When

co-expressed with HopZ1a and ZED1, the mutations ZAR1W150A

and ZAR1S152E completely abolished HopZ1a-induced oligo-

merization of ZAR1E11A/E18A in a BN-PAGE assay (Figure 3A),

indicating that these residues required for the oligomeric

complex of ZAR1–RKS1–PBL2UMP in vitro are also essential

for HopZ1a-induced oligomerization of ZAR1 in protoplasts.

The introduction of the ZAR1R149A/R297A mutation led to a

smaller oligomeric complexes with a molecular mass of

~700 kDa irrespective of HopZ1a or HopZ1aC216A (Figure 3A),

suggesting that the mutation ZAR1R149A/R297A resulted in

aberrant complex formation in protoplasts that no longer

respond to the effector.

Oligomerization of ZAR1 plays crucial roles in AvrAC-induced

cell death and disease resistance (Wang et al., 2019b). To

further evaluate the impact of these mutations on HopZ1a-

induced cell death, we co-expressed indicated constructs in

Arabidopsis protoplasts. ZAR1W150A and ZAR1S152E mutant pro-

teins showed a reduction of HopZ1a-induced cell death

compared with wild-type ZAR1, and ZAR1R149A/R297A

completely lost cell-death-triggering activity (Figure 3B). To

determine the role of these mutations on HopZ1a-induced dis-

ease resistance, we challenged wild-type, zar1, and representa-

tive transgenic lines complemented with wild-type ZAR1,

ZAR1W150A, ZAR1S152E, and ZAR1R149A/R297A with P. syringae

hopZ1a. These transgenic lines were selected because

they have been fully characterized and tested for resistance

to X. campestris campestris avrAC (Wang et al., 2019b).

The ZAR1W150A, ZAR1S152E, and ZAR1R149A/R297A lines

were significantly more susceptible to P. syringae hopZ1a

compared with wild-type lines (Figure 3C), indicating that the

HopZ1a-induced normal oligomerization activity of ZAR1 is

necessary for ZAR1-mediated disease resistance. The lack of

cell-death activity and disease resistance function for

ZAR1R149A/R297A further confirm that the aberrant aggregation

at about 700 kDa is non-functional.

(zed1 background) carrying the indicated ZED1 variants (G) and T2

transgenic lines (zar1 background) carrying the indicated ZAR1 variants

(H) were inoculated with P. syringae hopZ1a, and bacterial population in

the leaf was determined 3 days after inoculation. Box plots represent 16

and 24 data points from two (G) or three (H) independent experiments,

each of which contains eight plants. Colors indicate independent exper-

iments. Different letters indicate significant difference atP < 0.05 (one-way

ANOVA, Tukey’s post-test).


A B

C

Figure 3. Oligomerization of ZAR1 Is Critical for HopZ1a-Induced Immunity.(A) ZAR1 residues required for resistosome assembly in vitro are essential for HopZ1a-induced oligomerization in vivo. zar1 protoplasts expressing

indicated proteins were incubated for 12 h, and total protein was subjected to BN-PAGE. All assays were performed three times, and a representative

photograph is shown.

(B) Oligomerization of ZAR1 is required for HopZ1a-induced cell death in protoplasts. ZAR1 mutants were co-expressed with HopZ1a and ZED1 in zar1

protoplasts, and cell viability was measured by the Cell Titer-Glo Luminescent Cell Viability Assay. Data are presented as mean ± SE. Different letters

indicate significant difference at P < 0.05 (n = 3, one-way ANOVA, Tukey’s post-test, three independent experiments).

(C) Compromising oligomerization of ZAR1 impairs HopZ1a-induced antibacterial immunity. Transgenic lines were inoculated with P. syringae hopZ1a,

and bacterial population in the leaf was determined 3 days after inoculation. Box plots represent 24 data points from three biological replicates, each of

which contains eight technical replicates. Colors indicate biological replicates. Different letters indicate significant difference at P < 0.05 (one-way

ANOVA, Tukey’s post-test).


N-Terminal a Helix of ZAR1 Is Critical for HopZ1a-Induced Immunity

In the ZAR1 resistosome, the very N-terminal a1 helix of ZAR1

forms a funnel-shaped structure that is required for AvrAC-

induced cell death and PM association of ZAR1. However, the

a1 helix does not appear to be necessary for ZAR1 oligomeriza-

tion in vitro (Wang et al., 2019b). We tested various a1 helix

variants for ZAR1 oligomerization in vivo by using BN-PAGE. Ara-

bidopsis protoplasts of the zar1 background were transfected

with ZAR1F9A/L10A/L14A, which carried mutations in residues

located at the outer surface of the funnel-shaped structure, and

ZAR1D10, which lacked the first 10 residues of the a1 helix.

Both ZAR1F9A/L10A/L14A and ZAR1D10 retained the ability to form

oligomeric complex as indicated by BN-PAGE assay, although

the amount was less compared with ZAR1E11A/E18A (Figure 4A).

Thus the a1 helix did not appear to be required for ZAR1

oligomerization in vivo, which is consistent with our previous

in vitro study (Wang et al., 2019b).

We next asked whether the a1 helix mutations affect HopZ1a-

induced cell death as they did to the AvrAC-induced cell death

(Wang et al., 2019b). zar1 protoplasts transfected with ZAR1F9A/


L10A/L14A, ZAR1D10, or ZAR1E11A/E18A along with HopZ1a

showed much less cell death compared with those expressing

wild-type ZAR1 and HopZ1a (Figure 4B). To further examine the

effect of thesemutations onHopZ1a-induced disease resistance,

we challenged transgenic lines of zar1 background comple-

mented with wild-type ZAR1, ZAR1F9A/L10A/L14A, ZAR1D10, or

ZAR1E11A/E18A with P. syringae hopZ1a. These lines accumulate

similar amounts of ZAR1 protein and have been tested for resis-

tance to X. campestris campestris avrAC (Wang et al., 2019b).

The ZAR1F9A/L10A/L14A, ZAR1D10, and ZAR1E11A/E18A lines were

significantly more susceptible than the wild-type ZAR1 line

(Figure 4C), indicating that the a1 helix plays a critical role in

not only AvrAC-specified disease resistance but also HopZ1a-

specified resistance.

In summary, the results in the present study indicate that both

AvrAC and HopZ1a induce oligomerization of ZAR1 in vivo, which

can be detected by using BN-PAGE or gel filtration. These assays

are probably also suitable for analyses of other NLR proteins

in vivo. Indeed, an independent study showed that BN-PAGE

can be used to detect the oligomerization NLR protein RPP7

when co-expressed with an immune activating allele of RPW8

A B

C

Figure 4. The N-Terminal a1 Helix of ZAR1 Is Critical for HopZ1a-Induced Immunity.(A) Functional analysis of the N-terminal a1 helix of ZAR1 in HopZ1a-induced ZAR1 oligomerization. zar1 protoplasts expressing indicated proteins were

incubated for 12 h, and total protein was subjected to BN-PAGE. All assays were performed three times, and a representative photograph is shown.

(B) The a1 helix of ZAR1 is essential for HopZ1a-induced cell death in protoplasts. ZAR1 mutants were co-expressed with HopZ1a and ZED1 in zar1

protoplasts, and cell viability was measured by the Cell Titer-Glo Luminescent Cell Viability Assay. Data are presented as mean ± SE. Different letters

indicate significant difference at P < 0.05 (n = 3, one-way ANOVA, Tukey’s post-test, three independent experiments).

(C) The a1 helix of ZAR1 is required for HopZ1a-induced bacterial resistance. Transgenic lines were inoculated with P. syringae hopZ1a, and bacterial

population in the leafwasdetermined3daysafter inoculation.Boxplots represent 24data points from threebiological replicates, eachofwhich contains eight

technical replicates. Colors indicate biological replicates. Different letters indicate significant difference at P < 0.05 (one-way ANOVA, Tukey’s post-test).


protein (Li et al., 2020). Our results also support that structural

requirements for HopZ1a-induced ZAR1 oligomerization and im-

munity are highly consistent with ZAR1–RKS1–PBL2UMP resisto-

some assembly in vitro. Thus, ZAR1 resistosome formation in vivo

is important for HopZ1a- and AvrAC-triggered immunity.

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana plants used in this study include Col-0, zed1, zar1-1,

and zar1 transgenic lines complemented with various mutants (Lewis

et al., 2010; Wang et al., 2019a, 2019b). The plants used for

protoplast transfection and pathogen inoculation were grown in soil with

a photoperiod of 10 h of white light and 14 h of darkness at 23�C for 4–

5 weeks. The intensity of white light was 90 mE m�2 s�1 provided by

white fluorescent bulbs.

Constructs, Transgenic Plants, and Protoplast Transformation

To generate ProZED1:ZED1-HA transgenic plants with mutant variants,

we PCR amplified the full-length genomic DNA fragments containing pro-

moter and coding sequence of ZED1 fromCol-0 genomic DNA and cloned

them into pCAMBIA1300 vector. The constructs of ZED1 mutations were

generated by site-directed mutagenesis. These constructs were intro-

duced into zed1 mutant plants by Agrobacterium tumefaciens-mediated

transformation. Transgenic plants of T1 generation were identified for

transgene expression by anti-HA immunoblot.

Constructs of AvrAC, PBL2, RKS1, ZED1, HopZ1a, and ZAR1 were under

control of the 35S promoter and have been reported previously (Wang

et al., 2015, 2019a, 2019b). New ZED1 mutant constructs were

generated by site-directed mutagenesis, and all these genes were cloned

to pUC19-35S-HA-RBS or pUC19-35S-FLAG-RBS for protoplast trans-

fection as previously described (He et al., 2007).

Blue Native PAGE Assay

To determine ZAR1 oligomerization in protoplasts, we performed BN-

PAGE using the Bis-Tris Native PAGE system (Invitrogen) according to

the manufacturer’s instructions. In brief, protoplasts expressing indicated

plasmids were incubated for 12 h, and total protein was extracted with 13

Native PAGE Sample Buffer (Invitrogen) containing 1% digitonin and pro-

tease inhibitor cocktail. Protein samples containing 0.25% Coomassie

G-250 were loaded and run on a Native PAGE 3%–12% Bis-Tris gel.

The proteins were then transferred to polyvinylidene difluoride mem-

branes using NuPAGE Transfer Buffer, followed by immunoblot analysis

with the desired antibodies.

Gel-Filtration Assay

For the gel-filtration assay, Arabidopsis protoplasts were transfected with

the indicated plasmids and incubated with 1 mM LaCl3 for 12 h. Total



protein was then isolatedwith protein extraction buffer (50mMHEPES [pH

7.5], 150 mM NaCl, 1 mM EDTA, 0.3% Triton X-100, 1 mM dithiothreitol

[DTT], protease inhibitor cocktail). Protein samples were filtered through

a 0.22-mm low-protein binding filter (Millipore) and analyzed by gel filtra-

tion. An AKTA Purifier system (GE Healthcare) was used to perform these

experiments and a Superdex 200 Increase 10/300 GL column (GE Health-

care) was used at a flow rate of 0.4 ml/min. The buffer used in elution con-

tained 50mMHEPES (pH 7.5), 150mMNaCl, 1mMEDTA, and 1mMDTT.

The eluted fractions were analyzed by SDS–PAGE and detected by anti-

HA immunoblot.

Co-immunoprecipitation Assay

For the co-immunoprecipitation assay, protoplasts were transfected

with the indicated plasmids and incubated for 12 h. Total protein

was extracted with protein extraction buffer (50 mM HEPES [pH 7.5],

150 mM KCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM DTT, protease

inhibitor cocktail). Fifty microliters of anti-FLAG M2 agarose (Sigma)

was incubated with total protein for 2 h at 4�C, washed six times

with protein extraction buffer, and eluted with 60 ml of 0.5 mg/ml 33

FLAG peptide (Sigma) for 1 h at 4�C. Immunoprecipitates were sepa-

rated on a 10% SDS–PAGE gel and detected by the desired

antibodies.

Protoplast Viability Assay

For the protoplast viability assay, the zed1 or zar1 protoplasts trans-

fected with the indicated plasmids were incubated for 12 h as

previously described (Wang et al., 2019a, 2019b). Cell viability was

determined by the Cell Titer-Glo Luminescent Cell Viability Assay accord-

ing to the manufacturer’s instructions (Promega, G7570). ATP-based

luminescence intensity were measured by the EnSpire Multimode plate

Reader (PerkinElmer). The experimentally treated cells were normalized

against the control, assigned as 100%, to calculate the percentage of

cell survival.

Pathogen Strains and Inoculations

The bacterial strain P. syringae DC3000 carrying hopZ1a, which was orig-

inally isolated from P. syringae pv. syringae A2 (Lewis et al., 2008), was

used in this work. For the bacterial growth assay, 4-week-old Arabidopsis

plants were infiltrated with bacteria at 13 106 colony-forming units/ml by

a needleless syringe. The bacterial number in leaves was determined at 3

days after inoculation.

ACCESSION NUMBERSSequences of genes described in this work can be found in The Arabidop-

sis Information Resource using the following accession numbers: ZAR1

(TAIR: AT3G50950) and ZED1 (TAIR: AT3G57750).

SUPPLEMENTAL INFORMATIONSupplemental Information can be found online at Molecular Plant Online.

FUNDINGThe work was supported by grants from National Natural Science Foun-

dation of China (31521001), Ministry of Science and Technology of the

People’s Republic of China (2016YFD0100601), the Chinese Academy

of Sciences international cooperation key project grant GJHZ1311,

and the State Key Laboratory of Plant Genomics (SKLPG2016B-2) to

J.-M.Z.

AUTHOR CONTRIBUTIONSJ.-M.Z. designed the research; M.H. and J.Q. performed the experiments;

J.-M.Z. and G.B. wrote the manuscript.

ACKNOWLEDGMENTSNo conflict of interest declared.


Received: January 18, 2020

Revised: March 2, 2020

Accepted: March 11, 2020

Published: March 16, 2020

REFERENCESAde, J., DeYoung, B.J., Golstein, C., and Innes, R.W. (2007). Indirect

activation of a plant nucleotide binding site–leucine-rich repeat

protein by a bacterial protease. Proc. Natl. Acad. Sci. U S A

104:2531–2536.

Bastedo, D.P., Khan, M., Martel, A., Seto, D., Kireeva, I., Zhang, J.,

Masud, W., Millar, D., Lee, J.Y., Lee, A.H., et al. (2019).

Perturbations of the ZED1 pseudokinase activate plant immunity.

PLoS Pathog. 15:e1007900.

Baudin, M., Hassan, J.A., Schreiber, K.J., and Lewis, J.D. (2017).

Analysis of the ZAR1 immune complex reveals determinants for

immunity and molecular interactions. Plant Physiol. 174:2038–2053.

Cesari, S., Moore, J., Chen, C., Webb, D., Periyannan, S., Mago, R.,

Bernoux, M., Lagudah, E.S., and Dodds, P.N. (2016). Cytosolic

activation of cell death and stem rust resistance by cereal MLA-

family CC-NLR proteins. Proc. Natl. Acad. Sci. U S A 113:10204–

10209.

Chung, E.H., da Cunha, L., Wu, A.J., Gao, Z., Cherkis, K., Afzal, A.J.,

Mackey, D., and Dangl, J.L. (2011). Specific threonine

phosphorylation of a host target by two unrelated type III effectors

activates a host innate immune receptor in plants. Cell Host Microbe

9:125–136.

Cui, H., Tsuda, K., and Parker, J.E. (2015). Effector-triggered immunity:

from pathogen perception to robust defense. Annu. Rev. Plant Biol.

66:487–511.

Dangl, J.L., and McDowell, J.M. (2006). Two modes of pathogen

recognition by plants. Proc. Natl. Acad. Sci. U S A 103:8575–8576.

Dodds, P.N., and Rathjen, J.P. (2010). Plant immunity: towards an

integrated view of plant-pathogen interactions. Nat. Rev. Genet.

11:539–548.

Dodds, P.N., Lawrence, G.J., Catanzariti, A.M., Teh, T., Wang, C.I.,

Ayliffe, M.A., Kobe, B., and Ellis, J.G. (2006). Direct protein

interaction underlies gene-for-gene specificity and coevolution of the

flax resistance genes and flax rust avirulence genes. Proc. Natl.

Acad. Sci. U S A 103:8888–8893.

Feng, F., and Zhou, J.M. (2012). Plant-bacterial pathogen interactions

mediated by type III effectors. Curr. Opin. Plant Biol. 15:469–476.

Feng, F., Yang, F., Rong, W., Wu, X., Zhang, J., Chen, S., He, C., and

Zhou, J.-M. (2012). A Xanthomonas uridine 50-monophosphate

transferase inhibits plant immune kinases. Nature 485:114–118.

Fu, Z.Q., andDong, X. (2013). Systemic acquired resistance: turning local

infection into global defense. Annu. Rev. Plant Biol. 64:839–863.

Gutierrez, J.R., Balmuth, A.L., Ntoukakis, V., Mucyn, T.S.,

Gimenez-Ibanez, S., Jones, A.M., and Rathjen, J.P. (2010). Prf

immune complexes of tomato are oligomeric and contain multiple

Pto-like kinases that diversify effector recognition. Plant J. 61:507–518.

Guy, E., Lautier, M., Chabannes, M., Roux, B., Lauber, E., Arlat, M., and

Noel, L.D. (2013). xopAC-triggered immunity against Xanthomonas

depends on Arabidopsis receptor-like cytoplasmic kinase genes PBL2

and RIPK. PLoS One 8:e73469.

He, P., Shan, L., and Sheen, J. (2007). The use of protoplasts to study

innate immune responses. In Plant-Pathogen Interactions, P.C.

Ronald, ed. (Totowa, NJ: Humana), pp. 1–9.

van der Hoorn, R.A., and Kamoun, S. (2008). From guard to decoy: a

new model for perception of plant pathogen effectors. Plant Cell

20:2009–2017.

http://refhub.elsevier.com/S1674-2052(20)30066-6/sref1


























































Hu, Z., Zhou, Q., Zhang, C., Fan, S., Cheng, W., Zhao, Y., Shao, F.,

Wang, H.-W., Sui, S.-F., and Chai, J. (2015). Structural and

biochemical basis for induced self-propagation of NLRC4. Science

350:399–404.

Jones, J.D., and Dangl, J.L. (2006). The plant immune system. Nature

444:323–329.

Jones, J.D., Vance, R.E., and Dangl, J.L. (2016). Intracellular innate

immune surveillance devices in plants and animals. Science

354:aaf6395.

El Kasmi, F., Chung, E.H., Anderson, R.G., Li, J., Wan, L., Eitas, T.K.,

Gao, Z., and Dangl, J.L. (2017). Signaling from the plasma-

membrane localized plant immune receptor RPM1 requires self-

association of the full-length protein. Proc. Natl. Acad. Sci. U S A

114:E7385–E7394.

Khan, M., Subramaniam, R., and Desveaux, D. (2016). Of guards,

decoys, baits and traps: pathogen perception in plants by type III

effector sensors. Curr. Opin. Microbiol. 29:49–55.

Kofoed, E.M., and Vance, R.E. (2011). Innate immune recognition of

bacterial ligands by NAIPs determines inflammasome specificity.

Nature 477:592.

Kourelis, J., and van der Hoorn, R.A.L. (2018). Defended to the nines: 25

Years of resistance gene cloning identifies nine mechanisms for R

protein function. Plant Cell 30:285–299.

Krasileva, K.V., Dahlbeck, D., and Staskawicz, B.J. (2010). Activation of

an Arabidopsis resistance protein is specified by the in planta

association of its leucine-rich repeat domain with the cognate

oomycete effector. Plant Cell 22:2444–2458.

Laflamme, B., Dillon, M.M., Martel, A., Almeida, R.N., Desveaux, D.,

and Guttman, D.S. (2020). The pan-genome effector-triggered

immunity landscape of a host-pathogen interaction. Science

367:763–768.

Lewis, J.D., Abada, W., Ma, W., Guttman, D.S., and Desveaux, D.

(2008). The HopZ family of Pseudomonas syringae type III effectors

require myristoylation for virulence and avirulence functions in

Arabidopsis thaliana. J. Bacteriol. 190:2880–2891.

Lewis, J.D., Wu, R., Guttman, D.S., and Desveaux, D. (2010). Allele-

specific virulence attenuation of the Pseudomonas syringae HopZ1a

type III effector via the Arabidopsis ZAR1 resistance protein. PLoS

Genet. 6:e1000894.

Lewis, J.D., Lee, A.H.-Y., Hassan, J.A., Wan, J., Hurley, B., Jhingree,

J.R., Wang, P.W., Lo, T., Youn, J.-Y., Guttman, D.S., et al. (2013).

The Arabidopsis ZED1 pseudokinase is required for ZAR1-mediated

immunity induced by the Pseudomonas syringae type III effector

HopZ1a. Proc. Natl. Acad. Sci. U S A 110:18722–18727.

Li, L., Habring, A., Wang, K., and Weigel, D. (2020). Atypical resistance

protein RPW8/HR triggers oligomerization of the NLR immune receptor

RPP7 and autoimmunity. Cell Host Microbe 27:405–417.e6.

Liu, C., Cui, D., Zhao, J., Liu, N.,Wang, B., Liu, J., Xu, E., Hu, Z., Ren, D.,

Tang, D., et al. (2019). Two Arabidopsis receptor-like cytoplasmic

kinases SZE1 and SZE2 associate with the ZAR1-ZED1 complex and

are required for effector-triggered immunity. Mol. Plant 12:967–983.

Maekawa, T., Kufer, T.A., and Schulze-Lefert, P. (2011). NLR functions

in plant and animal immune systems: so far and yet so close. Nat.

Immunol. 12:817–826.

Mestre, P., and Baulcombe, D.C. (2006). Elicitor-mediated

oligomerization of the tobacco N disease resistance protein. Plant

Cell 18:491–501.

Monaghan, J., and Zipfel, C. (2012). Plant pattern recognition receptor

complexes at the plasma membrane. Curr. Opin. Plant Biol.

15:349–357.

Ravensdale, M., Bernoux, M., Ve, T., Kobe, B., Thrall, P.H., Ellis, J.G.,

and Dodds, P.N. (2012). Intramolecular interaction influences binding

of the flax L5 and L6 resistance proteins to their AvrL567 ligands. PLoS

Pathog. 8:e1003004.

Schultink, A., Qi, T., Bally, J., and Staskawicz, B. (2019). Using forward

genetics in Nicotiana benthamiana to uncover the immune signaling

pathway mediating recognition of the Xanthomonas perforans

effector XopJ4. New Phytol. 221:1001–1009.

Seto, D., Koulena, N., Lo, T., Menna, A., Guttman, D.S., and Desveaux,

D. (2017). Expanded type III effector recognition by the ZAR1 NLR

protein using ZED1-related kinases. Nat. Plants 3:17027.

Tang, D., Wang, G., and Zhou, J.M. (2017). Receptor kinases in plant-

pathogen Interactions: more than pattern recognition. Plant Cell

29:618–637.

Wang, G., Roux, B., Feng, F., Guy, E., Li, L., Li, N., Zhang, X., Lautier,

M., Jardinaud, M.F., Chabannes, M., et al. (2015). The decoy

substrate of a pathogen effector and a pseudokinase specify

pathogen-induced modified-self recognition and immunity in plants.

Cell Host Microbe 18:285–295.

Wang, J.,Wang, J., Hu,M.,Wu, S., Qi, J., Wang, G., Han, Z., Qi, Y., Gao,

N., Wang, H.W., et al. (2019a). Ligand-triggered allosteric ADP release

primes a plant NLR complex. Science 364:eaav5868.

Wang, J., Hu,M.,Wang, J., Qi, J., Han, Z.,Wang, G., Qi, Y.,Wang, H.W.,

Zhou, J.M., and Chai, J. (2019b). Reconstitution and structure of a

plant NLR resistosome conferring immunity. Science 364:eaav5870.

Wang, W., Feng, B., Zhou, J.-M., and Tang, D. (2019c). Plant immune

signaling: advancing on two frontiers. J Integr Plant Biol 62:2–24.

Yang, J., Zhao, Y., Shi, J., and Shao, F. (2013). Human NAIP and mouse

NAIP1 recognize bacterial type III secretion needle protein for

inflammasome activation. Proc. Natl. Acad. Sci. U S A 110:14408–

14413.

Zhou, J.M., and Chai, J. (2008). Plant pathogenic bacterial type III

effectors subdue host responses. Curr. Opin. Microbiol. 11:179–185.































































































Molecular Plant, Volume 13

Supplemental Information

Bacterial Effectors Induce Oligomerization of Immune Receptor ZAR1

In Vivo

Meijuan Hu, Jinfeng Qi, Guozhi Bi, and Jian-Min Zhou

Bacterial effectors induce oligomerization of immune receptor ZAR1 in vivo

Meijuan Hu1,2,3, Jinfeng Qi1,2,3, Guozhi Bi1,*, and Jian-Min Zhou1,2,*

1State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental

Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences,

Beijing 100101, P. R. China

2CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of

Sciences, Beijing 100049, P. R. China

3These authors contributed equally to this article

*Correspondence: Guozhi Bi ([email protected]), Jian-Min Zhou

([email protected])

Short Summary

The bacterial pathogen effectors AvrAC and HopZ1a induce oligomerization of the

Arabidopsis NLR protein ZAR1 in protoplasts. Structural requirements for ZAR1

resistosome assembly in vitro are also essential for HopZ1a-induced ZAR1

oligomerization in vivo and disease resistance in plants, providing evidence that the

ZAR1 resistosome forms in vivo during immune activation.



ZAR1-FALG

PBL2-FLAG

RKS1-FLAG

AvrAC-FLAG

LaCl3

+

+

+

-

0

+

+

+

-

1.5

+

+

+

-

5

+

+

+

+

0

+

+

+

+

1.5

+

+

+

+

5 mM

ZAR1

AvrAC

PBL2

RKS1 40

kDa

55

100

LaCl3 0 1 2 5 10 mM

16/16 0/16 0/16 0/16 0/16

0/16 0/16 0/16 0/16 0/16

Pst DC3000 (hopZ1a)

Col-0

zar1

Supplemental Figure 1. LaCl3 blocks HopZ1a-induced cell death and partially affects AvrAC-induced protein degradation.(A) LaCl3 blocks HopZ1a-induced cell death. Col-0 and zar1 plants were infiltrated with P. syringae DC3000 hopZ1a (1 × 108 cfu/mL) and indicated concerntration of LaCl3, and Macroscopic HR in leaves was recorded 5 h after inoculation. (B) LaCl3 inhibits AvrAC-induced protein degradation. Protoplasts expressing indicated proteins were incubated with LaCl3 for 12 h and total protein was subjected to SDS-PAGE and detected by immunoblotting with anti-FLAG antibody.

A B

zed1

Col-0 - WT I24E G29E

55

55Rubisco

kDa

ZED1-HA40

Supplemental Figure 2. Accumulation of ZED1, ZEDI24E and ZED1G29E proteins in T1 transgenic plants.Positive T1 plants carrying ZED1 variant transgenes were pooled, and total protein was isolated from the indicated transgenic lines and anti-HA immunoblot was done to detect the accumulation of ZED1-HA protein. Upper and lower bands are non-specific cross-reacting proteins. Ponceau staining of Rubisco indicate loading of protein.

Supplemental Figure 3. ZED1 mutants impaired in ZAR1-interaction fail to confer antibacterial resistance.Col-0, zed1, and T2 transgenic lines (zed1 background) carrying the indicated transgenes were inoculated with P. syringae DC3000 hopZ1a, and bacterial growth assay was performed as in Fig. 2G. Boxplots represent 24 data points from a single experiment containing eight plants/line. Colors indicate independent experiments. Different letters indicate significant difference at P < 0.05. (one-way ANOVA, Tukey post-test).

Ba

cte

ria

[lo

g10

(CF

U c

m-2

)]

3

4

5

6

7

8

L1 L2 L1 L1L2 L2

WT I24E G29E

a

b

a

b

c

a

b

bc

Supplemental Figure 4. ZAR1 mutants impaired in ZED1-interaction fail to confer antibacterial resistance(related to Figure 2H).Col-0, zar1, and T2 transgenic lines (zar1 background) carrying the indicated transgenes were inoculated with P. syringae DC3000 hopZ1a, and bacterial population in the leaf was determined 3 d after inoculation. Boxplots represent 8 data points from one replicate of this experiment shown in Figure 2H with green dots. Different letters indicate significant difference at P < 0.05. (one-way ANOVA, Tukey post-test).

3

4

5

6

7

8

a

b

a

b

c

bB

acte

ria

[lo

g10

(CF

U c

m-2

)]

Bacterial Effectors Induce Oligomerization of Immune ...Bacterial Effectors Induce Oligomerization...

Documents

Transcript of Bacterial Effectors Induce Oligomerization of Immune ...Bacterial Effectors Induce Oligomerization...