
==== Front
Nat Commun
Nat Commun
Nature Communications
2041-1723
Nature Publishing Group UK London

39256395
52204
10.1038/s41467-024-52204-w
Article
A secreted fungal laccase targets the receptor kinase OsSRF3 to inhibit OsBAK1–OsSRF3-mediated immunity in rice
Duan Yuhang 12
Wang Zhaoyun 1
Fang Yuan 1
Pei Zhangxin 3
Hu Hong 2
http://orcid.org/0000-0002-2265-6997
Xu Qiutao 45
Liu Hao 2
http://orcid.org/0000-0002-5492-516X
Chen Xiaolin 2
Luo Chaoxi 2
Huang Junbin 2
http://orcid.org/0000-0002-2414-1671
Zheng Lu luzheng@mail.hzau.edu.cn

2
http://orcid.org/0000-0001-9848-5496
Chen Xiaoyang cxy084@ahau.edu.cn

1
1 https://ror.org/0327f3359 grid.411389.6 0000 0004 1760 4804 Anhui Province Key Laboratory of Crop Integrated Pest Management, Anhui Agricultural University, Hefei, China
2 grid.35155.37 0000 0004 1790 4137 State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China
3 Wuhan Institute of Landscape Architecture, Wuhan, China
4 grid.256609.e 0000 0001 2254 5798 State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, China
5 https://ror.org/023b72294 grid.35155.37 0000 0004 1790 4137 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China
10 9 2024
10 9 2024
2024
15 789128 11 2023
28 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
The identification effector targets and characterization of their functions are crucial for understanding pathogen infection mechanisms and components of plant immunity. Here, we identify the effector UgsL, a ustilaginoidin synthetase with a key role in regulating virulence of the rice false smut fungus Ustilaginoidea virens. Heterologous expression of UgsL in rice (Oryza sativa) enhances plant susceptibility to multiple pathogens, and host-induced gene silencing of UgsL enhances plant resistance to U. virens, indicating that UgsL inhibits rice immunity. UgsL interacts with STRUBBELIG RECEPTOR KINASE 3 (OsSRF3). Genome editing and overexpression of OsSRF3 demonstrate that OsSRF3 plays a pivotal role in the resistance of rice to multiple pathogens. Remarkably, overexpressing OsSRF3 enhances resistance without adversely affecting plant growth or yield. We show that BRASSINOSTEROID RECEPTOR-ASSOCIATED KINASE 1 (OsBAK1) interacts with and phosphorylates OsSRF3 to activate pathogen-triggered immunity, inducing the mitogen-activated protein kinase cascade, a reactive oxygen species burst, callose deposition, and expression of defense-related genes. UgsL interferes with the phosphorylation of OsSRF3 by OsBAK1. Furthermore, UgsL mediates OsSRF3 degradation by facilitating its association with the ubiquitin-26S proteasome. Our results reveal that OsSRF3 positively regulates immunity in rice and that UgsL mediates its degradation, thereby inhibiting the activation of OsBAK1–OsSRF3-mediated immune pathways.

The Ustilaginoidea virens effector UgsL is shown to interact with receptor kinase OsSRF3. OsSRF3 positively regulates immunity in rice and UgsL mediates its degradation, thereby inhibiting the activation of OsBAK1–OsSRF3-mediated immune responses in rice.

Subject terms

Plant immunology
Effectors in plant pathology
Fungal host response
Pathogens
https://doi.org/10.13039/501100001809 National Natural Science Foundation of China (National Science Foundation of China) 32302302 Chen Xiaoyang Fundamental Research Funds for the Central Universities of China (2662023PY006)issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Plants are constantly being threatened by various pathogens, sometimes leading to plant diseases. Plant immune systems have evolved to fend off attack from pathogens via two layers of defense: pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) induced by the perception of highly conserved structural motifs in pathogens, and effector-triggered immunity induced by pathogen effector proteins1. PTI is activated when pattern recognition receptors (PRRs) located at the plant plasma membrane recognize PAMPs2,3. Plant PRRs comprise receptor‐like kinases (RLKs) and receptor‐like proteins (RLPs)4, which induce downstream immune responses including reactive oxygen species (ROS) bursts, calcium ion (Ca2+) influx, mitogen‐activated protein kinase (MAPK) cascades, and defense gene activation5,6. PRRs in Arabidopsis (Arabidopsis thaliana) include the leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) FLAGELLIN SENSING 2 (FLS2), EF‐TU RECEPTOR (EFR), PEP RECEPTORs (PEPRs), and BRASSINOSTEROID RECEPTOR-ASSOCIATED KINASE 1 (BAK1). Notably, the bacterial effectors AvrPto and AvrPtoB from Pseudomonas syringae pv. tomato target PRRs such as FLS2 and EFR7. Fungal effectors also target RLKs: for instance, the core effector necrosis-inducing secreted protein 1 from Colletotrichum spp. directly inhibits the kinase activity of host plasma membrane‐associated proteins BAK1 and BOTRYTIS-INDUCED KINASE 1 (BIK1)8. Despite the prevalence of phytopathogen virulence effectors that block host cell recognition of PAMPs by PRRs, interfere with host PTI, and inhibit basal defense9, the mechanism by which fungal effectors interfere with host PTI has not been fully elucidated.

In Arabidopsis, the STRUBBELIG-RECEPTOR FAMILY (SRF) RLKs belong to a monophyletic subfamily (LRR-V) and are encoded by nine genes (SRF1 to SRF9)10. SRFs function in various developmental programs including ovule, petal, carpel formation, and root hair patterning11. SRFs are also involved in plant responses to environmental stimuli: SRF6 expression is activated by heat stress and fungal infection; SRF7 is strongly induced by sulfate limitation and brassinosteroid treatment; and SRF3 functions in genetic incompatibility, which is linked to the RECOGNITION OF PERONOSPORA PARASITICA 1-mediated plant-pathogen immune response12. SRF3 participates in the sensing and transduction of early iron-deficiency signals, thereby triggering Ca2+ signaling and callose synthase activity in root tips to regulate plant growth, iron homeostasis, and bacterial-induced immune responses13. However, the function of SRFs in other plants remains unknown, especially the mechanisms involved in SRF-mediated regulation of plant immunity and pathogen interactions.

The fungus Ustilaginoidea virens causes rice false smut (RFS), a devastating disease of rice (Oryza sativa). RFS not only causes substantial yield losses (up to 40%), but also produces mycotoxins in false smut balls, including ustiloxins, ustilaginoidins, and sorbicillinoids, which are toxic to plants and animals and threaten human health14. Introducing genetically encoded resistance in plants is an environmentally friendly, economical approach for controlling plant diseases. To breed resistant rice varieties, it is crucial to explore resistance gene resources. However, the effectiveness of this technique is limited by the long, labor-intensive procedures of conventional breeding and the scarcity of major resistance genes15. RFS is the only floral organ disease in rice. Therefore, the U. virens–rice pathosystem can serve as a model for investigating invasion mechanisms as well as the interactions that occur between plant hosts and grain-infecting pathogens. Studying the functions of U. virens effectors to elucidate the molecular mechanisms underlying rice–U. virens interactions could help identify disease-resistance genes16. An increasing number of effectors have been shown to play critical roles in infection. The effector Chitin-binding protein 1 (UvCBP1) interacts with the rice scaffold protein RECEPTOR FOR ACTIVATED C KINASE 1A (OsRACK1A), competing for interaction with the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN B (OsRBOHB). This decreases the phosphorylation of OsRBOHB and lowering the production of ROS in rice, resulting in enhanced susceptibility to disease17. The effector small cysteine-rich effector 6 acts as a tyrosine phosphatase that dephosphorylates the negative defense regulator OsMPK6 to enhance its accumulation, thereby inhibiting rice immunity14. The effector SCRE1 is a competitive inhibitor of the interaction between SUCROSE NONFERMENTING-1 (SNF1)-RELATED PROTEIN KINASE A (OsSnRK1A) and the ATPase XA21 binding protein 24 (OsXB24), thereby inhibiting the OsSnRK1A–OsXB24-mediated phosphorylation cascade and ATPase activity, and decreasing rice immunity18. The secreted effector protein UvSec117 interacts with the rice histone deacetylase OsHDA701, recruiting more OsHDA701 to enter the nucleus to clear the histone H3 modification acetylation of lysine 9 (H3K9ac), thereby affecting the expression of disease-resistance genes regulated by H3K9ac and inhibiting rice immunity19. The effector Uv1809 enhances deacetylation mediated by the histone deacetylase SILENT INFORMATION REGULATION-RELATED 2 (OsSRT2), thereby decreasing acetylation levels of histone H4 at lysine 5 (H4K5ac) and lysine 8 (H4K8ac) in rice and disrupting the activation of defense genes20. The subtilase Protease 1a (UvPr1a) interacts with and directly degrades the rice disease-resistance-related protein SUPPRESSOR OF G2 ALLELE OF skp1 (OsSGT1), thereby influencing the OsSGT1-regulated rice immune response21. Despite this progress, our understanding of the molecular mechanisms whereby U. virens effectors suppress rice immunity remains limited.

The copper-containing polyphenol oxidases laccases are widely distributed in higher plants, fungi, insects, and bacteria. Plant laccases function in lignin degradation and oxidize a wide range of substrates and are therefore commonly used to degrade environmental pollutants22. Many genes in plant pathogenic fungi encode laccase isoenzymes with different biological functions. In Botrytis cinerea, the laccase lcc2 can degrade the plant-derived antitoxin of grapevine (Vitis vinifera) and promote infection23. In Colletotrichum gloeosporioides, the laccase LAC1 regulates virulence, mycelium growth, conidiation, appressorium formation, pigmentation, melanin biosynthesis, extracellular hydrolase secretion, and the utilization of exogenous nutrients24. In U. virens, the first step of ustilaginoidin biosynthesis is catalyzed by the polyketide synthase UvPKS1, which forms nor-rubrofusarin from acetyl-CoA and malonyl-CoA. The U. virens enzymes UgsJ (a methyltransferase), UgsL (a laccase), UgsO (an oxidoreductase), and UgsT (a major facilitator superfamily transporter) are responsible for the methylation of ustilaginoidin derivatives, monomer dimerization, redox modification, and mycotoxin transport, respectively25. Although some fungal laccases are typically secreted proteins, to date, there are no reports of laccases that function as effectors. Studying the mechanism underlying the interactions of fungal laccases with plants will enhance our understanding of the functions of fungal laccase.

In this study, we identified UgsL, a ustilaginoidin synthetase, as an effector that is important for hyphal growth and virulence of U. virens. Heterologous expression of UgsL in plant cells suppressed the programmed cell death (PCD) induced by the mouse proapoptotic protein BAX and the Phytophthora infestans PAMP INF1. We also identified the receptor kinase OsSRF3 as an UgsL-interacting protein. Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats)/(CRISPR-associated nuclease 9)-mediated gene editing and OsSRF3 overexpression, we demonstrate that OsSRF3 is important for the resistance of rice against multiple pathogens. We reveal that OsBAK1 interacts with and phosphorylates OsSRF3 to activate PTI responses, whereas UgsL promotes OsSRF3 degradation through the ubiquitin-26S proteasome, thereby inhibiting OsBAK1–OsSRF3-mediated immune pathways.

Results

UgsL is essential for the virulence of U. virens

We previously identified the secreted laccase UgsL from previously reported secretome and transcriptome datasets of U. virens during infection19,26. Reverse-transcription quantitative PCR (RT-qPCR) analysis revealed that UgsL is significantly upregulated during rice infection by U. virens (Fig. 1a), suggesting that UgsL plays an important role in the U. virens–rice interaction. UgsL encodes a 653-amino acid (aa) protein, containing a signal peptide (SP) and three copper (Cu)-oxidase Pfam motifs (Supplementary Fig. 1a). Phylogenetic analysis suggested that UgsL homologs are widely distributed among fungi (Supplementary Fig. 1b). Therefore, we generated UgsL deletion mutants in U. virens (ΔugsL-1 and ΔugsL-2) and a complementation strain (CΔugsL-1) using homologous recombination mediated by Agrobacterium (Agrobacterium tumefaciens) transformation. We confirmed all transformants by PCR and Southern blot analyses (Supplementary Fig. 2). The deletion of UgsL resulted in a lower mycelial growth rate than those observed for the wild-type strain HWD-2 and the complemented strain CΔugsL-1 (Fig. 1b), but we observed no significant differences in conidial production among genotypes (Fig. 1c). The ΔugsL mutants had significantly lower laccase activity (Fig. 1d), did not produce smut balls on rice spikelets, and completely lost virulence (Fig. 1e). In plant infection assays, we observed numerous hyphae spreading on the surfaces of rice spikelets for all tested strains at 1 day post inoculation (dpi), as evidenced by scanning electron microscopy. At 6 dpi, we observed hyphae for the HWD-2 and CΔugsL-1 strains in the inner spaces of rice spikelets, with fully infected anther filaments. By contrast, the ΔugsL mutants did not extend into the spikelets and failed to produce observable hyphae on the filament surface (Fig. 1f). We generated three UgsL mutants: C∆ugsLM1−1, in which the conserved HWHG sequence was mutated to AAAA (A: Ala); C∆ugsLM2-1, in which the SIDEH sequence was mutated to AAAAA; and C∆ugsLM3-1, in which the WKYKS sequence was mutated to AAAAA. After Flag IP, the laccase activity of equal amounts of UgsLM1-Flag, UgsLM2-Flag, and UgsLM3-Flag protein significantly lower than that of UgsL-Flag, indicating that UgsL is a laccase and these variant proteins lacked laccase activity (Supplementary Fig. 3a). C∆ugsLM1-1, C∆ugsLM2-1, and C∆ugsLM3-1 showed significantly reduced mycelial growth and virulence compared to the complemented strain CΔugsL-1 (Supplementary Fig. 3b, c). These results indicate that the laccase UgsL is important for vegetative growth and is required for U. virens virulence.Fig. 1 UgsL is essential for U. virens virulence.

a RT-qPCR analysis of UgsL transcript levels in rice spikelets during U. virens infection. Data are means ± SD from three biological replicates. b Colony morphology (left) and diameter (right) of the wild-type strain HWD-2, ΔugsL mutants, and the CΔugsL−1 complementation strain grown on PSA medium for 14 days at 28 °C. Data are means ± SD from three biological replicates. c Conidial production of the U. virens strains grown in PSB medium with shaking at 180 rpm for 7 days. Data are means ± SD from six biological replicates. d Laccase activity of the U. virens strains. Data were collected from two independent experiments for each treatment with five samples. e Virulence assay of the U. virens strains on rice spikelets at 21 dpi (left); Mean number of rice smut balls per panicle (middle), data were collected from three independent experiments for each treatment with 10 panicles; Diseased spikelet rate per panicle (%) (right), data were collected from three independent experiments for each treatment with 5 panicles. f Scanning electron microscopy observation of infection progression in inoculated rice spikelets at 1 and 6 dpi. Scale bars, 50 μm (1 dpi) and 100 μm (6 dpi). Data in (d) and (e) were shown as boxplots of the number of indicated biological samples displaying the maximum and minimum, first and third quantiles, and the median. In (a–e), different lowercase letters indicate significant differences at P < 0.05 (one-way ANOVA followed by two-sided LSD test for multiple-comparisons). P values are shown in the Source Data file. Source data are provided as a Source Data file.

We used a yeast secretion system to assess the role of the UgsL SP. We cloned the sequence encoding the 23-aa SP upstream of and in-frame with the sequence of the yeast invertase gene Suc2 carried by the pSUC2 vector and used this construct to transform yeast (Komagataella pastoris) strain YTK12, which lacks Suc2 activity. Transformants that accumulated UgsLSP and the positive control Avr1bSP grew on YPRAA medium containing raffinose as the sole carbon source. The transformants also secreted invertase, as evidenced by the formation of the red product 1,3,5-triphenylformazan (TPF) in a 2,3,5-triphenyltetrazolium chloride (TTC) assay. By contrast, transformants harboring the empty vector failed to grow on YPRAA medium and did not produce TPF in the TTC assay (Supplementary Fig. 4a). These results indicate that UgsL is a secreted protein of U. virens.

BAX and INF1 serve as markers of plant immune activation and are often used to screen for suppressors of plant immunity8,19. To investigate the role of UgsL in the host immune response, we generated the pVX-UgsL construct and introduced it into Agrobacterium strain GV3101 for Agrobacterium-mediated infiltration of Nicotiana benthamiana leaves. We observed no PCD in the leaves of N. benthamiana plants infiltrated with Agrobacterium cells harboring UgsL alone, whereas N. benthamiana leaves infiltrated with constructs harboring BAX or INF1 did show cell death. We also assessed the role of UgsL in inhibiting the PCD triggered by BAX or INF1 by co-infiltrating the relevant constructs into N. benthamiana leaves, revealing that UgsL suppresses the PCD induced by BAX and INF1. As a negative control, co-infiltrating the BAX or INF1 construct with the empty vector pVX harboring the 3 × Flag sequence did not abolish BAX- or INF1-induced PCD (Supplementary Fig. 4b, c). These results suggest that UgsL suppresses the plant immune response.

UgsL is a key enzyme in ustilaginoidin biosynthesis25. Here, the exogenous application of ustilaginoidins during infection partially restored the virulence of the ∆ugsL-1 mutant (Supplementary Fig. 5a–c). To investigate the role of the UgsL SP in U. virens infection, we generated a UgsL construct lacking the SP sequence and introduced it into the ΔugsL-1 mutant. The resulting CΔugsLΔSP-1 transformant produced significantly fewer (5–20) rice smut balls than CΔugsL-1 (50–80, Supplementary Fig. 5d), whereas there was no difference in mycelial growth (Supplementary Fig. 5e), indicating that the SP in UgsL is important for U. virens virulence. We used HPLC (high-performance liquid chromatography) to measure the ustilaginoidin contents in smut balls produced by C∆ugsL-1 and CΔugsLΔSP-1 (Supplementary Fig. 5f). The ustilaginoidin content of CΔugsLΔSP-1 was lower than that of C∆ugsL-1, indicating that the UgsL SP functions in ustilaginoidin biosynthesis.

Overall, these results indicate that the laccase activity of effector UgsL is required for the development and virulence of U. virens, and that ustilaginoidin is involved in UgsL mediated virulence of U. virens.

UgsL is a cytoplasmic effector

To investigate whether UgsL is delivered into plant cells, we generated the RP27-UgsL-GFP construct and transformed it into the rice blast fungus Magnaporthe oryzae strain P131. We then inoculated rice leaf sheaths ectopically co-expressing UgsL-GFP and PWL2-RFP with the engineered M. oryzae strain. We detected clear fluorescence in the biotrophic interfacial complex (BIC), a plant-derived, membrane-rich structure that develops at the tips of M. oryzae primary infection hyphae, at 30 h post inoculation (hpi). By contrast, we observed uniformly distributed green fluorescence within invasive hyphae, but not in the cells of rice leaf sheaths infected by M. oryzae expressing GFP alone (Supplementary Fig. 6a, b). We examined the localization of UgsL in floral tissues by immunofluorescence assay. Green fluorescence signals for UgsL were present in infected hyphae and stamen filament cells at 6 dpi with U. virens (Supplementary Fig. 6c), suggesting that UgsL can be secreted into rice cells.

To examine the subcellular localization of UgsL in plant cells, we generated the 35S-UgsL-GFP construct and introduced it into N. benthamiana leaves via Agrobacterium-mediated infiltration. The UgsL-GFP fluorescence signals co-localized with the signals from red fluorescent protein (RFP) in the nucleus, plasma membrane, and cytoplasm (Supplementary Fig. 7a). However, when we infiltrated N. benthamiana leaves with the 35S-UgsLΔSP-GFP construct, we observed GFP fluorescence in the nucleus and at the plasma membrane, a pattern similar to that N. benthamiana harboring the empty vector expressing GFP alone (Supplementary Fig. 7a). After inducing plasmolysis to separate the plasma membrane from the cell wall, we observed UgsL-GFP fluorescence in the intracellular space, but not in the apoplastic space (Supplementary Fig. 7b). UgsL-GFP fusion protein also showed clear nuclear, plasma membrane, and cytoplasm localization when transiently expressed in rice protoplasts (Supplementary Fig. 7c), suggesting that UgsL is a cytoplasmic effector.

Host-induced gene silencing of UgsL increases RFS resistance

We activated host-induced gene silencing (HIGS) of UgsL by creating transgenic plants in the japonica rice cv. Nipponbare (NPB) background expressing an RNA interference (RNAi) construct against the UgsL transcript (UgsL-HIGS; Supplementary Fig. 8). At 21 dpi with U. virens, an average of 10 smut balls per panicle were present on the spikelets of rice plants transformed with the empty vector (EV, pDS1301). However, UgsL-HIGS transgenic rice plants displayed strong resistance to U. virens, with an average of 1 to 3 smut balls per panicle (Fig. 2a). Notably, the smut balls of UgsL-HIGS plants contained significantly lower levels of ustilaginoidins compared to those of EV control plants (Fig. 2b). To confirm the notion that the resistance to RFS observed in infected UgsL-HIGS plants was caused by in planta silencing of UgsL, we collected infected spikelets from T2 plants at 6 dpi and measured UgsL transcript levels by RT-qPCR. Indeed, the relative UgsL transcript levels were significantly lower in the UgsL-HIGS lines than in EV control plants (Fig. 2c), indicating that resistance to RFS in the UgsL-HIGS lines is mediated by in planta silencing of UgsL. To determine whether the silencing of UgsL was caused by specific small interfering RNAs (siRNAs) produced in UgsL-HIGS plants, we extracted and sequenced the siRNAs that accumulated in the UgsL-HIGS-L1 line. We detected high levels of siRNAs matching the UgsL transcript, with a size distribution between 18 and 30 nucleotides (nt) and with 21-nt siRNAs being the most abundant (Fig. 2d). In fluorescence in situ hybridization (FISH) assays targeting these siRNAs, we observed fluorescence signals in rice flower tissues and U. virens infection hyphae in infected UgsL-HIGS-L1 plants at 6 dpi, but not in the EV control plants (Fig. 2e). These results indicate that siRNAs targeting UgsL were produced in the UgsL-HIGS lines and were transferred into fungal cells to diminish UgsL transcript levels during U. virens infection. Together, these results suggest that silencing of UgsL in rice promotes resistance to U. virens, indicating that UgsL functions as a key virulence effector during infection.Fig. 2 Silencing of UgsL via HIGS enhances rice resistance to U. virens.

a Resistance assay of UgsL-HIGS and empty vector (EV, pDS1301) transgenic rice plants against U. virens (left). Mean number of rice smut balls measured in the resistance assay (right). Data were collected from three independent experiments for each treatment with 10 panicles. b Ustilaginoidin contents from the smut balls of UgsL-HIGS and EV plants. Data are means ± SD from five independent experiments. c Relative UgsL expression during U. virens infection in the T3 transgenic rice plants at 6 dpi assessed by RT-qPCR. Data are means ± SD from three independent experiments. d Length distribution and abundance of siRNAs targeting UgsL in the T3-L2 transgenic line. e Visualization of siRNAs targeting UgsL in infected UgsL-HIGS and EV spikelets at 6 dpi by fluorescence in situ hybridization (FISH) using a specific probe that targets the most siRNA. An, anther; hy, U. virens hyphae. Scale bars, 20 μm. Data in (a–c) were shown as boxplots of the number of indicated biological samples displaying the maximum and minimum, first and third quantiles, and the median. Data in (a–c) are analyzed by one-way ANOVA followed by two-sided LSD test for multiple comparisons, and the adjusted P values were shown. Source data are provided as a Source Data file.

Heterologous expression of UgsL increases the susceptibility of rice to multiple pathogens

To further confirm that UgsL inhibits the plant immune response, we generated transgenic rice plants heterologously expressing UgsL. These 35S:UgsL transgenic plants (HE-1 and HE−2) grew as well as transgenic EV (35S:EV) control plants in the absence of pathogen challenge. However, 35S:UgsL plants produced significantly more smut balls than EV plants following inoculation with U. virens strain HWD-2 (Fig. 3a). We then evaluated resistance against M. oryzae strain ZB25. The 35S:UgsL plants were more susceptible to M. oryzae than the EV plants (Fig. 3b). Likewise, the 35S:UgsL plants were more susceptible to infection by Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99, with lesions in 35S:UgsL leaves ~2.5 times longer than those in EV leaves (Fig. 3c). Virulence assays of the ΔugsL−1 knockout mutant on 35S:UgsL and EV transgenic rice plants revealed that UgsL promotes the virulence of U. virens, as the 35S:UgsL plants produced a few (up to five) smut balls, whereas EV plants produced none (Supplementary Fig. 9). In addition, these assays provided genetic evidence that UgsL is a virulence effector. These results suggest that the heterologous expression of UgsL enhances the susceptibility of rice plants to multiple pathogens.Fig. 3 Heterologous expression of UgsL in rice increases rice susceptibility to multiple pathogens and inhibits the PTI response.

a Resistance assay of 35S:UgsL and EV plants to U. virens (left); Mean number of rice smut balls measured in the resistance assay (right), data were collected from three independent experiments for each treatment with 10 panicles. b Disease symptoms (left) and relative fungal biomass (right) of the 35S:UgsL and EV plants after spray-inoculation with M. oryzae. Relative fungal biomass was determined using RT-qPCR for M. oryzae MoPot2 and normalized to rice OsUBQ1. c Disease symptoms (left) and lesion lengths (right) of 35S:UgsL and EV plants after inoculation with Xoo. Data were collected from three independent experiments for each treatment with five leaves. d ROS burst in 35S:UgsL and EV rice protoplasts challenged with 8 nM chitin or water. RLU, relative luminescence unit. Data are means ± SD from five independent experiments. e 3,3’-diaminobenzidine (DAB) staining for the detection of H2O2 levels in leaves of 35S:UgsL and EV plants. f Activation of MAPK signaling in 35S:UgsL and EV plants (treated with 8 nM chitin) detected by immunoblotting with the phospho-p44/42 MAPK antibody at the indicated times. Intensity was quantified by densitometry using ImageJ and indicate relative phosphorylation levels of MPK3 and MPK6 normalized to OsActin abundance. Data are means ± SD from three independent experiments. g Callose deposition in 35S:UgsL and EV plants. h Relative OsPR1a, OsPR10b, and OsRBOHA transcript levels in 35S:UgsL and EV rice spikelets infected with U. virens at 1 dpi by RT-qPCR. Data are means ± SD from three independent experiments. Scale bars, 1 cm (b, c, e) and 100 μm (g). Data in (a–c) were shown as boxplots of the number of indicated biological samples displaying the maximum and minimum, first and third quantiles, and the median. In (a–c, f, h,) data are analyzed by one-way ANOVA followed by two-sided LSD test for multiple comparisons, and the adjusted P values were shown. Source data are provided as a Source Data file.

Heterologous expression of UgsL in rice inhibits the PTI response

To investigate whether UgsL inhibits the PTI response, we examined the responses of 35S:UgsL and EV plants to the PAMP elicitor chitin. We immersed individual rice protoplasts in 8 nM chitin solution and measured ROS production using a luminol chemiluminescence assay. Heterologous expression of UgsL resulted in a significant drop in ROS production compared to that in EV plants (Fig. 3d). After infection with M. oryzae strain ZB25, we detected lower H2O2 levels in the cells of 35S:UgsL plants compared to EV plants using 3, 3’-diaminobenzidine (DAB) staining (Fig. 3e). When exogenous chitin was applied to rice leaves, the MAPK pathway was suppressed in 35S:UgsL compared to EV rice leaves, as determined by immunoblot analysis with anti-Phospho-p44/42 antibody (Fig. 3f). In addition, callose deposition in the rice cell wall was reduced in 35S:UgsL plants compared to EV plants (Fig. 3g). We also determined that the defense-related genes PATHOGENESIS-RELATED 1b (OsPR1b), OsPR10a, and RESPIRATORY BURST OXIDASE HOMOLOG A (OsRBOHA) in rice spikelets infected with U. virens at 1 dpi were significantly downregulated in 35S:UgsL plants compared to EV plants (Fig. 3h), suggesting that UgsL inhibits PTI in rice plants.

Heterologous expression of UgsL interferes with rice metabolism

To identify metabolites with suppressed or increased production in response to UgsL, we conducted metabolomics analysis of EV and 35S:UgsL (HE-1) rice spikelets. After detection by LC-MS (liquid chromatography-tandem mass spectrometry), 11,300 peaks were extracted from 12 samples of 2 groups and 3 quality control (QC) samples. PCA and orthogonal partial least squares discriminant analysis (OPLS-DA) score plot analysis indicated that the data quality was reliable and that the distribution of 35S:UgsL and EV metabolites was significantly different (Supplementary Fig. 10a, b). The differentially expressed metabolites (DEMs) based on VIP > 1 and P < 0.05 are shown in Supplementary Data 1. We detected a total of 353, including 136 upregulated and 217 downregulated DEMs (Supplementary Fig. 10c, d). Some DEMs that could induce plant disease resistance: betaine, coumarin, α-linolenic acid (jasmonic acid biosynthesis pathways), phenylalanine (salicylic acid biosynthesis pathways)27. We annotated the DEMs by KEGG pathway analysis, and the DEMs were enriched in autophagy, linoleic acid metabolism, zeatin biosynthesis, biosynthesis of various plant secondary metabolites, various aa metabolism, cutin, suberine, and wax biosynthesis (Supplementary Data 1, Supplementary Fig. 10e). Gene set enrichment analysis (GSEA) showed that phenylpropanoid biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and flavonoid biosynthesis pathways were significantly enriched in our dataset (Supplementary Fig. 10f). These results indicate that UgsL interferes with rice metabolism.

UgsL interacts with rice OsSRF3 in vitro and in vivo

To further explore the role of UgsL in plant infection, we used UgsL as bait in a two-hybrid (Y2H) screen of a cDNA library prepared from RNA of U. virens-infected rice spikelets at 6 dpi. We identified 33 putative UgsL-interacting proteins (Supplementary Table 1). Among these interactors, we obtained several clones for the rice receptor kinase OsSRF3. OsSRF3 contains a SP, a STRUBBELIG (SUB) domain, six leucine-rich repeats (LRRs), a transmembrane domain, a proline-rich region, and a putative C-terminal cytoplasmic kinase domain, which is highly similar to Arabidopsis SRF3 (Supplementary Fig. 11). In Arabidopsis, SRF3 plays an important role in regulating plant growth, iron homeostasis, and bacterial-induced immune responses13. OsSRF3 transcript levels were markedly higher after infection with U. virens, and OsSRF3 localized to the plasma membrane (Supplementary Fig. 12), suggesting that OsSRF3 is involved in rice resistance to U. virens. Therefore, we selected OsSRF3 as a putative target of UgsL for characterization.

We confirmed the interaction between UgsL and OsSRF3 using a targeted Y2H assay (Fig. 4a). We also performed in vitro pull-down assays with recombinant UgsL-glutathione S-transferase (GST) and OsSRF3-His proteins purified from Escherichia coli. We detected OsSRF3-His in the protein samples pulled down by UgsL-GST loaded onto glutathione beads (Fig. 4b), indicating that UgsL-GST and OsSRF3-His interact in vitro. To test this interaction in vivo, we performed co-immunoprecipitation (Co-IP) assays with proteins extracts from rice protoplasts co-expressing UgsL-GFP and OsSRF3-Flag. UgsL was among the proteins that co-precipitated with OsSRF3 following IP with an anti-Flag antibody (Fig. 4c). We also validated the interaction between UgsL and OsSRF3 in a luciferase complementation imaging (LCI) assay in N. benthamiana leaves (Fig. 4d). Finally, we co-expressed OsSRF3-nYFP and UgsL-cYFP fusion constructs in rice protoplasts and performed a bimolecular fluorescence complementation (BiFC) assay. We observed fluorescence from reconstituted yellow fluorescent protein (YFP) at the plasma membrane of protoplasts (Fig. 4e, Supplementary Fig. 13a). These results demonstrate that UgsL interacts with OsSRF3 at the plasma membrane of plant cells.Fig. 4 UgsL physically interacts with OsSRF3 and knockout of OsSRF3 decreases rice resistance against multiple pathogens.

a Yeast two-hybrid (Y2H) analysis of the interaction between UgsL and OsSRF3. BD, pGBKT7; AD, pGADT7. b GST pull-down assay to detect the interaction between UgsL and OsSRF3. c Co-immunoprecipitation (Co-IP) assay to detect the interaction of UgsL with OsSRF3. d Luciferase complementation imaging (LCI) assay of the interaction between UgsL and OsSRF3 in N. benthamiana epidermal cells. e Bimolecular fluorescence complementation (BiFC) assays to detect the interaction between UgsL and OsSRF3. Scale bars, 5 µm. f Morphology and yield traits of the wild-type Nipponbare (NPB) and Ossrf3 rice mutants at the mature stage following growth in field conditions. g Resistance assay of NPB and Ossrf3 plants against U. virens (left). Number of rice smut balls measured in the resistance assay (right). Data were collected from three independent experiments for each treatment with 10 panicles. h Disease symptoms (left) and relative fungal biomass (right) of NPB and Ossrf3 plants after spray inoculation with M. oryzae. i Disease symptoms (left) and lesion length (right) of NPB and Ossrf3 plants after inoculation with Xoo. Data were collected from three independent experiments for each treatment with five leaves. j Disease symptoms (left) and lesion area (right) of NPB and Ossrf3 plants after inoculation with R. solani. The leaf lesion area was measured using ImageJ software. Scale bars, 1 cm (h, i, j). Data in (f–j) were shown as boxplots of the number of indicated biological samples displaying the maximum and minimum, first and third quantiles, and the median. Data are analyzed by one-way ANOVA followed by two-sided LSD test for multiple comparisons, and the adjusted P values were shown. Source data are provided as a Source Data file.

OsSRF3 positively regulates multi-pathogen resistance

To assess the role of OsSRF3 in pathogen resistance, we generated OsSRF3-knockout mutants (Ossrf3) using CRISPR/Cas9 gene editing (Supplementary Fig. 14a). The gross morphology and yield traits of these Ossrf3 plants were similar to those of the wild-type NPB (Fig. 4f). However, after inoculation with the wild-type U. virens strain HWD-2, the Ossrf3 mutants produced more smut balls on their spikelets than NPB (Fig. 4g). We also evaluated Ossrf3 and NPB plants for resistance against M. oryzae. Following spray-inoculation with M. oryzae strain ZB25, the Ossrf3 mutants were more susceptible to M. oryzae than NPB (Fig. 4h). Inoculation of the Ossrf3 mutants and NPB with Xoo strain PXO99 using the scissor-clipping method produced similar results, with lesions on Ossrf3 leaves being 2.5 times longer than those on NPB leaves (Fig. 4i). Likewise, the Ossrf3 mutants were more susceptible to infection by Rhizoctonia solani strain HG81 than NPB (Fig. 4j). These results indicate that knock out of OsSRF3 in rice enhances its susceptibility to multiple pathogens.

To test whether OsSRF3 promotes disease resistance in rice, we generated OsSRF3-overexpressing (SRF3-OE) transgenic rice plants (Supplementary Fig. 14b, c). These SRF3-OE plants appeared morphologically similar to the transgenic control EV plants (Supplementary Fig. 14d). However, SRF3-OE plants were more resistant to infection by U. virens, M. oryzae, Xoo, and R. solani than EV control plants (Fig. 5a–d). Together, these results indicate that OsSRF3 promotes broad-spectrum resistance against rice pathogens.Fig. 5 Overexpressing OsSRF3 in rice enhances rice resistance against multiple pathogens and OsSRF3 activates PTI responses.

a Resistance assay of SRF3-OE and EV plants against U. virens (left). Number of rice smut balls measured in the resistance assay (right). Data were collected from three independent experiments for each treatment with 10 panicles. b Disease symptoms (left) and relative fungal biomass (right) of SRF3-OE and EV plants after spray-inoculation with M. oryzae. c Disease symptoms (left) and lesion length (right) of SRF3-OE and EV plants after inoculation with Xoo. Data were collected from three independent experiments for each treatment with five leaves. d Disease symptoms (left) and lesion area (right) of SRF3-OE and EV plants after inoculation with R. solani. The leaf lesion area was measured using ImageJ software. e ROS burst in SRF3-OE and EV plants challenged with 8 nM chitin or water. RLU, relative luminescence unit. Data are means ± SD from five independent experiments. f DAB staining to detect H2O2 levels in leaves of SRF3-OE and EV plants. g Activation of MAPK signaling in SRF3-OE−1 and EV plants (treated with 8 nM chitin) was detected by immunoblotting with the phospho-p44/42 MAPK antibody at the indicated times. Intensity was quantified by densitometry using ImageJ and indicate relative phosphorylation levels of MPK3 and MPK6 normalized to OsActin abundance. Data are means ± SD from three independent experiments. h Callose deposition in SRF3-OE and EV plants. (i) Relative OsPR1b, OsPR10a, OsRBOHA, and OsPAL1 transcript levels in SRF3-OE and EV rice spikelets were evidenced by RT-qPCR. Data are means ± SD from three independent experiments. Scale bars (b–d, f), 1 cm. Data in (a–d) were shown as boxplots of the number of indicated biological samples displaying the maximum and minimum, first and third quantiles, and the median. In (a–d, g, i) data are analyzed by one-way ANOVA followed by two-sided LSD test for multiple comparisons, and the adjusted P values were shown. Source data are provided as a Source Data file.

OsSRF3 is involved in PTI

To investigate whether OsSRF3 is involved in the PTI response, we examined the responses of SRF3-OE and EV plants to the PAMP elicitor chitin. We immersed individual rice protoplasts in 8 nM chitin solution and measured the production of ROS using a luminol chemiluminescence assay. ROS production was higher in SRF3-OE plants than in EV plants (Fig. 5e). Following M. oryzae infection, we also observed increased H2O2 levels in SRF3-OE plants compared to EV plants based on DAB staining (Fig. 5f). After applying exogenous chitin, the MAPK pathway was activated in SRF3-OE compared to EV plants (Fig. 5g). In addition, callose deposition at the cell wall was increased in SRF3-OE plants compared to EV plants (Fig. 5h). In rice spikelets infected with U. virens at 1 dpi, the PTI-associated genes OsPR1b, OsPR10a, OsRBOHA, and PHENYLALANINE AMMONIA-LYASE 1 (OsPAL1) were significantly upregulated in SRF3-OE plants compared to EV plants (Fig. 5i). These results suggest that OsSRF3 activates PTI in rice.

UgsL accelerates the degradation of OsSRF3 during infection

Laccase are a group of enzymes that can oxidize and degrade a variety of proteins and small molecules24. To investigate the effect of the laccase UgsL on OsSRF3, we performed a cell-free degradation assay by incubating equal amounts of recombinant OsSRF3-His with total protein extracts from the spikelets of the rice cultivar WX98 inoculated with the wild-type U. virens strain HWD-2 or the ∆ugsL−1 mutant. In the presence of ATP, we detected the degradation of OsSRF3-His over time (30–60 min) in both mixtures (Fig. 6a). However, the rate of OsSRF3-His degradation was lower when co-incubated with protein extracts from ∆ugsL-1-infected rice spikelets vs. extracts from HWD-2-infected rice spikelets (Fig. 6a), suggesting that UgsL accelerates the degradation of OsSRF3. Under the same conditions, the rate of OsSRF3-His degradation increased when co-incubated with proteins from 35S:UgsL (HE-1) spikelets vs. proteins from EV spikelets (Fig. 6b). To confirm that UgsL accelerates OsSRF3 degradation in vivo, we transfected SRF3-OE-1 rice protoplasts with constructs harboring GFP, UgsL-GFP, UgsLM1-GFP, UgsLM2-GFP or UgsLM3-GFP. OsSRF3 levels were lower in SRF3-OE-1 rice protoplasts expressing UgsL-GFP compared to UgsLM1-GFP, UgsLM2-GFP, UgsLM3-GFP or GFP (Fig. 6c), and UgsL-GFP did not promotes OsSRF6 or OsSRF7 degradation (Supplementary Fig. 15a). These results indicate that UgsL specifically promotes the degradation of OsSRF3 in vivo, and that the laccase activity of UgsL affects this degradation. However, we did not observe OsSRF3 degradation when we co-incubated OsSRF3-His and UgsL-GST, or OsSRF3-His and GST in an in vitro reaction (Fig. 6d), indicating that the UgsL-mediated degradation of OsSRF3 requires a plant-derived component.Fig. 6 UgsL promotes OsSRF3 degradation and inhibits OsSRF3-mediated PTI responses in rice.

a Total proteins were extracted from spikelets of rice cv. WX98 inoculated with U. virens (WX98 + HWD-2) or the ∆ugsL−1 mutant (WX98 + ∆ugsL-1), and equal amounts of OsSRF3-His and total proteins were incubated for the indicated times after adding 10 mM ATP. OsSRF3-His protein levels were detected by immunoblot. b Total proteins were extracted from spikelets of HE−1 and EV plants, and equal amounts of OsSRF3-His and total proteins were incubated for the indicated times after adding 10 mM ATP. OsSRF3-His protein levels were detected by immunoblotting. c UgsL-GFP, UgsLM1-GFP, UgsLM2-GFP, UgsLM3-GFP or the empty GFP vector were individually transfected in protoplasts of SRF3-OE−1 plants, and OsSRF3-Flag protein levels were detected by immunoblotting. Data are means ± SD from three independent experiments. d Immunoblots showing the equal amounts of OsSRF3-His and UgsL-GST, or OsSRF3-His and GST incubated for the indicated times. e The degradation of OsSRF3-His by total protein extracts from spikelets of rice cv. WX98 inoculated with U. virens (WX98 + HWD-2) is inhibited by MG132. f Immunoblots of the indicated protein mixtures (OsSRF3-His and WX98 + HWD-2) (Input) or proteins co-purified with UgsL-GST from these protein mixtures (His IP) were detected. g GFP, UgsL-GFP, UgsLM3-GFP was co-expressed with OsSRF3-Flag in rice protoplasts. After Flag IP, anti-Ub antibody was used to quantitatively detect the level of ubiquitination modification on OsSRF3. h ROS burst in protoplasts of SRF3-OE−1 rice protoplasts challenged with 8 nM chitin and UgsL-GST. RLU, relative luminescence unit. Data are means ± SD from five independent experiments. i Rice plants were incubated with purified OsSRF3-His, UgsL-GST, or OsSRF3-His + UgsL-GST, and activated MAPK signaling was detected by immunoblotting. Data are means ± SD from three independent experiments. In (a–e) numbers below each lane indicate OsSRF3 levels normalized to OsActin abundance. In (c, i) different lowercase letters indicate significant differences at P < 0.05 (one-way ANOVA followed by two-sided LSD test for multiple-comparisons). P values are shown in the Source Data file. Source data are provided as a Source Data file.

UgsL enhances the interaction of OsSRF3 with the ubiquitin-26S proteasome

In eukaryotes, proteins are primarily degraded through the ubiquitin-26S proteasome pathway or the autophagy pathway28. We investigated whether UgsL promotes the degradation of OsSRF3 through the 26S proteasome pathway during fungal infection by testing the effect of MG132 (an inhibitor of the 26S proteasome) on OsSRF3 degradation. While OsSRF3 was degraded when incubated with protein extracts from infected rice spikelets as a source of UgsL, the degradation of OsSRF3 was suppressed by treatment with MG132 (Fig. 6e), indicating that the 26S proteasome is involved in its degradation. We then investigated the effects of UgsL on the interaction between OsSRF3 and the 26S proteasome via in vitro pull-down assays. We co-incubated recombinant OsSRF3-His with total protein extracts from infected rice spikelets in the presence or absence of UgsL-GST for 60 min before adding anti-His beads for a His pull-down assay. Following elution of the proteins that bound to the anti-His beads, we looked for proteins in the 26S proteasome using an anti-20S proteasome antibody. Regardless of whether UgsL-GST was added to the mixtures, we detected proteins of the 26S proteasome among proteins pulled down with OsSRF3-His (Fig. 6f). However, the presence of UgsL increased the amount of 26S proteasome proteins that co-purified with OsSRF3-His (Fig. 6f). Finally, an in vivo ubiquitination assay confirmed that OsSRF3 is subject to ubiquitin-mediated degradation, and the laccase activity of UgsL affects its promotion the ubiquitination degradation of OsSRF3 (Fig. 6g). These results suggest that UgsL accelerates the degradation of OsSRF3 by enhancing its association with the ubiquitin-26S proteasome.

UgsL inhibits OsSRF3-mediated PTI responses

To investigate whether UgsL inhibits OsSRF3-mediated PTI responses, we examined the responses of the SRF3-OE-1 transgenic line to the PAMP elicitor chitin with or without UgsL-GST. We immersed SRF3-OE-1 rice protoplasts in 8 nM chitin solution and measured ROS levels using a luminol chemiluminescence assay. ROS production decreased in SRF3-OE-1 rice protoplasts in the presence of UgsL (Fig. 6h). In rice leaves incubated with recombinant OsSRF3-His alone, the MAPK pathway was activated (Fig. 6i). However, in rice leaves co-incubated with recombinant OsSRF3-His and UgsL-GST proteins, the phosphorylation levels of MPK3 and MPK6 were lower than in rice leaves incubated with OsSRF3-His alone (Fig. 6j), suggesting that UgsL inhibits OsSRF3-mediated PTI responses in rice plants.

OsBAK1 physically interacts with OsSRF3

To explore the function of OsSRF3 in the PTI response, we performed a Y2H assay and identified 16 OsSRF3-interacting proteins (Supplementary Table 2). Notably, the brassinosteroid receptor kinase OsBAK1 was one of the interactors of OsSRF3. Therefore, we performed in vitro pull-down assays with recombinant OsBAK1-GST and OsSRF3-His proteins purified from E. coli. We detected OsSRF3-His among the proteins pulled down by OsBAK1-GST on glutathione beads (Fig. 7a), indicating that OsBAK1-GST and OsSRF3-His interact in vitro. To confirm this interaction in vivo, we conducted Co-IP assays using protein extracts from rice protoplasts co-expressing the OsBAK1-GFP and OsSRF3-Flag constructs. We detected OsBAK1 among the proteins immunoprecipitated with OsSRF3 using an anti-Flag antibody (Fig. 7b). We validated the interaction between OsBAK1 and OsSRF3 with a LCI assay in N. benthamiana (Fig. 7c). Finally, we co-expressed constructs harboring OsSRF3-nYFP and OsBAK1-cYFP constructs in rice protoplasts and performed a BiFC assay, resulting in the reconstitution of YFP fluorescence at the plasma membrane of the protoplasts (Fig. 7d, Supplementary Fig. 13b). Collectively, these results demonstrate that OsBAK1 interacts with OsSRF3 in vitro and in vivo.Fig. 7 OsBAK1 interacts with OsSRF3 and overexpressing OsBAK1 in rice enhances resistance against U. virens.

a GST pull-down assay to detect the interaction between OsBAK1 and OsSRF3. b Co-IP assay of the interaction between OsBAK1 and OsSRF3. c LCI assay of the interaction between OsBAK1 and OsSRF3 in N. benthamiana epidermal cells. d BiFC assays to detect the interaction between OsBAK1 and OsSRF3. Scale bar, 5 µm. e Relative OsBAK1 transcript levels in BAK1-OE plants by RT-qPCR and plant morphology of BAK1-OE plants. Data are means ± SD from three independent experiments. f Resistance assay of BAK1-OE and EV plants against U. virens (left); Diseased panicle rate (%) (middle), data are means ± SD from three independent experiments; Number of rice smut balls measured in the resistance assay (right), data were collected from three independent experiments for each treatment with 10 panicles. Data was shown as boxplots of the number of indicated biological samples displaying the maximum and minimum, first and third quantiles, and the median. g OsBAK1 phosphorylates OsSRF3 in vitro. An in vitro kinase assay was performed by incubating recombinant OsBAK1 and OsSRF3. Migration of OsSRF3 in an SDS-polyacrylamide gel containing Phos-Tag acrylamide was analyzed by immunoblotting with or without treatment with bacterial alkaline phosphatase (BAP). h Phosphorylation of OsSRF3 in BAK1-OE and EV rice protoplasts. OsSRF3-GFP was transfected in BAK1-OE and EV protoplasts and immunoprecipitated with anti-GFP resin. Phosphorylation of OsSRF3-GFP was detected with a phospho-Ser/Thr antibody. White asterisks represent the phosphorylation of OsSRF3. Numbers below each lane indicate relative phosphorylation of OsSRF3 levels normalized to OsSRF3-GFP abundance. Data in (e, f) are analyzed by one-way ANOVA followed by two-sided LSD test for multiple comparisons, and the adjusted P values were shown. Source data are provided as a Source Data file.

UgsL interferes with the phosphorylation of OsSRF3 by OsBAK1

To assess the function of OsBAK1 in rice resistance to U. virens, we constructed transgenic rice plants overexpressing OsBAK1 (BAK1-OE-5 and BAK1-OE-7) (Fig. 7e). These plants exhibited growth defects, which is consistent with a previous report29. In addition, the BAK1-OE plants were more resistant to U. virens than EV plants (Fig. 7f). The identification of OsBAK1 as an interactor of OsSRF3 suggested that OsBAK1 might phosphorylate OsSRF3. To test this possibility, we performed an in vitro phosphorylation assay using purified recombinant OsSRF3-His, GST, and OsBAK1-GST protein. Indeed, OsBAK1 directly phosphorylated OsSRF3 in vitro (Fig. 7g). To assess the link between OsBAK1 expression and OsSRF3 phosphorylation in vivo, we transfection protoplasts isolated from rice BAK1-OE and EV plants with the OsSRF3-GFP construct. The phosphorylation of OsSRF3-GFP was much stronger in BAK1-OE protoplasts than in EV protoplasts (Fig. 7h), indicating that OsBAK1 phosphorylates OsSRF3 in vivo. To identify the residues in OsSRF3 that are phosphorylated by OsBAK1, we incubated OsSRF3-His with OsBAK1-GST in an in vitro phosphorylation assay and then analyzed the tryptic peptides of OsSRF3-His by LC-MC. Two residues in OsSRF3 were identified as phosphorylation sites: Thr661 and Ser670 (Suplementary Fig. 16). We performed in vitro kinase assays using purified OsSRF3T661A-His, OsSRF3S670A-His, and OsSRF3T661A/S670A-His mutant proteins, confirming that Thr661 and Ser670 in OsSRF3 are the major residues phosphorylated by OsBAK1 (Fig. 8a). To explore how the phosphorylation of OsSRF3 affects its stability, we co-expressed OsSRF3-Flag, OsSRF3T661A-Flag, OsSRF3S670A-Flag, OsSRF3T661A/S670A-Flag, and UgsL-GFP in rice protoplasts, finding that the phosphorylation of OsSRF3 at Ser670 promotes its stability (Fig. 8b). To examine the importance of Thr661 and Ser670 phosphorylation for the function of OsSRF3 in plant immunity, we generated transgenic rice expressing OsSRF3, OsSRF3T661D, and OsSRF3S670D in the srf3-1 mutant background. Following spray inoculation with M. oryzae strain ZB25, the srf3-1/OsSRF3S670D plants were more resistant to M. oryzae than srf3-1, srf3-1/OsSRF3, and srf3-1/ OsSRF3T661D plants and more susceptible to M. oryzae than OE-1 plants (Fig. 8c). These results indicate that OsBAK1-mediated phosphorylation of OsSRF3 at Ser670 promotes the stability and immune function of OsSRF3.Fig. 8 UgsL interferes with the phosphorylation of OsSRF3 by OsBAK1.

a In vitro kinase assays to show the phosphorylation levels of different OsSRF3-His variants when these mutant proteins were incubated with OsBAK1-GST, and migration of OsSRF3 in an SDS-polyacrylamide gel containing Phos-Tag acrylamide was analyzed by immunoblotting. White asterisks represent the phosphorylation of OsSRF3. b Different OsSRF3-Flag variants were co-expressed with UgsL-GFP or GFP in rice protoplasts, and the degradation rates of OsSRF3 mutant proteins were detected by immunoblotting. Data were collected from three independent experiments. c Disease symptoms (Top) and relative fungal biomass (Bottom) of OE-1, srf3-1, srf3−1/SRF3, srf3−1/SRF3T661D, and srf3-1/SRF3S670D plants after spray-inoculation with M. oryzae. Different lowercase letters indicate significant differences at P < 0.05. d Western blots of the indicated protein mixtures (Input) or proteins co-purified with UgsL-MBP from these mixtures (GST IP) were detected. e UgsL disturbs the interaction between OsBAK1 and OsSRF3. f UgsL interferes with the phosphorylation of OsSRF3 by OsBAK1. g Relative OsBAK1 transcript levels in SRF3-OE−1 and BAK1-KD plants by RT-qRCR. Data are means ± SD from three independent experiments. h Disease symptoms (left) and relative fungal biomass (right) of SRF3-OE−1 and BAK1-KD plants after spray-inoculation with M. oryzae. i Resistance assay of SRF3-OE-1 and BAK1-KD plants against U. virens (left). Number of rice smut balls measured in the resistance assay (right). j ROS burst in SRF3-OE-1 and BAK1-KD-1 protoplasts challenged with 8 nM chitin or water. RLU, relative luminescence unit. Data are means ± SD from five independent experiments. k Callose deposition in SRF3-OE-1 and BAK1-KD plants. l Relative OsPR1b and OsRBOHA transcript levels in SRF3-OE-1 and BAK1-KD rice leaves were evidenced by RT-qPCR. Data are means ± SD from three independent experiments. Data in (c, h, i) were shown as boxplots of the number of indicated biological samples displaying the maximum and minimum, first and third quantiles, and the median. In (c, g–i, l) data are analyzed by one-way ANOVA followed by two-sided LSD test for multiple comparisons, and the adjusted P values were shown. Source data are provided as a Source Data file.

Because both UgsL and OsBAK1 interacted with OsSRF3, and UgsL did not interacts with OsBAK1 (Supplementary Fig. 15b, c), we reasoned that these proteins might compete with each other to bind OsSRF3. To test this possibility, we performed in vitro pull-down assays using purified OsSRF3-His, UgsL-MBP, and OsBAK1-GST fusion proteins. When we incubated the equal amounts of OsSRF3-His and OsBAK1-GST proteins with GST beads and added increasing amounts of UgsL protein to the OsBAK1–OsSRF3 mixture, the amount of OsSRF3 that bound to OsBAK1 gradually decreased (Fig. 8d), indicating that UgsL and OsBAK1 compete for binding with OsSRF3. We also investigated whether UgsL interferes with the interaction between OsBAK1 and OsSRF3 in vivo by performing LCI assays, finding that UgsL reduced the interaction between OsBAK1 and OsSRF3 (Fig. 8e). Finally, adding UgsL-MBP and UgsLM3-MBP decreased the phosphorylation level of OsSRF3 (Fig. 8f), indicating that OsBAK1-mediated phosphorylation of OsSRF3 is affected by the presence of UgsL. Taken together, these results indicate that OsBAK1 interacts with and phosphorylates OsSRF3, while UgsL interferes with the phosphorylation of OsSRF3 by OsBAK1.

OsSRF3-mediated disease resistance is dependent on OsBAK1

To explore whether OsSRF3-mediated disease-resistance is dependent on OsBAK1, we constructed SRF3-OE-1 plants in which OsBAK1 was silenced via RNAi. The expression of OsBAK1 was significantly reduced in SRF3-OE-1 plants compared to OsBAK1 knockdown (BAK1-KD-1 and BAK1-KD-2) plants (Fig. 8g). We then evaluated SRF3-OE-1 and BAK1-KD plants for resistance against M. oryzae. Following spray-inoculation with M. oryzae strain ZB25, the BAK1-KD plants were more susceptible to M. oryzae than SRF3-OE-1 plants (Fig. 8h). Likewise, the BAK1-KD plants were more susceptible to infection by U. virens strain HWD-2 than SRF3-OE-1 plants (Fig. 8i). These results indicate that OsSRF3-mediated disease-resistance is dependent on OsBAK1.

To investigate whether OsBAK1 regulates OsSRF3-mediated PTI responses, we incubated SRF3-OE-1 and BAK1-KD rice protoplasts in 8 nM chitin and measured ROS levels using a luminol chemiluminescence assay. ROS production decreased in BAK1-KD compared to in SRF3-OE-1 protoplasts (Fig. 8j). In addition, callose deposition at the rice cell wall was reduced in BAK1-KD plants compared to SRF3-OE-1 plants (Fig. 8k). In the leaves of rice plants infected with M. oryzae at 1 dpi, the PTI-associated genes OsPR1b and OsRBOHA were significantly downregulated in BAK1-KD plants compared to SRF3-OE-1 plants (Fig. 8l). These results indicate that OsBAK1 regulates OsSRF3-mediated PTI responses in rice.

Discussion

Plants have complex, precise immune systems to defend themselves against pathogen attack1. Although many immune components have been identified in various plant species, our understanding of the molecular mechanisms behind plant immunity remains limited. Pathogen effectors suppress plant immunity by targeting various immune components30,31. Therefore, identifying host targets of pathogen effectors will increase our understanding of plant immune signaling. U. virens secretes multiple, diverse effectors to promote its infection of host plants15. Elucidating the functions of these U. virens effectors may help elucidate the molecular mechanisms underlying the rice–U. virens interaction and might help uncover molecular probes to explore disease resistance-related genes16. In this study, we demonstrated that UgsL is essential for the virulence of U. virens (Fig. 1e), and that laccase activity is required for the development and virulence of U. virens (Supplementary Fig. 3). In addition, UgsL suppressed BAX- and INF-induced PCD in N. benthamiana leaves (Supplementary Fig. 4b), and heterologous expression of UgsL in rice enhanced plant susceptibility to multiple pathogens (Fig. 3). Various cytoplasmic effectors secreted by M. oryzae and U. virens are transported to host cells via the BIC15,20,32. Using the M. oryzae secretory system, we determined that UgsL-GFP produced from transgenic M. oryzae co-accumulated with PWL2 in the BIC32. An immunofluorescence assay revealed that UgsL was translocated into rice floral cells (Supplementary Fig. 6). Significantly fewer smut balls were produced on rice spikelets inoculated by the CΔugsLΔSP-1 strain (harboring a UgsL construct encoding a protein lacking the SP) compared to the complemented CΔugsL-1 strain (Supplementary Fig. 5d), indicating that UgsL has other functions beside serving as an effector. Virulence assays of the ΔugsL-1 knockout mutant on 35S:UgsL and EV transgenic rice plants revealed that UgsL promotes U. virens virulence, as the 35S:UgsL plants produced few smut balls, whereas EV plants produced none (Supplementary Fig. 10). This result provides genetic evidence that UgsL is a virulence effector. Taken together, we conclude that UgsL is a key virulence effector that inhibits rice immunity.

Plant fungal diseases lead to yield losses and reduce the quality of agricultural products. These fungi also produce mycotoxins that threaten human and animal health33. Mycotoxins are involved in fungal virulence: for example, the Fusarium mycotoxin most commonly present in cereal grains, deoxynivalenol (DON), is a key virulence factor in F. graminearum infection34. In this study, we showed that the ustilaginoidin synthetase UgsL is essential for the virulence of U. virens. Interestingly, exogenous application of ustilaginoidins (UA) during infection with ∆ugsL-1 partially restored the virulence of this mutant, indicating that ustilaginoidins are directly involved in the virulence of U. virens (Supplementary Fig. 5a–c). These results support the notion that ustilaginoidins are an important virulence factor of U. virens. In this study, we found that the laccase activity of UgsL secreted by U. virens can regulate rice immunity. In addition, UgsL also functions in ustilaginoidin biosynthesiss. We speculated that UgsL has a dual function in virulence of U. virens, which can directly inhibit the immunity of rice and also affect the content of ustilaginoidins to regulate the virulence of U. virens. The roles of ustilaginoidins in host immune regulation, environmental microorganism colonization or pathogen signaling recognition need to be determined.

HIGS was recently developed as an alternative strategy to control fungal diseases, whereby siRNAs produced by the host plant target and silence selected genes in the invading pathogen35. For example, HIGS of the F. graminearum genes SIX Gene Expression 1, F. graminearum GTI1/PAC2 1, and Sterile 12 conferred resistance to Fusarium head blight in wheat (Triticum aestivum), while also decreasing DON production36. Similarly, our findings suggest that HIGS against UgsL could be an effective strategy to diminish the severity of RFS and ustilaginoidins in rice (Fig. 2). We conclude that UgsL plays a major role in plant infection by U. virens and represents an exciting candidate gene for HIGS-based approaches to enhance U. virens resistance in rice.

To elucidate how UgsL suppresses the plant immune system, we performed a Y2H screen to identify UgsL-interacting proteins (Supplementary Table 1), leading to the identification of OsSRF3 as a putative target of UgsL in rice. Multiple approaches verified this interaction, such as targeted Y2H, GST pull-down, Co-IP, LCI, and BiFC assays (Fig. 4a–e). In Arabidopsis, SRF3 is involved in bacterial-induced immune responses as well as sensing and transducing early iron-deficiency signals, thereby triggering Ca2+ signaling and callose biosynthesis in root tips to regulate growth and iron homeostasis13. In the current study, knocking out OsSRF3 in rice enhanced rice susceptibility to M. oryzae, U. virens, R. solani, and Xoo (Fig. 4f–j). By contrast, overexpressing OsSRF3 in rice enhanced resistance to these pathogens (Fig. 5a–d). Remarkably, these enhancements in resistance were achieved without adverse effects on plant growth or yield. OsSRF3 mediates the typical PTI responses, activating MAPK signaling and the ROS burst, leading to callose deposition, and inducing the expression of defense-related genes (Fig. 5e–i). Laccases can oxidize and degrade a variety of proteins and small molecules24. The above findings prompted us to investigate whether UgsL alters the stability of OsSRF3. Interaction between UgsL and OsSRF3 likely result in the degradation of OsSRF3 in infected rice spikelets, given that OsSRF3 degradation was accelerated in the presence of UgsL in rice cells (Fig. 6a–d). Treatment with MG132 decreased UgsL-mediated OsSRF3 degradation in rice cells (Fig. 6e). These finding suggest that OsSRF3 degradation occurs through the 26S proteasome pathway, UgsL increases the association of OsSRF3 with the ubiquitin-26S proteasome, and the activity of the laccase UgsL affects the degradation rate of OsSRF3 (Fig. 6c, f, g). Furthermore, we showed that UgsL inhibits the PTI response in rice plants (Fig. 3d–h), as well as their OsSRF3-mediated PTI response (Fig. 6h, i). Taken together, we revealed that OsSRF3 positively regulates plant immunity via key PTI-related components and that UgsL inhibits plant immunity by promoting OsSRF3 degradation through the ubiquitin-26S proteasome pathway during infection.

Laccase is a copper-containing polyphenol oxidase that acts on a wide range of substrates. Fungal laccase is able to oxidize substrates to form free radical intermediates, which are then involved in oxidative coupling or bond breaking, ultimately leading to oxidation, decomposition, or polymerization of the substrates37. These indicate that some copper-containing polyphenol oxidases laccases might function on oxidative modification of amino acids, the change of the spatial structure of the enzyme, or the interaction with the substrate binding site38. We hypothesized that laccase UgsL might affect the stability or activity of OsSRF3 by oxidative modifications.

In Arabidopsis, BAK1 interacts with FLS2 or PEP1 RECEPTORs (PEPRs) and is a PTI co-receptor39,40. The FLS2–BAK1 complex associates with and phosphorylates BIK141. The BAK1-induced phosphorylation of BRASSINOSTEROID INSENSITIVE 1 (BRI1) activates the kinase activity of BRI1 and promotes brassinosteroid signaling42. Upon activation with the flagellin peptide Flg22, BAK1 phosphorylates CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1), which functions in brassinosteroid signal transduction, to activate CERK1 and protect plants from multiple fungal infections43. BAK1 also phosphorylates BAK-TO-LIFE 2 (BTL2) to suppress its activity and inactivate autoimmune signaling44. Thus, BAK1-mediated phosphorylation of its interacting receptor kinases functions as a switch that activates or inactivates signaling. In rice, OsBAK1 positively regulates plant defense against the bacterial pathogen Xoo strain PXO9945. Here, overexpressing OsBAK1 in rice enhanced resistance to RFS (Fig. 7f). Therefore, we revealed a previously unknown role of OsBAK1. OsBAK1 interacts with and phosphorylates OsSRF3 (Fig. 7g–h). OsBAK1-mediated phosphorylation of OsSRF3 at Ser670 promotes the stability and immune function of OsSRF3 (Fig. 8b–c), and OsSRF3 acts downstream of OsBAK1 in plant defense (Fig. 8g–l). The phosphorylation signaling pathway in vivo is transient and complex, and the OsSRF3 protein has abundant phosphorylation sites (Supplementary Fig. 16). In this situation, we can not directly confirm that OsBAK1 directly phosphorylate these two sites of OsSRF3 protein in vivo. However, the sites identified by in vitro mass spectrometry are indeed involved in the OsSRF3 mediated PTI signaling pathway and rice immunity. With the development of protein mass spectrometry technology, there will be a more comprehensive and clearer supplement to the phosphorylation events of OsSRF3 in the future.

Several plant kinases can phosphorylate specific substrates to regulate different downstream pathways and influence various phenotypes46. Notably, we identified 16 putative OsSRF3-interacting proteins by Y2H, including MAPK kinase kinase ε (OsMAPKKKε), OsRBOHB, and RECEPTOR-LIKE CYTOPLASMIC KINASE 185 (OsRLCK185) (Supplementary Table 2). Our results point to OsSRF3 as a candidate transmitter that links the PRR OsBAK1 with MAP kinase cascades. How OsSRF3 transmits a signal to the MAP kinase cascade remains to be addressed. OsSRF3 may directly pass the signal to MAPKKK, a starting point for activating MAP kinases47, possibly via an interaction and/or phosphorylation. RBOHs are NADPH oxidases that produce ROS48. RBOHs are phosphorylated by receptor kinases to ensure ROS production17. For example, the RLCK PBS1-LIKE KINASE 13 phosphorylates the NADPH oxidase RBOHD to enhance ROS production in Arabidopsis49. However, the underlying mechanism through which OsSRF3 phosphorylates RBOHs to enhance ROS production remains unclear. OsSRF3 is degraded by the ubiquitination pathway, and further research can be conducted on the key E3 ligase involved in its degradation. In addition to being phosphorylated by OsBAK1, we also identified 12 other phosphorylation sites in OsSRF3 (Supplementary Fig. 16), indicating the presence of other interacting regulated protein kinases. Thus, it will be interesting to screen for the upstream activated receptor kinases or other modifying enzymes of OsSRF3, as well as the downstream OsSRF3-phosphorylated substrates to investigate how OsSRF3 activates PTI signaling in plants.

We propose a working model for how UgsL targets OsSRF3 to suppress host plant immunity (Fig. 9). During U. virens infection in rice, several U. virens-secreted PAMPs are perceived by the OsBAK1–OsSRF3 complex or by other PRRs. Following PAMP perception, OsBAK1 phosphorylates OsSRF3 to activate PTI signaling and enhance rice immunity. As a pathogenic strategy, the effector UgsL is secreted by U. virens into plant cells where it interacts with OsSRF3 and promotes its degradation via the ubiquitin-26S proteasome, thereby inhibiting OsBAK1–OsSRF3-mediated PTI responses and suppressing plant immunity. Moreover, our study highlights the critical role of OsSRF3 as a component in orchestrating multi-pathogen resistance, further underscoring its importance in plant defense. Thus, OsSRF3 represents a valuable gene resource for disease-resistance breeding in rice, which has important theoretical and practical applications.Fig. 9 A working model illustrating how UgsL manipulates OsSRF3 to suppress rice immunity during U. virens infection.

During infection, some U. virens-secreted PAMPs are perceived by the OsBAK1–OsSRF3 complex at the rice plasma membrane. After perceiving PAMPs, OsBAK1 phosphorylates OsSRF3 to activate MAPK signaling and the ROS burst, and induces the expression of defense-related genes, thereby enhancing plant immunity (a). In turn, the effector UgsL is secreted by U. virens and transported into host cells where it interacts with OsSRF3 and promotes its degradation via the ubiquitin-26S proteasome, OsBAK1-mediated phosphorylation of OsSRF3 is also suppressed by UgsL, thereby inhibiting OsBAK1–OsSRF3-mediated immune pathways (b).

Methods

Plant materials, strains, and inoculation assays

The 347 bp coding sequence of UgsL was cloned into the HIGS vector pDS1301. The full-length coding sequence of UgsL, OsSRF3 or OsBAK1 was cloned into the overexpression vector pU1301-Flag. The sgRNA (ACTTGGATCACCGG and TTAGGCAGCCTGGG) of OsSRF3 was cloned into the CRISPR/Cas9 vector pRGEB32. Transgenic rice (Oryza sativa) plants were generated in the NPB background, and rice transformation was performed by Wuhan Tianwen Biotechnology Co., Ltd (Wuhan, China). The coding sequences of OsSRF3 as well as OsSRF3T661D and OsSRF3S670D (produced by site-directed mutagenesis) were subcloned into the pU1301-Flag vector. The mutated sequences were overexpressed in the srf3−1 (without Cas9) line via Agrobacterium-mediated transformation.

Rice protoplast isolation was performed as described previously50. NPB rice seedlings were grown on Murashige and Skoog (MS) medium in a growth chamber in the dark for 14 d. The seedlings were cut into approximately 0.3-mm strips and soaked in cell-wall digestion buffer (0.75% Macerozyme R-10, 1.5% Cellulase R-10, 0.6 M D-mannitol, 10 mM N-morpholinoethanesulfonate (MES), 0.1% BSA, and 3.4 mM CaCl2 at pH 5.7; Sigma-Aldrich, USA) for 3 h. After digestion, the strips were washed with W5 solution and filtered through an 80-mm mesh. The protoplasts were centrifuged for 3 min at 1000 × g and washed with W5. The supernatant was removed, and the protoplasts were resuspended in MMG solution (0.6 MD-mannitol, 15 mM MgCl2, and 4 mM MES at pH 5.7) at a concentration of 5 × 106 cells/mL.

Protoplast transfection was performed using the polyethylene glycol (PEG)-mediated method50. The plasmids and protoplasts were mixed, and the same volume of PEG solution (0.6 M D-mannitol, 100 mM CaCl2, and 40% [w/v] PEG 4000) was added. The mixture was incubated at 28 °C for 15 min, and W5 was added to terminate the reaction. The protoplasts were collected by centrifugation and resuspended in W5. Following incubation at 28 °C in the dark for 16–24 h, the protoplasts were used for assay.Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99, Magnaporthe oryzae strain ZB25, Ustilaginoidea virens strain HWD-2, and Rhizoctonia solani strain HG81 were used for rice inoculation. Rice plants were inoculated with Xoo PXO99 at the late tillering stage by the leaf clipping method, and lesion length was scored at 14 days post inoculation (dpi). For spray inoculation, rice seedlings were sprayed with M. oryzae ZB25 conidial suspensions (5 × 104 conidia/mL) in 0.5% (v/v) Tween 20 at the four-leaf stage. The inoculated rice seedlings were placed in an artificial climate incubator with a relative humidity of 100% and a temperature of 28 °C, and disease lesions were examined at 7 dpi. Rice plants were inoculated at the booting stage with 2 mL of a U. virens HWD-2 mycelial/spore suspensions (1 × 106 conidia/mL) using a syringe. The inoculated plants were placed in a greenhouse with a relative humidity of 100% and a temperature of 28 °C, the number of smut balls per panicle was counted at 21 dpi27. The leaves of 4-week-old rice plants were inoculated with R. solani HG81, and lesion areas were scored at 3 dpi and measured using ImageJ software.

Deletion and complementation of UgsL in U. virens

U. virens transformation was carried out as previously described51. Briefly, about 1200 bp of flanking sequences upstream and downstream of UgsL were ligated into the knockout vector pGKO. An approximately 4-kb complementation fragment was ligated into the complementation vector pNeo3300III (FLAG tags). Agrobacterium (Agrobacterium tumefaciens) strain EHA105 was transformed with the pGKO-UgsL, pNeo3300III-UgsLM1, pNeo3300III-UgsLM2, or pNeo3300III-UgsLM3 vector. The transformed U. virens strains were identified by Southern blot and PCR analyses. Southern blotting was performed using an Amersham Gene Images Alkphos Direct Labeling and Detection System (GE Healthcare, UK).

Assays examining the function of the SP of UgsL

For the yeast invertase secretion assay, the 69-bp sequence encoding the SP of UgsL was cloned into the pSUC2 vector. The pSUC2 (negative control), pSUC2-Avr1bSP (positive control), and pSUC2-UgsLSP vectors were individually transformed into yeast (Komagataella pastoris) strain YTK12. The transformants were subjected to invertase secretion assays in synthetic defined (SD) medium lacking tryptophan (SD–Trp) or YPRAA medium. Invertase activity was detected based on the reduction of TTC to the insoluble red precipitate TPF.

Assays for the suppression of BAX- or INF1-induced cell death by UgsL

The full-length coding sequence of UgsL was ligated with pVX (FLAG tags) to generate the recombinant vector pVX-UgsL. pVX-UgsL, pVX-BAX, pVX-INF1, and empty pVX vectors were introduced into Agrobacterium strain GV3101 via electroporation. Cultures harboring each construct were resuspended in infiltration buffer (0.2 mM acetylacetone, 10 mM MgCl2, and 0.5 mM MES) to a final OD600 of 0.8 and infiltrated into the leaves of 20-day-old Nicotiana benthamiana plants. To investigate the suppression of INF1- or BAX-induced PCD, N. benthamiana leaves were infiltrated with Agrobacterium cultures carrying pVX-UgsL, and then the same areas were infiltrated 24 h later with Agrobacterium carrying pVX-BAX or pVX-INF1; the extent of PCD was assessed at 4 dpi.

Small RNA sequencing, FISH, and immunofluorescence assays

Total RNA extracted from the spikelets of UgsL-HIGS transgenic rice lines at the booting stage was subjected to small RNA sequencing. RNA libraries were constructed with an NEB Next Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Inc., Ipswich, MA, USA) and sequenced on an Illumina HiSeq instrument by Wuhan IGENEBOOK Biotechnology Co., Ltd (Wuhan, China). Unique reads were mapped to the corresponding target fragment of UgsL.

For fluorescence in situ hybridization (FISH) assays, a 21-bp oligonucleotide probe (GCCTAGCTCTGGCCAT AGGCG), which was the most common siRNA sequence identified by small RNA sequencing, was labeled with digoxigenin (DIG). Infected spikelets at 6 dpi were placed in fixative solution (Servicebio, Wuhan, China) for 6 h, embedded in paraffin, and cut into sections. The paraffin sections were dewaxed with xylene, rehydrated in a graded ethanol series, and digested with proteinase K (20 µg/mL) at 37 °C for 30 min. After washing with phosphate-buffered saline, prehybridization solution was added to the mixture. Following incubation at 37 °C for 1 h, the paraffin sections were covered with prehybridization solution. DIG-labeled probes were added to the solution, and hybridization was performed at 37 °C for 16 h. After hybridization, sections were washed and sealed, and an anti-DIG antibody conjugated to horseradish peroxidase (anti-DIG-HRP) was added to the samples. The samples were incubated at 37 °C for 1 h, washed five times with PBS, and treated with Alexa Fluor 594 Tyramide (Thermo Fisher Scientific, USA). The fluorescence of the DIG-labeled siRNA was analyzed under a laser-scanning confocal microscope. DAPI (4’-6-diamidino-2-phenylindole) was used to stain the nucleus.

For the immunofluorescence assay, rice spikelets infected with U. virens strain CΔugsL-1 were sampled at 6 dpi and immediately fixed in 4% paraformaldehyde solution for 2 d, dehydrated in an ethanol series, and embedded in paraffin. U. virens-infected stamen filaments were incubated with mouse UgsL-Flag antibody. Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody was used to detect UgsL-Flag localization under a Zeiss LSM 510 Meta confocal microscope (LSCM) (Carl Zeiss, Jena, Germany).

Untargeted-metabolomics analysis

Rice spikelets from EV and 35S:UgsL (HE−1) plants were ground into a powder in liquid nitrogen, and 60 mg tissue samples were first mixed with 600 µl of ultrapure water (70% methanol, containing 4 μg/mL metabolite internal standards) and sonicated for 30 min. The samples were centrifuged at 13,000 g at 4 °C for 20 min and transferred to a new injection vial for LC-MS analysis. LC-MS analysis was performed as described previously52. All LC-MS raw data were subjected to baseline filtering, peak recognition, integration, retention time correction, peak alignment, and normalization using Progression QI 3.0 metabolomics processing software. PCA, OPLS-DA, and hierarchical cluster analysis were performed using MetaboAnalyst 5.0 software. Metabolic pathways were analyzed using KEGG and GSEA.

Yeast two-hybrid (Y2H) assay

The coding sequence of UgsL (without the SP) was cloned into pGBKT7 to generate the bait vector pGBKT7-UgsL. mRNA from rice spikelets 6 dpi with U. virens was used to construct a cDNA library in the prey vector pGADT7. Library screening for UgsL interactors was performed as described19. To analyze the interaction of UgsL and OsSRF3 in yeast, pGADT7-OsSRF3, and pGBKT7-UgsL vectors were co-transformed into yeast strain Y2H Gold. Positive clones were selected on SD medium lacking Trp, Leu, and His (SD−3), and confirmed on SD medium lacking Trp, Leu, His, and Ade (SD−4) containing X-α-Gal (Clontech, USA). The interaction between BD-53 and AD-T was used as the positive control, while the interaction between BD-UgsL and AD was used as the negative control.

Co-immunoprecipitation (Co-IP) assays

The coding sequences of UgsL or OsBAK1 was cloned into pRTVcGFP (GFP tags), and the coding sequence of OsSRF3 was ligated into pRTVcFLAG (Flag tags). The resulting constructs encoding UgsL-GFP and OsSRF3-Flag, OsBAK1-GFP, and OsSRF3-Flag fusion proteins were co-expressed in rice protoplasts. Total proteins were extracted from the protoplasts at 16 h after transformation and incubated with anti-Flag M2 affinity gel (Yeasen Biotech, Shanghai, China). Proteins eluted from the gels were analyzed by immunoblotting with anti-GFP or anti-Flag antibodies.

GST pull-down assays

The coding sequences of UgsL (without the SP) or OsBAK1 was cloned into pGEX-2TK (GST tags), and the coding sequence of OsSRF3 was cloned into pET28a (His tags). These constructs were individually introduced into Escherichia coli BL21 (DE3) cells. OsBAK1-GST or UgsL-GST fusion protein was purified from E. coli cells and incubated with 100 µL of glutathione-agarose beads (GE Healthcare, UK) at 4 °C for 4 h with shaking. Following centrifugation at 13,000 × g for 2 min at 4 °C, the beads were collected and washed three times with PBS. The beads were then incubated with recombinant OsSRF3-His protein at 4 °C for 2 h with shaking and washed three times with PBS. The beads were boiled for 5 min at 100 °C in 40 µL SDS sample loading buffer, and proteins released from the beads were analyzed by immunoblotting with anti-His or anti-GST antibodies.

Bimolecular fluorescence complementation (BiFC) assays

The coding sequence of UgsL or OsBAK1 was ligated into pRTVcVn, and the coding sequence of OsSRF3 was ligated into pRTVnVn. The resulting constructs encoding UgsL-cYFP and OsSRF3-nYFP, OsBAK1-cYFP, and OsSRF3-nYFP were co-expressed in rice protoplasts. The fluorescence signals in the protoplasts were examined under a LSCM at 16 h after transformation.

Firefly luciferase complementation imaging (LCI) assay

The coding sequence of UgsL or OsBAK1 was ligated into pCAMBIA1301-nLuc, and the coding sequence of OsSRF3 was ligated into pCAMBIA1301-cLuc. Agrobacterium strain GV3101 cells were transformed individually with UgsL-nLuc, OsSRF3-cLuc, OsBAK1-nLuc, or OsSRF3-cLuc. The appropriate combination of cultures was resuspended in infiltration buffer to a final OD600 of 1.6, mixed at a 1:1 ratio (v/v), and infiltrated into the leaves of N. benthamiana plants. At 2 days after infiltration, the N. benthamiana leaves were infiltrated with 1 mM luciferin (Gold Biotechnology, USA). Bioluminescence images were taken with a Chemi-Image System (Tanon 5200Multi, China).

Subcellular localization of UgsL and OsSRF3 in plants

The coding sequence of UgsL or OsSRF3 was cloned into pCNG (GFP tags) and introduced into Agrobacterium strain GV3101. Agrobacterium cultures harboring each construct were resuspended in an infiltration buffer to a final OD600 of 1.0 and infiltrated into the leaves of N. benthamiana plants. Fluorescence was examined under a confocal microscope at 2 days after infiltration.

The coding sequences of UgsL and OsSRF3 were cloned into pRTVcGFP and pRTVcRFP, respectively. The fusion construct was used to transform or co-transform protoplasts, and fluorescence was examined under a confocal microscope 20 h after transformation.

Immunoblot analysis

Proteins were extracted from the samples in lysis buffer (0.1% SDS, 1% Triton X−100, 10 mM DDT, 2 mM EDTA, 0.02 M Tris-HCl, and 1% protease inhibitor). The proteins were then separated by 12% (w/v) SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, USA) using wet transfer at 85 V for 1 h with a BioRad electroblotting apparatus. The membranes were blocked in Tris-buffered saline with 0.1% (v/v) Tween 20 (TBST) containing 10% (w/v) non-fat dry milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: anti-GFP (1:5000 dilution, AE012, ABclonal, China), anti-Flag (1:5000 dilution, AE005, ABclonal, China), anti-His (1:5000 dilution, AE003, ABclonal, China), anti-GST (1:5000 dilution, AE001, ABclonal, China), anti-actin (1:5000 dilution, AE009, ABclonal, China), anti-MBP (1:5000 dilution, AE016, ABclonal, China), anti-Phospho-Ser/Thr (1:1000 dilution, 9381S, Cell Signaling Technology, USA) and anti-20S (1:1000 dilution, ab22673, Abcam, UK), or anti-Phospho-p44/42 (1:1000 dilution, 9101S, Cell Signaling Technology, USA). The membranes were incubated with primary antibodies in TBST with 5% (w/v) non-fat dry milk at room temperature for 2 h with shaking and washed six times (5 min each) with TBST. The membranes were incubated with goat anti-mouse or goat anti-rabbit IgG H&L (HRP) (1:10000 dilution, ab6721, ab6728, Abcam, UK) secondary antibody in TBST with 5% (w/v) non-fat dry milk at room temperature for 1 h with shaking. The membranes were washed six times (5 min each) with TBST, and the signals were detected using Pierce ECL Western blotting substrate (Thermo Fisher Scientific, USA) in a ChemiDoc XRS+ system (Bio-Rad, USA).

Protein purification and degradation assays

Recombinant UgsL-GST, UgsL-MBP, UgsLM3-MBP, OsBAK1-GST, OsSRF3-His, OsSRF3T661A-His, OsSRF3S670A-His, and OsSRF3T661A/S670A-His fusion proteins were produced in E. coli BL21 (DE3) cells. Protein production was induced by adding of 0.1 M isopropyl β-D−1-thiogalactopyranoside (IPTG). The cells were harvested by centrifugation and crushed under ultra-high pressure using a JN-3000PLUS machine (JNBIO, Nanjing, China). Recombinant UgsL-MBP and OsSRF3-His proteins were purified with MBPSep Dextrin Agarose Resin 6FF (Yeasen Biotech, Shanghai, China) and HisPur Ni-NTA Resin (Thermo Scientific, USA), respectively. Recombinant UgsL-GST, OsBAK1-GST or GST proteins were purified with GSTrap Resin (Thermo Scientific, USA). Total proteins isolated from rice spikelets and OsSRF3-His recombinant protein were used for the degradation assays at 30 °C for 0, 30, and 60 min. In brief, a final concentration of 10 mM ATP was added to a mixture containing equal amounts of OsSRF3-His and crude protein extracts isolated from rice spikelets. Detection with an anti-actin antibody was used as a loading control. To inhibit 26S proteasome activity, a final concentration of 50 μM MG132 was added to the reaction mixtures. Immunoblotting of these reaction mixtures with an anti-His antibody was performed to estimate the amount of remaining OsSRF3-His protein.

Detection of ROS accumulation and callose deposition

Rice protoplasts were placed into a 96-well plate, and 100 μL of assay solution (8 nM chitin, 10 μg/mL horseradish peroxidase, and 50 μΜ luminol) was added to each well using a multi-channel pipette. Chemiluminescence was measured at 30 s intervals over a period of 30 min in a SPARK−10M microplate reader (TECAN, Switzerland). Five biological replicates were used per sample. Distilled water was used as the mock control.

For DAB (3,3’-diaminobenzidine tetrahydrochloride hydrate, Sigma-Aldrich, USA) staining assays, the M. oryzae-infected rice leaves were stained with 50 mL DAB solution (50 mg DAB, 0.05% [v/v] Tween 20, 0.5 mM NaH2PO4) and placed under a vacuum for 5 min to remove air from the samples. The leaves were incubated in DAB solution for 8 h on a low-speed shaker and cleared in glycerol:lactic acid:ethanol (1:1:3, v/v/v) for 12 h with frequent changes with fresh solution. The leaves were observed under a light microscope.

The leaves of 2-week-old rice seedlings were treated with 8 nM chitin by vacuum infiltration for 30 min and incubated at 28 °C for 24 h. The leaves were fixed in ethanol:acetic acid (3:1, v/v) for 6 h with frequent changes with fresh solution. The leaves were washed three washes times and incubated in staining solution (0.01% [w/v] aniline blue, 150 mM K2HPO4) for 4 h on an end-over-end shaker. The leaves were then observed under a LSCM using a UV light source (340–380 nm).

RT-qPCR

Total RNA was extracted using TRIzol reagent (Vazyme Biotech, Nanjing, China). First-strand cDNA synthesis was carried out with a cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Quantitative reverse-transcription-PCR was performed with TransStart® Tip Green qPCR SuperMix (TransGen Biotech). Transcript levels were normalized using the rice ubiquitin gene (OsUBQ1) or U. virens b-tubulin gene (UvTub1). At least three biological replicates were tested per treatment.

Statistical analyses

The data are presented as means ± standard deviation (SD). Statistically significant differences between control and experimental groups were determined by one-way ANOVA with two-sided least significant difference or Tukey’s/Dunnett’s multiple-comparison test or t test. P < 0.05 was considered as statistically significant. Data analysis was conducted using GraphPad Prism Software (Version 8.0.1).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information

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Description of Additional Supplementary Files

Supplementary Data 1

Reporting Summary

Source data

Source data

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-024-52204-w.

Acknowledgements

We are grateful to Prof. Wenxian Sun (China Agricultural University) for providing the gene deletion vector pGKO. We are grateful to Prof. Meng Yuan (Huazhong Agricultural University) for providing the Xoo strain PXO99. This work was financially supported by the National Natural Science Foundation of China (32302302 and 32302382) and the Fundamental Research Funds for the Central Universities of China (2662023PY006).

Author contributions

X.Y.C. and L.Z. conceived the project. Y.H.D. and Z.Y.W. conducted the most experiments. Y.F. H.H. and Z.X.P. performed data analyses. H.L., X.L.C., Q.T.X., L.Z., C.X.L., and J.B.H. gave critical suggestions for the project. Manuscript was prepared by X.Y.C., and all authors read and approved the contents of this paper.

Peer review

Peer review information

Nature Communications thanks Yoji Kawano, Wen-Ming Wang, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

Accession numbers for the genes mentioned in this article are: UgsL (Uv8b_2091); UvTub1 (Uv8b_900); OsSRF3 (Os02g07960); OsBAK1 (Os08g07760); OsPAL1 (Os02g41630); OsPR1b (Os01g28450); OsPR10a (Os12g36880); OsRBOHA (Os01g53294); OsUBQ1 (Os03g13170). The data of untargeted-metabolomics that have been deposited into CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb) with accession number CNP0005733. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Yuhang Duan, Zhaoyun Wang.
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