Article

Perception of viral infections and initiation

of antiviral defence in rice

https://doi.org/10.1038/s41586-025-08706-8 Yu Huang1,11,12, Jialin Yang2,3,12, Xi Sun1,3,12, Jiahao Li1,3, Xiaoqiang Cao4,5, Shengze Yao1,
Yanhong Han3, Changtian Chen3, Linlin Du6, Shuo Li6, Yinghua Ji6, Tong Zhou6, He Wang7,
Received: 23 January 2024
Jia-jia Han8, Wenming Wang7, Chunhong Wei1, Qi Xie4,5,9, Zhirui Yang10 ✉ & Yi Li1,3 ✉
Accepted: 27 January 2025

Published online: xx xx xxxx

Crop production faces persistent threats from insect-vector-borne viral diseases1,2.
Open access Recent advancements have revealed the intricate immune mechanisms that plants
Check for updates deploy against viral pathogens3–8. However, the molecular mechanisms through which
plant hosts recognize viral infections and initiate antiviral defence at disease onset have
not been elucidated. Here, through the natural infection of rice by inoculation with
insect vectors carrying the natural forms of viruses, we show that viral coat proteins
are perceived by the RING1–IBR–RING2-type ubiquitin ligase (RBRL), initiating the first
step of the natural antiviral response in rice. RBRL subsequently targets an adaptor
protein of the transcriptional repression complex of the jasmonate pathway, NOVEL
INTERACTOR OF JAZ 3 (NINJA3), for degradation through the ubiquitination system,
inducing jasmonate signalling and activating downstream antiviral defence. We further
show that this phenomenon is a universal molecular mechanism used by rice plants
to perceive viral infections and initiate antiviral signalling cascades. This approach is
important not only for obtaining a deeper understanding of virus–host interactions
but also for further disease resistance breeding.

Rice (Oryza sativa) is a staple food for approximately half the global chewing insects and mechanical damage22. The JA pathway func-
population, and its production is facing a serious threat of numerous tions in a classical relief-of-repression model. In the absence of JA-Ile,
insect-vector-transmitted diseases, such as rice stripe disease, caused jasmonate-ZIM domain ( JAZ) proteins recruit TOPLESS (TPL) and
by rice stripe virus (RSV)9–12. TOPLESS-RELATED (TPR) corepressors through NINJA proteins, ulti-
RSV, a member of the Tenuivirus genus, is transmitted by the small mately suppressing the transcription of JA-responsive genes23,24. When
brown planthopper (Laodelphax striatellus Fallén)13. The vector insects JA-Ile is synthesized, interactions between JAZ repressors and the F-box
use their needle-like piercing mouthparts to deliver virions into host protein CORONATINE INSENSITIVE 1 (COI1) trigger JAZ degradation
cells while sucking plant nutrients14. This process differs from that used through the 26S proteasome, liberating JA-responsive transcription
by animal viruses, which enter host cells through specific receptors15. factors to modulate gene transcription17,25–27. Although considerable
To date, no receptor for plant viruses has been identified16. efforts have been made to elucidate the biosynthesis and transduction of
We have shown that the RSV coat protein (CP) is an effector that jasmonate signals28–30, study of the turnover of NINJA proteins is lacking.
triggers the accumulation of jasmonic acid ( JA) and subsequently Ubiquitination regulates eukaryotic cellular processes, such as
upregulates the transcription of the antiviral RNA silencing core signal transduction, immune responses and apoptosis31. Ubiquitina-
factor ARGONAUTE 18 (AGO18) through the JA-responsive MYB tran- tion is coordinated by ubiquitin-activating enzymes (E1s), ubiquitin-
scription factor ( JAMYB)17. AGO18 functions as a decoy, sequestering conjugating enzymes (E2s) and ubiquitin ligases (E3s)32. E3 ligases,
microRNA168 (miR168) and miR528 away from AGO118–20. This action including really interesting new genes (RINGs; topologically similar
leads to the release of target genes AGO1 and L-ascorbate oxidase (AO), U-box E3s), homologous to the E6-AP C terminus (HECTs) and the
thereby strengthening the antiviral defence18,19. However, there is lim- more recently recognized RING1–IBR–RING2 (RBR)-type E3 ligases,
ited information concerning the intricate mechanism through which are closely associated with viral infection33–36. In mammals, RBR-type
rice perceives RSV infection and orchestrates this signalling cascade E3 ligases exhibit a RING/HECT hybrid-like function; they bind to E2s
to activate the antiviral response17. through RING1 and catalyse ubiquitin transfer through the forma-
JA and its derivatives are present throughout the plant kingdom21 and tion of an obligate thioester-linked ubiquitin (Ub) with the conserved
have pivotal roles in plant defence against necrotrophic pathogens, cysteine residue in RING237,38. Despite their evolutionary persistence,

1
State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Peking University, Beijing, P. R. China. 2Academy for Advanced Interdisciplinary Studies, Peking
University, Beijing, P. R. China. 3State Key Laboratory of Agricultural and Forestry Biosecurity, Vector-borne Virus Research Center, Institute of Plant Virology, Fujian Agriculture and Forestry
University, Fuzhou, P. R. China. 4Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, P. R. China. 5University of
Chinese Academy of Sciences, Beijing, P. R. China. 6Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, P. R. China. 7State Key Laboratory of Crop Gene Exploration
and Utilization in Southwest China, Sichuan Agricultural University, Chengdu, P. R. China. 8Institute of Biodiversity, School of Ecology and Environmental Science Yunnan University, Kunming,
P. R. China. 9National Center of Technology Innovation for Maize, State Key Laboratory of Crop Germplasm Innovation and Molecular Breeding, Syngenta Group China, Beijing, P. R. China.
10
State Key Laboratory of Plant Environmental Resilience, College of Biological Science, China Agricultural University, Beijing, P. R. China. 11Present address: Sichuan Institute of Edible Fungi,
Sichuan Academy of Agricultural Sciences, Chengdu, P. R. China. 12These authors contributed equally: Yu Huang, Jialin Yang, Xi Sun. ✉e-mail: rynn_yang@cau.edu.cn; liyi@pku.edu.cn

Nature | www.nature.com | 1
Article
the regulatory and biological roles of plant RBR-type E3 ligases have genes related to defence, JA signalling and reactive oxygen species
largely not been explored39–41. (ROS) pathway (Extended Data Fig. 2g–p and Supplementary Table 2).
Here we show that rice perceives RSV infection through the RBR-type Taken together, these results indicated that OsRBRL might act as a
E3 ligase RBRL, which then orchestrates the ubiquitination-mediated sensor of viral CP and exert a positive regulatory influence on JA bio-
degradation of NINJA3, a transcriptional repressor. This process initi- synthesis and JA signalling.
ates JA signalling followed by RNA-silencing-mediated antiviral defence,
offering insights into the intricate antiviral responses of rice.
RBRL is required in antiviral defence
Subsequently, we investigated whether the interaction between RSV CP
OsRBRL perceives RSV infection and OsRBRL was correlated with CP-mediated rice antiviral defence. We
To better understand how RSV CP triggers JA signalling-coupled anti- performed natural infection of rice lines with RSV-carrying small brown
viral defence, we analysed the subcellular localization of RSV CP in planthoppers and compared disease symptoms in NPB, rbrl-mutant
RSV-infected wild-type rice NPB (O. sativa subsp. japonica cv. Nip- and RBRL-OE lines (Fig. 2a,b). Notably, compared with the NPB plants,
ponbare) and CP-overexpressing (CP-OE) plants. Notably, RSV CP the rbrl-mutant plants exhibited more pronounced stunting after RSV
was partially localized in the nucleus, suggesting that it may func- infection, whereas the RBRL-OE plants displayed less stunting (Fig. 2a).
tion as a signalling factor for viral perception (Fig. 1a,b; immuno­blot The disease symptoms were subsequently categorized based on their
source data are provided in Supplementary Fig. 1). We performed severity on leaves (Extended Data Fig. 3a). Compared with the NPB
immunoprecipitation–mass spectrometry (IP–MS) assays using the plants, rbrl plants displayed fewer symptomless (grade N) leaves and
nuclear fractions shown in Fig. 1b to identify proteins interacting with more severe symptoms (grade III). Conversely, RBRL-OE plants showed
CP. Among the CP-interacting proteins, an RBR-type ubiquitin ligase, more symptomless (grade N) leaves, and fewer typical (grade II) and
named OsRBRL, was found in the prey lists from RSV-infected NPB and severe symptoms (grade III) (Fig. 2b and Supplementary Table 3). Con-
CP-OE samples (Extended Data Fig. 1a and Supplementary Table 1). sistent with these phenotypic observations, the accumulation of RSV
Phylogenetic analysis of OsRBRL identified an OsRBRL-like protein in CP and viral RNAs was significantly increased in the rbrl mutants and
the same subclade as RBRL. A conserved motif analysis revealed that substantially decreased in the RBRL-OE lines (Fig. 2c,d).
RBRL and RBRL-like proteins possess the classic RING1–IBR–RING2 A previous study showed that RSV CP activates the JA signalling path-
domains (Extended Data Fig. 1b,c). way, thereby inducing the expression of OsAGO1817. CP-OE plants exhib-
The interaction between CP and OsRBRL was further validated by ited higher OsAGO18 protein levels compared with NPB plants, and the
co-immunoprecipitation (co-IP), luciferase complementation imaging expression of OsAGO18 and CP was correlated18. Thus, the accumulation
(LCI) and pull-down assays (Fig. 1c,d and Extended Data Fig. 2a–c). The of OsAGO18 serves as a marker for the rice antiviral defence. Notably,
LCI assays showed that CP did not interact with OsRBRL-like (Fig. 1d). RSV-infected rbrl plants accumulated more CP but less OsAGO18 com-
Microscale thermophoresis (MST) assays further showed a strong bind- pared with RSV-infected NPB plants (Fig. 2c). Conversely, RSV-infected
ing affinity between CP and RBRL with a dissociation constant (Kd) of RBRL-OE plants showed lower CP and higher OsAGO18 levels (Fig. 2c).
5.28 nM (Extended Data Fig. 2d). Moreover, LCI assays showed that These results strongly suggest that OsRBRL bridges RSV CP and the
CP interacts with RBRL through the C-terminal Ariadne (Ari) domain, JA-signalling-coupled antiviral RNA interference (RNAi) pathway.
N-terminal domain (NTD) and RBR domain (Extended Data Fig. 2e,f). To further confirm that the perception of CP by OsRBRL is essential
Importantly, we observed that the expression of OsRBRL mRNA for activating downstream antiviral signalling, we overexpressed CP
and OsRBRL protein was induced by RSV infection and CP overexpres- in the rbrl-mutant background, generating two lines: CP-OE/rbrl 1 and
sion (Fig. 1e–h and Extended Data Fig. 1d). However, transcriptome CP-OE/rbrl 2. These CP-OE/rbrl lines displayed moderate resistance
deep sequencing (RNA-sequencing, RNA-seq) data indicated that the compared with NPB plants, whereas their antiviral responses were
expression levels of OsRBRL-like did not significantly change after RSV weakened compared with those of the CP-OE plants17 (Fig. 2e,f and
infection (Extended Data Fig. 1d). Moreover, OsRBRL-like did not inter- Supplementary Table 3). Furthermore, the levels of viral RNA and accu-
act with CP (Fig. 1d); we therefore focused on OsRBRL for further study. mulated CP in the CP-OE/rbrl lines were between those in the NPB and
CP-OE plants17 (Fig. 2g,h). Notably, the accumulation of OsAGO18 also
followed this pattern (Fig. 2h). As RSV CP accumulated significantly
OsRBRL initiates the JA signalling pathway higher in RSV-infected NPB plants than in CP-OE plants (Extended Data
To further investigate the function of OsRBRL, we obtained the rbrl- Fig. 3b), we propose that a low dose of CP is sufficient to be sensed by
knockout mutant and RBRL-overexpressing (RBRL-OE) rice lines RBRL for initiating the JA–AGO18 antiviral signalling. A comparison of
(Extended Data Fig. 2g,l). The transcript levels of CM-LOX1, CM-LOX2, the CP-OE/rbrl lines with the rbrl-mutant lines revealed that overexpres-
OsAOS2 and OsJMT1 (involved in JA biosynthesis), as well as those of sion of CP partially rescued the sensitive phenotypes of the rbrl-mutant
OsCOI1A, OsCOI1B, OsCOI2, OsJAMYB, OsAGO18 and multiple OsJAZ plants after RSV infection, suggesting that natural antiviral signalling
genes (involved in JA signalling), were downregulated in the rbrl is initiated in rice plants not entirely, but mainly through OsRBRL.
mutants but upregulated in the RBRL-OE plants compared with the A previous study showed that the expression of OsAGO18 is regulated
NPB plants (Fig. 1i). Immunoblotting results confirmed these find- by JAMYB and inhibited by JAZ617. To determine whether OsRBRL affects
ings, showing lower protein levels of CM-LOX2 and AOS2 in rbrl lines the activation of antiviral immunity by regulating OsAGO18 transcrip-
and higher levels in RBRL-OE lines than in NPB plants (Fig. 1j). The JA tion, we co-expressed 35S::YFP-JAMYB, 35S::MYC-JAZ6, AGO18pro::LUC
levels were significantly decreased in the rbrl mutant but substantially and 35S::HA-RBRL in tobacco leaves or rice protoplasts (a list of the
increased in the RBRL-OE plants (Fig. 1k,l). In particular, after RSV infec- pri­mers used to construct these vectors is provided in Supplementary
tion, the JA content was higher in RBRL-OE rice plants and lower in rbrl Table 5). Consistent with previous reports, JAZ6 inhibited the transcrip-
mutants than in NPB plants (Fig. 1k,l), indicating that RBRL contributes tional activity of the OsAGO18 promoter17. However, increasing the
to RSV-induced JA accumulation. To further elucidate the broad role of expression of 35S::HA-RBRL significantly derepressed AGO18pro::LUC,
OsRBRL in regulating the response of rice plants to viral infection, we and the activation of AGO18pro::LUC was specifically rescued by the
performed RNA-seq and comparative transcriptome analysis in NPB, co-expression of 35S::HA-RBRL but not 35S::HA-RBRL-like (Extended
rbrl and RBRL-OE plants with or without RSV infection. We identified Data Fig. 3c–e).
numerous potential RBRL-regulated differentially expressed genes Moreover, co-IP assays using the nuclear fraction of mock-inoculated
(DEGs). Gene Ontology (GO) analysis revealed high enrichments of and RSV-infected RBRL-OE samples demonstrated that CP interacts

2 | Nature | www.nature.com
 a b c d
Input IP: anti-GFP CP–nLUC CP–nLUC
NPB, mock CP-OE RSV-infected NPB
GFP + – + – + +
cLUC–RBRL cLUC–GUS cps
YFP–CP – + – +

t
pu

pu

pu
53,204

in

in

in
HA–RBRL + + + + kDa 49,091

IP eop sm
sm

IP eop sm
sm

IP eop sm
sm
N leo m

N leo m

N leo m
IB: 70 44,978

l la
la

l la
la

l la
la
uc s

uc s

uc s
uc p

uc p

uc p
N pl a

N la

N pl a
anti-HA 40,865

op
C al

C l

C al
o

o
ta
36,751

yt

yt

yt
t

t
To

To

To
kDa
70 32,638
40
Anti-FBPA 55 28,525
35 24,412
IB:
20,299
15 Anti-H3 anti-GFP 16,185
GUS–nLUC CP–nLUC
+ + 12,072
35 ** ** Anti-CP 25 cLUC–RBRL cLUC–RBRL-like
Mock RSV
NPB

e f g h i rbrl RBRL-OE
NPB –3
P = 4.45 × 10 NPB 1 2 1 2
3 P = 1.47 × 10
–3 Mock RSV 1.4
kDa NPB CP-OE CM-LOX1

Relative expression of RBRL

Relative expression of RBRL

kDa CM-LOX2
35 Anti-CP 1.3 OsAOS2
35 Anti-HA (CP)
OsJMT1
2 OsCOI1A
1.2
70 OsCOI1B
Anti-RBRL 70 OsCOI2
1.1 Anti-RBRL OsJAZ6
1 OsJAZ8
1.0 3.2
1.0 1.0 1.3 OsJAZ10
OsJAZ12
40 Anti-actin 40 Anti-actin OsPR10
0 0.9 OsJAMYB
Mock RSV NPB CP-OE OsAGO18
log10[FC]
NPB
–1 0 1

j rbrl RBRL-OE
k NPB, mock NPB, RSV l –5
P = 6.55 × 10
rbrl 1, mock rbrl 1, RSV –3
kDa NPB 1 2 1 2 P = 5.45 × 10 Mock
rbrl 2, mock rbrl 2, RSV 60 –10
140 P = 2.06 × 10 RSV
RBRL-OE 1, mock RBRL-OE 1, RSV

Contents of JA (pg mg FW)

–10
95 Anti-CM-LOX2 RBRL-OE 2, mock RBRL-OE 2, RSV 50 P = 1.73 × 10

–1
800 40
1.0 0.5 0.5 1.5 2.6 JA
700 30
54 m/z 209
Anti-AOS2 600 20
42 500
Density

15
1.0 0.5 0.4 1.1 1.6
400
300 10
70 Anti-HA
200 5
0 0 0 1.0 2.7 100
0 0
42 Anti-actin 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0

PB

2
rl

rl

E
N

rb

rb

-O

-O
Retention time (min)

RL

RL
RB

RB
Fig. 1 | OsRBRL perceives CP and activates JA biosynthesis and signalling. cps, signal counts per second. e,g,i, RT–qPCR results showing the expression
a, Photographs of mock-inoculated and RSV-infected wild-type NPB rice plants. levels of OsRBRL (e and g) or genes related to JA synthesis and signalling (i) in
The photographs were captured at 4 weeks post-infection (w.p.i.). Scale bar, the indicated plants. f,h,j, Immunoblotting analysis of the indicated protein
10 cm. b, IP–MS assays conducted with the nuclear fraction of mock-inoculated levels in the indicated plants. Actin was used as a sample processing control.
NPB plants, CP-OE plants and RSV-infected NPB plants. FBPA was used as a The analyses were repeated three to five times with similar results. k,l, LC–MS
cytoplasmic marker, and histone H3 as a nuclear marker. c, Co-IP assays analysis (k) and quantification (l) of the JA concentrations in the NPB, rbrl and
illustrating the interaction between RSV CP and OsRBRL in N. benthamiana RBRL-OE plants with or without RSV infection. Rice plants were collected at
leaves through YFP–CP and HA–RBRL. GFP was used as a negative control. 3 w.p.i. Statistical analysis was performed using two-sided Student’s t-tests
IB, immunoblotting. The analyses in b and c were repeated two to three times (e and g) and two-way analysis of variance (ANOVA) (l), the P values in l indicate
with similar results. d, LCI assays demonstrating the interaction between RSV the interactions between genotype and treatment. All P values are shown in the
CP and OsRBRL. cLUC–GUS and GUS–nLUC were used as negative controls. figure. Data are mean ± s.d. n = 3 independent biological samples.

with RBRL in the nuclear fraction (Extended Data Fig. 4a). RSV CP reconstituted in bacteria42. The results confirmed that OsRBRL pos-
and OsRBRL were colocalized in the nucleoplasm and cytoplasm of sesses E3 ligase activity (Fig. 3a). Sequence analysis of the RBRL pro-
the rice protoplasts (Extended Data Fig. 4b). Transient expression of tein showed that OsRBRL is a member of the Ariadne subfamily of
OsRBRL containing a nuclear export signal (NES) at the N terminus RBR-type E3 ligases (Fig. 3b and Extended Data Fig. 1b). In humans,
(OsRBRL(NES)) in rice protoplasts showed higher virus accumula- these ligases typically undergo autoinhibition. Removing the Ariadne
tion compared with transient expression of OsRBRL (Extended Data domain activates the human homologue of Ariadne (HHARI)37. HHARI
Fig. 4c,d). These data suggest that OsRBRL mediates antiviral defence and the mammalian homologue of ari-2 (TRIAD) are activated after
in the nucleus. binding to neddylated Cullin–RING ligase complexes39,43. However,
Taken together, these results suggest that the CP-induced rice anti- research on the mechanism underlying their activation is limited39.
viral defence requires OsRBRL to relieve the repression of JA signal- We observed that removing the NTD and Ariadne domains of OsRBRL
ling and therefore promote the expression of JA-responsive genes and resulted in instability of the OsRBRL RBR domain when expressed
OsAGO18. independently in plant cells (Fig. 3c). Moreover, self-ubiquitination
assays in the bacterial system revealed that the conserved cysteine
residue (Cys305) in the RING2 domain is essential for the activity of
CP enhances OsRBRL’s E3 ligase activity RBRL, and RSV CP increased the activity of RBRL (Fig. 3d,e). Semi-in
To elucidate the molecular function of RBRL, we performed self- vivo degradation assays revealed that, compared with the control, RSV
ubiquitination assays using the plant ubiquitination cascade CP promoted the degradation of RBRL (Fig. 3f). These results indicate

Nature | www.nature.com | 3
Article
a b c
120
III II I N RSV

rbrl RBRL-OE
100
NPB 1 2 1 2
kDa

Percentage of symptoms (%)

80 35 Anti-CP

1.0 2.1 2.8 0.7 0.6

60
130
Anti-AGO18
100
40 1.0 0.4 0.3 1.6 1.5

70
Anti-HA
20
NPB 1 2 1 2 NPB 1 2 1 2
0 0 0 1.0 2.7
rbrl RBRL-OE rbrl RBRL-OE
0 Anti-actin
Mock RSV 40
NPB 1 2 1 2
rbrl RBRL-OE

d e f
–2

120
P = 3.45 × 10

III II I N

100
–2

4
–5

P = 2.97 × 10
–2
P = 2.62 × 10

P = 2.66 × 10

Percentage of symptoms (%)

Relative expression level of viral RNAs

80
3

60
–4
–4

P = 2.91 × 10
P = 8.47 × 10

2
–4

–5
P = 4.94 × 10

P = 4.12 × 10

40

1
20
1 2 1 2
PB

PB
rl

rl
E

E
rb

rb
O

O
N

N
P-

P-

0
CP-OE/rbrl CP-OE/rbrl
C

0
1 2 1 2
PB

PB

1 2
rl

rl

rl

rl

Mock RSV

PB

rl
E
N

N
rb

rb

rb

rb

RBRL-OE RBRL-OE

rb
O
N

P-
CP-OE/rbrl

C
RNA1 RNA3

g h
2.0
Mock RSV
Relative expression level of viral RNAs

a
a CP-OE/rbrl CP-OE/rbrl
E

E
O

O
1.5
PB

PB
P-

P-
rl

rl
1 2 1 2
rb

rb
kDa kDa
N

b b Anti-CP Anti-CP
35 35
1.0
c cd c c
d 0 1.0 0 2.0 2.4 1.0 0.5 1.6 0.7 0.7

0.5
d 130 130
Anti-AGO18 Anti-AGO18
100 100

0 1.0 3.2 0.6 1.2 1.4 1.0 2.0 0.1 1.3 1.5
1 2 1 2
PB

PB
rl

rl
E

E
rb

rb
O

O
N

N
P-

P-

CP-OE/rbrl CP-OE/rbrl Anti-actin Anti-actin

C

40 40
RNA1 RNA3

Fig. 2 | The CP-induced natural antiviral defence requires RBRL. a, Images of CP-OE/rbrl plants. Scale bar, 10 cm. f, The percentages of RSV-infected NPB,
mock-inoculated or RSV-infected NPB, rbrl and RBRL-OE plants. Scale bar, 10 cm. CP-OE, rbrl and CP-OE/rbrl plants with various disease symptom grades. Data
b, The percentages of RSV-infected NPB, rbrl and RBRL-OE plants with various are mean ± s.d. n = 3 independent biological experiments. g, RT–qPCR analysis
disease symptom grades. Data are mean ± s.d. n = 3 independent biological of RSV RNA accumulation in the specified plants. Statistical analysis was
experiments. c, Immunoblotting analysis of the RSV CP and AGO18 levels in the performed using one-way ANOVA with Tukey’s multiple-comparison test
specified plants. OsRBRL expressed in RBRL-OE lines 1 and 2 was labelled with (different letters represent significantly different groups; P < 0.05). Data are
a 1× HA tag at the N terminus. Actin was used as a sample processing control. mean ± s.d. n = 3 independent biological samples. h, Immunoblotting analysis
d, RT–qPCR analysis of RSV RNA accumulation in the specified plants. Statistical of the RSV CP and AGO18 levels in the specified plants. All of the mock-inoculated
analysis was performed using two-sided Student’s t-tests, and all P values are rice plants were 6 weeks old and all of the RSV-infected plants were collected at
shown in the figure. Data are mean ± s.d. n = 3 independent biological samples. 4 w.p.i. Actin was used as a sample processing control. All of the experiments
e, Images showing mock-inoculated or RSV-infected NPB, CP-OE, rbrl and were repeated two to four times with similar results.

that the natural state of RBRL is partially autoinhibited, as reported for or RBRL-OE rice plants, nor in degradation assays performed in bacte-
homologues, and that the CP–RBRL interaction could activate the E3 rial systems or tobacco leaves (Extended Data Fig. 4e–g), suggesting
ligase activity of RBRL. that CP is not the substrate of RBRL. The CP–RBRL interaction may
Although E3 ligase usually interacts with and targets viral pro- promote the degradation of a substrate to trigger downstream anti-
teins for degradation, CP is not ubiquitinated in RSV-infected NPB viral defence.

4 | Nature | www.nature.com
a b c e
UBA1-S – + + + + RBRL NTD RBR domain Ariadne
UBC8-S + – + + + RBRL-RBR RBR domain
RBRL–MYC + + – + + UBA1-S – + + + + +
0 100 200 300 400 500 aa HA–RBRL HA–RBRL-RBR
Flag–Ub + + + – + UBC8-S + – + + + +

DM 2

32
kDa

M SO

M SO
Low-complexity region RING1 IBR RING2 Ariadne RBRL–MYC + + – + + +

3
G1

G1
DM
130 Anti-MYC Flag–Ub + + + – + +
OsRBRL kDa
RBRL–MYC GST–CP – – – – – +
100 + Ubn OsRBRL-like 70 HA–RBRL kDa
Anti-HA Anti-MYC
70 RBRL–MYC AtARI3
Human ARI2 25 HA–RBRL-RBR 130
RBRL–MYC
170 Exposure time: 1 s 1.0 3.5 + Ub
Mouse ARI2 n
130 100
100 RING2
70 HA–RBRL 70
70 OsRBRL CPKCSKPIEKNGGCNLVHC-KCGQCLCWLC RBRL–MYC
OsRBRL-like CPKCSKPIEKNGGCNHVRC-KCGQCLCWLC Anti-HA
55
40 AtARI3 CPKCSKPIQKRDGCNLMTC-KCGQHFCWLC HA–RBRL-RBR 170
25 130
Anti-Flag Human ARI2 CPKCNICIEKNGGCNHMQCSKCKHDFCWMC Exposure time: 100 s 100
35 Mouse ARI2 CPKCNICIEKNGGCNHMQCSKCKHDFCWMC
40 Anti-actin 70
55 Anti-Flag
25
40
d f HA–RBRL 35
RBRL(WT) CPKCSKPIEKNGGCNLVHCKCGQCLCWLC
25
15 RBRL(CCAA) APKASKPIEKNGGCNLVHCKCGQCLCWLC

G –CP

G CP

G CP

P
C
15 Flag–Ub

P–

P–

P–
RBRL(C305A) CPKCSKPIEKNGGANLVHCKCGQCLCWLC

YF P

FP

FP

YF P
P
10 His–Flag–Ub 10

F
YF

YF
G
E1 + E2 + Ub 0 0 10 10 20 20 40 40 Min
kDa 170 UBA1-S + Ub
170 UBA1-S + Ub RBRL RBRL RBRL 130 UBA1-S
130 kDa (WT) (CCAA) (C305A) 70 Anti-HA 100
UBA1-S
100 70
70 1.0 0.9 0.6 0.4 0.2 0.1 0.1 0
Anti-MYC 55 Anti-S
55 170 70 40
Anti-S YFP–CP
40 55
130 RBRL–MYC + Ubn UBC8-S
35 35 + Ub
0.25
0.26
0.17
0.26
0.12
0.06

UBC8-S + Ub Anti-GFP
100 25
25
25 GFP UBC8-S
70 RBRL–MYC 15
UBC8-S
55 70 GST–CP
15 Rubisco
55 anti-GST

Fig. 3 | RBRL acts as an active E3 ligase and CP enhances its activity. The RBRL–MYC + Ubn /RBRL–MYC ratios are listed. e, CP enhanced the ubiquitin
a, Autoubiquitination analysis of OsRBRL. The bacterial lysates from Escherichia ligase activity of OsRBRL. The bacterial lysates from E. coli strains expressing
coli strains expressing UBA1-S (E1), UBC8-S (E2), Flag–Ub and RBRL–MYC or from UBA1-S, UBC8-S, Flag–Ub, RBRL–MYC and GST–CP or from strains lacking one
strains lacking one of these components were analysed by immunoblotting with of these components were analysed by immunoblotting using anti-MYC (row 1),
anti-MYC (top), anti-Flag (middle) or anti-S (bottom) antibodies. b, The domain anti-Flag (row 2), anti-S (row 3) and anti-GST (row 4) antibodies. f, Time-course
compositions of OsRBRL, OsRBRL-like, Arabidopsis thaliana ARI3 (AtARI3), analysis of CP-promoted OsRBRL self-degradation. The OsRBRL degradation
human ARI2 and mouse ARI2. An asterisk indicates the reported active site of assay was performed by mixing cell extracts from separately infiltrated GFP,
human ARI2. c, The effect of MG132 on the stability of the full-length OsRBRL YFP–CP and HA–RBRL samples, and GFP was used as a negative control. Ponceau S
and the RBR domain of OsRBRL. Actin was used as a sample processing control. staining (bottom) of the Rubisco protein is shown as a loading control. All of the
d, Autoubiquitination of OsRBRL and its corresponding point mutants. experiments were repeated two to four times with similar results.

detected in these plants through the anti-NINJA3 antibody (Fig. 4d).

OsRBRL targets NINJA3 for degradation These results suggested that OsNINJA3 might be a substrate of OsRBRL
To identify the mechanisms underlying OsRBRL-mediated modulation and could be degraded after RSV infection.
of the JA signalling pathway, we conducted IP–MS assays focusing on To test our hypothesis, we conducted protein degradation assays in
potential ubiquitination substrates. The results revealed that OsNINJA3, tobacco leaves. Increasing the expression of HA–RBRL rather than HA–
an adaptor protein that links JAZ proteins to their corepressor TPL/TPR, RBRL-like, the protein level of YFP–NINJA3 but not that of YFP–NINJA4
is a potential substrate (Extended Data Fig. 5a,b and Supplementary decreased (Extended Data Fig. 6a). Furthermore, we validated these
Table 4). Phylogenetic analysis revealed that rice NINJA1/2/3 proteins findings using a semi-in vivo system. In the presence of the HA–RBRL
belong to the same subclade as most NINJA proteins in monocotyle- protein, the protein level of YFP–NINJA3, but not that of YFP–NINJA4,
donous plants (Extended Data Fig. 5c). Notably, this subclade is closely was significantly decreased (Fig. 4e,f). We further showed that the
related to the ABA INSENSITIVE FIVE BINDING PROTEINS (AFPs) found degradation of YFP–NINJA3 was significantly attenuated by MG132,
in dicot plants (Extended Data Fig. 5c). Moreover, rice NINJA4 and maize suggesting that the OsRBRL-mediated degradation of OsNINJA3
NINJA7/8 proteins fall within the same subclade as the NINJA proteins occurs through the 26S proteasome (Extended Data Fig. 6b). More­
from Arabidopsis and tobacco (Extended Data Fig. 5c). Conserved over, increasing the level of RSV CP promoted the proteolysis of NINJA3
motif analysis showed that NINJA proteins mainly contain four con- but not that of NINJA4 by activating RBRL (Extended Data Fig. 6c).
served motifs: EAR, NINJA-B, ZBD core and ZBD NTE motifs (Extended As the 26S proteasome primarily recognizes ubiquitinated pro-
Data Fig. 5c). We subsequently examined the interactions between teins and a potential ubiquitinated form of OsNINJA3 was detected
OsRBRL and OsNINJA proteins through yeast two-hybrid (Y2H), LCI in RSV-infected rice (Fig. 4d), we examined whether OsRBRL directly
and pull-down assays. The results indicated that OsRBRL interacted ubiquitinates OsNINJA3. Ubiquitination assay in the bacterial system42
with OsNINJA1/2/3 but not OsNINJA4 (Fig. 4a–c and Extended Data showed that ubiquitination of OsNINJA3 occurred only in the pres-
Fig. 5d–h). The full-length OsNINJA1/2/3 proteins interacted mainly ence of E1, E2, OsRBRL and ubiquitin (Fig. 4g). Importantly, increased
with the NTD and RBR domains of RBRL, while the full-length RBRL pro- ubiquitination of OsNINJA3 was observed when RSV CP was added to
tein interacted with the ZBD NTE motif- and NINJA-B motif-containing the system (Extended Data Fig. 6d).
domains of the NINJA3 protein (Extended Data Fig. 5f–h). Notably, To confirm the above results, we co-expressed NINJA3-GFP with
RSV-infected rice plants showed significantly decreased protein lev- HA-RBRL or empty vector (EV) in NPB protoplasts. The NINJA3–GFP
els of OsNINJA3, and several higher-molecular-mass proteins were signal was substantially weaker in cells co-expressing RBRL than in

Nature | www.nature.com | 5
Article
a b c e HA–RBRL
YFP–NINJA4 YFP–NINJA3

L
BR

BR
Input PD: anti-MBP Time (h) 0 0.5 1 2 3 0 0.5 1 2 3

-R

-R
AD RBRL–nLUC RBRL–nLUC kDa kDa

AD

AD

AD
+ + GST + – – – – + – – – – 100 70 Anti-GFP
cLUC–NINJA1 cLUC–NINJA2 GST–NINJA1 – + – – – – + – – – 1.0 1.1 1.1 1.1 1.1 1.0 0.6 0.5 0.4 0.3
BD GST–NINJA2 – – + – – – – + – –
70 70 Anti-HA
GST–NINJA3 – – – + – – – – + –
GST–NINJA4 – – – – + – – – – + 55 55
BD-NINJA1 MBP–HA–RBRL + + + + + kDa Rubisco
+ + + + +
130
f
BD-NINJA2 * * * *100 *
YFP–NINJA4 + RBRL YFP–NINJA3 + RBRL
70 * * –2 –3
8 × 10 1 × 10 –2 –3

IB: anti-GST
55 120 P = 3.0 P = 4.9 = 1.40 × 10 = 1.78 × 10
P P
40 100

remaining (%)
BD-NINJA3

YFP–NINJAs
RBR–nLUC RBR–nLUC 80
35
+ +
cLUC–NINJA3 cLUC–NINJA4 * 25 60
BD-NINJA4 40
IB: anti-MBP 130 20
SD-L-W SD-L-W-H-Ade
118
230
341
452
564
675
786
898
1,009
1,120
1,232
0
0 0.5 1.0 2.0 3.0
cps
Time (h)

d g – + + + + + UBA1-S h i j
+ – + + + + UBC8-S GFP Chl. Merge
P = 2.04 × 10
–19 NINJA3–GFP
+ + – + + + RBRL–MYC 10.00
NPB kDa EV HA–RBRL

Relative intensity of GFP

+ + + + – + MBP–NINJA3–HA 8.00

(fluorescence relative
kDa + + + – + + Flag-Ub 6.00 65
kDa Mock RSV EV Anti-GFP
54

to chlorophyll)
4.00
Anti-MYC Anti-HA

NINJA3–GFP
MBP–NINJA3–HA + Ubn
95 2.00 1.0 0.3
MBP–NINJA3–HA
65 65
170 0.03 Anti-HA
54
95 RBRL–MYC + Ubn 0.02
100 HA–RBRL 42 Anti-actin
65 RBRL–MYC 0.01
NINJA3
70
190 0
ACTIN
Anti-NINJA3 95 EV HA–RBRL
65
Anti-Flag

40 * 42 k GFP Chl. Merge l m

1.0 0.2 23 15 NINJA3–GFP

Relative intensity of GFP

–4
P = 4.76 × 10

(fluorescence relative
kDa NPB rbrl
Anti-CP NPB
35 10 His–Flag–Ub

to chlorophyll)
65
NINJA3–GFP

190 UBA1-S + Ub 10 Anti-GFP

54
Anti-actin 95 UBA1-S
40 65 1.0 2.6
5
Anti-S

42 42 Anti-actin
UBC8-S + Ub rbrl
NINJA3
23
0 ACTIN
10 UBC8-S NPB rbrl

Fig. 4 | RBRL targets NINJA3 for degradation. a–c, Y2H (a), LCI (b) and GFP–NINJA3 in NPB rice protoplasts, NPB rice protoplasts transformed with
pull-down (PD) (c) assays analysing the interactions between OsNINJAs and empty vector or 35S::HA-RBRL overexpression vector (h–j) and rbrl rice
OsRBRL. d, Immunoblotting of OsNINJA3 in mock-inoculated or RSV-infected protoplasts (k–m) was analysed using confocal microscopy (h,k). Scale bars,
NPB plants (indicated by the asterisk). The red line denotes higher-molecular- 5 μm (h and k). i,l, The NINJA3–GFP signal was quantified and normalized to the
mass derivatives of OsNINJA3. Actin was used as a processing control. e, Time- chlorophyll fluorescence. n = 81 and 87 cells (i) and 74 and 71 cells (l) over two
course analysis of RBRL-promoted NINJA3 degradation. YFP–NINJA4 was used independent biological experiments. For the box plots in i and l, the centre line
as a negative control. Ponceau S staining of the Rubisco proteins was used as shows the median, the box limits show the upper and lower quartiles, the
a sample processing control. f, The intensities of the bands shown in e were whiskers show the minimum to the maximum, and the points show individual
quantified using ImageJ. Relative values were calculated by comparison with values. j,m, Immunoblotting analysis of the NINJA3–GFP levels in h and k. Actin
the values at 0 h (set to 1.0). Data are mean ± s.d. n = 3 independent biological was used as a loading control (middle). NINJA3 and ACTIN mRNA levels were
samples. g, Ubiquitination of NINJA3 by RBRL. Bacterial lysates from the analysed using RT–PCR (bottom). For f, i and l, statistical analysis was performed
indicated E. coli strains were analysed by immunoblotting. h–m, The NINJA3– using two-sided Student’s t-tests; all P values are shown in the figures. All of the
GFP signal was affected by OsRBRL in rice protoplasts. Fluorescence of experiments were repeated two to four times with similar results.

cells expressing the EV (Fig. 4h,i). Consistently, immunoblotting indi- Collectively, these results provided strong evidence showing that,
cated that HA–RBRL significantly reduced NINJA3–GFP levels while after RSV infection, CP activates the E3 ligase activity of OsRBRL to
the NINJA3 mRNA level was equivalent across various co-transforma- mediate the ubiquitination and subsequent degradation of OsNINJA3
tion conditions (Fig. 4j). We extended this analysis using rice proto- through the ubiquitination system, therefore inducing jasmonate
plasts extracted from the NPB and rbrl mutants. The signal intensity signalling. These findings revealed a mechanism for relieving plant
of NINJA3–GFP was substantially higher in protoplasts from the rbrl RBR-type E3 ligase autoinhibition and the turnover mechanism of
mutants than in those from the NPB plants (Fig. 4k,l). Notably, the OsNINJA3.
protein levels of NINJA3–GFP were consistent with the fluorescence
results (Fig. 4m). Importantly, the protein levels of OsNINJA3 were
lower in the RBRL-OE lines and greater in the rbrl-mutant lines compared NINJA3 suppresses antiviral JA signalling
with those in the NPB plants (Extended Data Fig. 6e). Furthermore, Although AFP2 and AFP3 have been shown to not interact with JAZ1 in
with RSV infection, higher-molecular-mass proteins (ubiquitinated Arabidopsis24, the afp2 mutant presented phenotypes similar to those
OsNINJA3) accumulated in the NPB and RBRL-OE lines but not in the of the Arabidopsis jaz-D (decuple JAZ mutant) mutant44, indicating
rbrl-mutant plants (Extended Data Fig. 6e). This is possibly due to that AFPs in dicotyledonous plants may be involved in the regulation
the CP-enhanced RBRL-mediated ubiquitination of NINJA3 and the of JA signalling. Rice NINJA1 interacts with most OsJAZs and inhibits JA
irregular proteasome assembly in RSV-infected plants (Extended Data signalling by recruiting of OsTPR145, suggesting that NINJAs or AFPs of
Fig. 6d,f). monocotyledonous crop species may have evolved different functions

6 | Nature | www.nature.com
a b c d
NPB ninja3
NINJA3 CP
CM-LOX1 RBRL

E
O
ninja3

PB
CM-LOX2

P-
kDa

C
WT OsAOS2 kDa NPB 1 2 Anti-
OsJMT1 40
18 28 138 130 NINJA3
OsCOI1A Anti-CM-LOX2
OsCOI1B 100 TPL 1.0 0.3
ninja3 1 OsCOI2 1.0 2.4 2.5 Anti-
5 bp deletion OsJAZ6 NINJA3
18 28 138 35 HA (CP)
OsJAZ8 130 Anti-OsAGO18
OsJAZ10 JAZs
JAZs Anti-
ninja3 2 TCC OsJAZ12 1.0 2.3 2.3 40 actin
deletion OsPR10
18 28 Anti-actin JAMYB or other TFs
OsJAMYB 40
OsAGO18 Genes in JA signalling or
* Premature termination
log2[FC] RNA-silencing pathways
–3 0 3

e f g h
120 4
III II I N

Relative expression level of viral RNAs

100 –2 ninja3 NINJA3-OE
P = 1.88 × 10
Percentage of symptoms (%)

–6
P = 6.84 × 10
3 –5
kDa NPB 1 2 1 2
P = 2.91 × 10
80 Anti-
–3 35 CP
P = 4.64 × 10
60 2 P = 4.50 × 10
–3
–4
1.0 0.1 0.1 1.5 1.6
P = 8.09 × 10
P = 4.39 × 10
–3 55 Anti-
–6
40 P = 4.50 × 10 40 MYC
1 1.0 2.2
20 Anti-
40 actin
NPB 1 2 1 2 NPB 1 2 1 2
0
ninja3 NINJA3-OE ninja3 NINJA3-OE 0
NPB 1 2 1 2 NPB 1 2 1 2
Mock RSV NPB 1 2 1 2
ninja3 NINJA3-OE ninja3 NINJA3-OE
ninja3 NINJA3-OE
RNA1 RNA3

Fig. 5 | NINJA3 negatively regulates JA signalling and compromises rice RSV-infected NPB, ninja3 and NINJA3-OE plants with different disease symptom
antiviral defence. a, Construction of ninja3-knockout (CRISPR–Cas9) lines. grades. Data are mean ± s.d. n = 3 independent biological experiments.
The sgRNA sequence that specifically targets OsNINJA3 is indicated, and the g, RT–qPCR analysis of RSV RNA accumulation in the indicated plants. Statistical
PAM is highlighted in red. The deletions in the sgRNA target sites in ninja3 analysis was performed using two-sided Student’s t-tests; all P values are shown
lines led to premature termination of the translation of OsNINJA3. b, RNA-seq in the figure. Data are mean ± s.d. n = 3 independent biological samples.
analysis of the expression levels of genes related to JA synthesis and signalling h, Immunoblotting analysis of the RSV CP levels in the indicated plants.
in the NPB and ninja3 rice lines. c, Immunoblotting analysis of the indicated OsNINJA3 expressed in NINJA3-OE lines 1 and 2 was labelled with a 3× MYC tag at
protein levels in the NPB and ninja3 rice lines. d, Schematic of RBRL and NINJA3 the N terminus. Actin in c, d and h was used as a sample processing control. All
in regulating gene expression and immunoblot analysis of the OsNINJA3 levels of the rice plants were collected at 4 w.p.i. All of the experiments were repeated
in the indicated plants. e, Photographs of mock-inoculated or RSV-infected two to four times with similar results. TFs, transcription factors.
NPB, ninja3 and NINJA3-OE plants. Scale bar, 10 cm. f, The percentages of

in regulating plant growth and stress responses. To examine the role of OsNINJA3 exacerbated the RSV infection symptoms and disease
OsNINJA3 in antiviral defence, we knocked out OsNINJA3 to generate progression (Fig. 5e,f and Supplementary Table 3). These plants exhib-
ninja3-mutant lines (Fig. 5a) and performed RNA-seq analysis of NPB ited more severe symptoms and increased viral RNA and CP accumula-
and ninja3 mutants with or without RSV infection. We identified 9,942 tion (Fig. 5g,h).
DEGs between NPB and ninja3 mutants under mock-inoculated and Given that OsRBRL interacts with OsNINJA1/2/3, to verify the roles
RSV-infected conditions (Extended Data Fig. 7a–c). GO analysis revealed of OsNINJA1 and OsNINJA2 in rice antiviral defence, we also obtained
that these DEGs were enriched in biological processes associated with a ninja1-mutant (modd-2)46 (Extended Data Fig. 9a), along with the
cell division, ROS metabolic processes, response to JA stimulus, RNAi OsNINJA2-knockout mutant (Extended Data Fig. 9e) and OsNINJA2
and abscisic acid signalling pathways (Extended Data Fig. 7d). Consist- overexpression (NINJA2-OE) lines. In response to natural RSV infec-
ently, the absence of NINJA3 led to the activation of JA biosynthesis- and tion, the disease symptoms of the ninja1 mutants were similar to those
signalling-responsive genes, including the rice antiviral defence marker of the wild-type Dongjing (DJ) plants (Extended Data Fig. 9b,c and
gene OsAGO18 (Fig. 5b,c). Supplementary Table 3). Compared with that in DJ plants, CP accu-
We further demonstrated through Y2H and LCI assays that OsNINJA3 mulation in ninja1 mutants was not significantly different (Extended
interacts with various OsJAZ proteins, including OsJAZ6 (Extended Data Data Fig. 9d). Compared with the NPB plants, the ninja2-mutant and
Fig. 8). OsNINJA1 has been reported to interact with TPR1/3 to inhibit NINJA2-OE rice plants presented similar disease symptoms and CP
downstream gene expression45,46. The formation of complexes with accumulation (Extended Data Fig. 9f–h and Supplementary Table 3).
OsJAZ proteins enables OsNINJA3 to inhibit JA-responsive gene expres- We also generated ninja1/2/3 triple mutants in the ninja3 single-mutant
sion by recruiting the corepressor TPL/TPR. Moreover, CP enhanced background (Extended Data Fig. 9i). The resistance of the ninja1/2/3
the ability of RBRL to reverse this inhibition by degrading the OsNINJA3 triple mutants to RSV infection was comparable to that of the ninja3
protein (Fig. 5d). The NINJA3 protein level was lower in the CP-OE rice single mutant (Extended Data Fig. 9j,k). These data indicated that, in
plants than in the NPB plants (Fig. 5d). contrast to OsNINJA3, OsNINJA1 and OsNINJA2 do not have a major role
We also investigated the role of OsNINJA3 in rice antiviral defence by in rice antiviral defence against RSV.
inoculating OsNINJA3-related rice lines with small brown planthopper- Previous studies have shown that OsAGO18 releases OsAO through
carrying viruses. The ninja3-mutant lines displayed milder disease competitive binding of miR528 to OsAGO1, thereby increasing ROS lev-
symptoms after infection with RSV compared with the NINJA3-OE lines els to defend against viruses. We therefore measured the levels of OsAO
and NPB plants (Fig. 5e,f and Supplementary Table 3). Moreover, the protein and ROS in NPB and rbrl- and ninja3-mutant plants. OsAO levels
levels of viral RNA and CP accumulation were significantly lower in were reduced in the rbrl mutants (Extended Data Fig. 9l) and increased
the ninja3-mutant lines (Fig. 5g,h). Conversely, overexpression of in the ninja3 mutants (Extended Data Fig. 9m) compared with those

Nature | www.nature.com | 7
Article
in the NPB plants. Consistently, the ROS levels were lower in the rbrl vast majority of studies on plant antiviral defence mechanisms have
mutants and higher in the ninja3 mutants (Extended Data Fig. 9n,o). used artificial infection methods, such as mechanical inoculation with
Overall, the absence or RBRL-mediated degradation of OsNINJA3 in vitro viral RNA transcripts or virions or infiltration of agrobacteria
relieves its repression of the JA signalling pathway, elevates the expres- containing engineered viral DNA. These methods often yield markedly
sion of OsAGO18 and, ultimately, strengthens the defence of rice hosts greater inoculum levels compared with those that occur under natural
against viral infection. infection, and the potential influence of these methods on the plant
immune response remains an unanswered question. Here we used
natural viral insect vectors to initiate infection, which best reflects
RBRL mediates broad-spectrum resistance the infection process under field conditions regarding the behaviour
Recent studies have shown that the activation of JA signalling and of viruses and the host response. This approach is important not only
the RNAi pathway mediates antiviral defence against different rice for the dissection of virus–host interactions but also for further design
viruses47,48. We wondered whether the perception of viral effectors by of disease-resistance breeding programs.
RBRL induces the initiation of the universal antiviral defence of plants. JA is a key immune hormone that enhances the resistance of plants
RNA-seq data from a previous study showed that rice dwarf virus (RDV) to diverse threats, from microbial pathogens to various herbivores51,52.
infection also activates JA biosynthesis and signalling in rice49 (Extended The core pathways governing the biosynthesis and signalling of JA
Data Fig. 10a). RDV is a member of the Phytoreovirus genus50. We tested in plants have been extensively studied in recent decades52,53. More­
whether RBRL could also interact with RDV-encoded proteins through over, the importance of post-translational modifications in the fine-
Y2H assays. The RDV coat proteins P2 and P9 interacted with RBRL tuning of JA responses across diverse spatiotemporal scales has just
(Extended Data Fig. 10b). MST assays showed that the Kd between P2 been revealed54–57. Here we identified a derepression mechanism for
and RBRL is 183.47 nM (Extended Data Fig. 10c). Like RSV CP, P2 and P9 JA-mediated antiviral signalling through the OsRBRL-mediated ubiqui-
are not substrates of RBRL (Extended Data Fig. 4h,i), indicating that tination and degradation of OsNINJA3. OsNINJA1 and OsNINJA2 display
the same mechanism of RBRL-mediated antiviral activity against RDV almost no effects on antiviral defence, although they also interact with
occurs. We therefore challenged NPB and JA signalling-related rice lines OsRBRL, which indicates that there is little redundancy among these
with RDV through natural infection experiments using viruliferous OsNINJA proteins during rice antiviral defence. These functional dif-
leafhoppers (Nephotettix cincticeps), the transmission vector of RDV. ferences among NINJA adaptors could be the result of evolutionary
We categorized the RDV-induced disease symptoms into four classes divergence.
based on severity (Extended Data Fig. 10d). Compared with the NPB Results from HHARI and TRIAD proteins, orthologues of OsRBRL,
plants, the coi1-13, jamyb and rbrl rice mutants were more susceptible indicate that binding by their interactors could release their autoinhibi-
to infection, whereas the JAMYB-OE and RBRL-OE rice lines showed tion by opening the mask of the Ariadne domain on the active site39,43.
increased resistance17 (Extended Data Fig. 10e–j and Supplementary RSV CP–RBRL interaction promotes RBRL self-ubiquitination and
Table 3). Consistently, the viral RNA and protein levels were increased RBRL-mediated ubiquitination of NINJA3 in bacterial systems. Thus,
in the rbrl mutants and decreased in the RBRL-OE rice plants (Extended CP appears to release the autoinhibition of OsRBRL, possibly through
Data Figs. 4j and 10k–l). These results underscore the pivotal role of interaction, thereby exposing the active site. Notably, we found that
OsRBRL in strengthening broad-spectrum rice antiviral defences. CP-mediated degradation and activation of RBRL varies with context,
Overall, our study revealed a fundamental mechanism by which rice similar to Keep on Going, a previously reported E3 ubiquitin ligase58.
plants respond to viral infections. This mechanism centres on the per- When co-expressing RBRL and CP in tobacco leaves, CP primarily pro-
ception of viral CP by the RBR-type E3 ligase RBRL, which functions as motes the degradation of RBRL. Meanwhile, in RSV-infected rice or
a sensor for the subsequent antiviral defence in the host. After RSV when co-expressing RBRL with CP and OsNINJA3 in tobacco leaves,
infection, CP is perceived by OsRBRL to facilitate the degradation of CP facilitates the degradation of NINJA3 by RBRL.
OsNINJA3, a negative regulator of the JA signalling pathway, and this Overall, our findings reveal a fundamental mechanism by which
degradation process is orchestrated by the ubiquitination activity of plant hosts perceive viral infection and initiate subsequent antiviral
OsRBRL. The degradation of OsNINJA3 activates the JA signalling path- defence responses through the RBR-type E3 ligase and will facilitate
way, leading to increased JA biosynthesis and upregulation of OsAGO18 diverse research in the broader field of plant immunity.
expression. These events occur independently of COI1-mediated JA
signalling responses. With the accumulation of JA, COI1-mediated JA
signalling establishes a positive-feedback loop, amplifying the expres- Online content
sion of OsAGO18 to a sufficiently high level. This increased expression of Any methods, additional references, Nature Portfolio reporting summa-
OsAGO18 enhances the host antiviral defence by alleviating the repres- ries, source data, extended data, supplementary information, acknowl-
sion of AGO1-mediated antiviral RNAi and promoting the upregula- edgements, peer review information; details of author contributions
tion of ROS levels by AO, as previously reported17–19 (Extended Data and competing interests; and statements of data and code availability
Fig. 10m). Thus, our study provides deeper insights into the intricacies are available at https://doi.org/10.1038/s41586-025-08706-8.
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Article
Methods Uncropped immunoblotting images are provided in Supplementary
Fig. 1.
Rice plants and growth conditions
We used the rice ecotype NPB or DJ as the wild-type control. The coi1-13 Nuclear‒cytoplasmic fractionation followed by an
mutant was identified by quantitative PCR with reverse transcription immunoprecipitation assay
(RT–qPCR) and phenotypic observation, consistent with previous To precisely screen for rice proteins that interact with RSV CP in the
reports59. The jamyb mutant was identified by sequencing, as previ- nucleus, we performed nuclear–cytoplasmic fractionation followed
ously described17. The ninja1 (modd-2) mutant was identified by geno- by IP–MS on RSV-infected NPB and CP-OE rice, with mock NPB rice used
typing using gene-specific and T-DNA-specific primers as described as a control. First, we conducted subcellular fractionation on the rice
previously46. All of the rice plants were cultivated in a greenhouse at materials with minor modifications as described previously63. The rice
28–32 °C and at a relative humidity of 60 ± 5%. materials were harvested and ground into powder in liquid nitrogen
at 4 weeks after RSV infection. The powder (approximately 1 g) was
N. benthamiana growth conditions resuspended in Honda buffer (0.4 M sucrose, 2.5% Ficoll, 5% dextran
N. benthamiana plants were cultivated in soil within a greenhouse under T40, 25 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 0.5% Triton X-100, 0.5 mM
a 16 h–8 h light–dark photoperiod at a constant temperature of 24 °C. PMSF, 10 mM β-mercaptoethanol, RNase inhibitor and Roche protease
For transient expression assays, the upper three leaves of 1-month-old inhibitor cocktail) at a ratio of 2 ml g−1. The homogenate was filtered
plants were used. twice through a double-layered Miracloth, and the supernatant was
subsequently centrifuged at 1,500g for 5 min at 4 °C. The supernatant
Vector construction and rice transformation was further centrifuged at 10,000g for 10 min at 4 °C to obtain the
The coding sequences of OsRBRL, RSV CP, OsNINJA2 and OsNINJA3 cytoplasmic fraction. The pellet containing the nuclear proteins was
were initially amplified by RT–PCR. Subsequently, the genes were indi- resuspended in three volumes of nuclear extraction buffer (500 mM
vidually cloned and inserted into the pCambia2300-Actin1-OCS and KCl, 20 mM Tris-HCl (pH 8.0), 0.5% Triton X-100, 25% glycerol, 1.5 mM
pCambia2300-Actin1-3×MYC vectors, resulting in the generation of MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol (DTT), cocktail and 1 mM
the Actin1::HA-OsRBRL, Actin1::HA-CP, Actin1::3×MYC-OsNINJA2 and PMSF) and incubated for 30 min at 4 °C. Insoluble nuclear residues were
Actin1::3×MYC-OsNINJA3 constructs. The rbrl-, ninja2-, ninja3- and then sedimented at 10,000g at 4 °C for 10 min, and the supernatant
ninja1/2/3-knockout lines were generated using CRISPR–Cas9 accord- was collected for the IP assay. The nuclear extract was diluted with
ing to established protocols60. These constructs were subsequently four volumes of dilution buffer (20 mM Tris-HCl (pH 8.0), 500 mM KCl,
introduced into wild-type NPB plants or rbrl 2 mutant plants through 0.5% Triton X-100, 1 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, cocktail and
Agrobacterium tumefaciens-mediated transformation using a process 1 mM PMSF). The mixture was precleared with protein G for 30 min,
facilitated by BioRun and BIOGLE GeneTech. The rice lines overexpress- and 1:100 anti-CP antibody was then added and incubated for 2 h, fol-
ing OsRBRL, RSV CP and OsNINJA3 were verified by immunoblotting. lowed by the addition of 1:200 protein G and further incubation for
The genome-edited mutants were confirmed by sequencing and ana- 2 h. The samples were washed three times with washing buffer (20 mM
lysed using SnapGene (https://www.snapgene.com). For transient Tris-HCl (pH 8.0), 500 mM KCl, 0.5% Triton X-100, 5% glycerol, 1 mM
expression assays, genes in recombinant binary vectors were initially MgCl2, 0.5 mM EDTA, 1 mM DTT, cocktail and 1 mM PMSF). Finally, the
amplified by RT–PCR and subsequently cloned and inserted into the bead-bound proteins were eluted in 2× Laemmli buffer and subjected
corresponding entry vectors pEASY-Blunt-Simple and pENTR/D-TOPO. to SDS–PAGE. For LC–MS/MS analysis, the gel was subjected to silver
These inserts were subsequently cut and either ligated or recombined staining to identify the candidate CP-interacting proteins.
into destination vectors. Detailed primer information is provided in
Supplementary Table 5. IP for LC‒MS/MS assay
IP–LC–MS/MS assays were performed as previously described with
Immunoblotting and quantification analysis some modifications64,65. To detect proteins that interact with RBRL,
Plant samples were homogenized in liquid nitrogen, and total pro- we performed IP-LC–MS/MS assays with transgenic plants that overex-
teins were extracted from equal weights of ground powder using the pressed HA-tagged versions of RBRL. In brief, rice samples were homog-
same volume of 2× Laemmli buffer (4% (w/v) SDS, 20% (v/v) glycerol, enized in liquid nitrogen. Total proteins were extracted from 200 mg of
10% (v/v) 2-mercaptoethanol, 0.004% (w/v) bromophenol blue and ground powder using IP buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl,
0.125 M Tris-HCl, pH 6.8) and subsequently boiled at 95 °C for 10 min. 4 mM MgCl2, 0.5% (v/v) NP-40, 5 mM DTT, 1 mM phenylmethylsulfonyl
The supernatants were collected by centrifugation at 12,000 rpm fluoride, 50 μM MG132 and 1× protease inhibitor cocktail) and then
for 5 min and separated by SDS–PAGE. The PageRuler Prestained incubated for 30 min at 4 °C with gentle rotation. Next, the samples
Protein Ladder (Thermo Fisher Scientific) and Prestained Protein were centrifuged at 12,000 rpm and 4 °C three times for 5 min each;
Ladder (Meilun Biotech) were used as molecular mass standards. The the supernatants were collected after each centrifugation step. The
proteins were subsequently transferred to nitrocellulose membranes resulting supernatant was transferred to a new tube containing 7.5 μl of
and detected with antibodies against CM-LOX2 (1:2,000)17, AOS2 HA-magnetic agarose (MBL) and incubated at 4 °C for 2.5 h with gentle
(1:2,000)17, RSV CP (1:5,000)61, RDV P2 (1:5,000)62, AGO18 (1:500)18, rotation. The samples were then washed three times with wash buffer
AO (1:500)19, RBRL (1:500), NINJA3 (1:500), MYC (ABclonal, 1:2,000), A (50 mM Tris-HCl (pH 7.4), 150 mM NaCl and 4 mM MgCl2). Finally, the
HA (ABclonal, 1:2,000), GFP/YFP (ABclonal, 1:2,000), GST (ABclonal, bead-bound proteins were eluted in 2× Laemmli buffer and subjected
1:2,000), MBP (ABclonal, 1:2,000), Flag (TransGen Biotech, 1:2,000), to SDS–PAGE. For LC–MS/MS analysis, the gel was subjected to silver
S (EarthOx, 1:2,000), FBPA (Beijing Protein Innovation, 1:2,000), staining to identify the candidate RBRL-interacting proteins.
histone H3 (Abcam, 1:2,000), cLUC (Sigma-Aldrich, 1:1,000) and
actin (CWBIO, 1:10,000). Actin was used as the loading control or LC‒MS/MS assay
sample processing control. Images from immunoblotting were col- First, the gel was cut into small pieces and placed into tubes for diges-
lected using the Molecular lmager ChemiDoc XRS+ (Bio-Rad) system. tion. A total of 400 μl of decolourization solution was added to each
The corresponding band intensities were quantified using ImageJ tube and shaken until completely decolorized, after which the liquid
(https://imagej.net/ij/). The band intensities for a particular protein was discarded. Next, 400 μl of acetonitrile was added, and the mixture
were normalized to those for actin or Rubisco. Relative values were was shaken until the gel pieces turned white; the liquid was then dis-
calculated by comparison with the first band on the left in each figure. carded. Next, 200 μl of 10 mM DTT and 25 mM NH4HCO3 were added,
and the mixture was incubated at 56 °C for 1 h. The mixture was cooled with an OD600 of 1.0. The infiltration procedures were performed as
to room temperature, the liquid was discarded, 200 μl of 55 mM IAM described above (see the ‘Transient expression in N. benthamiana
and 25 mM NH4HCO3 were added, and the mixture was incubated in leaves’ section). Leaves were harvested at 2 days after agroinfiltration,
the dark for 45 min. After the liquid in the tubes was discarded, the gels and total proteins were extracted with native extraction buffer (50 mM
were washed twice with 25 mM NH4HCO3. Next, 400 μl of acetonitrile Tris-MES (pH 8.0), 500 mM sucrose, 1 mM MgCl2, 10 mM EDTA, freshly
was added, and the mixture was shaken until the gel pieces turned added 5 mM DTT and 1× protease inhibitor cocktail)66. The mixture was
white. The liquid was discarded, and the gel pieces were crushed with a incubated at 4 °C for 30 min with gentle rotation and then centrifuged
pipette tip. For each tube, trypsin was added to 25 mM NH4HCO3 buffer at 12,000 rpm and 4 °C three times for 10 min each; the supernatants
at a trypsin ratio of 1:50 (w/w), and the mixture was subsequently incu- were collected after each centrifugation step. Cleared extracts were
bated at 37 °C overnight. Then, 200 μl of acetonitrile containing 0.1% immunoprecipitated using GFP-tagged or HA-tagged Nanoselector
formic acid was added to each tube, the tubes were shaken for 10 min Agarose (HUABIO) and incubated for 1 h at 4 °C with gentle rotation.
and the supernatant was then transferred to a clean tube. Next, 30 μl The samples were then washed three times with washing buffer B
of 0.1% formic acid was added to the gel, which was shaken for 10 min, (10 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.5 mM EDTA). Finally, the
then 200 μl of acetonitrile containing 0.1% formic acid was added and bead-bound proteins were eluted in 2× Laemmli buffer. The eluted
the mixture was shaken for another 10 min. The supernatant was col- proteins were boiled for 10 min, centrifuged, separated by SDS–PAGE
lected and dried using a vacuum centrifuge. Peptide samples were dis- and detected with antibodies against HA (ABclonal) and GFP (ABclonal).
solved in 0.1% formic acid solution to a concentration of approximately
0.1 μg μl−1. The mixture was centrifuged at 16,700g for 12 min, and the Protein expression and purification
supernatant was transferred to a MS injection vial to conduct the LC– The coding sequences of RSV CP, RDV P2, RDV P21–786, RDV P9, OsRBRL
MS/MS assay. LC–MS/MS analysis was performed using the EASY-nLC and four OsNINJA genes were inserted into the pHM4 (MBP tag, modified
1200 liquid chromatography system and a C18 column coupled with from pMAL-c2X Vector, New England Biolabs), pGEX4T1 (GST tag, GE
a Thermo Fusion Lumos mass spectrometer. The mobile phase for Healthcare), pACYCDuet42 or pCDFDuet42 vector and expressed as tag
liquid chromatography consisted of 0.1% formic acid (phase A) and fusion proteins (MBP–RBRL, GST–CP, GST–NINJA1, GST–NINJA2, GST–
80% acetonitrile/0.1% formic acid (phase B). The flow rate was set at NINJA3, GST–NINJA4, RBRL–MYC, MBP–P2–HA, MBP–P9–HA, MBP–
280 nl min−1. The LC–MS/MS results were processed using Proteome P21–786 and MBP–NINJA3–HA) in E. coli strain Transetta or BL21 (DE3;
Discoverer 2.2 software and the rice database to acquire the CP- and TransGen Biotech). The fusion proteins were purified using glutathione
RBRL-interacting proteins. Sepharose 4B beads (GE Healthcare) or amylose resin (New England
Biolabs) or detected by immunoblotting as previously described42.
Transient expression in N. benthamiana leaves
Agrobacterium-mediated transient expression assays were performed Pull-down assay
as previously described17 with minor modifications. The recombinant Equimolar amounts of MBP–RBRL on MBP tag Nanoselector Agarose
binary vectors were subsequently transformed into A. tumefaciens (HUABIO) were separately incubated with equal amounts of GST, GST–
strain GV3101 using the freeze–thaw method. Suspensions of Agrobac- CP, GST–NINJA1, GST–NINJA2, GST–NINJA3 or GST–NINJA4 in pull-down
terium cultures were adjusted to an optical density at 600 nm (OD600) of buffer (20 mM Tris-HCl (pH 7.5), 200 mM NaCl) at 4 °C for 1 h with gentle
1.0 and used to infiltrate leaves of N. benthamiana plants at the four- to rotation. The beads were then washed (five times for 5 min each) with
five-leaf stage through a 1-ml syringe without a needle. Leaf tissues were pull-down buffer (containing 2% (v/v) Triton X-100). The bound pro-
harvested at 2 days after agroinfiltration. teins were boiled in 2× Laemmli buffer, separated by SDS–PAGE, and
detected with antibodies against MBP (ABclonal) and GST (ABclonal).
LCI assay
LCI assays were also conducted as previously described17. The coding Virus inoculation
sequences of OsRBRL, RSV CP, four OsNINJA genes and OsRBRL-like, The virus inoculation procedures were performed as previously
and the RBR NTD domain, RBR RBR domain, RBR Ari domain, NINJA3 described17,67. In brief, 2-week-old rice plants were inoculated with two
EAR domain, NINJA3 NINJA-B domain, NINJA3 ZBD NTE domain and viruliferous (containing RSV or RDV) or virus-free (mock) insects. Then,
NINJA3 ZBD core domain and all OsJAZ genes were separately inserted 2 days after inoculation, the insects were removed, and the rice plants
into the pCambia1300-nLUC or pCambia1300-cLUC vectors. A list were returned to the greenhouse as described above (see the ‘Rice
of the primers used is provided in Supplementary Table 5. All of the plants and growth conditions’ section). The inoculated plants were
constructs were subsequently transformed into A. tumefaciens strain monitored weekly for the appearance of viral symptoms. The numbers
GV3101 using the freeze–thaw method. Four combinations of A. tume- of rice plants of each line with various disease symptom grades were
faciens suspensions were mixed, adjusted to a final concentration with recorded at 4 w.p.i. (Supplementary Table 3). Photographs of plants
an OD600 of 1.0 and coinfiltrated into four different regions on the same with representative symptoms were acquired at 2 or 4 w.p.i.; whole
N. benthamiana leaf. At 2 days after agroinfiltration, cells were infil- shoots were harvested for RT–qPCR and immunoblotting assays at
trated with 200 μM luciferin (Promega), and luciferase activity was 4 w.p.i.
detected using a low-light cooled charge-coupled device imaging appa-
ratus (NightOWL II LB983 with IndiGO software v.2.0.4.0). At least three RNA extraction and RT‒qPCR analysis
biological replicates were performed. Rice samples were homogenized in liquid nitrogen, and total RNA
was extracted from 100 mg of ground powder using TRIzol Reagent
Co-IP assay (Thermo Fisher Scientific) in accordance with the manufacturer’s
35S::YFP-CP was constructed by inserting the coding sequence of RSV CP instructions. Total RNA was treated with RQ1 RNase-free DNase (Pro-
into the pEarleyGate104 vector and was subsequently used to express mega) to remove traces of contaminating genomic DNA. The RNA was
YFP–CP. 35S::HA-RBRL was constructed by fusing the coding sequence subsequently reverse-transcribed using M-MLV reverse transcriptase
of OsRBRL into the pCambia2300 vector and was subsequently used to (Promega), oligo(dT)18 primer and recombinant RNasin ribonuclease
express HA–RBRL. pCambia1301-35S-GFP was used to express GFP. All inhibitor (Promega) according to the manufacturer’s instructions. The
of the constructs were subsequently transformed into A. tumefaciens resulting cDNA was used as the template for RT–PCR and RT–qPCR.
strain GV3101 using the freeze–thaw method. The indicated combina- RT–qPCR was performed using the SYBR Green Real-Time PCR Master
tions of A. tumefaciens suspensions were mixed to a final concentration Mix (Mei5 Biotech) according to the manufacturer’s instructions using
Article
the Bio-Rad CFX96 system with CFX Maestro 1.1 software. The level of glycol (PEG). Rice protoplasts were isolated from the leaf sheaths of
OsEF-1a expression was detected in parallel and used as the internal 10–14-day-old wild-type (NPB) or rbrl 2 plants. The leaf sheaths were
control. A list of the primers used is provided in Supplementary Table 5. initially cut into 0.5-mm pieces with sharp razor blades and then
submerged in enzyme solution (0.4 M d-mannitol, 20 mM MES-KOH
Phylogenetic analysis (pH 5.7), 20 mM KCl, 1.5% cellulase R10 (w/v), 0.7% macerozyme R10
The amino acid sequences of RBRL, other RBR family proteins, and (w/v), 0.1% bovine serum albumin and 10 mM CaCl2) for 7 h with shak-
NINJA family proteins were obtained from UniProt (https://www. ing (40 rpm) at 28 °C in the dark. Each sample was filtered through a
uniprot.org/). An unrooted, neighbour-joining tree was constructed 40-μm nylon mesh filter. After removal of the enzyme solution, the
using Molecular Evolutionary Genetic Analysis 11 (MEGA11). tissues were suspended in W5 solution (154 mM NaCl, 125 mM CaCl2,
5 mM KCl and 2 mM MES (pH 5.7)) and subsequently filtered through
Generation of rice RBRL and NINJA3 antibodies another 40-μm nylon mesh filter. The flow-through samples from the
Rabbit polyclonal antibodies were generated by HUABIO or ABclonal. above-mentioned filtrations were mixed and then centrifuged for 3 min
The synthetic peptides RBRL (DLHLRLPDDRPADC) and NINJA3 at 900 rpm to pellet the protoplasts. The protoplasts were resuspended
(STGKPLNGTVTQQS) were used to obtain rabbit polyclonal antibod- in W5 solution and incubated on ice for 30 min. The protoplasts were
ies to RBRL and NINJA3, respectively. The antisera were affinity-purified then centrifuged at 900 rpm for 3 min and resuspended at 3 × 105 cells
and used for immunoblotting. per ml in MMG solution (0.4 M d-mannitol, 15 mM MgCl2 and 4 mM
MES-KOH (pH 5.7)) for PEG-mediated transformation. In the transfor-
Sample preparation and JA quantitative assay mation, 110 μl of freshly prepared PEG-CaCl2 solution (0.2 M d-mannitol,
Hormone extractions and measurements of the JA concentration 100 mM CaCl2 and 40% (v/v) PEG 4000) and 10 μg (10 μl) of plasmid were
were performed as previously described68. In brief, rice shoots from gently mixed with 100 μl of protoplasts and then incubated for 15 min
2-week-old NPB, rbrl and RBRL-OE plants were separately collected at room temperature in the dark. After transformation, the cells were
and ground into fine powder. JA was extracted from 110 mg of ground washed with 10 volumes of W5 and then resuspended in W1 solution
powder using 400 μl of 10% methanol containing 1% acetic acid and (0.5 M d-mannitol, 4 mM MES-KOH (pH 5.7) and 20 mM KCl) overnight
then purified with a 0.22-μm nylon filter. The eluate was analysed by at 28 °C in the dark. The GFP signal was quantified and normalized to
ultra-high-performance liquid chromatography–triple quadrupole the chlorophyll fluorescence, as previously reported71. The observa-
MS (UPLC–MS/MS) on a mass spectrometer (UPLC 1290-MS/MS 6495). tion and quantification of each cell were performed under the same
set of confocal parameters and at the same scale using LSM710 (Zeiss).
In vivo and semi-in vivo protein degradation
In vivo and semi-in vivo protein degradation experiments were Dual-luciferase reporter system
conducted as previously described66. For in vivo protein degrada- The dual-luciferase reporter system was established as previously
tion experiments, agrobacterial strains containing 35S::HA-RBRL, described17. In brief, the coding sequences of OsRBRL and OsRBRL-like
35S::HA-RBRL-like, 35S::YFP-NINJA3, 35S::YFP-NINJA4, 35S::FLAG-GUSA were cloned and inserted into the pCambia2300-35S vector. A list of
(internal control) or 35S::MYC-CP were coinfiltrated at the indicated the primers used is provided in Supplementary Table 5. When conduct-
ratios. The infiltration procedures were performed as described above ing the assay in tobacco leaves, these two constructs were separately
(see the ‘Transient expression in N. benthamiana leaves’ section). Then, transformed into A. tumefaciens strain GV3101 using the freeze–thaw
2 days after infiltration, the samples were collected for analysis. For method. The indicated combinations of A. tumefaciens suspensions
semi-in vivo protein degradation experiments, agrobacterial strains were mixed and adjusted to a final concentration with an OD600 of 1.0.
containing 35S::HA-RBRL, 35S::YFP-NINJA3, 35S::YFP-NINJA4, 35S::GFP, The infiltration procedures were performed as described above (see the
35S::YFP-CP or pCambia2300 (empty vector) were infiltrated separately. ‘Transient expression in N. benthamiana leaves’ section). The expres-
The infiltration procedures were performed as described above (see sion levels of firefly and Renilla luciferases in N. benthamiana leaves
the ‘Transient expression in N. benthamiana leaves’ section). Then, were measured using the GLO-MAX 20/20 luminometer (Promega) at
2 days after infiltration, the samples were collected separately and 2 days after agroinfiltration. When conducting the assay in rice proto-
extracted using native extraction buffer (containing 10 μM ATP). An plasts, these two constructs were co-transformed into rice protoplasts
equal volume of the corresponding extracts was mixed together. along with 35S::myc-JAZ6, 35S::GFP, 35S::YFP-JAMYB, pCambia2300
A final concentration of 50 μM MG132 was added to the correspond- (empty vector) and 35S::AGO18pro:LUC at a fixed ratio. The expression
ing mixture. The mixtures were incubated at 4 °C with gentle rotation. levels of firefly and Renilla luciferases in rice protoplasts were meas-
The samples were removed at various timepoints, and the reaction ured using the GLO-MAX 20/20 luminometer (Promega) at 12 h after
was terminated by the addition of 2× Laemmli buffer and boiling for transformation. The ratio of firefly luciferase to Renilla luciferase (LUC/
5 min; finally, the samples were subjected to immunoblotting analysis. REN) was calculated to determine the final transcriptional activity.

Y2H assay Virus isolation and rice protoplast infection

The coding sequences of 14 OsJAZ genes, 4 OsNINJA genes, 12 RDV RSV was isolated and purified from rice plants infected with RSV, with
proteins and OsRBRL were separately cloned and inserted into the some modifications compared to previous studies10. Approximately
pDEST-GBKT7 or pDEST-GADT7 vector. A list of the primers used is 10 g of leaves that had been stored at −80 °C were blended in 40 ml of
provided in Supplementary Table 5. Yeast transformation was per- phosphate buffer (0.1 M, pH 7.5) containing 0.1% 2-mercaptoethanol,
formed as described by the vector manufacturer (Clontech, Mountain 1% Triton X-100 and 0.01 M EDTA. The homogenate was then filtered
View). Different combinations of constructs were cotransformed into through two layers of Miracloth (Millipore), mixed with 20% (v/v) chlo-
yeast AH109 cells. All yeast transformants were subsequently grown on roform and stirred for 15 min at room temperature. After centrifuga-
SD/−Leu/−Trp and subsequently transferred to SD/−Leu/−Trp/−His/− tion at 4 °C, 5,000g for 20 min for clarification, the supernatant was
Ade medium for interaction tests. adjusted to 6% PEG 6,000 and 0.1 M NaCl in an ice bath for 4 h, and then
rotated at 4 °C overnight. The resulting precipitate was collected by
Transient expression in rice protoplasts centrifugation at 4 °C, 5,000g for 20 min and the pellets were dissolved
Transient expression in rice protoplasts was conducted as previously in 0.01 M phosphate buffer at pH 7.5. After centrifugation at 4 °C, 5,000g
described, with some modifications17,65,69,70. In brief, the corresponding for 10 min, the supernatant was further centrifuged at 4 °C, 100,000g
plasmids were cotransformed into protoplasts using polyethylene for 2 h. The resulting pellets were dissolved in 0.01 M phosphate buffer
at pH 7.5. Carefully laid the resuspended pellet solution on top of 20% the nucleus, a NES corresponding to amino acids 371 to 387 of Arabi-
glycerol cushion. The virus particles were pelleted by centrifugation at dopsis thaliana RTL2 (KKAESSSAYHMIRALRK) was introduced into
4 °C, 100,000g for 2 h. After centrifugation, discarded the supernatant the vector Ubi::RBRL-GFP. After sequencing verification, the modified
and dissolved the pellets in 0.01 M phosphate buffer at pH 7.5. The construct was transformed into rice protoplasts with RSV particles (see
purified virus particles were characterized by 10% SDS–PAGE, and the the ‘Transient expression in rice protoplasts’ and ‘Virus isolation and
virus concentration was determined using the Bradford assay with BSA rice protoplast infection’ sections).
as a standard. The purified particles were then stored at −80 °C. When
conducting rice protoplast infection, 1 μg RSV particles, 10 μg corre- Proteasome assembly assay
sponding plasmid and 110 μl freshly prepared PEG-CaCl2 solution were The proteasome assembly assays were conducted as previously
gently mixed with 100 μl of protoplasts and then incubated for 15 min described73. The aboveground parts of the mock- and RSV-infected
at room temperature in the dark. After transfection, the cells were NPB plants were collected at 4 w.p.i. The total protein extracted with
washed with 10 volumes of W5 and then resuspended in W1 solution buffer F (50 mM Tris-HCl (pH 7.5), 25 mM NaCl, 2 mM MgCl2, 1 mM
overnight at 28 °C in the dark. The infected protoplasts were collected EDTA, 2 mM DTT, 5 mM ATP and 5% glycerol) was used for native PAGE,
at 18 h after infection and the RSV RNAs and RSV CP accumulation were followed by standard western blotting. Proteasome extracted from
analysed using RT–qPCR and immunoblotting. one-month-old A. thaliana Col-0 ecotype were used as a positive control
and the marker. The anti-PAG1 antibody (1:1,000)73 was used to detect
Protein expression and ubiquitination assay in E. coli the assembly of proteasome.
The protein expression and ubiquitination assays conducted in the
E. coli strain BL21 (DE3) were performed according to previous studies42. Histochemical staining of H2O2 and O2·−
In brief, different combinations of the indicated expression vectors Hydrogen peroxide (H2O2) and superoxide (O2·−) in rice leaves were
were transformed into the E. coli strain BL21 (DE3), and the strains were detected using the 3,3′-diaminobenzidine (DAB, Sigma-Aldrich) and
cultured in Luria–Bertani liquid medium at 37 °C. Then, 0.5 mM IPTG nitro blue tetrazolium (NBT, Sigma-Aldrich) staining methods, with
was added to the medium when the absorbance at 600 nm reached minor modifications based on previously described protocols19. In brief,
0.4–0.6 to induce the expression of the target proteins. The bacte- rice leaves approximately 1 cm in length were immersed in either 10 mM
ria were further cultured at 28 °C for 10–12 h and then stored at 4 °C Tris-HCl buffer (pH 6.5) containing DAB (1 mg ml−1) for H2O2 detection
overnight. The crude proteins were extracted and analysed by western or 50 mM sodium phosphate buffer (pH 7.0) containing NBT (0.05%)
blotting with the corresponding antibodies. for O2·− detection. The samples were incubated in the dark at 37 °C
for 16 h. After incubation, the leaves were bleached with a solution of
MST assay ethanol and acetic acid (3:1) at 70 °C for 60 min to remove chlorophyll.
The MST assays were conducted as previously described17. To assess Finally, the leaves were washed 4–5 times with 75% ethanol until clear
the binding affinity between OsRBRL and RSV CP or RDV P2, we first and photographed through a stereomicroscope under uniform light-
labelled the GST–CP and MBP–P21–786 protein with the red fluorescent ing conditions.
dye NHS according to the instructions of the Monolith Series Protein
Labeling Kit RED-NHS 2nd Generation (NanoTemper Technologies). The H2O2 determination in rice seedlings
NHS-labelled GST–CP and MBP–P21–786 protein was gradually diluted To quantify H2O2 levels in rice seedlings, a standardized protocol was
until its fluorescence intensity ranged between 800 and 1,000 under used. Fresh rice leaves (100 mg) were weighed and ground into a fine
20% LED power. The initial concentration of the MBP–OsRBRL protein powder using liquid nitrogen. The powder was mixed with 1 ml of
was 258 µM. It was subjected to 12 rounds of 1:1 serial dilution to a final 50 mM sodium phosphate buffer (pH 7.4) and incubated on ice for
concentration of 0.126 µM. After brief incubation in the dark, the sam- 30 min to facilitate H2O2 extraction. After incubation, the mixture was
ples were loaded into MST standard capillaries. The measurements centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatant was
were performed with 20% MST power on a Microscale Thermophoresis carefully transferred to a new tube. The H2O2 concentration was then
Monolith NT.115 instrument (NanoTemper Technologies). The assays determined using the Amplex Red Hydrogen Peroxide/Peroxidase
were repeated at least twice for each affinity measurement. Data analy- Assay Kit (Invitrogen, A22188) according to the manufacturer’s instruc-
ses were performed using the MO.Affinity analysis software provided tions. The absorbance was measured at approximately 560 nm using
by the manufacturer. a BioTek Microplate Reader (BioTek Cytation5), and the H2O2 content
was calculated by comparing the sample absorbance to a standard
Transcriptome sequencing curve of known H2O2 concentrations.
The aboveground parts of the mock- and RSV-infected rice plants
were collected at 4 w.p.i. The plant material was thoroughly ground Quantification and statistical analysis
in liquid nitrogen and approximately 0.1 g of the sample was used for The data from the JA quantitative assays were analysed using two-way
RNA extraction (see the ‘RNA extraction and RT–qPCR analysis’ sec- ANOVA. The data from the RT–qPCR analysis, dual-luciferase reporter
tion). Library construction and paired-end RNA-seq were performed system and protein degradation assay were analysed using Stu-
by Biomarker Technologies. FastQC software was used to assess the dent’s t-tests or one-way ANOVA with Tukey’s multiple-comparison
quality of the raw sequencing reads. After adapters and low-quality test. The above statistical analyses were performed with GraphPad
reads were removed, the clean reads were mapped to the rice genome Prism (v.7.0). All descriptive statistics of the JA quantitative analysis,
(MSU Rice Genome Annotation Project Database v.7.0, https://rice. RT–qPCR analysis, dual-luciferase reporter system and RSV disease
uga.edu/download_osa1r7.shtml) using TopHat. Gene expression lev- symptom classification are shown as the mean ± s.d. The number
els were calculated as fragments per kilobase per million reads. The (n) of biological replicates is indicated in each legend. For immuno-
multiple-testing-adjusted P value (false-discovery rate < 0.01) with blotting quantification, the band intensities were quantified using
an absolute fold change ≥ 2 was used to determine whether individual ImageJ. No statistical method was used to predetermine sample size.
genes were significantly differentially expressed. The sample sizes were determined from experimental trials and pre-
vious publications on similar experiments. Blinding and randomi-
RBRL nuclear exclusion experiments zation were used. For example, the virus-infection assay, different
We conducted an RBRL nuclear-export experiment based on those rice lines were numbered; the investigator was blinded to the group
performed in previous studies72. To prevent RBRL from localizing within allocation during the experiments including the inoculation of
Article
viruliferous small brown planthopper, the infection rates statistics, 64. Yu, F. et al. ESCRT-I component VPS23A is targeted by E3 ubiquitin ligase XBAT35 for
proteasome-mediated degradation in modulating ABA signaling. Mol. Plant 13,
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Reporting summary
Further information on research design is available in the Nature Port-
Acknowledgements We thank D. Xie, C. Li, Y. Ning, J. Zhou, W. Wang, S. Ding and F. Qu for
folio Reporting Summary linked to this article. their insights and suggestions; Z. He for providing the coi1-13 seeds used in this study; D. Lu for
supplying the Duet expression vectors used to reconstitute the plant ubiquitination cascade
in E. coli; F. Qin for supplying the pGreenII vectors used in this study; X. Zhou, J. Wu and F. Cui
for providing the RSV CP antibody used in this study; L. Xiong for providing the rice seeds of
Data availability the ninja1 T-DNA mutants and wild-type DJ; the staff at the National Center for Protein Sciences
OsRBRL (LOC_Os03g42760), OsRBRL-like (LOC_Os03g42780), OsNINJA1 and the Core Facilities at the School of Life Sciences at Peking University for their support with
the MS experiments and UPLC–MS/MS procedures. We acknowledge the contributions of the
(LOC_Os03g11550), OsNINJA2 (LOC_Os07g41160), OsNINJA3 (LOC_
staff at HUABIO (Hangzhou, China) and ABclonal Biotechnology (Wuhan, China) to generating
Os03g30570) and OsNINJA4 (LOC_Os05g48500) are available from the antibodies used in this research. Financial support for this study was provided by the
the Rice Genome Annotation Project Database (https://rice.uga.edu/). Ministry of Science and Technology (2021YFA1300702) and the Natural Science Foundation
of China (31530062 and 32090010).
RNA-seq raw data have been deposited at the Genome Sequence Archive
in National Genomics Data Center, China National Center for Bioinfor- Author contributions Y. Huang, J.Y., Z.Y. and Y.L. designed the experiments. Y. Huang, J.Y., Z.Y.
mation/Beijing Institute of Genomics, Chinese Academy of Sciences and X.S. performed most of the experiments. J.L., L.D., S.L., Y.J. and T.Z. helped conducting
(CRA020855) and are publicly accessible. Uncropped immunoblotting virus inoculation assays. H.W. helped with rice plant cultivation and seed multiplication. J.-j.H.
helped with conducting proteasome assembly assays. Z.Y., Y.L., X.C., S.Y., Y. Han, C.C. and Q.X.
images are provided in Supplementary Fig. 1. Source data are provided contributed to experimental data analysis. Y. Huang, J.Y., Z.Y. and Y.L. wrote the manuscript.
with this paper. Y. Huang, J.Y., X.S., W.W., C.W., Q.X., Z.Y. and Y.L. revised the manuscript. All of the authors
discussed the results and commented on the manuscript.
59. Yang, D.-L. et al. Plant hormone jasmonate prioritizes defense over growth by interfering
with gibberellin signaling cascade. Proc. Natl Acad. Sci. USA 109, E1193 (2012). Competing interests The authors declare no competing interests.
60. Miao, J. et al. Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res. 23,
1233–1236 (2013). Additional information
61. Fu, S. et al. Rice stripe virus interferes with S-acylation of remorin and induces its Supplementary information The online version contains supplementary material available at
autophagic degradation to facilitate virus infection. Mol. Plant 11, 269–287 (2018). https://doi.org/10.1038/s41586-025-08706-8.
62. Jin, L. et al. Rice dwarf virus P2 protein hijacks auxin signaling by directly targeting the Correspondence and requests for materials should be addressed to Zhirui Yang or Yi Li.
rice OsIAA10 protein, enhancing viral infection and disease development. PLoS Pathog. Peer review information Nature thanks Alain Goossens, Zuhua He and the other, anonymous,
12, e1005847 (2016). reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are
63. Liu, C. et al. Arabidopsis ARGONAUTE 1 binds chromatin to promote gene transcription in available.
response to hormones and stresses. Dev. Cell 44, 348–361 (2018). Reprints and permissions information is available at http://www.nature.com/reprints.
Extended Data Fig. 1 | See next page for caption.
Article
Extended Data Fig. 1 | Phylogenetic analysis of RBR-type E3 ligases. a, Scores (RBRL_ORYSJ) and its homologue, OsRBRL-like protein (RBRL-like_ORYSJ), are
and intensities of the CP protein and its interacting protein RBRL identified by highlighted in red. c, Sequence alignment of OsRBRL and OsRBRL-like proteins.
mass spectrometry in different samples. b, Phylogenetic analysis of RBR-type The RING1, IBR, RING2, and Ariadne domains are indicated by horizontal lines.
E3 ligases in rice and other species. An unrooted, neighbour-joining tree was d, Expression analysis of OsRBRL and OsRBRL-like via transcriptome sequencing
constructed by aligning RBR-type E3 ligase sequences from rice and other (RNA-seq) data. FPKM, fragments per kilobase of exon per million reads
species. The proteins are colour-coded dark red for rice (Oryza sativa), yellow mapped. Statistical analysis was performed via two-sided Student’s t tests,
for soybean (Glycine max), light green for Arabidopsis (Arabidopsis thaliana), and all p values are shown in the figure. The data are the mean ± s.d. n = 3
dark green for tobacco (Nicotiana tabacum), light purple for mouse independent biological samples.
(Mus musculus) and dark purple for human (Homo sapiens). Rice OsRBRL
Extended Data Fig. 2 | See next page for caption.
Article
Extended Data Fig. 2 | OsRBRL interacts with RSV CP and plays a role in JA the translation of OsRBRL. h, Volcano plots representing the fold changes in
signalling pathway. a, Co-IP assays of YFP-CP and HA-RBRL revealed that RSV the expression of differentially expressed genes (DEGs) in the NPB versus rbrl
CP interacts with OsRBRL in N. benthamiana leaves. GFP was used as a negative comparisons with or without the virus (FDR < 0.01, fold change ≥ 2.0). i, Venn
control. IP, immunoprecipitation. IB, immunoblotting. b, In vitro pull-down diagram representing DEGs regulated by OsRBRL. j, Hierarchical clustered
assays illustrating the interaction between RSV CP and OsRBRL. GST served as heatmap of 2167 DEGs (1271 upregulated genes and 896 downregulated genes,
a negative control. PD, pull-down. c, Protein detection of different transient left) and 492 DEGs (287 upregulated genes and 205 downregulated genes,
expression combinations in the LCI assays (Fig. 1d). The indicated proteins right). k, Gene Ontology (GO) analysis showing 20 representative enrichment
were detected via the corresponding antibodies. d, MST assays showing the terms of all the DEGs shown in (i). The significance of the GO terms was using
binding affinity between RSV CP and OsRBRL. The data are the mean ± s.d. adjusted p < 0.05 (Fisher’s exact test). l, RT–qPCR analysis of the OsRBRL
n = 3 independent biological samples. e, LCI assays showing that the RSV CP expression level in the NPB and RBRL-OE plants. Statistical analysis was
interacts with the N-terminal domain (NTD), RBR domain, and Ariadne domain performed via two-sided Student’s t tests, and all p values are shown in the
of OsRBRL. cLUC-RBRL-like served as a negative control. cps, signal counts figure. The data are the mean ± s.d. n = 3 independent biological samples.
per second. f, Protein detection of different transient expression combinations m, Venn diagram representing DEGs regulated by OsRBRL. n, Volcano plots
in the LCI assays (e). The indicated proteins were detected via the corresponding representing the fold change in expression of the DEGs in the NPB versus
antibodies. The red asterisks indicate the target protein bands. The analyses in RBRL-OE comparisons with or without the virus (FDR < 0.01, fold change ≥ 2.0).
a-c and f were repeated two to three times with similar results. g, Generation of o, Hierarchical clustered heatmap of 1685 DEGs (686 upregulated genes and
rbrl-knockout (CRISPR/Cas9) lines. The guide RNA (gRNA) sequence targeting 999 downregulated genes, left) and 2872 DEGs (1961 upregulated genes and
OsRBRL is indicated, and the protospacer-adjacent motif (PAM) is highlighted in 911 downregulated genes, right). p, GO analysis showing 20 representative
red. In rbrl line, an ‘A’ deletion and a ‘C’ deletion in the gRNA target site resulted enrichment terms of all the DEGs shown in (m). The significance of the GO
in premature translational termination of OsRBRL. In rbrl line 2, a ‘T’ insertion terms was using adjusted p < 0.05 (Fisher’s exact test).
and a 3-bp deletion in the gRNA target site resulted in premature termination of
Extended Data Fig. 3 | OsRBRL promotes the transcription of OsAGO18 in A schematic diagram of the constructs is shown in (c). EV: empty vector. The
N. benthamiana leaves and rice protoplasts. a, Photographs of RSV-infected activities of firefly luciferase (LUC) and Renilla luciferase (REN) were measured
NPB plants with different degrees of disease symptoms. The photographs were sequentially, and the LUC/REN ratio was calculated as the final transcriptional
taken at 4 w.p.i. Scale bars, 10 cm (left panel) and 2 cm (right panel). N, no activity. The data are the mean ± s.d. n = 3 (left panels in d and e) or 4 (right
noticeable symptoms; I, mild symptoms on the fourth or fifth leaf; II, typical panels in d and e) independent biological samples. Statistical analysis was
yellow stripes on the third leaf and curl or death of the fourth leaf; III, curl or performed via one-way analysis of variance (ANOVA) with Tukey’s multiple
death of the third leaf. b, Immunoblotting analysis of the RSV CP levels in the comparison test (different letters represent significantly different groups;
specified plants. All the mock-inoculated rice plants were 6-week-old and p < 0.05). Immunoblotting analysis of all the proteins in the corresponding
all the RSV-infected plants were collected at 4 w.p.i. Actin served as sample dual-luciferase assays were shown in the lower panels. Rubisco and Actin
processing controls. The analysis was repeated four times with similar results. served as sample processing controls.
c-e, Dual-luciferase assays in N. benthamiana leaves (d) and rice protoplasts (e).
Article

Extended Data Fig. 4 | See next page for caption.

Extended Data Fig. 4 | OsRBRL colocalizes with CP in rice, and its biological represent significantly different groups; p < 0.05). e, Full gel graph of the
function depends on its nuclear localization, but OsRBRL does not immunoblotting analysis of CP in the indicated plants; no ubiquitinated bands
ubiquitinate viral coat proteins. a, IP assays were conducted with the of CP were detected. f. Full gel graph of the immunoblotting analysis of CP in
nuclear fraction of mock-inoculated and RSV-infected RBRL-OE plants. FBPA the bacterial ubiquitination system (related to Fig. 3e). g. Full gel graph of the
served as a cytoplasmic marker, and histone H3 served as a nuclear marker. immunoblotting analysis of CP in the semi-in vivo degradation experiments
Mock-inoculated RBRL-OE plants served as a negative control. b, Subcellular (related to Fig. 3f). h, Ubiquitination analysis of P2 by RBRL. Bacterial lysates
localization of GFP-RBRL and mCherry-CP in rice protoplasts. Scale bars, from E. coli strains expressing UBA1-S (E1), UBC8-S (E2), Flag-Ub, MBP-P2 and
10 μm. c, RBRL(NES) was observed to be localized in the cytoplasm via confocal RBRL-MYC or from strains lacking one of these components were analysed by
microscopy. scale bars, 10 μm. d, RT–qPCR analysis (upper panel) of RSV RNAs immunoblotting with an anti-MBP antibody to determine the expression of P2.
accumulation in the rice protoplast transfected with RBRL-GFP, RBRL(NES)-GFP i, Ubiquitination analysis of P9 by RBRL. Bacterial lysates from E. coli strains
or HA-RBRL-like, together with RSV. Immunoblotting analysis (lower panel) of expressing UBA1-S (E1), UBC8-S (E2), Flag-Ub, MBP-P9 and RBRL-MYC or from
RSV CP accumulation, RBRL-GFP, RBRL(NES)-GFP and HA-RBRL-like expression strains lacking one of these components were analysed by immunoblotting
in the indicated samples. Actin served as loading controls. Mock, the protoplasts with an anti-MBP antibody to determine the expression of P9. j, Full gel graph
did not transfect with RSV. Data are shown as mean ± s.d. n = 3 independent of the immunoblotting analysis of RDV P2 levels in the indicated plants; no
biological samples. Statistical analysis was performed via one-way analysis of ubiquitinated bands of P2 were detected (related to Extended Data Fig. 10l).
variance (ANOVA) with Tukey’s multiple comparison test (different letters All the experiments were repeated two to four times with similar results.
Article

Extended Data Fig. 5 | See next page for caption.

Extended Data Fig. 5 | Identification of potential substrates of OsRBRL and Conserved motif analysis was performed via the online software program
functional domains involved in the interaction between OsRBRL and MEME (http://meme-suite.org/tools/meme). d, Protein detection of different
OsNINJA3. a-b, HA-RBRL and its interaction proteins purified with anti-HA transient expression combinations in the LCI assays (Fig. 4b). The indicated
magnetic agarose from proteins extracts of RBRL-OE rice lines were detected proteins were detected via the corresponding antibodies. The red asterisks
by immunoblotting with anti-HA antibody (a) and silver staining (b). OsRBRL indicate the target protein bands. e, Y2H assays showing that the N-terminal
expressed in RBRL-OE lines was labelled with a 1× HA tag at the N-terminus. domain (NTD) of OsRBRL primarily interacts with OsNINJA1/2/3. OsNINJA4
Wild-type NPB plants served as the negative control. Red arrows indicate the served as a negative control. f, LCI assays showing that the N-terminal domain
specific RBRL band. Scores of the RBRL protein and its interacting protein (NTD) of OsRBRL primarily interacts with OsNINJA1/2/3. cLUC-GUS served
NINJA3 identified by mass spectrometry in different samples. c, Phylogenetic as a negative control. cps, signal counts per second. g, Protein detection of
analysis was conducted using the full-length protein sequences of OsNINJA3 different transient expression combinations in the LCI assays (f). The indicated
and NINJA family proteins from the indicated plant species. The proteins are proteins were detected via the corresponding antibodies. The red asterisks
colour-coded red for rice (Oryza sativa subsp. japonica), orange for wheat indicate the target protein bands. The analyses in a, b, d and g were repeated at
(Triticum aestivum), yellow for maize (Zea mays), light green for Arabidopsis least two times with similar results. h, LCI assays showing that the NINJA-B and
(Arabidopsis thaliana), dark green for tobacco (Nicotiana tabacum), and grey ZBD NTE regions but not the EAR motif of OsNINJA3 interact with OsRBRL. cps,
for soybean (Glycine max). Rice NINJA proteins are highlighted in red. signal counts per second.
Article

Extended Data Fig. 6 | See next page for caption.

Extended Data Fig. 6 | OsRBRL mediates the degradation of OsNINJA3 internal control. The mRNA expression levels of the target genes OsNINJA3 and
through the 26S proteasome. a, The in vivo degradation of NINJA3 was ACTIN were analysed via RT–PCR. The numbers at the top indicate the ratio
assessed by determining the YFP-NINJA3 protein level via co-infiltration of the concentrations of Agrobacterium used in co-infiltration. YFP-NINJA4
experiments with increasing amounts of HA-RBRL. Flag-GUSA served as the (right panel) served as a negative control. d, CP promotes the ubiquitination of
internal control. The mRNA levels of ACTIN, NINJA3 and NINJA4 were analysed NINJA3 by OsRBRL. The bacterial lysates from E. coli strains expressing UBA1-S,
via reverse transcriptase–polymerase chain reaction (RT–PCR) to ensure that UBC8-S, Flag-Ub, RBRL-MYC, MBP-NINJA3-HA and GST-CP or from strains
equal amounts of NINJA3 or NINJA4 were transcribed across the co-infiltration lacking one of these components were analysed by immunoblotting with an
conditions. The numbers at the top denote the ratios of the concentrations anti-HA antibody to detect the ubiquitination of OsNINJA3. e, Immunoblotting
of Agrobacterium used for co-infiltration. YFP-NINJA4 (middle panel) and HA- analysis of OsNINJA3 levels in the plants indicated by the asterisk. The red line
RBRL-like (right panel) served as negative controls. b, Effect of the proteasome denotes the ubiquitinated form of NINJA3. Actin served as sample processing
inhibitor MG132 on OsNINJA3 degradation by OsRBRL. MG132 was added to the controls. f, Proteasome assembly in mock-treated and RSV-infected NPB
corresponding protein mixture samples at a final concentration of 50 μM to plants were detected with anti-PAG1 antibody via native polyacrylamide gel
prevent protein degradation through the 26S proteasome. Dimethyl sulfoxide electrophoresis (PAGE). Arabidopsis (Col-0 ecotype) proteasome was also
(DMSO) was used as a control. Ponceau S staining of the Rubisco proteins detected as a positive control and the marker. Proteasome and half-baked
served as sample processing controls. c, The in vivo degradation of OsNINJA3 proteasome are indicated. Actin was used as the loading control. All the
was conducted by determining the YFP-NINJA3 protein level via co-infiltration experiments were repeated two to four times with similar results.
experiments with increasing amounts of MYC-CP. Flag-GUSA was used as the
Article

Extended Data Fig. 7 | Transcriptome analysis of NPB and ninja3 with or (4013 upregulated genes and 4038 downregulated genes, left) and 4179 DEGs
without RSV infection. a, Volcano plots representing the fold change in the (2582 upregulated genes and 1597 downregulated genes, right). d, GO analysis
expression of the DEGs in the NPB versus ninja3 comparisons with or without showing 19 representative enrichment terms of all the DEGs shown in (b). The
the virus (FDR < 0.01, fold change ≥ 2.0). b, Venn diagram representing DEGs significance of the GO terms was using adjusted p < 0.05 (Fisher’s exact test).
regulated by OsNINJA3. c, Hierarchical clustered heatmap of 8051 DEGs
Extended Data Fig. 8 | NINJA3 interacts with most rice JAZ proteins to The indicated proteins were detected via the corresponding antibodies. The
regulate JA signal transduction. a, Analysis of the interactions between red asterisks indicate the target protein bands. The analysis was repeated two
OsJAZs and OsNINJA3 via Y2H assays. b, Analysis of the interactions between to three times with similar results. d, Summary of the Y2H (a) and LCI (b) assays
OsJAZs and OsNINJA3 via LCI assays. cps, signal counts per second. c, Protein of the interaction between OsNINJA3 and OsJAZs. The strength of each
detection of different transient expression combinations in the LCI assays (b). interaction was rated as strong (++), weak (+) or undetectable (−).
Article

Extended Data Fig. 9 | See next page for caption.

Extended Data Fig. 9 | NINJA1 and NINJA2 are not involved in rice antiviral n = 3 independent biological experiments. h, Immunoblotting analysis of the RSV
immunity, and the OsRBRL-OsNINJA3 module regulates the accumulation CP levels in the specified plants. i, Generation of ninja1/2/3-knockout (CRISPR/
of downstream AO and ROS. a, Characterization of ninja1 T-DNA insertion Cas9) lines. The guide RNA (gRNA) sequences targeting the indicated OsNINJAs
mutants. Schematic representation of the OsNINJA1 gene and the position of are indicated, and the protospacer-adjacent motifs (PAMs) are highlighted in
the T-DNA insertion. F1, R1, and T1 represent the positions of the two primer red. A 188-bp deletion in NINJA1, a 5-bp deletion in NINJA2 and a 5-bp deletion in
pairs used to identify whether T-DNA insertion had occurred. b, Images NINJA3 resulted in premature translational termination of OsNINJA1, OsNINJA2
showing mock-inoculated or RSV-infected Dongjing (DJ) and ninja1 plants. and OsNINJA3. j, Images showing mock-inoculated or RSV-infected NPB, ninja3
Scale bar, 10 cm. c, Percentages of RSV-infected Dongjing (DJ) and ninja1 and ninja1/2/3 plants. Scale bar, 10 cm. k, Immunoblotting analysis of the
plants with various disease symptom grades. The data are the mean ± s.d. n = 3 RSV CP levels in the specified plants. Rice plants were all collected at 4 w.p.i.
independent biological experiments. d, Immunoblotting analysis of the RSV l, Immunoblotting analysis of the AO levels in mock-inoculated and RSV-infected
CP levels in the specified plants. e, Generation of ninja2-knockout (CRISPR/ NPB and rbrl plants. m, Immunoblotting analysis of the AO levels in mock-
Cas9) lines. The guide RNA (gRNA) sequence targeting OsNINJA2 is indicated, inoculated and RSV-infected NPB and ninja3 plants. Actin in d, h, k, l, and m
and the protospacer-adjacent motif (PAM) is highlighted in red. In ninja2 served as sample processing controls. n, In situ detection of leaf ROS levels via
line 1, a ‘T’ insertion in the gRNA target site resulted in premature translational DAB and NBT staining in the indicated rice plants. o, Quantification of H2O2
termination of OsNINJA2. In ninja2 line 2, a 65-bp deletion in the gRNA target levels in the indicated rice plants. Statistical analysis was performed via two-
site resulted in premature termination of the translation of OsNINJA2. f, Images sided Student’s t tests, and all p values are shown in the figure. The data are the
showing mock-inoculated or RSV-infected NPB, ninja2 and NINJA2-OE plants. mean ± s.d. n = 3 independent biological samples. The analyses in a, d, h, and
Scale bar, 10 cm. g, Percentages of RSV-infected NPB, ninja2 and NINJA2-OE k-n were repeated two to three times with similar results.
plants with various disease symptom grades. The data are the mean ± s.d.
Article

Extended Data Fig. 10 | See next page for caption.

Extended Data Fig. 10 | Jasmonate signalling mediates a broad-spectrum The data are the mean ± s.d. n = 3 independent biological experiments.
immune response against rice viruses. a, Expression analysis of genes related g, Images of mock-inoculated and RDV-infected NPB, jamyb, and JAMYB-OE
to JA synthesis and JA signalling in mock-inoculated and RDV-infected wild-type plants. Scale bar, 10 cm. h, Percentages of RDV-infected NPB, jamyb, and JAMYB-
Zhonghua11 rice plants via transcriptome sequencing data from a previous OE plants with various disease symptom grades. The data are the mean ± s.d.
study. The numbers are the Z scores of the FPKM values for each gene. FPKM, n = 3 independent biological experiments. i, Images showing mock-inoculated
fragments per kilobase of transcript per million. b, Analysis of the interactions and RDV-infected NPB, rbrl, and RBRL-OE plants. Scale bar, 10 cm. j, Percentages
between RDV proteins and OsRBRL via Y2H assays. c, MST assays showing the of RDV-infected NPB, rbrl, and RBRL-OE plants with various disease symptom
binding affinity between RDV P2 N-terminus and OsRBRL. The data are the grades. The data are the mean ± s.d. n = 3 independent biological experiments.
mean ± s.d. n = 3 independent biological samples. d, Photographs of RDV- k, RT–qPCR analysis of RDV RNA accumulation in the indicated plants. Statistical
infected NPB plants with different grades of disease symptoms. The photographs analysis was performed via two-sided Student’s t tests, and all p values are
were taken at 4 w.p.i. Scale bars, 10 cm (upper panel) and 2 cm (bottom panel). shown in the figure. The data are the mean ± s.d. n = 3 independent biological
N, no noticeable symptoms; I, typical yellow stripes on the leaves; II, typical samples. l, Immunoblotting analysis of RDV P2 levels in the indicated plants.
yellow stripes on the leaves and mild chlorosis; III, typical yellow stripes on the OsRBRL expressed in RBRL-OE lines 1 and 2 was labelled with a 1× HA tag at the
leaves and severe chlorosis. From Grade N to Grade III, the dwarfism of the RDV- N-terminus. Actin served as loading controls. The analysis was repeated five
infected rice plants became increasingly severe. e, Images of mock-inoculated times with similar results. m, A proposed working model for initiation of the
and RDV-infected NPB and coi1-13 plants. Scale bar, 10 cm. f, Percentages of antiviral immune response via RBRL. Rice plants were all collected at 4 w.p.i.
RDV-infected NPB and coi1-13 plants with different disease symptom grades.
 natureportfolio|reportingsummary
Correspondingauthor(s): LiYi
Lastupdatedbbya
y uthor(s):Jan17,2025

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ANDvariation(e.g.standarddeviation)orassociatedestimateso ofuncertainty(e.g.confidenceintervals)
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Softwareandcode
Policyinformationaboutavailabilityo
ofcomputercode
Datacollection Thefluorescencesignalwasdetectedusingaconfocalmicroscopy(LSM710,Zeiss).
ImagesfromimmunoblottingwerecollectedwithMolecularlmager®ChemiDoc™MXRS+(Bio-Rad).
Theluciferaseactivitywasdetectedusingalow-lightcooledcharge-coupleddeviceimagingapparatus(NightOWLIIILB983withIndiGO
softwareversion2.0.4.0).
TheluciferaseactivitywasmeasuredusingaGLO-MAX20/20luminometer(Promega).
Bio-RadCFX96withCFXMaestro1.1softwarewasusediinq n RT-PCRanalysis.
Forendogenousphytohormonequantification,JAa A nalysiswasperformedoonama
n ssspectrometer(UPLC1290-MS/MS6495).
EASY-nLC1200liquidchromatographysystemandaC18columncoupledwithaThermoFusionLumosmassspectrometerwereusedt to
conductIP-LC-MS/MSassays.
TheMSTassayswereperformedoonaMi n croscaleThermophoresisMonolithNT.115instrument(NanoTemperTechnologies,Munich,
Germany).
BioTekCytation5wasusedttoq o uantifytheH2O2levelstogetherwiththeAmplexRedHydrogenPeroxide/PeroxidaseAssayKit.
Dataanalysis Imageanalysis:ImageJ(version1.45).
Statisticalanalysis:GraphPadPrism(version7.0).
Phylogenetictree:MEGA11.
April2023

Conservedmotifanalysis:onlineMEMEwebsite(http://meme-suite.org/tools/meme).
Rawsequencedataqualitycontrol:FastQCsoftware(version0.12.0).
TheLLC‒MS/MSresultswereprocessedviaProteomeDiscoverer2.2software.
MSTdataanalyseswereperformedusingtheMO.Affinityanalysis3software.
1
 Sequenceanalysis:SnapGene(version4.1.9).
Formanuscriptsutilizingcustomalgorithmsorsoftwarethatarecentraltotheresearchbutnotyetdescribedinpublishedliterature,softwaremustbemadeavailabletoeditorsand
reviewers.Westronglyencouragecodedepositioninacommunityrepository(e.g.GitHub).SeetheNaturePortfolioguidelinesforsubmittingcode&softwareforfurtherinformation.

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Data
Policyinformationaboutavailabilityofdata
Allmanuscriptsmustincludeadataavailabilitystatement.Thisstatementshouldprovidethefollowinginformation,whereapplicable:
-Accessioncodes,uniqueidentifiers,orweblinksforpubliclyavailabledatasets
-Adescriptionofanyrestrictionsondataavailability
-Forclinicaldatasetsorthirdpartydata,pleaseensurethatthestatementadherestoourpolicy
Allsourcedataassociatedwiththisworkareavailableaspartofthemanuscript’ssupplementaryinformationorasdetailedbelow.OsRBRL(LOC_Os03g42760),
OsRBRL-like(LOC_Os03g42780),OsNINJA1(LOC_Os03g11550),OsNINJA2(LOC_Os07g41160),OsNINJA3(LOC_Os03g30570),OsNINJA4(LOC_Os05g48500)are
availablefromtheRiceGenomeAnnotationProjectDatabase(https://rice.uga.edu/).RNA-seqrawdatahavebeendepositedattheGenomeSequenceArchivein
NationalGenomicsDataCenter,ChinaNationalCenterforBioinformation/BeijingInstituteofGenomics,ChineseAcademyofSciences(GSAaccession:CRA020855)
thatarepubliclyaccessibleathttps://ngdc.cncb.ac.cn/gsa.UncroppedimmunoblottingimagesareprovidedinSupplementaryFig.1.

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FPleiaesesldele-csttpheoencebiefloiwtcr e p o r ti n g
hatisthebestfitforyourresearch.Ifyouarenotsure,readtheappropriatesectionsbeforemakingyourselection.
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LAlilsftes ci e n c e ss tu d yd e sig n
udiesmustdiscloseonthesepointsevenwhenthedisclosureisnegative.
Samplesize Thesamplesizeandtheresultsofstatisticalanalysesaredescribedintherelevantfiguresormethodsection.Samplesizewasdetermined
basedonexperimentaltrialsandpreviouspublicationsonsimilarexperiments(Yangetal.,2020,CellHost&Microbe,28,89-103;Wangetal.,
2020,NatureCommunications,11,5830;Yaoetal.,2022,ScienceAdvances,8,eabm0660;Yaoetal.,2019,MolecularPlant,12,1114-1122;
Wuetal.,2017,NaturePlants,3,16203;Wangetal.,2014,NatureCommunications,5,4768).
Dataexclusions Thereisapre-establishedcriteria.Fortheseexperimentswealwayschosetheplantsinthesamegrowthstatustoanalysisinfectionrates,
genesexpressions,hormonecontentsandsoon.Otherplantswillbeexcluded.
Replication Allexperimentswererepeatedatleasttwoorthreetimes,andthenumberofindependentexperimentsorbiologicalreplicatesisindicatedin
thefigurelegends.
Randomization Allsampleswerearrangedrandomlyintoexperimentalgroups.Plantsforexperimentsweregrownsidebysidetominimizeunexpected
environmentalvariationsduringgrowth.
Blinding Fordifferentricelinesexperiments,wefirstlynumberedtheselinesbyonepartnerandthefollowinganalysisweredonebyothermembers
toavoidsubjectiveinfluence.Forexample,thevirus-infectionassay,differentricelineswerenumberedatfirst,theinvestigatorwasblinded
tothegroupallocationduringtheexperimentsincludingtheinoculationofviruliferoussmallbrownplanthopper,theinfectionratesstatistics,
andvirusRNAsqRT-PCRanalysis.
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systemormethodlistedisrelevanttoyourstudy.Ifyouarenotsureifalistitemappliestoyourresearch,readtheappropriatesectionbeforeselectingaresponse.

natureportfolio|reportingsummary
Materials&experimentalsystems Methods
n/a Involvedinthestudy n/a Involvedinthestudy
8 Antibodies 8 ChIP-seq
8 Eukaryoticcelllines 8 Flowcytometry
8 Palaeontologyandarchaeology 8 MRI-basedneuroimaging
8 Animalsandotherorganisms
8 Clinicaldata
8 Dualuseresearchofconcern
8 Plants

Antibodies
Antibodiesused anti-CM-LOX2(custom-developedbyABclonal®Technology,China,generatedinYangetal.,2020,CellHost&Microbe,DOI:
10.1016/j.chom.2020.05.001,andwaspreservedbyourlaboratory);
anti-AOS2(custom-developedbyABclonal®Technology,China,generatedinYangetal.,2020,CellHost&Microbe,DOI:10.1016/
j.chom.2020.05.001,andwaspreservedbyourlaboratory);
anti-RSVCP(providedbyDr.JianxiangWufromZhejiangUniversity,generatedinFuetal.,2018,MolecularPlant,DOI:10.1016/
j.molp.2017.11.011);
anti-RDVP2(custom-developedbyABclonal®Technology,China,generatedinJinetal.,2016,PLoSPathogens,DOI:10.1371/
journal.ppat.1005847,andwaspreservedbyourlaboratory);
anti-AGO18(custom-developedbyABclonal®Technology,China,generatedinWuetal.,2015,eLife,DOI:10.7554/eLife.05733,and
waspreservedbyourlaboratory);
anti-AO(custom-developedbyIntegratedR&DServices-WuXiAppTec,generatedinWuetal.,2017,NaturePlants,DOI:10.1038/
nplants.62016.203,andwaspreservedbyourlaboratory);
anti-RBRL(custom-developedbyABclonal®Technology,China,generatedinthisstudy);
anti-NINJA3(custom-developedbyABclonal®Technology,China,generatedinthisstudy);
anti-MYC(ABclonal,Wuhan,China,Cat#AE010);
anti-HA(ABclonal,Wuhan,China,Cat#AE008);
anti-GFP/YFP(ABclonal,Wuhan,China,Cat#AE012);
anti-GST(ABclonal,Wuhan,China,Cat#AE001);
anti-MBP(ABclonal,Wuhan,China,Cat#AE016);
anti-FLAG(TransGenBiotech,Beijing,China,Cat#HT201);
anti-S(EarthOx,Cat#E022130);
anti-OsFBPA(BeijingProteinInnovation,Beijing,China,Cat#AbP80247-A-SE);
anti-HistoneH3(Abcam,Cat#ab1791);
anti-cLUC(Sigma-Aldrich,America,Cat#L2164);
anti-Actin(CWBIO,Beijing,China,Cat#CW0096M);
anti-PAG1(generatedinHanetal.,2019,NewPhytologist,DOI:10.1111/nph.15471,andwaspreservedbyourlaboratory).
Validation Validationstatementsandexperimentscanbeobtainedfromthefollowingwebsitesandpublications:
Anti-CM-LOX2(DOI:10.1016/j.chom.2020.05.001),species:Oryzasativasubsp.japonica,application:immunoblotting;
anti-AOS2(DOI:10.1016/j.chom.2020.05.;01),species:Oryzasativasubsp.japonica,application:immunoblotting;
anti-RSVCP(DOI:10.1016/j.chom.2020.05.001),species:Ricestripevirus,application:immunoblottingandimmunoprecipitation;
anti-RDVP2(DOI:10.1093/mp/ssu007),species:Ricedwarfvirus,application:immunoblotting;
anti-AGO18(DOI:10.7554/eLife.05733),species:Oryzasativasubsp.japonica,application:immunoblotting;
anti-AO(DOI:10.1038/nplants.62016.203),species:Oryzasativasubsp.japonica,application:immunoblotting;
anti-RBRL(generatedinthisstudy),species:Oryzasativasubsp.japonica,application:immunoblotting;
anti-NINJA3(generatedinthisstudy),species:Oryzasativasubsp.japonica,application:immunoblotting;
anti-MYC(https://abclonal.com.cn/catalog/AE010),application:immunoblotting;
anti-HA(https://abclonal.com.cn/catalog/AE008),application:immunoblotting;
anti-GFP/YFP(https://abclonal.com.cn/catalog/AE012),application:immunoblotting;
anti-GST(https://abclonal.com.cn/catalog/AE001),application:immunoblotting;
anti-MBP(https://abclonal.com.cn/catalog/AE016),application:immunoblotting;
anti-FLAG(https://www.transgen.com/antibody_tag/371.html),application:immunoblotting;
anti-S(EarthOx,Cat#E022130,https://earthox.net/product-category/life-science-products/antibodies/),application:immunoblotting;
anti-OsFBPA(http://www.proteomics.org.cn/product/818.html),species:Oryzasativasubsp.japonica,application:immunoblotting;
anti-HistoneH3(https://www.abcam.cn/products/primary-antibodies/histone-h3-antibody-nuclear-marker-and-chip-grade-
ab1791.html),species:Oryzasativasubsp.japonica,application:immunoblotting;
April2023

anti-cLUC(https://www.sigmaaldrich.cn/CN/zh/product/sigma/l2164),application:immunoblotting;
anti-Actin(https://www.cwbio.com/goods/index/id/10113),species:Oryzasativasubsp.japonicaandNicotianabenthamiana,
application:immunoblotting.
anti-PAG1(DOI:10.1111/nph.15471),species:Oryzasativasubsp.japonicaandArabidopsisthaliana,application:immunoblotting.
3
Dualuseresearcho
ofconcern

natureportfolio|reportinggsummaary
Policyinformationaboutdualuseresearcho
ofconcern
Hazards
Couldtheaccidental,deliberateo
orrecklessmisuseo
ofagentso
ortechnologiesgeneratedi
int
n hework,ortheapplicationo
ofinformationpresented
int
n hemanuscript,poseathreatto:
No Yes
8 Publichealth
8 Nationalsecurity
8 Cropsand/orlivestock
8 Ecosystems
8 Anyothersignificantarea
Experimentsoofconcern
Doestheworkinvolveanyo
oftheseexperimentso
ofconcern:
No Yes
8 Demonstratehowttoro enderavaccineineffective
8 Conferresistancettot
o herapeuticallyusefulantibioticsoorantiviralagents
8 Enhancethevirulenceoofapathogenoorrenderanonpathogenvirulent
8 Increasetransmissibilityoofapathogen
8 Alterthehostrangeoofapathogen
8 Enableevasionoofdiagnostic/detectionmodalities
8 Enabletheweaponizationoofabiologicalagentortoxin
8 Anyotherpotentiallyharmfulcombinationoofexperimentsandagents

Plants
Seedstocks Thewild-typeNPBwerepreservedbbyoy urlaboratory.Thewild-typeDJ
DJwasprovidedbbyDr
y .LizhongXiongfromtheNationalKey
LaboratoryoofCropGeneticImprovement,HubeiHongshanLaboratory,HuazhongAgriculturalUniversity.
Novelplantgenotypes TheoverexpressionricelinesoofOsRBRL,RSVCP,OsNINJA2andOsNINJA3weregeneratedthroughtransgenicmethods.The
knockoutricelinesoofOsRBRL,OsNINJA1,OsNINJA2andOsNINJA3weregeneratedusingCRISPR/Cas9.

Authentication Thecoi1-13mutantwasidentifiedthroughqRT‒PCRandphenotypicobservation,whichiisc sonsistentwithpriorreports.Thejamyb

mutantwasidentifiedbbys
yequencing,asps reviouslydescribed.Theninja1(modd-2)mutantwasidentifiedbbyg
y enotypingusinggene-
specificandT-DNA-specificprimersaasd
s escribedpreviously.ThericelinesoverexpressingOsRBRL,RSVCP,andOsNINJA3were
verifiedbbyi
ymmunoblotting.Thegenome-editedmutantswereconfirmedbbys yequencingandanalyzedusingSnapGene(https://
www.snapgene.com). April2023