The rice XA21 receptor confers immunity to the Gram-negative bacterial pathogen, _Xanthomonas oryzae_ pv. _oryzae_ (_Xoo_) upon recognition of the conserved microbial signature AxY^S^22. Here, we demonstrate that the intracellular kinase domain of XA21 translocates to the nucleus upon AxY^S^22-mediated perception and that this translocation event is required for XA21-mediated immunity

Chang-Jin Park

Pamela C. Ronald

English

Nature Precedings

1 
 
Ligand induced cleavage and nuclear localization of the rice XA21 immune receptor 
 
Chang-Jin Park and Pamela C. Ronald* 
 
 
Department of Plant Pathology and the Genome Center, University of California Davis, Davis, 
CA 95616, USA. 
 
* To whom correspondence should be addressed.  
E-mail: pcronald@ucdavis.edu 
Tel: +1 530-752-1654 
Fax: +1 530-752-6088  
  
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2
2 
 
Abstract 
The rice XA21 receptor confers immunity to the Gram-negative bacterial pathogen, 
Xanthomonas oryzae pv. oryzae (Xoo) upon recognition of the conserved microbial signature 
AxY
S
22. Here, we demonstrate that the intracellular kinase domain of XA21 translocates to the 
nucleus upon AxY
S
22-mediated perception and that this translocation event is required for 
XA21-mediated immunity.  
  
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2
3 
 
Plants and animals perceive conserved microbial signatures via plasma membrane and 
cytoplasmically localized receptors
1
. Such immune receptors, often called pattern recognition 
receptors, include animal TLRs (Toll-like receptors) and plant receptor kinases. These receptors 
require serine-threonine kinases of the non-arginine aspartate (non-RD) subclass to transduce the 
immune response
2
. Whereas RD kinases are regulated by autophosphorylation of the activation 
segment, a centrally located loop that sits close to the catalytic center, very little is known about 
non-RD kinase activation. 
In plants, well-studied immune receptors include rice XA21 (Xanthomonas resistance 21), 
Arabidopsis FLS2 (flagellin sensitive 2) and EFR (elongation factor Tu receptor). In animals, 
studies have focused on TLR1, 3, 5, 6, 7, 8, and 9, which signal through non-RD interleukin-1 
receptor-associated kinases 1 (IRAK1), and TLR3 and TLR4, which signal through non-RD 
receptor interacting protein 1 (RIP1) kinases
1-3
. A general theme that has emerged from these 
studies is that non-RD kinase activity is at least partially dispensable for the innate immune 
response in both plants and animals
1
 and that the kinases function partly as phosphorylation-
mediated scaffold proteins that recruit different signaling components
3
. Furthermore, for XA21, 
the inactive state is promoted by autophosphorylation of specific residues in the juxtamembrane 
domain by an associated ATPase
4
. These results suggest that non-RD kinases are activated in a 
manner distinctly different from the well-characterized RD kinases. 
Similar to the plant immune receptors, all members of the epidermal growth factor 
receptor (EGFR) family have an extracellular ligand binding domain, a transmembrane domain, 
and a cytoplasmic kinase domain. Many of these receptors require a nuclear translocation step 
for their signal transductions. For example, in response to binding their corresponding ligands, 
the intact protein or the intracellular domain of EGFR, ErbB-2 (v-erb-a erythroblastic leukemia 
viral oncogene homolog 2), ErbB-3, and ErbB-4 are translocated to the nucleus
5,6
. ErbB-2 and 
ErbB-4 carry proline-rich carboxyl termini that contain intrinsic transcription activity and 
function as transcriptional regulators in the nucleus
7-9
. EFGR interacts with the transcription 
factors STAT3 (signal transducer and activator of transcription 3), STAT5, and E2F1 (E2F 
transcription factor-1). Each of these transcription factors is then regulates expression of target 
genes 
10-12
. Such nuclear translocation events have not been reported for receptor kinases 
governing the innate immune response.  
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2
4 
 
Rice XA21 confers immunity to the Gram-negative bacterium Xanthomonas oryzae pv. 
oryzae (Xoo)
13
. XA21 binds a conserved sulfated peptide, called AxY
S
22 peptide, derived from 
the Xoo Ax21 (activator of XA21-mediated immunity) protein
14
. We previously reported that 
upon binding to AxY
S
22, XA21 undergoes a proteolytic cleavage, producing a 110 kDa N-
terminal cleavage product in transgenic rice plants expressing a N-terminal Myc-tagged XA21 
(Myc-XA21)
14-16
. Here we show that a 70 kDa C-terminal cleavage product, corresponding to 
the kinase domain fused to cyan fluorescent protein (CFP), is also released after infection of 
transgenic rice plants carrying a C-terminal CFP-tagged XA21 (XA21-CFP) 
17
 (data not shown).  
To localize the XA21 intracellular domain in vivo, we investigated the cellular 
distribution of C-terminal green fluorescent protein (GFP) tagged XA21 (XA21-GFP) in rice 
protoplasts. Consistent with our previous results
15
, XA21-GFP is primarily localized to the 
plasma membrane in the absence of AxY
S
22 (Fig. 1a). GFP alone localizes to both the nucleus 
and cytoplasm (data not shown). In contrast, treatment with AxY
S
22 (Fig. 1a) or with 
supernatants prepared from Xoo
14
 (data not shown), triggers significant accumulation of the 
XA21-GFP inside the nucleus (24.1% ± 3.8 of the protoplasts accumulated XA21-GFP protein in 
the nucleus, data not shown). Nuclear localization was not observed in protoplasts treated with 
water or the biologically inactive peptide, AxY22
14
 (1.2% ± 0.1 and 1.8% ± 0.7, respectively) 
(Fig. 1a). These results indicate that the XA21 intracellular domain translocates to the nucleus in 
protoplasts after treatment with biological active AxY
S
22.  
To investigate if the XA21 intracellular domain translocates to the nucleus in mature 
plants, we generated stable transgenic rice plants expressing biologically active XA21-GFP (Ubi-
XA21-GFP) (data not shown). After Xoo infection or AxY
S
22 treatment, we observed a 
significant increase of the XA21 intracellular domain in the nuclei-enriched fraction (Fig. 1b and 
c). In contrast, the intracellular domain was not detected in nuclei-enriched fractions after mock 
infection and was not detected in nuclei-depleted fractions. These results indicate that AxY
S
22 
recognition triggers nuclear translocation of the XA21 intracellular domain in planta. 
Because XA21 does not carry a proline-rich domain indicative of intrinsic transcription 
activity, we hypothesized that the nuclear-localized XA21 interacts with transcription factors to 
control transcriptional reprogramming. To test hypothesis, we assessed if the rice transcription 
factor OsWRKY62, which negatively regulates XA21-mediated immunity
18
, interacts with the 
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2
5 
 
XA21 intracellular domain in plant cells. Indeed, using bimolecular fluorescence 
complementation (BiFC) assays, we showed that the XA21 intracellular domain and 
OsWRKY62 interact exclusively in the nucleus of rice protoplasts (data not shown).   
We next assessed whether the nuclear localization of the XA21 intracellular domain is 
critical for XA21-mediated immunity. For this purpose, we generated transgenic rice plants 
expressing XA21 fused to a C-terminal nuclear export signal (NES)
19
  (Ubi-XA21-GFP-NES) 
and inoculated with Xoo (data not shown). Out of twelve independently transformed rice plants 
expressing Ubi-XA21-GFP-NES, all displayed significantly enhanced susceptibility to Xoo 
compared with the control transgenic rice plants expressing Ubi-XA21-GFP, lacking the NES. 
The NES did not affect the expression or stability of XA21-GFP (data not shown). The enhanced 
susceptibility in Ubi-XA21-GFP-NES was quantified by lesion length and growth curve analysis 
(Fig. 2 and data not shown). Rice plants expressing XA21-GFP-NES under the control of its 
native promoter (Native-XA21-GFP-NES) were also susceptible compared to resistant Native-
XA21-GFP, lacking NES (data not shown). These results demonstrate that nuclear translocation 
of the XA21 intracellular domain is required for XA21-mediated immunity.  
The observation that the non-RD kinase domain of a plant immune receptor is cleaved 
and localized to the nucleus where it interacts with a transcriptional regulator has not previously 
been reported. However, studies of animal TLRs and their associated non-RD kinases have 
demonstrated a similar nuclear localization requirement for activation of TLR-mediated immune 
responses. For example, upon challenge with lipopolysaccharide or interleukin-1, IRAK1 
localizes to the nucleus and binds to NF-κB-responsive elements located within the IκB-α 
promoter to regulate IκB-α transcription20-23. Nuclear-localized IRAK1 also binds promoters of 
STAT3 and interleukin-10 upon lipopolysaccharide challenge and subsequently regulates 
interleukin-10 gene expression
22,24
.  
Together these results suggest a model where non-RD kinases translocate to the nucleus 
following recognition of conserved microbial signatures where they directly interact with 
transcription factors to regulate the immune response.   
  
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2
6 
 
Figure legends 
Figure 1. Nuclear translocation of XA21-GFP after treatment with AxY
S
22 peptide or Xoo.  
(a) XA21-GFP was expressed in rice protoplast cells using polyethylene glycol (PEG)-mediated 
transformation. AxY
S
22 or AxY22 peptides were applied 16 hours after transformation.  
Expression of the introduced genes was observed one hour after peptide treatment. NLS-DsRed 
(nuclear localization signal-Discosoma sp. red fluorescent protein) was co-transformed as a 
nucleus marker. Images were collected with a Leica True Confocal Scanner SPE confocal 
microscope and coded in green and red for GFP and DsRed, respectively. Arrowheads mark the 
nuclei. Scale bar, 10 μm.  
 (b) Western blot analysis of XA21-GFP proteins extracted from transgenic rice plants 
expressing XA21-GFP under the control of the Ubi promoter (Ubi-XA21-GFP, homozygous 5-5-
4). Equal amount of total proteins (75 μg) were extracted from leaf discs of Kitaake and Ubi-
XA21-GFP plants three hours after Mock, Xoo, or AxY
S
22 treatment. XA21-GFP and C-
terminal cleavage product, detected with an anti-GFP antibody, displayed bands of 
approximately 170 and 70 kDa, respectively. Equal loading of total proteins was confirmed by 
Coomassie blue staining of proteins (lower panel). 
(c) Western blot analysis of XA21-GFP extracted from nuclei-depleted and nuclei-enriched 
fractions from Ubi-XA21-GFP line (homozygous 5-5-4) detected by the anti-GFP antibody. 
Nuclei-enriched (100 μg) and nuclei-depleted (100 μg) fractions were prepared from total 
proteins in (b). XA21-GFP and C-terminal cleavage product-GFP displayed bands at 
approximately 170 and 70 kDa, respectively. Cytolsolic phosphoenolpyruvate carboxylase 
(PEPC) and nuclear histone H3 protein were used as cytosolic and nuclear markers, respectively. 
Figure 2. Nuclear export of XA21 disrupts XA21-mediated immunity. 
Transgenic plants expressing XA21 carrying a nuclear export signal (Ubi-XA21-GFP-NES) 
developed long lesion lengths after Xoo inoculation. Rice leaves were photographed twelve days 
after inoculation (DAI) with Xoo. From left to right: Kitaake, Ubi-XA21-GFP (homozygous 5-5-
4), Ubi-XA21-GFP-NES (7-22-1 and 8-21-1), and Native-XA21-GFP (9-7-2) rice plants. 
  
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2
7 
 
References 
1 Ronald, P. C. & Beutler, B. Plant and animal sensors of conserved microbial signatures. 
Science 330, 1061-1064, (2010). 
2 Dardick, C. & Ronald, P. Plant and animal pathogen recognition receptors signal through 
non-RD kinases. PLoS Pathog. 2, e2, (2006). 
3 Barton, G. M. & Medzhitov, R. Toll signaling: RIPping off the TNF pathway. Nat. 
Iimmunol. 5, 472-474, (2004). 
4 Chen, X. et al. An ATPase promotes autophosphorylation of the pattern recognition 
receptor XA21 and inhibits XA21-mediated immunity. Proc. Natl Acad. Sci. USA 107, 
8029-8034, (2010). 
5 Wang, Y. N., Yamaguchi, H., Hsu, J. M. & Hung, M. C. Nuclear trafficking of the 
epidermal growth factor receptor family membrane proteins. Oncogene 29, 3997-4006, 
(2010). 
6 Carpenter, G. & Liao, H. J. Trafficking of receptor tyrosine kinases to the nucleus. Exp. 
Cell Res. 315, 1556-1566, (2009). 
7 Lin, S. Y. et al. Nuclear localization of EGF receptor and its potential new role as a 
transcription factor. Nat. Cell Biol. 3, 802-808, (2001). 
8 Ni, C. Y., Murphy, M. P., Golde, T. E. & Carpenter, G. γ-Secretase cleavage and nuclear 
localization of ErbB-4 receptor tyrosine kinase. Science 294, 2179-2181, (2001). 
9 Wang, S. C. et al. Binding at and transactivation of the COX-2 promoter by nuclear 
tyrosine kinase receptor ErbB-2. Cancer Cell 6, 251-261, (2004). 
10 Hanada, N. et al. Co-regulation of B-Myb expression by E2F1 and EGF receptor. Mol. 
Carcinog. 45, 10-17, (2006). 
11 Hung, L. Y. et al. Nuclear epidermal growth factor receptor (EGFR) interacts with signal 
transducer and activator of transcription 5 (STAT5) in activating Aurora-A gene 
expression. Nucleic Acids Res. 36, 4337-4351, (2008). 
12 Lo, H. W. et al. Nuclear interaction of EGFR and STAT3 in the activation of the 
iNOS/NO pathway. Cancer Cell 7, 575-589, (2005). 
13 Song, W. Y. et al. A receptor kinase-like protein encoded by the rice disease resistance 
gene, Xa21. Science 270, 1804-1806, (1995). 
14 Lee, S. W. et al. A type I-secreted, sulfated peptide triggers XA21-mediated innate 
immunity. Science 326, 850-853, (2009). 
15 Park, C. J. et al. Overexpression of the Endoplasmic Reticulum Chaperone BiP3 
Regulates XA21-Mediated Innate Immunity in Rice. PLoS One 5, e9262, (2010). 
16 Xu, W. H. et al. The autophosphorylated Ser686, Thr688, and Ser689 residues in the 
intracellular juxtamembrane domain of XA21 are implicated in stability control of rice 
receptor-like kinase. Plant J. 45, 740-751, (2006). 
17 Park, C. J. et al. Ectopic expression of rice Xa21 overcomes developmentally controlled 
resistance to Xanthomonas oryzae pv. oryzae. Plant Sci. 179, 466-471, (2010). 
18 Peng, Y. et al. OsWRKY62 is a negative regulator of basal and Xa21-mediated defense 
against Xanthomonas oryzae pv. oryzae in rice. Mol. Plant 1, 446-458, (2008). 
19 Matsushita, T., Mochizuki, N. & Nagatani, A. Dimers of the N-terminal domain of 
phytochrome B are functional in the nucleus. Nature 424, 571-574, (2003). 
20 Bol, G., Kreuzer, O. J. & Brigelius-Flohe, R. Translocation of the interleukin-1 receptor-
associated kinase-1 (IRAK-1) into the nucleus. FEBS Lett. 477, 73-78, (2000). 
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2
8 
 
21 Liu, G., Park, Y. J. & Abraham, E. Interleukin-1 receptor-associated kinase (IRAK) -1-
mediated NF-κB activation requires cytosolic and nuclear activity. FASEB J. 22, 2285-
2296, (2008). 
22 Huang, Y., Li, T., Sane, D. C. & Li, L. IRAK1 serves as a novel regulator essential for 
lipopolysaccharide-induced interleukin-10 gene expression. J. Biol. Chem. 279, 51697-
51703, (2004). 
23 Su, J., Richter, K., Zhang, C., Gu, Q. & Li, L. Differential regulation of interleukin-1 
receptor associated kinase 1 (IRAK1) splice variants. Mol. Immunol. 44, 900-905, (2007). 
24 Huang, Y. S., Misior, A. & Li, L. W. Novel role and regulation of the interleukin-1 
receptor associated kinase (IRAK) family proteins. Cell. Mol. Immunol. 2, 36-39, (2005). 
 
 
  
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2
9 
 
Acknowledgments 
We thank Dr. Benjamin Schwessinger and Dr. Benoit Lefebvre for critical reading of the 
manuscript, Patrick E. Canlas for generation of transgenic rice plants, and Randy Ruan for lab 
and green house management. This work was supported by NIH GM59962.  
Authorship 
C.J.P and P.C.R. designed the project. C.J.P. performed experiments. C. J. P. and P.C.R. wrote 
the paper.  
Conflict of Interest 
The authors declare no competing financial interests. 
  
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2
10 
 
 
 
Figure 1. Nuclear translocation of XA21-GFP after treatment with AxY
S
22 peptide or Xoo.  
(a) XA21-GFP was expressed in rice protoplast cells using polyethylene glycol (PEG)-mediated 
transformation. AxY
S
22 or AxY22 peptides were applied 16 hours after transformation.  
Expression of the introduced genes was observed one hour after peptide treatment. NLS-DsRed 
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2
11 
 
(nuclear localization signal-Discosoma sp. red fluorescent protein) was co-transformed as a 
nucleus marker. Images were collected with a Leica True Confocal Scanner SPE confocal 
microscope and coded in green and red for GFP and DsRed, respectively. Arrowheads mark the 
nuclei. Scale bar, 10 μm.  
 (b) Western blot analysis of XA21-GFP proteins extracted from transgenic rice plants 
expressing XA21-GFP under the control of the Ubi promoter (Ubi-XA21-GFP, homozygous 5-5-
4). Equal amount of total proteins (75 μg) were extracted from leaf discs of Kitaake and Ubi-
XA21-GFP plants three hours after Mock, Xoo, or AxY
S
22 treatment. XA21-GFP and C-
terminal cleavage product, detected with an anti-GFP antibody, displayed bands of 
approximately 170 and 70 kDa, respectively. Equal loading of total proteins was confirmed by 
Coomassie blue staining of proteins (lower panel). 
(c) Western blot analysis of XA21-GFP extracted from nuclei-depleted and nuclei-enriched 
fractions from Ubi-XA21-GFP line (homozygous 5-5-4) detected by the anti-GFP antibody. 
Nuclei-enriched (100 μg) and nuclei-depleted (100 μg) fractions were prepared from total 
proteins in (b). XA21-GFP and C-terminal cleavage product-GFP displayed bands at 
approximately 170 and 70 kDa, respectively. Cytolsolic phosphoenolpyruvate carboxylase 
(PEPC) and nuclear histone H3 protein were used as cytosolic and nuclear markers, respectively. 
  
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2
12 
 
 
Figure 2. Nuclear export of XA21 disrupts XA21-mediated immunity. 
Transgenic plants expressing XA21 carrying a nuclear export signal (Ubi-XA21-GFP-NES) 
developed long lesion lengths after Xoo inoculation. Rice leaves were photographed twelve days 
after inoculation (DAI) with Xoo. From left to right: Kitaake, Ubi-XA21-GFP (homozygous 5-5-
4), Ubi-XA21-GFP-NES (7-22-1 and 8-21-1), and Native-XA21-GFP (9-7-2) rice plants. 
 
N
at
ur
e 
Pr
ec
ed
in
gs
 : 
hd
l:1
01
01
/n
pr
e.
20
12
.6
77
6.
1 
: P
os
te
d 
11
 J
an
 2
01
2


A receptor kinase-like protein encoded by the rice disease resistance gene,

A type I-secreted, sulfated peptide triggers XA21-mediated innate immunity.

An ATPase promotes autophosphorylation of the pattern recognition receptor XA21 and inhibits XA21-mediated immunity.

Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2.

Co-regulation of B-Myb expression by E2F1 and EGF receptor.

Differential regulation of interleukin-1 receptor associated kinase 1 (IRAK1) splice variants.

Dimers of the N-terminal domain of phytochrome B are functional in the nucleus.

Ectopic expression of rice Xa21 overcomes developmentally controlled resistance to Xanthomonas oryzae pv.

Interleukin-1 receptor-associated kinase (IRAK) -1-mediated NF-κB activation requires cytosolic and nuclear activity.

IRAK1 serves as a novel regulator essential for lipopolysaccharide-induced interleukin-10 gene expression.

Novel role and regulation of the interleukin-1 receptor associated kinase (IRAK) family proteins.

Nuclear epidermal growth factor receptor (EGFR) interacts with signal transducer and activator of transcription 5 (STAT5) in activating Aurora-A gene expression.

Nuclear interaction of EGFR and STAT3 in the activation of the iNOS/NO pathway.

Nuclear localization of EGF receptor and its potential new role as a transcription factor.

Nuclear trafficking of the epidermal growth factor receptor family membrane proteins.

OsWRKY62 is a negative regulator of basal and Xa21-mediated defense against Xanthomonas oryzae pv. oryzae in rice.

Overexpression of the Endoplasmic Reticulum Chaperone

Plant and animal pathogen recognition receptors signal through non-RD kinases.

Plant and animal sensors of conserved microbial signatures.

The autophosphorylated Ser686, Thr688, and Ser689 residues in the intracellular juxtamembrane domain of XA21 are implicated in stability control of rice receptor-like kinase.

Toll signaling: RIPping off the TNF pathway.

Trafficking of receptor tyrosine kinases to the nucleus.

Translocation of the interleukin-1 receptorassociated kinase-1 (IRAK-1) into the nucleus.

γ-Secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase.

Ligand induced cleavage and nuclear localization of the rice XA21 immune receptor

http://precedings.nature.com/documents/6776/version/1

Ligand induced cleavage and nuclear localization of the rice XA21 immune receptor

Abstract

Similar works

Full text

Available Versions

Nature Precedings