
==== Front
Plant Commun
Plant Commun
Plant Communications
2590-3462
Elsevier

S2590-3462(24)00209-8
10.1016/j.xplc.2024.100939
100939
Research Article
Leafhopper salivary carboxylesterase suppresses JA-Ile synthesis to facilitate initial arbovirus transmission in rice phloem
Chi Yunhua 12
Zhang Hongxiang 12
Chen Siyu 1
Cheng Yu 1
Zhang Xiaofeng 1
Jia Dongsheng 1
Chen Qian 1
Chen Hongyan 1
Wei Taiyun weitaiyun@fafu.edu.cn
1∗
1 Vector-borne Virus Research Center, Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
∗ Corresponding author weitaiyun@fafu.edu.cn
2 These authors contributed equally to this article.

09 5 2024
09 9 2024
09 5 2024
5 9 10093927 11 2023
16 4 2024
1 5 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Plant jasmonoyl-L-isoleucine (JA-Ile) is a major defense signal against insect feeding, but whether or how insect salivary effectors suppress JA-Ile synthesis and thus facilitate viral transmission in the plant phloem remains elusive. Insect carboxylesterases (CarEs) are the third major family of detoxification enzymes. Here, we identify a new leafhopper CarE, CarE10, that is specifically expressed in salivary glands and is secreted into the rice phloem as a saliva component. Leafhopper CarE10 directly binds to rice jasmonate resistant 1 (JAR1) and promotes its degradation by the proteasome system. Moreover, the direct association of CarE10 with JAR1 clearly impairs JAR1 enzyme activity for conversion of JA to JA-Ile in an in vitro JA-Ile synthesis system. A devastating rice reovirus activates and promotes the co-secretion of virions and CarE10 via virus-induced vesicles into the saliva-storing salivary cavities of the leafhopper vector and ultimately into the rice phloem to establish initial infection. Furthermore, a virus-mediated increase in CarE10 secretion or overexpression of CarE10 in transgenic rice plants causes reduced levels of JAR1 and thus suppresses JA-Ile synthesis, promoting host attractiveness to insect vectors and facilitating initial viral transmission. Our findings provide insight into how the insect salivary protein CarE10 suppresses host JA-Ile synthesis to promote initial virus transmission in the rice phloem.

This study reports that the leafhopper salivary protein CarE10 directly binds to rice JAR1 and promotes its degradation by the proteasome system to suppress JA-Ile synthesis, while virus-mediated increase in CarE10 secretion promotes host attractiveness to insect vectors and facilitates initial viral transmission.

Key words

leafhopper
salivary protein CarE10
JAR1 degradation
JA-Ile synthesis
rice phloem
initial viral transmission
Published: May 9, 2024
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pmcIntroduction

Many devastating plant, animal, and human viral pathogens are horizontally transmitted by arthropod insects (Mayer et al., 2017; Eigenbrode et al., 2018). Arthropod-borne viruses (arboviruses) establish their initial infection in the insect midgut, then spread to the hemolymph and ultimately into the salivary glands, from which virions are introduced into susceptible hosts together with saliva (Hogenhout et al., 2008; Wei and Li 2016). Salivary gland cells of insects are filled with abundant apical plasmalemma-lined cavities where saliva is stored (Wei and Li 2016; Mao et al., 2017). Piercing-sucking insects, such as leafhoppers, planthoppers, aphids, and whiteflies, persistently transmit numerous viral pathogens mixed with their saliva into the plant phloem (Hogenhout et al., 2008; Jia et al., 2018; Chen et al., 2021a). Notably, rice arboviruses have developed diverse strategies to traverse the apical plasmalemma into insect salivary cavities and thereby move with saliva to establish the initial infection in hosts. For example, the reovirus rice dwarf virus and the bunyavirus rice stripe virus hijack small vesicles, referred to as exosomes, to traverse the apical plasmalemma into the saliva-storing cavities in leafhopper or planthopper vectors (Chen et al., 2021a; Lu et al., 2022). Furthermore, the reovirus rice gall dwarf virus (RGDV) induces the formation of virus-associated vesicles constructed from viral nonstructural protein Pns11 that attach to the apical plasmalemma to induce an exocytosis-like process for viral release into salivary cavities in leafhopper vectors (Mao et al., 2017). However, the molecular mechanisms by which arboviruses manipulate insect vectors and infected plants remain poorly understood.

Arboviruses have co-evolved with insect vectors to optimize their horizontal transmission to hosts (Jin et al., 2018). Insect salivary components modulate host immune responses and facilitate the horizontal transmission of arboviruses to hosts (Conway et al., 2014; Surasombatpattana et al., 2014; Sun et al., 2020). Plant phloem is formed with highly evolved sieve tubes comprising sieve elements, companion cells, and parenchyma cells and is the unique ecological niche for various devastating pests that have evolved mechanisms to access it (Jiang et al., 2019). Phloem-feeding insects secrete watery or gelling saliva containing specific enzymes and/or effectors to interfere with plant defense responses in the phloem (Jiang et al., 2019). Phloem plugging caused by callose deposition on sieve plates is a crucial defense mechanism against the ingestion of phloem sap by insects (Hao et al., 2008; Chen et al., 2013). The roles of insect saliva in promoting the horizontal transmission of nonpersistent and persistent transmitted viruses into the phloem have been extensively studied (Gray Stewart and Banerjee, 1999; Ali et al., 2006; Li et al., 2020). For example, aphid salivary carbonic anhydrase II facilitates infection of nonpersistent transmitted viruses such as cucumber mosaic virus and turnip mosaic virus (Guo et al., 2023). Furthermore, infection of the salivary glands of leafhopper vectors by the persistent transmitted virus RGDV inhibits the secretion of calcium-binding protein, a universal insect salivary protein, into the rice phloem, thus increasing cytosolic Ca2+ and callose deposition on the sieve plates (Wu et al., 2022). This process stimulates viruliferous leafhoppers to secrete more saliva into the rice plants, thereby enhancing horizontal virus transmission (Wu et al., 2022). Currently, how insect saliva components facilitate persistent insect-to-plant virus transmission remains largely unknown.

Many plant defense responses to phloem-feeding insects are regulated by the jasmonate (JA) signaling pathway (Lortzing and Steppuhn 2016; Wang et al., 2019; Yang et al., 2020). The precursor of JA, α-linolenic acid, is modified by various enzymes, such as 13-lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase, to form the intermediate compound 12-oxophytodienoic acid, which is further processed to produce JA (Schaller and Stintzi 2009; Kombrink 2012). The F-box protein COI1 subsequently recruits JA ZIM-domain proteins (JAZs), key repressors of JA signaling, for ubiquitination and degradation (Sheard et al., 2010). Jasmonate resistant 1 (JAR1), classified in the GH3 family of plant enzymes, encodes a JA-amino acid (aa) synthetase that catalyzes the synthesis of JA-isoleucine (JA-Ile) by conjugating Ile to JA (Fukumoto et al., 2013; Pratiwi et al., 2017). JA-Ile is a major active conjugated form of JA and plays a vital role in JA signaling (Tamogami et al., 2008; Fukumoto et al., 2013). Virus-encoded proteins directly interact with and modify key proteins in the JA signaling pathway to increase host attractiveness to insect vectors (Wu et al., 2017; Pan et al., 2021). However, how insect salivary proteins serve as effectors to suppress the host JA signaling pathway, thereby increasing host attractiveness to insect vectors and promoting horizontal arbovirus transmission in the phloem, remains unknown.

JA and JA-Ile are important defense signaling molecules that can induce the insect resistance of plants and activate the detoxification ability of insects (Li et al., 2002; War et al., 2012). For example, the corn earworm Helicoverpa zea uses JA to activate its cytochrome P450, which is associated with detoxification, ultimately protecting H. zea against toxins produced by its host plants (Li et al., 2002; Elzaki et al., 2019). Cytochrome P450, carboxylesterase (CarE), glutathione-S-transferase (GST), and acetylcholinesterase are the main insect detoxification enzymes (Ramsey et al., 2010; Hilliou et al., 2021). CarEs are the third major family of detoxification enzymes and are associated with detoxification of various xenobiotics, including insecticides (Satoh and Hosokawa 2006; Cai et al., 2009; Li et al., 2021). Insect detoxification enzymes have a high catalytic efficiency and potentially hydrolyze endogenous plant metabolites, enabling insects to adapt to host defense responses (Després et al., 2007; Heckel, 2014). However, whether insect detoxification enzymes can be secreted as salivary components to modulate host JA signaling and how arboviruses mediate the regulation of detoxification enzyme secretion to promote horizontal virus transmission into the plant phloem by insect vectors remain unknown.

RGDV is a destructive phloem-inhabiting viral pathogen transmitted by the phloem-feeding rice leafhopper Recilia dorsalis (Omura et al., 1980; Yi et al., 2021). RGDV was first described in Thailand in 1979 and has caused severe epidemics in southern China and Southeast Asia (Omura et al., 1980; Mao et al., 2019). Insect salivary gland cells are filled with abundant apical plasmalemma-lined cavities where saliva is stored (Wei and Li, 2016; Mao et al., 2017). RGDV infection induces the formation of virus-associated filaments constructed from viral nonstructural protein Pns11 within the salivary glands of R. dorsalis (Mao et al., 2017). Such filaments can attach to the apical plasmalemma to induce the formation of vesicles for viral secretion into salivary cavities in the leafhopper vectors (Mao et al., 2017). In this study, we reveal that one CarE ortholog (CarE10) is specifically expressed in the salivary glands of leafhoppers and serves as an important salivary protein. RGDV infection activates and promotes the secretion of CarE10 via sequestration into Pns11-induced vesicles in the salivary cavities and then into the rice phloem to directly bind to and impair the enzyme activity of OsJAR1, which is required for JA-Ile synthesis. The reduced JA-Ile levels in rice seedlings upon viruliferous leafhopper feeding promote host attractiveness to insect vectors. We thus uncover a previously undescribed phenomenon in which leafhopper CarE10, as a salivary effector, manipulates host attractiveness to insect vectors by suppressing JA-Ile synthesis, thereby facilitating horizontal virus transmission by insect vectors.

Results

Feeding activity of viruliferous leafhoppers reduces rice JA-Ile levels to promote host attractiveness to insect vectors

Initially, we investigated how Pns11-formed vesicles in the salivary cavities carried RGDV viral particles into the rice phloem during feeding of viruliferous leafhoppers on rice seedlings. Approximately 30 nonviruliferous or viruliferous fifth-instar leafhopper nymphs were allowed to feed on a single rice seedling in a glass tube (Figure 1A). The insect-fed rice leaf sheaths were then collected at 0, 12, 24, 36, and 72 h after insect infestation. First, the insect-fed rice leaf sheaths were processed for immunofluorescence microscopy. The vascular bundles of the rice seedlings were differentiated into phloem on the abaxial side and xylem on the adaxial side (Figure 1B). The major outer capsid protein P8 and the nonstructural protein Pns11 of RGDV appeared in the sieve tube cells of the rice phloem to establish initial infection after viruliferous leafhopper feeding for 24 h; by contrast, the viroplasm protein Pns9 of RGDV, which was used to monitor viral propagation (Zheng et al., 2015), initially appeared in the sieve tube cells of the phloem after leafhopper feeding for 72 h (Figure 1B). We found that almost all Pns11 and P8 fluorescent signals were colocalized on discrete puncta in infected phloem areas (Figure 1C). Western blot assays confirmed that Pns11 and P8 initially appeared in rice samples at 24 h, whereas Pns9 was initially detected after 72 h of viruliferous leafhopper feeding (Figure 1D). These findings suggest that RGDV establishes an initial infection in the rice phloem as early as 24 h and then induces the formation of viroplasms to support viral replication and assembly of progeny virions after 72 h of viruliferous vector feeding.Figure 1 Effects of initial transmission of RGDV into rice phloem by viruliferous leafhoppers on rice defense responses and insect feeding behavior.

(A) Insect feeding devices. A rice seedling at the two-leaf stage and the tested leafhoppers were placed in a glass tube.

(B) Immunofluorescence detection of RGDV P8, Pns11, and Pns9 in cross sections of rice seedlings fed upon by 30 nonviruliferous or viruliferous fifth-instar leafhopper nymphs for 24 or 72 h. Rice tissue sections were immunolabeled with Pns11-FITC (green), P8-rhodamine (red), or Pns9-rhodamine (red). The vascular bundles were differentiated into phloem on the abaxial side and xylem on the adaxial side. Arrows indicate Pns9 puncta or the colocalization of Pns11 and P8. Bars, 20 μm.

(C) The mean percentage of P8-positive puncta colocalized with Pns11 in rice phloem tissues. Data are presented as means (±SEM) of three independent biological replicates, each containing 30 phloem tissues (two-tailed t-test).

(D) Western blot assay showing the levels of Pns11, P8, and Pns9 in rice seedlings fed upon by 30 viruliferous leafhoppers for 0, 12, 24, and 72 h detected using Pns11-, P8-, and Pns9-specific antibodies. The relative band intensities quantified with ImageJ are shown below. Coomassie-blue-stained gels demonstrate the loading control of the proteins. Data represent three replicates of 30 leafhoppers each.

(E and F) A Y-tube dual-choice assay (E) was used to test the attractiveness of RGDV-infected and healthy rice plants to nonviruliferous or viruliferous fifth-instar leafhopper nymphs. The number of insects (F) was recorded at 3, 6, 12, and 24 h after insects were released. For each tested time point, data are presented as means (±SEM) of five independent biological replicates, each containing 90 leafhoppers (two-tailed t-test).

(G and H) Concentrations of JA (G) and JA-Ile (H) in rice seedlings exposed to 30 nonviruliferous and viruliferous fifth-instar leafhopper nymphs for 0.5 and 1 h, as detected by UPLC–MS/MS. Samples without insect infestation served as the control (mock). Means (±SEM) represent three independent biological replicates (one-way ANOVA with Tukey’s multiple-comparisons test). Different letters denote statistically significant differences (p < 0.01). ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns, not significant. V−, nonviruliferous; V+, viruliferous; Xy, xylem; Ph, phloem; Mes, mesophyll.

We carefully compared the feeding tendencies of nonviruliferous and viruliferous fifth-instar leafhopper nymphs on healthy or RGDV-infected rice plants using a Y-tube olfactometer bioassay, recording the numbers of insects at 3, 6, 12, and 24 h after insect release (Figure 1E). The nonviruliferous nymphs showed a significant tendency to feed on virus-infected rice plants rather than healthy controls from 6 to 24 h after insects were released (Figure 1F). Interestingly, viruliferous nymphs exhibited a tendency to feed on healthy controls rather than virus-infected rice plants at 6 and 12 h, but this tendency was not obvious at 24 h after insect release (Figure 1F). Overall, nonviruliferous nymphs tended to feed on virus-infected rice plants, whereas viruliferous nymphs tended to feed on healthy rice plants. Thus, healthy plants are more attractive to viruliferous insects than to nonviruliferous insects. These results indicate that initial feeding by viruliferous leafhoppers improves the attractiveness of rice hosts to insect vectors, thereby promoting RGDV transmission.

In general, the defense responses of plants to insect pests are regulated by the phytohormones JA and JA-Ile (Tamogami et al., 2008; Fukumoto et al., 2013). JA and JA-Ile concentrations were significantly higher in rice seedlings after 0.5–1 h of nonviruliferous leafhopper nymph feeding than in control seedlings without insect feeding (Figure 1G and 1H). However, JA and JA-Ile concentrations were significantly lower in rice seedlings after 0.5–1 h of viruliferous nymph feeding relative to nonviruliferous controls (Figure 1G and 1H). These data demonstrate that suppression of JA and JA-Ile levels upon viruliferous leafhopper feeding reduces the defense responses of rice plants to insect pests, thereby facilitating the attractiveness of the hosts to insect vectors.

Secretion of the salivary protein CarE10 by leafhoppers into rice phloem suppresses JA-Ile production

Eight CarEs were identified from R. dorsalistranscriptome data, and tissue-specific RT–qPCR analysis showed that CarE6, CarE9, and CarE10 were expressed in the salivary glands. However, only CarE10 was highly expressed in the salivary glands but not in other organs (Figure 2A; Supplemental Figure 1). The CarE10 cDNA contains a 1506-bp open reading frame (ORF) encoding a 502-aa polypeptide with a predicted molecular mass of 55 kDa that contains carboxylesterase conserved domains and a putative secretory signal peptide at the N-terminal 250-aa region (Supplemental Figure 2; Supplemental Table 1). Sequence alignment showed that the N-terminal 250-aa region of CarE10 shared 39.7%–47.49% aa homology with corresponding regions of the other seven CarEs, whereas the C-terminal region of CarE10 only shared 13.52%–33.13% aa homology with corresponding regions of the other seven CarEs (Supplemental Figure 3A; Supplemental Table 2). We therefore used the polypeptide AKGGIKDAPWMTYVT at aa positions 320–334 of CarE10, which shared a low aa sequence homology with the other CarEs, to generate a specific antibody against CarE10 (Supplemental Figure 3A). Western blot assays showed that the CarE10 antibody specifically reacted with the ∼55-kDa protein (Supplemental Figure 3B). Furthermore, CarE10 was specifically detected in the salivary glands but not in the midgut, ovaries, testes, or residual bodies (Figure 2B), further confirming that the CarE10 antibody did not react with other CarEs present in these tissues.Figure 2 Leafhopper salivary CarE10 interacts with OsJAR1 and inhibits JA-Ile synthesis in rice.

(A) Relative mRNA expression levels of CarE10 in different insect organs were measured by RT–qPCR, with the transcript level of EF1α as the internal reference for normalization. Data are presented as means (±SEM) of five independent biological replicates, each containing 30 organs (one-way ANOVA with Tukey’s multiple-comparisons test). Different letters indicate a statistically significant difference (p < 0.01).

(B) Protein levels of CarE10 in different insect organs were measured by western blot assay using CarE10 antibody. Insect histone H3 was used as the loading control. The relative band intensities quantified with ImageJ are shown below. Data represent three replicates, each containing 30 organs.

(C) Immunofluorescence microscopy image showing that CarE10 was specifically expressed in the type III secretory cells of principal salivary glands, as determined by immunolabeling with CarE10-FITC (green). Immunofluorescence microscopy image of the salivary glands immunolabelled with pre-immune antibody conjugated to FITC served as the control.

(D) The mean percentage of type III and IV secretory cells with CarE10-specific puncta. Data are presented as means (±SEM) of three independent biological replicates, each containing 30 secretory cells (two-tailed t-test).

(E) Salivary cavities in type III cells, as observed by electron microscopy.

(F and G) Salivary glands were immunolabeled with CarE10 antibody (F) or pre-immune antibody (G) as a primary antibody, followed by treatment with goat anti-rabbit IgG conjugated with 15-nm-diameter gold particles as a secondary antibody. Red arrows indicate gold particles.

(H) The mean number of gold particles labeled by CarE10 antibody or pre-immune antibody in salivary cavities. Data are presented as means (±SEM) of three independent biological replicates, each containing 30 random 0.25-μm2 fields of salivary cavities (two-tailed t-test).

(I) Immunofluorescence detection of CarE10 (arrow) in cross sections of rice seedlings exposed to 30 nonviruliferous leafhoppers for 24 h. Non-infested rice samples served as the mock control. Rice tissue sections were immunolabeled with CarE10-FITC (green).

(J) Accumulation of CarE10 in rice seedlings after feeding by 30 leafhoppers for 24 h as determined by a western blot assay with CarE10 antibody. Non-infested rice leaves served as a control (mock). Coomassie-blue-stained gel for Rubisco was used as the loading control. Data represent three biological replicates.

(K) An interaction between OsJAR1 and CarE10 was detected by yeast two-hybrid assay. DDO, SD-Trp-Leu medium; QDO, SD-Trp-Leu-His-Ade medium with 2.5 mM 3-AT.

(L) GST pull-down assays were used to test the interaction of CarE10 with OsJAR1. CarE10 was fused with GST as the bait, and OsJAR1 was fused with His as the prey. The baits or the GST control were incubated with cell lysate that expressed His-fused protein. Input and affinity-isolation samples were detected by western blot assay using antibodies against GST or His.

(M and N) The mRNA expression and enzyme activity levels of CarE10 in 30 dsGFP- and dsCarE10-treated leafhoppers as determined by (M) an RT–qPCR assay and (N) a carboxylesterase activity assay, respectively. Data are presented as means (±SEM) of three independent biological replicates, each containing 30 leafhoppers (two-tailed t-test).

(O–Q) mRNA expression levels of OsJAR1, JAR concentrations, and JA-Ile concentrations in rice seedlings after feeding by 30 dsGFP- or dsCarE10-treated leafhoppers for 0.5 and 1 h as measured by RT–qPCR assay (O), the Plant JAR ELISA Kit (P), and UPLC–MS/MS (Q), respectively. Data are presented as means (±SEM) of three independent biological replicates (two-tailed t-test). ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns, not significant. V−, nonviruliferous. Wb, whole bodies; Sg, salivary glands; Mg, midguts; Te, testes; Ov, ovaries; Res, residues except salivary glands; APL, apical plasmalemma; Cv, cavity; Sc, salivary cytoplasm; Xy, xylem; Ph, phloem. Bars, 50 μm (C), 10 μm (E), 500 nm (F and G), and 20 μm (I).

Immunofluorescence microscopy showed that CarE10 was exclusively present in the type III secretory cells of the salivary glands but not in the type IV secretory cells of the salivary glands, midgut, ovaries, or testes (Figure 2C; Supplemental Figure 4). We found that almost all type III secretory cells tested contained CarE10 (Figure 2D); type III cells have been reported to be the most abundant of the six secretory cell types and to produce a high volume of watery saliva (Chen et al., 2021b). Immunoelectron microscopy revealed that CarE10 was distributed in the whole secretory cells more abundantly in the salivary cavities where saliva is stored (Figure 2E, 2F, and 2H). By contrast, immunofluorescence and immunoelectron microscopy showed that the pre-immune antibody did not react with the components within the salivary cavities (Figure 2C–2G and 2H). Thus, we identified a new leafhopper salivary protein carboxylesterase.

We next asked whether CarE10 could be secreted from the salivary glands into the plant phloem during leafhopper feeding on rice plants. Approximately 30 nonviruliferous leafhoppers were allowed to feed on a single rice seedling in a glass tube. The rice leaf sheaths inoculated with insects were processed for immunofluorescence microscopy, and we observed that CarE10 appeared in the sieve tube cells of the rice phloem after 24 h of leafhopper feeding. By contrast, the specific fluorescent signals of CarE10 did not appear in rice phloem in the absence of insect feeding (Figure 2I). Western blot assays confirmed that the CarE10 antibody specifically detected CarE10 in rice plants after 24 h of leafhopper feeding but did not detect rice proteins in the absence of leafhopper feeding (Figure 2J). CarE10 and rice carboxylesterases (CXEs) have a low aa sequence homology (5.99% to 13.15%), with CareE10 and CXE1 showing the highest homology of 13.15% (Supplemental Figure 5; Supplemental Table 3), thus excluding the possibility that CarE10 antibody could react with rice CXEs in the absence of leafhopper feeding. Thus, the salivary protein CarE10 can be secreted into the rice phloem.

We next determined whether leafhopper salivary protein CarE10 served as an effector to suppress the JA signaling pathway in rice. First, we performed yeast two-hybrid assays to identify key factors in the rice JA signaling pathway that could interact directly with CarE10. These assays revealed that CarE10 specifically interacted with rice JAR1 (OsJAR1) but not with other rice proteins required for the early steps of the JA signaling pathway (Figure 2K; Supplemental Figure 6). GST pull-down assays further confirmed that CarE10 interacted directly with OsJAR1 (Figure 2L). Yeast two-hybrid assays also showed that rice CXE1 did not interact with OsJAR1 (Supplemental Figure 7). Thus, rice CXEs are not involved in the regulation of OsJAR1.

Because the C-terminal 250-aa region of CarE10 shared low aa homology with the corresponding regions of the other seven CarEs, we investigated the functional role of CarE10 during insect feeding on host plants by microinjecting third-instar nymphs with in vitro-synthesized double-stranded RNA (dsRNA) targeting this region of CarE10 (dsCarE10); in vitro-synthesized dsRNA targeting GFP (dsGFP) was injected in the same manner as a control. Four days after microinjection of dsCarE10, the mRNA expression levels of CarE10, but not the other seven CarEs, were significantly reduced (Figure 2M; Supplemental Figure 8), causing a reduction of at least 65% in the carboxylesterase activity levels of insect salivary glands (Figure 2N). Plant JA-Ile synthesis is regulated by JAR1 enzyme activity (Schaller and Stintzi 2009; Kombrink 2012; Fukumoto et al., 2013; Pratiwi et al., 2017). Feeding of dsCarE10-treated leafhoppers on rice plants did not affect the transcript level of OsJAR1, but it significantly increased the JAR concentration, thus increasing JA-Ile synthesis compared with that of rice samples inoculated with dsGFP-treated controls (Figure 2O–2Q). We therefore speculated that the direct interaction of CarE10 with OsJAR1 affects OsJAR1 protein stability, presumably impairing OsJAR1 enzyme activity for the synthesis of JA-Ile.

Direct interaction with the salivary protein CarE10 impairs the enzyme activity of OsJAR1

To confirm the effect of CarE10 on the enzyme activity of OsJAR1, mixtures of purified OsJAR1 proteins (0.25 mg/ml) with different concentrations of purified CarE10 proteins (0.1, 0.25, and 0.5 mg/ml) were incubated with an in vitro JA-Ile synthesis reaction solution for 10 min at 25°C. There was a gradual reduction in OsJAR1 enzyme activity as the CarE10 concentration increased (Figure 3A), but the concentration of OsJAR1 did not change significantly (Figure 3B). Thus, the CarE10–OsJAR1 interaction may prevent the binding of OsJAR1 to its substrate, ultimately impairing OsJAR1 enzyme activity. JAR1s of wheat (Triticum aestivum L.) contain three conserved motifs, namely SSGTSQGRPK, YGSSE, and YRLGD, that are involved in ATP/AMP binding and are required for enzymatic catalysis of JA-Ile synthesis (Pratiwi et al., 2017; Tuan et al., 2022). JAR1 of rice (O. sativa) contains similar conserved catalytic sites, namely SSGTTHGKPK, YGASE, and YRLGD (Supplemental Figure 9A). Yeast two-hybrid assays showed that CarE10 specifically interacted with the OsJAR1 domain that contained the conserved catalytic sites but not with other OsJAR1 domains (Supplemental Figure 9B). These results suggested that direct binding of CarE10 to the conserved catalytic sites of OsJAR1 negatively regulates OsJAR1 enzyme activity, thereby impairing the synthesis of JA-Ile.Figure 3 Leafhopper salivary CarE10 impairs the enzyme activity of OsJAR1.

(A) OsJAR1 activity was detected in an in vitro JA-Ile synthesis solution system in the presence of different concentrations of purified His-CarE10 protein (0.1, 0.25, and 0.5 mg/ml) and 0.25 mg/ml purified His-OsJAR1 protein. Reaction solutions with different concentrations of purified His-GFP protein (0.1, 0.25, and 0.5 mg/ml) served as controls.

(B) Concentrations of OsJAR1 were measured by the JAR ELISA Kit after the in vitro JA-Ile synthesis reaction.

(C and D) Protein levels of OsJAR1-Myc and CarE10-His (C) and JAR concentrations (D) after co-infiltration of OsJAR1-Myc and CarE10-His into N. benthamiana leaves.

(E) In vivo assay showing the effects of MG132 treatment or the CarE10–OsJAR1 interaction on OsJAR1 protein stability. MG132 (100 μM) or an equal volume of DMSO (negative control) was infiltrated into leaves transiently co-expressing OsJAR1-Myc and CarE10-His for 12 h before harvesting. Ratios of Agrobacteriumculture mixtures containing the OsJAR1-Myc plasmid and the CarE10-His plasmid in (C)–(E) are indicated.

(F) CarE10 increased the ubiquitination level of OsJAR1. A Co-immunoprecipitation assay for OsJAR1 ubiquitination in N. benthamiana leaves co-expressing CarE10-His, OsJAR1-Myc, and Ub-HA was performed via western blotting using the indicated antibodies. Data in (A), (B), and (D) are presented as means (±SEM) of three independent biological replicates (two-tailed t-test). ∗∗p < 0.01; ∗p < 0.05; ns, not significant.

We next used the in vitro system of protein transient expression in Nicotiana benthamiana leaves to determine how CarE10 suppresses OsJAR1 content in planta. Various concentrations of Agrobacterium cells containing CarE10-His and OsJAR1-Myc were mixed to achieve the desired ratios of CarE10 to OsJAR1. Co-infiltration of N. benthamiana leaves with different ratios ofCarE10-His and OsJAR1-Myc revealed that increased protein accumulation of CarE10 was correlated with reduced protein accumulation of OsJAR1, thereby decreasing JAR concentrations (Figure 3C and 3D). These results suggested post-translational degradation of OsJAR1. The 26S proteasome system is one of the major pathways for protein degradation (Ravid and Hochstrasser, 2008). To examine the potential involvement of the proteasome in CarE10-induced OsJAR1 reduction, we infiltrated 100 μM MG132, a proteasome inhibitor, into N. benthamiana leaves transiently co-expressing OsJAR1-Myc and CarE10-His for 12 h. OsJAR1 protein levels were recovered in MG132-treated leaves compared with DMSO-treated controls (Figure 3E), confirming the involvement of the proteasome pathway in CarE10-induced OsJAR1 degradation.

The ubiquitin (Ub)-26S proteasome system is one of the major protein degradation pathways (Ravid and Hochstrasser, 2008). To visualize Ub modification of OsJAR1, we transiently expressed CarE10-His, OsJAR1-Myc, and Ub-HA in N. benthamiana leaves and then immunoprecipitated the substrate protein OsJAR1-Myc with anti-Myc beads from the extracted cellular proteins. After immunoprecipitation of OsJAR1-Myc, detection with an HA antibody revealed that the presence of CarE10-His enhanced the level of Ub chains in the immunoprecipitated OsJAR1-Myc (Figure 3F). This result suggested that OsJAR1 undergoes ubiquitin-mediated degradation and that CarE10 promotes OsJAR1 degradation by the 26S proteasome pathway.

Viral infection activates and facilitates secretion of the leafhopper salivary protein CarE10 into the rice phloem

We next examined the association between CarE10 expression and RGDV infection in vector salivary glands. RT–qPCR and western blot assays showed that RGDV infection significantly increased CarE10 expression in the salivary glands at the mRNA and protein levels (Figure 4A and 4B). Accordingly, carboxylesterase activity was also significantly increased in the salivary glands of viruliferous leafhoppers (Figure 4C). We then investigated whether virus-activated CarE10 facilitated viral infection in vector salivary glands. Third-instar nymphs of viruliferous leafhoppers were microinjected with dsRNA that targeted CarE10 or GFP (dsCarE10 or dsGFP). As expected, knockdown of CarE10 expression effectively reduced carboxylesterase activity and viral protein accumulation in the salivary glands of viruliferous leafhoppers (Figure 4D–4F). These results suggest that increased carboxylesterase activity promotes viral infection of vector salivary glands.Figure 4 Viral infection activates and promotes CarE10 secretion into vector salivary cavities.

(A–C) mRNA and protein levels of CarE10, and carboxylesterase activity levels in the salivary glands of 30 nonviruliferous and viruliferous leafhoppers as determined by RT–qPCR (A), western blotting (B), and carboxylesterase activity assays (C).

(D and E) mRNA expression levels of CarE10 and carboxylesterase activity levels in the salivary glands of 30 dsGFP- and dsCarE10-treated viruliferous leafhoppers as determined by RT–qPCR (D) and carboxylesterase activity (E) assays.

(F) Western blot assay showing the levels of Pns11, P8, and CarE10 in the salivary glands of 30 dsGFP- and dsCarE10-treated viruliferous leafhoppers.

(G) Immunofluorescence microscopy showing the association of CarE10 with Pns11 fluorescent signals (arrow) in virus-infected secretory cells. Virus-infected or virus-free salivary glands and dsGFP- or dsCarE10-treated virus-infected salivary glands were immunostained with CarE10-rhodamine (red) and Pns11-FITC (green).

(H) Mean percentages of Pns11 colocalized with CarE10 in virus-infected secretory cells.

(I–K) Immunoelectron microscopy showing the association of CarE10 with Pns11-induced vesicles in virus-infected salivary cavities. RGDV-infected (I) and RGDV-free (J) salivary glands and RGDV-infected midgut (K) were immunolabeled with CarE10 antibody as the primary antibody, followed by 15-nm gold-particle-conjugated IgG as the secondary antibody. Red arrows indicate gold particles. The boxed area in (I-I) has been enlarged to show the association of CarE10 with Pns11-formed vesicles (I-II).

(L) Mean number of gold particles labeled by CarE10 antibody in Pns11-formed vesicles in salivary cavities or midgut epithelial cells. Data in (A) and (C)–(E) are presented as means (±SEM) of three or five independent biological replicates, each containing 30 insects (two-tailed t-test). Data in (H) and (L) are presented as means (±SEM) of three replicates, each containing 30 secretory cells and virus-infected vesicles, respectively (two-tailed t-test). The proteins in (B) and (F) were detected by western blotting with CarE10, Pns11, P8, or histone H3 antibodies. The relative band intensities quantified with ImageJ are shown below. The data for western blotting represent three biological replicates. ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01. V−, nonviruliferous. V+, viruliferous. Vi, virions; Ve, vesicle; Sc, secretory cytoplasm; Sg, salivary gland; APL, apical plasmalemma; Me, midgut epithelium; Mg, midgut; Cv, cavity. Bars, 20 μm (G), 500 nm (I–I), 100 nm (I-II, J, and K).

Immunofluorescence microscopy revealed abundant accumulation of CarE10 within virus-infected areas of secretory cells in the salivary glands; by contrast, CarE10 was sparsely distributed throughout the cytoplasm in virus-free regions of the secretory cells (Figure 4G). In virus-infected regions, CarE10 was closely associated with Pns11-labeled puncta (Figure 4G), and more than 90% of Pns11 fluorescent signals colocalized with CarE10 in virus-infected regions (Figure 4H). Furthermore, knockdown of CarE10 expression significantly reduced such colocalized foci in virus-infected secretory cells of salivary glands (Figure 4G). Immunoelectron microscopy showed that the CarE10 antibody specifically recognized Pns11-induced vesicles along the apical plasmalemma or within virus-infected salivary cavities (Figure 4I). In addition, the CarE10 antibody specifically reacted with the saliva component in virus-infected or virus-free salivary cavities (Figure 4I and 4J). However, CarE10 antibody did not react with Pns11-induced vesicles in RGDV-infected midgut epithelial cells of viruliferous leafhoppers (Figure 4K and 4L), further suggesting that the increased CarE10 contents in RGDV-infected salivary glands were accompanied by Pns11-induced vesicles into the salivary cavities during viral infection.

The association of CarE10 with Pns11 in vector salivary cells implies a possible interaction between the two proteins. Yeast two-hybrid and GST pull-down assays demonstrated that CarE10 directly interacted with Pns11 (Figure 5A and 5B). We then used an in vitro baculovirus expression system to investigate the relationship between CarE10 and Pns11 in Sf9 cells. When expressed individually, Pns11 formed filamentous structures and CarE10 was diffused throughout the cytoplasm of Sf9 cells (Figure 5C). However, co-expression led to recruitment of CarE10 into the filamentous structures of Pns11 (Figure 5C). Thus, bridging of CarE10 to the filamentous structures of Pns11 is mediated by the CarE10–Pns11 interaction.Figure 5 Direct interaction of CarE10 with Pns11 of RGDV promotes co-secretion of Pns11, virions, and CarE10 into the rice phloem.

(A) An interaction between Pns11 and CarE10 was detected by yeast two-hybrid assay. DDO, SD-Trp-Leu medium; QDO, SD-Trp-Leu-His-Ade medium with 2.5 mM 3-AT.

(B) GST affinity-isolation assay showing CarE10–Pns11 interaction. CarE10 fused with GST was used as the bait, and Pns11 fused with His was used as the prey. The baits or GST control were incubated with cell lysate that expressed His-fused protein. Input and affinity-isolation samples were detected using antibodies against GST or His in the western blot assay.

(C) At 48 hpi, Sf9 cells expressing CarE10-His or Pns11-Myc alone or co-expressing CarE10-His and Pns11-Myc were fixed and immunolabeled with His-FITC (green) or Myc-rhodamine (red). Arrows indicate the colocalization of CarE10 with Pns11. Bars, 5 μm.

(D–F) Immunofluorescence microscopy showing the colocalization of CarE10 with P8 or Pns11 (arrows) in cross sections of rice seedlings after viruliferous leafhopper feeding for 24 h (F). Rice seedlings inoculated with nonviruliferous leafhoppers for 24 h (E) or without insect infestation (D) served as the controls. Rice tissue sections were immunolabeled with CarE10-FITC (green), P8-rhodamine (red), or Pns11-rhodamine (red). Xy, xylem; Ph, phloem. Bars, 20 μm.

(G) Mean percentage of CarE10-positive puncta colocalized with P8 or Pns11 in infected phloem tissues. Data are presented as means (±SEM) of three independent biological replicates, each containing 30 phloem tissues (one-way ANOVA with Tukey’s multiple-comparisons test). Different letters indicate a statistically significant difference (p < 0.01).

(H) Western blot assay showing the levels of Pns11, P8, and CarE10 in rice seedlings after feeding by 30 nonviruliferous or viruliferous leafhoppers for 24 h. Rice seedlings without insect infestation served as the control (mock).

(I) Western blot assay showing the levels of Pns11, P8, and CarE10 in saliva secreted by 200 dsGFP- and dsCarE10-treated viruliferous leafhoppers and collected by membrane feeding.

(J) Viral transmission rates of 30 dsGFP- and dsCarE10-treated viruliferous leafhoppers. Six days after microinjection of dsRNA, 30 dsGFP- and dsCarE10-treated viruliferous insects were placed on healthy rice seedlings to test viral transmission rates, calculated as the percentage of RT–PCR-positive plants out of the total number of plants. Data are presented as means (±SEM) of three independent biological replicates, each containing 30 insects (two-tailed t-test).

(K and L) JAR concentrations (K) and OsJAR1 accumulation levels (L) in rice seedlings inoculated with 30 nonviruliferous and viruliferous leafhoppers as detected with the Plant JAR ELISA Kit and western blot assays, respectively. Rice seedlings without insect infestation served as the control (mock).

(M and N) The concentrations of JAR (M) and JA-Ile (N) in rice seedlings after feeding by 30 dsGFP- or dsCarE10-treated viruliferous leafhoppers. Data in (K), (M), and (N) are presented as means (±SEM) of three independent biological replicates. For (K), statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparisons test. Different letters indicate a statistically significant difference (p < 0.01). For (M) and (N), statistical analysis was performed using a two-tailed t-test. ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns, not significant. Coomassie-blue-stained gel for Rubisco (H and L) or insect GAPDH (I) was used as the loading control. V−, nonviruliferous; V+, viruliferous.

We next investigated whether Pns11-induced vesicles carried CarE10 into the rice phloem during vector feeding on host plants. Approximately 30 nonviruliferous or viruliferous fifth-instar leafhopper nymphs were allowed to feed on a single rice seedling. The fed areas were collected after 24 h of insect infestation and processed for immunofluorescence microscopy. CarE10 appeared in limited areas of the rice phloem after nonviruliferous leafhopper feeding (Figure 5D and 5E). However, almost all CarE10 fluorescence signals were observed to colocalize with Pns11 and P8 in extensive areas of the rice phloem after viruliferous leafhopper feeding (Figure 5F and 5G). As expected, western blot assays confirmed the presence of greater CarE10 contents in rice seedlings inoculated with viruliferous leafhoppers than in nonviruliferous controls at 24 h (Figure 5H). Furthermore, the contents of CarE10, Pns11, and P8 in the saliva secreted from dsCarE10-treated viruliferous leafhoppers were also lower than the corresponding contents in saliva of dsGFP-treated controls (Figure 5I). This phenomenon eventually led to a decrease in viral transmission rates by dsCarE10-treated viruliferous leafhoppers (Figure 5J). All these results suggest that viral infection activates CarE10 expression and carboxylesterase activity and that more CarE10 is accompanied by RGDV Pns11-induced vesicles to traverse the apical plasmalemma into the salivary cavities and finally into the rice phloem to facilitate establishment of initial viral infection during vector feeding.

OsJAR1 protein accumulation and JAR concentration in rice plants were significantly reduced upon viruliferous leafhopper feeding for 1.5 h (Figure 5K and 5L). However, feeding of dsCarE10-treated viruliferous leafhoppers on rice plants significantly increased JAR concentrations, thereby increasing JA-Ile synthesis compared with that in rice samples inoculated with dsGFP-treated controls (Figure 5M and 5N). Thus, viruliferous leafhoppers potentially secrete more CarE10 into the rice phloem to directly interact with OsJAR1 and impair its activity, inhibiting JA-Ile synthesis.

CarE10 overexpression in rice plants promotes viral transmission by suppressing JAR1-mediated JA-Ile synthesis

To further characterize the function of CarE10 in impairing OsJAR1 activity in synthesis of JA-Ile, we generated transgenic rice plants that constitutively overexpressed CarE10 under the control of the 35S promoter (OECarE10), which were screened by RT–qPCR and western blot assays (Figure 6A–6D). Transgenic lines #1 and #2, which accumulated high levels of CarE10, were chosen for further analyses. OECarE10 transgenic plants exhibited a dwarf phenotype compared with the wild-type (WT) rice controls (Figure 6B). To compare viral transmission efficiency, WT and OECarE10 transgenic plants were fed upon by viruliferous leafhoppers for 2 days and then collected for testing of viral titers. Western blot assays revealed a greater accumulation of viral proteins in OECarE10 transgenic lines, and this led to an increase in viral transmission rates by viruliferous leafhoppers compared with those observed for WT plants (Figure 6E and 6F). CarE10 overexpression in rice plants thus enhanced viral transmission by insect vectors.Figure 6 Overexpression of CarE10 in transgenic rice plants suppresses OsJAR1-mediated JA-Ile synthesis and thus promotes host attractiveness to insect vectors.

(A and B) Growth status (A) and mean plant height (B) of two CarE10-His-overexpressing transgenic lines (OECarE10-1 and OECarE10-2) compared with those of WT rice.

(C) Transcript levels of CarE10 in two transgenic rice lines (OECarE10-1 and OECarE10-2) as determined by RT–qPCR. The transcript level of rice actin served as the internal reference for normalization. WT rice was used as the control.

(D) Protein levels of CarE10 in different transgenic rice lines as determined by western blot assay.

(E) Protein levels of RGDV P8 and Pns11 in OECarE10-1 and OECarE10-2 transgenic and WT rice plants after feeding by 30 viruliferous leafhoppers for 48 h, as determined by western blot assay.

(F) OECarE10 lines promoted RGDV transmission by leafhoppers, as determined by transmission rates. Means (±SEM) from 40 viruliferous leafhoppers individually feeding on one WT, OECarE10-1, or OECarE10-2 rice seedling are shown.

(G) OsJAR1 levels in OECarE10-1 and OECarE10-2 transgenic and WT rice plants with or without feeding by 30 nonviruliferous insects, as determined by western blot assays.

(H) JAR concentrations in OECarE10-1 and OECarE10-2 transgenic and WT rice plants after feeding by 30 nonviruliferous insects for 0.5 or 1 h as determined with the Plant JAR ELISA Kit.

(I and J) Concentrations of JA (I) and JA-Ile (J) in OECarE10-1 and OECarE10-2 transgenic and WT rice plants after feeding by 30 nonviruliferous leafhoppers for 1 h as determined by UPLC–MS/MS.

(K) A Y-tube device was used to examine the feeding preference of 90 nonviruliferous fifth-instar leafhopper nymphs between WT and OECarE10-1 or OECarE10-2 transgenic rice plants. The numbers of insects were recorded at 3, 6, 12, and 24 h after insect release. Means (±SEM) in (B), (C), (F), and (H)–(J) are based on three biological replicates. Significant differences were determined using two-tailed t-tests. Coomassie-blue-stained gels for Rubisco in (D), (E), and (G) demonstrate the loading amounts of the proteins. The relative intensities of the protein bands in (E) and (F) were quantified with ImageJ. The data represent three biological replicates. ∗∗∗∗p < 0.0001; ∗∗p < 0.01; ∗p < 0.05; ns, not significant.

There was a significant reduction in OsJAR1 protein accumulation and JAR concentration in OECarE10 transgenic rice lines without leafhopper feeding or upon feeding of nonviruliferous leafhoppers for 0.5–1 h (Figure 6G and 6H). However, CarE10 overexpression did not significantly affect JA concentration and decreased JA-Ile concentration upon feeding of viruliferous leafhoppers for 1 h (Figure 6I and 6J). Thus, CareE10 overexpression decreased OsJAR1 enzyme activity and led to reduced synthesis of JA-Ile, but not JA, in rice plants. Together, these results suggest that the greater CarE10 contents secreted by viruliferous leafhoppers into the rice phloem could directly associate with OsJAR1 and impair its enzyme activity for conjugation of JA to Ile.

We next performed feeding preference assays using OECarE10 transgenic rice lines. A Y-tube olfactometer bioassay showed that nonviruliferous leafhoppers preferred the OECarE10 transgenic rice lines to the WT controls from 3 to 24 h (Figure 6K). These results collectively demonstrate that CarE10 overexpression in rice plants enhances their attractiveness to insects by inhibiting JA-Ile synthesis, thereby enhancing viral transmission.

Discussion

Manipulation of plant host defense pathways during the horizontal transmission of arboviruses from the salivary glands to the plant phloem generally enhances viral transmission (Dáder et al., 2017; Wu et al., 2022). JA and JA-Ile are plant-produced signals that activate defense responses to insect vectors (Wasternack and Kombrink, 2010; Ballaré, 2011). Several plant virus–encoded proteins, such as cucumber mosaic virus 2b, tomato yellow leaf curl China virus βC1, barley yellow striate mosaic virus P6, and southern rice black-streaked dwarf virus P8, have been shown to directly bind and repress crucial proteins in the JA signaling pathway to attract insect vectors (Wu et al., 2017; Farooq et al., 2019; Zhang et al., 2019; Tungadi et al., 2020; Gao et al., 2022). The JA signaling pathway has received attention in connection with the salivary proteins secreted by phloem-feeding insects. For example, the whitefly salivary protein Bt56 directly elicits the salicylic acid-signaling pathway by interacting with the tobacco class II KNOTTED 1-like homeobox transcription factor, leading to downregulation of the JA defense pathway and reduced resistance to whitefly (Xu et al., 2019). Piercing-sucking insects can horizontally transmit numerous viral pathogens together with their saliva into the plant phloem (Chen et al., 2021a). Insect salivary proteins can serve as effectors to antagonize host immune responses; by contrast, it has not been clear whether insect salivary proteins directly bind to and repress crucial proteins in the host JA signaling pathway and facilitate the horizontal transmission of arboviruses into the plant phloem.

In this study, we reveal that a leafhopper salivary protein, the detoxification enzyme CarE10, directly binds to OsJAR1 and impairs its enzyme activity, thereby suppressing JA-Ile synthesis by blocking the conversion of JA to JA-Ile (Figure 7). JA-Ile is a major active form of jasmonate during defense against insect feeding (Tamogami et al., 2008; Fukumoto et al., 2013). Feeding of nonviruliferous leafhoppers on rice plants rapidly boosts JA and JA-Ile synthesis; however, viruliferous leafhoppers secrete more CarE10 than nonviruliferous controls into the rice phloem to suppress JA-Ile synthesis, thereby decreasing resistance to insect vectors (Figure 5H and 5N). The reduced JA-Ile accumulation upon viruliferous leafhopper feeding thus promotes host attractiveness to insect vectors, ultimately promoting horizontal virus transmission (Figure 7). How viruliferous leafhopper feeding leads to reduced synthesis of JA in rice plants is an interesting topic for future investigation. Our findings reveal a previously undescribed phenomenon in which a detoxification enzyme acts as an insect salivary effector to suppress host JA-Ile synthesis by impairing JAR1 enzyme activity. It is possible that suppression of plant JA-Ile synthesis by insect salivary CarEs is a conserved strategy exploited by additional arboviruses to facilitate initial virus transmission in the plant phloem.Figure 7 Proposed model for the suppression of JA-Ile synthesis mediated by leafhopper CarE10 to facilitate viral transmission by insect vectors.

RGDV infection activates and promotes the secretion of a newly identified leafhopper salivary protein, CarE10, by interacting with viral nonstructural protein Pns11, resulting in sequestration of CarE10 into Pns11-induced vesicles and finally into salivary cavities. CarE10 directly binds OsJAR1 and impairs its enzyme activity, which is required for the synthesis of JA-Ile in rice, thereby promoting host attractiveness to insect vectors to enhance horizontal virus transmission. APL, apical plasmalemma; Sc, secretory cytoplasm; Cv, cavity; SD, salivary duct.

CarEs belong to an ABH superfamily whose members function as carboxylic ester hydrolases of both xenobiotics and endogenous metabolites in insects (Gershater and Edwards 2007; Xu et al., 2021). Furthermore, insect detoxification enzymes potentially hydrolyze endogenous metabolites in plants, thereby adapting to the defense mechanisms of host plants (Schuler, 1996; Gatehouse, 2002; Lu et al., 2021). Previous studies have postulated that CarEs from Spodoptera littoralis degrade host plant volatiles, suggesting that CarEs are associated with insect detoxification of phytochemical toxins (Durand et al., 2010; Kong et al., 2010). RGDV infection in vector salivary glands activates the production of CarE10 and upregulates its detoxification enzyme activity (Figure 4A–4C). Notably, silencing of CarE10 strongly inhibits its detoxification enzyme activity and the effective viral infection of vector salivary glands (Figure 4E and 4F). Virus-mediated increases in CarE10 detoxification enzyme activity in vector salivary glands may ensure a stable insect cellular environment, ultimately promoting effective RGDV infection. RGDV infection of the vector salivary glands further activates the secretion of more CarE10 into the rice phloem, leading to an increase in CarE10 detoxification enzyme activity in rice (Figure 5E–5H). This process may also ensure a stable plant cellular environment and thus benefit initial virus infection in the plant host. Our study suggests that CarE10 detoxification enzyme activity in rice plants is beneficial for suppressing JA-Ile synthesis upon insect feeding, thus facilitating early horizontal virus transmission (Figure 5). Together, these findings reveal an previously unknown role of CarE10 enzyme activity in insect–virus–plant interactions, providing insight into how arboviruses establish initial insect-to-plant transmission.

Structurally, OsJAR1 contains conserved catalytic sites involved in the synthesis of JA-Ile in rice (Guranowski et al., 2007). The close association of CarE10 with the conserved catalytic sites of OsJAR1 may therefore prevent direct binding of the OsJAR1 catalytic sites to the JA substrate, impairing OsJAR1 enzyme activity (Supplemental Figures 9 and Figure 3A). Moreover, transient CarE10 expression enhances the ubiquitin-mediated degradation of OsJAR1 by the 26S proteasome system, indicating that the proteolytic degradation pathway is responsible for the reduced regulation of OsJAR1 level upon leafhopper feeding (Figure 3E and 3F). One explanation for this finding is that the stability of OsJAR1 is disrupted after association with CarE10, and this destabilization leads to premature degradation of the protein in vivo. However, it remains unknown whether insect CarE10 orthologs or paralogs also impair OsJAR1 activity. An interesting question is whether plant CXEs also directly target and impair OsJAR1 activity. The findings of this study exclude this possibility because rice CXEs have low aa homology with CarE10 and do not interact with OsJAR1 directly (Supplemental Figures 5 and 7). Overall, leafhopper salivary CarE10 plays a dual role by promoting ubiquitin-mediated degradation of OsJAR1 and binding to the OsJAR1 catalytic sites to affect its enzyme activity.

The cytoplasm of salivary secretory cells is largely occupied by intracellular salivary cavities in which saliva is stored (Sogawa, 1971). The apical plasmalemma of the salivary cavities forms a loose network, thus providing large surface areas in the secretory cells (Sugiya 2011). RGDV propagates to produce large amounts of virus-loaded Pns11 filaments that target most of the cavity plasmalemmas in virus-infected regions to induce an exocytosis-like process for viral secretion into the salivary cavities (Mao et al., 2017). In general, CarE10 also directly attaches to the actin-based apical plasmalemma and is secreted as a salivary component during insect feeding. The increased CarE10 contents do not compete with Pns11 filaments for attachment to the apical plasmalemma. Instead, the direct interaction of CarE10 with Pns11 leads to sequestration of CarE10 into Pns11-induced vesicles and finally into salivary cavities (Figures 4I and 5A–5C). Viruses in the salivary cavities typically move with the saliva flow to the canal in the stylets and are injected into sieve cells of the plant phloem as the piercing-sucking insect vectors feed on susceptible hosts (Hogenhout et al., 2008; Wei and Li 2016). RGDV infection activates and promotes the co-secretion of virions and CarE10 into salivary cavities, and CarE10, Pns11, and virions could thus arrive simultaneously at the rice phloem upon viruliferous R. dorsalis feeding (Figure 5E and 5F). In the initial viral infection sites of the rice phloem, both insect feeding and virions would activate strong defense responses, including callose deposition and JA signaling (Vincent et al., 2017). However, the increase in CarE10 contents effectively impairs OsJAR1 activity and suppresses JA-Ile-mediated host immune responses, thereby increasing plant attractiveness to viruliferous insects and promoting the accumulation of more virions. This process results in an optimal environment for viral propagation in the rice phloem.

In summary, we define the salivary protein CarE10 as an insect effector that downregulates rice JA-Ile synthesis and induces plant attractiveness to insects. Our results shed light on the tripartite interaction among plant viruses, host plants, and insect vectors and provide a potential target for the control of viral transmission in the future. Finally, our results offer fascinating evidence to show that insect vectors can regulate plant resistance responses to increase horizontal transmission of viruses into the plant phloem.

Materials and methods

Plants, insects, viruses, and antibodies

Viruliferous and nonviruliferous R. dorsalis colonies have been established in our laboratory and maintained in insect-proof greenhouses at 28°C ± 1°C with a 16-h/8-h light/dark cycle and 65% ± 5% relative humidity, as described previously (Wan et al., 2023). All rice plants were grown in the greenhouse at 28°C–30°C under a 10-h/14-h light/dark cycle. Rabbit polyclonal antibodies against viral proteins P8, Pns9, and Pns11; GAPDH; and histone H3 of R. dorsalis, as well as the pre-immune antibody, have been described previously (Mao et al., 2019; Chen et al., 2023). The CarE10 polypeptide (AKGGIKDAPWMTYVT) derived from R. dorsalis and the OsJAR1 polypeptide (PDFHQSLYCHLLCGLIYSEEV) derived from rice were conjugated to the carrier protein mariculture Keyhole Limpet Hemocyanin and injected into rabbits to generate antibodies against CarE10 and OsJAR1, respectively. The antibodies were produced by Genscript USA Innovation Company (Nanjing) and were approved by the Science Technology Department of Jiangsu Province, China, with approval number SYXK (Su) 2018-0015. Immunoglobulin (Ig) Gs were purified from specific polyclonal antisera and conjugated directly to fluorescein isothiocyanate (FITC) or rhodamine following the manufacturer’s instructions (Invitrogen). Rabbit polyclonal antibodies against 6× His tag and GST were purchased from Sangon Biotech (D110002 and D110271). The His-tag antibody conjugated to Alexa Fluor 488 (His-Alexa Fluor 488) was purchased from Thermo Fisher Scientific (MA1-135-A488).

Sequence alignment and phylogenetic analysis

The nucleotide and aa sequences of insect CarEs were downloaded from NCBI and used to generate multiple sequence alignments using MEGA 7.0. The signal peptide and motifs of CarEs were analyzed using SignalP-5.0 (https://services.healthtech.dtu.dk/service.php?SignalP-5.0) and ScanProsite (https://prosite.expasy.org/scanprosite/). The rice CXEs were selected from the China Rice Data Center (https://www.ricedata.cn/gene/) and used to produce multiple sequence alignments of CarE10 and rice CXEs with CLUSTALX. The nucleotide and aa sequences of OsJAR1 were also downloaded from the China Rice Data Center, and the conserved motifs were analyzed using DNAMAN.

Expression analysis of CarE10 in the salivary glands of R. dorsalis

To compare the mRNA expression levels of the CarEs (CarE1, CarE3, CarE4, CarE5, CarE6, CarE8, CarE9, and CarE10) in different tissues of R. dorsalis, whole bodies, salivary glands, midguts, female ovaries, and male testes were dissected from 30 leafhopper individuals, and total RNA was extracted from different tissues using the TRIzol reagent (Thermo Fisher Scientific, 15596026). The relative expression levels of CarEs in different leafhopper tissues were determined by RT–qPCR using 2× RealStar Fast SYBR qPCR Mix (High ROX; Genstar, A303) in the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). The primers used for RT–qPCR assays are shown in Supplemental Table 4. The transcript level of the housekeeping gene elongation factor 1 alpha (EF1α) of R. dorsalis (GenBank: AB836665) served as the internal reference for normalization of gene expression. Relative gene expression was estimated by the −ΔΔCT (cycle threshold) method (Wan et al., 2023). The experiments were replicated at least three times, and a pool of 30 leafhoppers was used for each replicate.

The protein levels of CarE10 in different leafhopper tissues were analyzed by western blot assays. Total proteins were extracted from whole bodies, salivary glands, midguts, female ovaries, male testes, and residues except salivary glands of 30 individual leafhoppers using radio immunoprecipitation assay lysis buffer (Thermo Fisher, 89901) and analyzed by western blot assays. Antibodies against CarE10 and histone H3 (0.5 μg/μl) served as the primary antibodies, and goat anti-rabbit IgG-peroxidase (Sigma-Aldrich, A0545) (0.5 μg/μl) served as the secondary antibody. The experiments were replicated at least three times, and a pool of 30 leafhoppers was used for each replicate. Bands for histone H3 demonstrated protein loading, and band intensities were quantified using ImageJ software.

To determine the effects of RGDV infection on mRNA and protein levels of CarE10 in vector salivary glands, total RNA and protein were extracted from the dissected salivary glands of 30 fifth-instar nymphs of nonviruliferous and viruliferous leafhoppers. The effects of RGDV infection on CarE10 expression were detected by RT–qPCR and western blot assays, as shown above.

Insect carboxylesterase activity and rice JAR concentration assays

The carboxylesterase activity of leafhopper salivary glands was measured using the carboxylesterase activity assay kit (Solarbio-BC0845) following the manufacturer’s instructions. Rice JAR concentrations were measured using the Plant JAR ELISA Kit (COIBO BIO, CB10437) following the manufacturer’s instructions. In brief, samples were collected and rapidly frozen with liquid nitrogen for homogenization. The samples were then centrifuged, and JAR concentration in the supernatant was measured at a wavelength of 450 nm using a microplate reader. Calibration standards were included with the samples to generate a standard curve of optical density versus JAR concentration.

Rice JA and JA-Ile concentration assays

Rice leaves were harvested at different time points after leafhopper feeding. JA and JA-Ile concentrations were analyzed by high-performance liquid chromatography–mass spectroscopy (UPLC–MS/MS) using labeled internal standards.

In vitro OsJAR1 activity assay

The in vitroassay for OsJAR1 JA-Ile synthesis activity has been described previously (Svyatyna et al., 2014). In brief, purified His-OsJAR1 proteins at a final concentration of 0.25 mg/ml were incubated with a mixture in the 400-μl reaction system. To examine the effect of CarE10 on OsJAR1 enzyme activity, purified His-CarE10 proteins at final concentrations of 0.1, 0.25, and 0.5 mg/ml were mixed in a reaction solution of 50 mM Tris–HCl (pH 8.6), 3 mM MgCl2, 3 mM ATP, 1 mM DTT, 1 mM JA, and 1 mM Ile for 10 min at 25°C. Purified His-GFP proteins at the same concentrations served as controls. JAR concentrations after enzymatic analysis were measured using the JAR ELISA Kit. The levels of synthesized JA-Ile in the mixture were evaluated by UPLC–MS/MS using labeled internal standards.

Transient expression of CarE10 and OsJAR1 in N. benthamiana

CarE10-His and OsJAR1-Myc were inserted into the destination vector pEarleygate101. Binary vectors were used to transform N. benthamiana via Agrobacterium tumefaciens GV3101. Agrobacteriumcultures were diluted to an optical density of 0.2–0.5 at 600 nm for agroinfiltration of N. benthamiana. The agroinfiltrated plants were maintained under normal growth conditions for 2–4 days. Various volumes of CarE10-His and OsJAR1-Myc Agrobacteriumcells were co-infiltrated into N. benthamiana leaves to test the effect of CarE10 on the protein level of OsJAR1. For in vitro analysis of OsJAR1 protein degradation by the 26S proteasome, MG132 (C2211, Sigma-Aldrich) was dissolved in DMSO and used at a concentration of 100 μM. The agroinfiltrated leaves were treated with MG132 or an equal volume of DMSO control solution for 12 h before sampling. Total proteins were extracted from N. benthamiana leaves for western blotting using anti-His or anti-Myc IgG antibody (0.5 μg/μl). JAR concentrations in the agroinfiltrated N. benthamiana were measured using the Plant JAR ELISA Kit.

Effect of viruliferous R. dorsalis feeding on protein levels and JAR concentrations in rice seedlings

Western blot assays were used to detect CarE10, OsJAR1, Pns11, P8, and Pns9 in rice seedlings inoculated with nonviruliferous and viruliferous leafhoppers. At least 30 nonviruliferous or viruliferous fifth-instar leafhopper nymphs were starved for 2 h and then fed on a single rice seedling at the two-leaf stage in a glass tube. The fed areas were collected at 0, 12, 24, 36, and 72 h after insect infestation to extract equal quantities of protein, and extracted proteins were analyzed by western blotting using antibodies against CarE10, OsJAR1, Pns11, P8, and Pns9 as primary antibodies and goat anti-rabbit IgG-peroxidase as the secondary antibody. The Rubisco large subunit was used as a loading control, as detected by staining with Coomassie brilliant blue. Rice seedlings without insect infestation served as the controls. Band intensities of proteins on the western blots were quantified with ImageJ software.

Similarly, rice seedlings were fed upon by 30 nonviruliferous, viruliferous, or dsGFP- or dsCarE10-treated nonviruliferous or viruliferous fifth-instar leafhopper nymphs for 0.5–3 h, then collected for measurement of JAR concentrations using the Plant JAR ELISA Kit.

Silencing of CarE10 in R. dorsalis

RNA interference was used to knock down the expression of CarE10. dsRNA targeting approximately 700-bp regions of the C-terminal 250-aa region of CarE10 (dsCarE10) and GFP (dsGFP) were synthesized in vitro using the T7 RiboMAX Express RNAi System (Promega Biotech) according to the manufacturer’s protocol. The primers used are listed in Supplemental Table 4. Preliminary experiments showed that third-instar leafhopper nymphs microinjected with in vitro-synthesized dsRNA required approximately 4 or more days to achieve a high interference efficiency, enabling the insects to develop into fifth instars or adults for subsequent experiments. Third-instar nymphs of nonviruliferous and viruliferous leafhoppers were microinjected with dsGFP or dsCarE10 (approximately 200 ng/leafhopper) using a Nanoject II Auto-Nanoliter Injector (Spring) and then placed on healthy rice seedlings. Gene-silencing efficiency was assessed by RT–qPCR and western blot assays 4 or 6 days after dsRNA microinjection. Pns11 and P8 of RGDV were used to monitor viral infection by western blot assays. Thirty individual insects in each dsRNA treatment group were randomly mixed for extraction of total RNA and protein. The carboxylesterase activity of 30 dsGFP- and dsCarE10-treated nonviruliferous insects was measured using the carboxylesterase activity assay kit. JA-Ile concentrations of rice seedlings were measured after 0.5 or 1 h of feeding by 30 dsGFP- or dsCarE10-treated nonviruliferous or viruliferous insects.

We also tested the ability of CarE10 from leafhoppers to facilitate horizontal virus transmission into the rice phloem. The carboxylesterase activity of 30 dsGFP- and dsCarE10-treated viruliferous leafhoppers was measured using the carboxylesterase activity assay kit. Saliva samples from 200 dsGFP- and dsCarE10-treated leafhoppers were collected by membrane feeding with 4% (v/v) sucrose for 24 h. Total proteins were extracted from the saliva samples, and levels of CarE10, P8, and Pns11 were detected using western blot assays.

To test the effect of insect CarE10 knockdown on JAR and JA-Ile concentrations in rice seedlings, 30 nonviruliferous, viruliferous, or dsGFP-treated or dsCarE10-treated viruliferous leafhoppers were allowed to feed on rice seedlings for 0.5–1 h, and plant JAR and JA-Ile concentrations were measured.

To investigate the effect of CarE10knockdown on viral transmission by insect vectors, 30 individual dsGFP- and dsCarE10-treated insects were transferred into glass tubes with single rice seedlings at 6 days post microinjection. The insects were allowed to feed on the seedlings for 48 h and were then individually analyzed by RT–PCR to determine whether or not they were viruliferous. Seedlings inoculated with viruliferous leafhoppers were subjected to RT–PCR detection 20 days later. The test was conducted with more than three replicates. The transmission rate of RGDV by leafhoppers was calculated on the basis of RT–PCR results from the tested plants.

CarE10-His-overexpressing transgenic rice lines

Full-length CarE10-His was cloned for construction of an expression vector through homologous recombination to generate transgenic rice plants overexpressing CarE10-His. The recombinant plasmid was electroporated into A. tumefaciens strain GV3101 for transformation into ZH11 (the widely used japonica rice cultivar Zhonghua 11). CarE10-His-overexpressing (OECarE10-His) transgenic rice lines were identified by RT–qPCR and western blot assays using CarE10 transcript levels and a His-tag antibody, respectively. WT ZH11 was used as the control, and the rice actin transcript served as an internal reference for normalization of gene expression levels. The western blot assays used a primary antibody against the His tag and goat anti-mouse IgG-peroxidase as the secondary antibody. Band intensities of proteins on the western blot were quantified using ImageJ software. The mean height of the transgenic rice lines was calculated from at least 10 rice plants.

We next assessed the effects of CarE10 overexpression in rice plants on viral transmission during leafhopper feeding. Thirty viruliferous fifth-instar leafhopper nymphs were starved for 3 h and transferred to a glass tube to feed on seedlings of transgenic or WT rice plants for 24 h. These leaves were collected and their total proteins extracted. Levels of RGDV P8 and Pns11 proteins were detected by western blot assays. Similarly, 30 nonviruliferous fifth-instar leafhopper nymphs were placed on transgenic and WT rice plants for 0.5–1 h, and JAR enzyme, JA, and JA-Ile concentrations were tested.

Insect attraction bioassay

The feeding preferences of 90 fifth-instar nymphs of viruliferous or nonviruliferous leafhoppers on healthy, diseased, and OECarE10 transgenic rice plants were determined by a Y-tube dual-choice assay as described by Koschier et al. (2000) (Figure 1E). The assay used a glass Y-tube (0.5-cm inner diameter) with a 5-cm base tube and two 5-cm branching arms at an angle of ca. 45° leading from the base tube. The Y-tube was placed in a brown light-proof box, and the surrounding light had even illumination to minimize visual distractions. Purified air was equally and continually pumped from the glass chambers to the olfactometer at 300 ml min−1. The Y-tube olfactometer was replaced after each replication to avoid positional bias. Approximately 90 nonviruliferous or viruliferous fifth-instar leafhopper nymphs were released from the net center, and their numbers on different types of treated rice plants were counted and analyzed at 3, 6, 12, and 24 h after insect release. Three replicates were performed for each treatment.

Immunofluorescence staining

The salivary glands from 30 nonviruliferous, viruliferous, or dsGFP- or dsCarE10-treated viruliferous leafhoppers were dissected, fixed in 4% (v/v) paraformaldehyde for 2 h, and permeabilized in 0.2% (v/v) Triton X-100 for 1 h. The salivary glands were immunolabeled with Pns11-specific IgG conjugated to FITC (Pns11-FITC), CarE10-specific IgG conjugated to rhodamine (CarE10-rhodamine), CarE10-specific IgG conjugated to FITC (CarE10-FITC), or pre-immune antibody conjugated to FITC (0.5 μg/μl). The midguts, salivary glands, ovaries, and testes from 30 nonviruliferous leafhoppers were dissected, fixed, permeabilized, and immunolabeled with CarE10-FITC (0.5 μg/μl). The samples were then examined using a Leica TCS SP5II confocal microscope.

To detect the release of target proteins in rice plants, at least 30 nonviruliferous or viruliferous fifth-instar leafhopper nymphs were starved for 2 h and then fed on a single rice seedling at the two-leaf stage in a glass tube. Hand-cut tissues of the feeding areas on the tested rice seedlings were collected at 0, 12, 24, 36, and 72 h after insect infestation. The samples were processed into 20-μm-thick sections using a cryostat (Leica CM1900). The tissue sections were fixed with 4% (v/v) paraformaldehyde for 12 h and permeabilized in 4% (v/v) Triton X-100 for 6 h. The tissues were then immunolabeled with CarE10-FITC, Pns11-specific IgG conjugated to rhodamine (Pns11-rhodamine), P8-specific IgG conjugated to rhodamine (P8-rhodamine), or Pns9-specific IgG conjugated to rhodamine (Pns9-rhodamine) (0.5 μg/μl). Rice seedlings without insect infestation served as controls. The fluorescent signals were visualized using a Leica TCS SP5II confocal microscope.

Baculovirus expression assay

CarE10-His and Pns11-Myc were expressed in the baculovirus vector according to the manufacturer’s instructions (Thermo Fisher Scientific). Sf9 cells were cultured and maintained in growth medium Sf900 III (Gibco, 12658019). In brief, the ORFs of CarE10-His and Pns11-Myc were amplified and cloned into the pDEST8 vector to construct recombinant baculoviruses. The primers used are listed in Supplemental Table 4. Recombinant bacmids were generated by transforming Escherichia coli DH10Bac with the recombinant baculovirus. The purified recombinant bacmids were then transfected into Sf9 cells using Cellfectin II. The cells were fixed in 4% (v/v) paraformaldehyde for 30 min and then permeabilized in 0.2% (v/v) Triton X-100 for 15 min at 48 h post infection (hpi). The cells were then immunolabeled with His-tag antibody conjugated to FITC (His-FITC) or Myc antibody conjugated to rhodamine (Myc-rhodamine) (0.5 μg/μl) and processed for immunofluorescence microscopy.

Immunoelectron microscopy

The midguts or salivary glands dissected from nonviruliferous and viruliferous leafhoppers were fixed in 2% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde for 2 h. The fixed samples were dehydrated in an ethanol gradient up to 100% and embedded in LR gold resin (Bioscience). Polymerization was allowed to proceed for 72 h at −20°C. Samples were sectioned on an ultramicrotome (LKB Nova) with a diamond knife, and the ultrathin sections were immunolabeled with CarE10-specific antibody or pre-immune antibody (0.5 μg/μl) as a primary antibody, followed by goat anti-rabbit IgG conjugated with 15-nm-diameter gold particles (0.5 μg/μl; Abcam) as a secondary antibody (Li et al., 2004). The sections were examined using an H-7650 Hitachi transmission electron microscope (Hitachi, Tokyo, Japan).

Yeast two-hybrid assay

The full-length ORFs of CarE10 and CXE1 were amplified by PCR and separately cloned into the bait vector pGKBT7, and the full-length ORFs of Pns11, OsJAR1, LOX1, LOX2, LOX5, AOS2, OPR3, JAZ1, and JAZ9 were amplified by PCR and separately cloned into the prey vector pGADT7. Different segments of OsJAR1 were also amplified and separately cloned into the prey vector pGADT7. The primers used are listed in Supplemental Table 4. Potential interaction pairs were co-transformed into the yeast strain AH109 according to the manufacturer’s instructions (Takara, Japan). The transformants were cultivated on Synthetic Dropout Medium (SD)/-Leu/-Trp (Double Dropout Supplements (DDO); Clontech, 630417) plates; positive clones were transferred and grown on SD/-Leu/-Trp/-His/-Ade/X-a-gal (Quadruple Dropout Supplements (QDO); Clontech, 630428) culture medium. The positive control pGBKT7-53/pGADT7-T and negative control pGBKT7-Lamp/GADT7-T were transfected in the same way. At the same time, plasmids of the linkage genes were co-expressed with the corresponding empty vector pGKBT7 or pGADT7 to verify its self-activation. Yeast cells were grown for 3 days at 30°C and photographed to record growth. All experiments were performed in triplicate.

GST pull-down assay

The CarE10 gene was cloned into the pGEX-4T-3 vector to construct a plasmid expressing the GST fusion protein as a bait (GST-CarE10). The Pns11 gene was cloned into a pEASY-Blunt E1 expression vector for fusion with a His tag. OsJAR1 was inserted into the pET-28a vector to obtain a His-OsJAR1 plasmid. The primers used are listed in Supplemental Table 4. All recombinant proteins were expressed in E. coli strain BL21 and purified. GST-CarE10 was bound to GST-Sepharose 4B beads (GE) for 3 h at 4°C. His-tag fusion proteins were separately added to the beads and incubated for 3 h at 4°C. The bead-bound proteins were separated by SDS–PAGE and detected by western blotting with His-tagged and GST-tagged antibodies (TransGen Biotech).

Co-immunoprecipitation assay for detecting OsJAR1 ubiquitination in N. benthamiana leaves

To investigate Ub modification of OsJAR1, full-length sequences of CarE10-His, OsJAR1-Myc, and Ub-HA were each amplified and cloned into the pEarleyGate 101 vector. The constructs were then transiently co-expressed in N. benthamiana leaves. At 48 h post infiltration, cell lysates were prepared, and the substrate protein OsJAR1-Myc was immunoprecipitated with anti-Myc beads (P2118-0.5ml, Beyotime Biotechnology). Ub-modified proteins were detected by immunoblotting with the indicated antibodies.

Data analysis

Data were analyzed using Microsoft Excel and GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). Percentage data were arcsine square-root transformed before analysis. Significant differences were analyzed using a two-tailed t-test for paired data, whereas multiple comparisons were performed by one-way ANOVA with Tukey’s multiple-comparisons test. Data represented three biological replicates. Band intensities of proteins on western blots were quantified using ImageJ software (version1.53e; https://imagej.nih.gov/ij/).

Funding

This work was supported by the 10.13039/501100012166 National Key Research and Development Program of China (2023YFD1400300 ), the 10.13039/501100003392 Natural Science Foundation of Fujian Province (2021J01065 and 2022J01127 ), and the 10.13039/501100001809 National Natural Science Foundation of China (nos. 618 31920103014 and 32202270 ).

Author contributions

H.Z. and T.W. designed the research. Y. Chi, S.C., Y. Cheng, X.Z., D.J., H.C., and Q.C. performed the research. X.Z., D.J., Q.C., H.Z., and T.W. analyzed the data. H.Z. and T.W. wrote the paper. All authors read and approved the manuscript.

Supplemental information

Document S1. Supplemental Figures 1–9 and Supplemental Tables 1–4

Document S2. Article plus supplemental information

Acknowledgments

No conflict of interest is declared.

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information is available at Plant Communications Online.
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