RH3 enhances antiviral defense by facilitating small RNA loading into Argonaute 2 at endoplasmic reticulum–chloroplast membrane contact sites

Huang, Juan; Du, Juan; Liu, Yan; Lu, Lu; Xu, Yanzhuo; Shi, Jianfei; Liu, Qing; Li, Qi; Liu, Yang; Chen, Yaqiu; Du, Meng; Zhao, Yiming; Huo, Liangxiao; Wang, Weiran; Ding, Chenxi; Wei, Liya; Wu, Jianguo; Yuan, Yao-Wu; Chen, Jinfeng; Li, Ruixi; Cui, Feng; Zhang, Xiaoming

doi:10.1038/s41467-025-57296-6

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Published: 25 February 2025

RH3 enhances antiviral defense by facilitating small RNA loading into Argonaute 2 at endoplasmic reticulum–chloroplast membrane contact sites

Juan Huang^1,2^na1,
Juan Du^1,2^na1,
Yan Liu^1,3,
Lu Lu¹,
Yanzhuo Xu^1,2,
Jianfei Shi^1,2,
Qing Liu^1,2,
Qi Li¹,
Yang Liu^1,2,
Yaqiu Chen¹,
Meng Du¹,
Yiming Zhao^1,2,
Liangxiao Huo⁴,
Weiran Wang⁴,
Chenxi Ding⁴,
Liya Wei⁴,
Jianguo Wu⁵,
Yao-Wu Yuan⁶,
Jinfeng Chen ORCID: orcid.org/0000-0002-5628-6322¹,
Ruixi Li ORCID: orcid.org/0000-0003-4763-5436⁷,
Feng Cui ORCID: orcid.org/0000-0001-6215-7159^1,2 &
…
Xiaoming Zhang ORCID: orcid.org/0000-0002-0033-0400^1,2,8

Nature Communications volume 16, Article number: 1953 (2025) Cite this article

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Abstract

While RNA silencing is crucial for plant resistance against viruses, the cellular connections between RNA silencing and antiviral responses in plants remain poorly understood. In this study, we aim to investigate this relationship by examining the subcellular localization of small RNA loading and viral replication in Arabidopsis. Our findings reveal that Argonaute 2 (AGO2), a key component of RNA silencing, loads small RNAs at the endoplasmic reticulum (ER)–chloroplast membrane contact sites (MCSs). We identify a chloroplast-localized protein, RNA helicase 3 (RH3), which interacts with AGO2 and facilitates the loading of small RNAs into AGO2 at these MCSs. Furthermore, we discover that MCSs serve as replication sites for certain plant viruses. RH3 also promotes the loading of viral-derived small RNAs into AGO2, thereby enhancing plant antiviral resistance. Overall, our study sheds light on the roles of RH3 in RNA silencing and plant antiviral defenses, providing valuable insights into the cytobiological connections between RNA silencing, viral replication, and antiviral immunity.

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Introduction

RNA silencing is a conserved gene regulation mechanism found across eukaryotes, mediating by small RNAs (sRNAs) associated with Argonaute (AGO) proteins, which are effectors of the RNA-induced silencing complex (RISC). These sRNAs, typically 20-30 nucleotides long, are processed from double-stranded RNAs (dsRNAs) by Dicer or Dicer-like (DCL) proteins^1,2. In plants, sRNAs originated from endogenous sources can be classified into different subgroups, including microRNAs (miRNAs), phased siRNAs (phasiRNAs), and heterochromatin siRNAs (hc-siRNAs)^3,4,5. Additionally, sRNAs can be generated from exogenous sources, such as viruses, and function in defense against these pathogens. During the replication of RNA viruses or transcription of DNA viruses, abundant dsRNAs are generated and processed into virus-derived small interfering RNAs (vsiRNAs)^3,6,7. Despite the well-established importance of RNA silencing in plant antiviral responses, the cytobiological connections between RNA silencing and plant antiviral immunities remain poorly understood.

The loading of sRNAs into specific AGO proteins is a crucial step in RNA silencing-mediated gene regulation^3,4. Different AGO proteins have been shown to load specific types of sRNAs at distinct subcellular localizations. For example, Arabidopsis AGO1 loads miRNAs in the nucleus and trans-acting siRNAs (ta-siRNA, a type of phasiRNAs) in the cytoplasm⁸, while AGO4 loads hc-siRNAs in the cytoplasm^9,10. AGO2 loads various types of sRNAs and is involved in host resistance to bacteria and DNA repair processes^11,12. AGO2 also associates with vsiRNAs and plays pivotal roles in the antiviral immune response^{13,14,15,16,17,18,19}. However, the subcellular localizations for sRNA loading into AGO2 remain undetermined.

Eukaryotic cells are characterized by the presence of membrane-bound organelles, which compartmentalize the cell and carry out specialized functions^20,21,22. The endoplasmic reticulum (ER) is the largest organelle and is involved in protein synthesis and folding, lipid and steroid synthesis, and calcium storage and release^23,24. Arabidopsis thaliana AGO1, an essential component of RNA silencing, has been shown to localize to the ER, where it inhibits the translations and cleavage miRNA target transcripts^25,26,27. Chloroplasts, which evolved from endosymbiotic cyanobacteria²⁸, lack typical RNA silencing machinery²⁹ but can provide necessary molecules^28,30 or spatial compartments for RNA silencing. Membrane contact sites (MCSs) between organelles, where different organelles are physically tethered but do not fuse, allow for communication and coordination between organelles^21,22,31. The ER and chloroplasts can form unique ER-chloroplast MCSs in plants^32,33,34,35, although their specific roles in biological processes still need to be investigated^36,37.

In this study, we investigated the subcellular localizations where sRNAs are loaded into AGO2 and their relationship with viral replication sites. Through immunoprecipitation (IP) coupled with mass spectrometry (MS) assays, we identified RH3, a DEAD-box RNA helicase localized in the chloroplast, interacting with AGO2 at ER–chloroplast MCSs. Further experiments involving microsomal membrane fractionation and sRNA pulldown assays revealed that AGO2 loads sRNAs at the ER–chloroplast MCSs, and RH3 facilitates their loading at the same sites. Additionally, protein co-localization and fluorescence in situ hybridization (FISH) assays demonstrated that the replication of certain viruses occurs at the ER–chloroplast MCSs. RH3 facilitates the loading of vsiRNAs into AGO2, thereby contributing to antiviral immunity. Together, this study provides valuable insights into the cytobiological aspects of RNA silencing in the context of host defense against viruses.

Results

Chloroplast-localized RH3 interacts with AGO2

To explore the sRNA loading mechanism and the involvement of chloroplast in RNA silencing, we employed a combination of AGO2 IP with MS to identify proteins associated with AGO2 (Supplementary Fig. 1). We performed IP-MS assay on pAGO2::HA-AGO2 in ago2-1 and p35S::3HA-GFP in Col-0, and identified 44 candidate proteins (fold change > 2, p < 0.05) (Supplementary Fig. 2 and Supplementary Data 1). Among these candidate proteins, we focused on RH3, a conserved chloroplast-localized DEAD-box RNA helicase protein in plants (Supplementary Figs. 2, 3). RNA helicases are known to participate in various RNA-related processes by modulating RNA or RNA-protein complex structures^38,39. Given the potential involvement of RNA helicases in sRNA loading, we selected RH3 for further analysis.

To investigate the interaction between RH3 and AGO2, we conducted co-immunoprecipitation (Co-IP) experiments in Nicotiana benthamiana plants. We used chloroplast-localized BFA1-FLAG as a control for RH3⁴⁰. The Co-IP assay successfully demonstrated the pull-down of RH3 by AGO2 (Fig. 1a, middle panel), indicating their interaction. Similarly, the reciprocal Co-IP experiment showed the pull-down of AGO2 by RH3 (Fig. 1a, right panel). Furthermore, Co-IP assays were performed in transgenic Arabidopsis plants expressing pAGO2::HA-AGO2 in the ago2-1 mutant background, with p35S::3HA-GFP in Col-0 plants as a control. The results showed that RH3 protein was only detected in HA-AGO2 IP fractions (Fig. 1b), providing additional evidence for the association between RH3 and AGO2.

**Fig. 1: Chloroplast-localized RH3 interacts with AGO2.**

To investigate the direct interaction between RH3 and AGO2, we conducted in vitro pulldown assays using recombinant proteins purified from Escherichia coli cell cultures (Supplementary Figs. 4, 5a). When FLAG-AGO2 was incubated with RH3-HA or glutathione S-transferase (GST)-HA, a strong RH3-HA signal was detected on the FLAG-AGO2 beads, while no GST-HA signal was observed (Fig. 1c). Moreover, deletion of residues 451-500 in RH3 resulted in a decreased interaction (Supplementary Fig. 5b–f). Additionally, the association of RH3 with AGO2 was reduced when the K459A, R460A, R463A, and R467A mutations were introduced simultaneously (Fig. 1d, e, and Supplementary Fig. 5g). Coexpressing HA-AGO2 with mRH3-FLAG (K459A, R460A, R463A and R467A) or with wild-type RH3-FLAG in N. benthamiana plants demonstrated that the accumulation of mRH3 in the HA-AGO2 groups decreased by 45.1% compared to that of wild-type RH3 (Fig. 1f, g), indicating the importance of these residues in the association between RH3 and AGO2. These findings provide evidence that AGO2 directly interacts with RH3.

RH3 interacts with AGO2 at chloroplast periphery

To investigate the subcellular localization where the RH3-AGO2 interaction occurs, we examined the localization patterns of RH3 and AGO2. While RH3 is predominantly localized in the chloroplast, a fraction of RH3 was observed to localize at the chloroplast periphery when transiently expressed in the Arabidopsis protoplasts (Supplementary Fig. 6a). This localization pattern was confirmed through immunofluorescence assays using an anti-FLAG antibody in pRH3::RH3-FLAG in rh3-4 plants (Fig. 2a). Co-labeling RH3 with Toc34, a marker for the chloroplast outer membrane⁴¹, further demonstrated the localization of RH3 at the chloroplast periphery (Fig. 2b). Immunoelectron microscopy assays also revealed the presence of RH3 particles in the envelope area of the chloroplast (Fig. 2c). Similarly, transient expression assays in Arabidopsis protoplasts (Supplementary Fig. 6a) and immunofluorescence assays using an anti-HA antibody in pAGO2::HA-AGO2 in ago2-1 plants (Fig. 2d) showed that AGO2 disperses around the chloroplast periphery.

To further examine the co-localization of RH3 and AGO2, we performed immunofluorescence assay using anti-FLAG and anti-AGO2 antibodies in pRH3::RH3-FLAG in rh3-4 plants. The specificity of the anti-AGO2 antibody was confirmed using a dual labeling assay (Supplementary Fig. 6b). The results showed that a portion of RH3 co-localized with AGO2 at the chloroplast periphery (Fig. 2e and Supplementary Fig. 6c, d). Furthermore, a bimolecular fluorescence complementation (BiFC) assay revealed an interaction between RH3 and AGO2 at the chloroplast periphery (Fig. 2f and Supplementary Fig. 7).

RH3 interacts with AGO2 at ER–chloroplast MCSs

We observed that the BiFC signal of the RH3-AGO2 interaction at the chloroplast periphery displayed an uneven distribution (Fig. 2f). By expressing the commonly used ER marker RFP-HDEL⁴², we found that the ER contacts the chloroplasts at discontinuous subdomains (Supplementary Fig. 8a). Considering the physical connection between the ER and the chloroplasts and the localization of Arabidopsis AGO1 to the ER²⁶, we hypothesized that the uneven localization of the RH3-AGO2 interaction at the chloroplast periphery related to ER-chloroplast contact sites.

To test this hypothesis, we first determined the potential subcellular localization of AGO2 with the ER. AGO2-YFP was co-expressed with RFP-HDEL in N. benthamiana. As shown in Fig. 3a, YFP-AGO2 signals exhibited a net-like pattern that was identical to RFP-HDEL in both epidermal and mesophyll cells. Immunofluorescence assays in transgenic plants expressing GFP-HDEL also showed that AGO2 associates with GFP-labeled ER signals, particularly at the chloroplast periphery (Fig. 3b and Supplementary Fig. 8b). These results demonstrate that AGO2 is distributed around the ER network.

**Fig. 3: RH3 interacts with AGO2 at ER-chloroplast MCSs.**

Next, we examined whether the interaction between RH3 and AGO2 occurs at the ER juxtaposed to the chloroplast. In epidermal cells lacking chloroplasts, the colocalization of RH3 and AGO2 (Fig. 3c) and the BiFC YFP signal of RH3-AGO2 (Fig. 3d) were observed at ER-mesh junctions, suggesting that the ER serves as the anchoring platform for their interaction. In mesophyll cells, the BiFC YFP signal of RH3-AGO2 aligned with the RFP-HDEL signal adjacent to the chloroplast periphery (Fig. 3e and Supplementary Fig. 8c). By imaging multiple focal planes within the same cell, we discovered that the BiFC YFP signal of RH3-AGO2 was present only at subdomains of the chloroplast periphery covered by the ER (marked by RFP-HDEL), and absent at regions of the chloroplast periphery not linked to the ER (Fig. 3e, upper panel and Supplementary Fig. 8e). Furthermore, the distance-intensity profile provided additional evidence for the association of the RH3-AGO2 interaction with the ER (Fig. 3f and Supplementary Fig. 8d).

To eliminate optical interference caused by clustered chloroplasts⁴³, the interaction signals of AGO2 and RH3 were further observed in epidermal cells containing very few chloroplasts. In these cells, the interaction signals of AGO2 and RH3 aligned perfectly with ER wrapping the chloroplast surface, while TOC64-III⁴⁴, an outer-membrane chloroplast protein, showed no colocalization with ER on certain chloroplasts (Fig. 3g). Furthermore, Z-stack scanning revealed that the appearance and intensity of the RH3–AGO2 BiFC signal corresponded to the RFP–HDEL signal at the chloroplast periphery (Supplementary Movie 1). By contrast, TOC64-III–CFP distribution and intensity were independent of RFP–HDEL (Supplementary Movie 2).

The 6K2 protein of TuMV has been reported to associate with ER-like membranes, localizing at the chloroplast periphery^45,46,47. To mimic the interaction between RH3 and AGO2 under physiological conditions, pRH3::RH3-cYFP and pAGO2::nYFP-HA-AGO2 were expressed in Arabidopsis seedling. The resulting interaction signal (YFP) co-localized with TuMV-6K2-mCherry at the chloroplast periphery (Fig. 3h and Supplementary Fig. 9). These findings collectively support the conclusion that RH3 interacts with AGO2 at ER–chloroplast MCSs.

RH3-AGO2 interaction promotes the loading of sRNAs

The interaction between RH3 and AGO2 suggests that RH3 may affect RNA silencing mediated by AGO2-associated sRNAs. To investigate the effect of RH3 on AGO2-associated sRNAs, we compared the sRNA profiles of Col-0 and rh3 mutant plants. Since the rh3 null mutant is embryonic lethality⁴⁸ and amiR-RH3 knockdown seedlings display a pale-green phenotype and growth cessation (Supplementary Fig. 10a), we utilized the rh3-4 weak allele⁴⁸ (Supplementary Fig. 10b, c) for deep sequencing analysis of sRNAs. AGO2 is known to predominantly associate with 21-nt sRNAs starting with a 5’-A¹². While the total 21-nt sRNAs (Supplementary Fig. 10d) and 21-nt sRNAs starting with a 5’-A (Supplementary Fig. 10e) were comparable between rh3-4 and Col-0 plants, the deep sequencing analysis of AGO2-bound sRNAs revealed that rh3-4 plants had fewer sRNAs loaded into AGO2 compared to Col-0 plants (Fig. 4a, Supplementary Fig. 10f and Supplementary Data 2). These sRNAs included miRNAs, miRNA*s, and ta-siRNAs (Fig. 4b and Supplementary Data 2). Northern blot analysis confirmed that the levels of AGO2-loaded sRNAs were decreased in the rh3-4 plants compared to Col-0 plants (Fig. 4c). Moreover when transgenic lines carrying pRH3::RH3-FLAG in the rh3-4 background (Supplementary Fig. 10g, h) were generated, the sRNAs associated with AGO2 in these lines were comparable to those in Col-0 plants (Fig. 4c).

To further investigate the role of RH3 in AGO2 loading, we examined the subcellular localization of sRNAs associated with AGO2 using a FISH assay in Col-0 plants. The results revealed punctate compartments with a miR393b* signal at the periphery of chloroplasts (Supplementary Fig. 11). RNA immunoprecipitation (RIP)-qPCR using an anti-AGO2 antibody on isolated microsomal membranes (which are enriched for the ER network) showed that the loading efficiency of sRNAs into AGO2 was significantly higher in the microsomal membrane fraction compared to the soluble fraction (Supplementary Fig. 12a–c). With Col-0 and rh3-4 plants, microsomal membrane fractions and immunoprecipitation assays revealed that the presence of RH3 and AGO2 in the microsomal membrane fractions and the levels of AGO2-loaded sRNAs in the rh3-4 microsomal membrane fraction were significantly lower compared to those in the Col-0 microsomal membrane fraction (Fig. 4d, and Supplementary Fig. 12d–f). These findings suggest that ER–chloroplast MCSs may serve as sites for the loading of sRNAs into AGO2 and further demonstrate that RH3 facilitates the loading of sRNAs into AGO2.

To investigate whether the effect of RH3 on sRNA loading into AGO2 is related to the abnormal chloroplast morphology⁴⁹, we performed experiments using N. benthamiana plants coexpressing HA-AGO2 and pre-miR393b with p35S::RH3-FLAG. We observed a significant improvement in the binding of miR393b* to AGO2 in the presence of transient expressed RH3 (Fig. 4e, f), although the chloroplast morphology was similar between samples (Supplementary Fig. 13a). Further analysis with the p35S::RH3-CFP-HA in Col-0 Arabidopsis plants showed that sRNAs associated with AGO2 were more abundant in the RH3-overexpressing plants than those in the Col-0 plants (Fig. 3g and Supplementary Fig. 13b). Notably, the p35S::RH3-CFP-HA in Col-0 transgenic plants did not display any obvious developmental defects (Supplementary Fig. 13c), and the chloroplast structures appeared normal (Supplementary Fig. 13d). These findings suggest that the role of RH3 in promoting sRNAs loading into AGO2 does not result from abnormal chloroplast morphology.

RH3 may indirectly inhibit the degradation of primary miRNA (pri-miRNA) via the retrograde signaling pathway⁵⁰. However, we observed that the accumulation of tested pre- and pri-miRNAs, as well as TAS2, in the rh3-4 mutant plants, was not significantly altered (Supplementary Fig. 13e–g). To further test this hypothesis, we performed in vitro loading assays (Supplementary Fig. 14a). The AGO2 protein, synthesized in a wheat germ extract system, was mixed with RH3 or GST proteins that was translated in an extract of evacuolated BY-2 protoplasts⁵¹ to perform in vitro loading assays. The loading of miR393b/miR393b*, miR391/miR391* or miR156a/miR156a* into AGO2 were determined (Supplementary Fig. 14b, c). As expected, miR393b* and miR391*, but not miR156a were associated with AGO2. Moreover, the association of miR393b* and miR391* with AGO2 was significantly promoted by RH3 (Fig. 4h, i). GST was used as control of RH3. Since the BYL extract does not contain intact chloroplasts required for the retrograde signaling, the in vitro loading assays together with the unchanged levels of pri-/pre-miRNA demonstrate that RH3 does not promotes the level of sRNAs loaded into AGO2 through retrograde signaling.

To determine whether RH3 promotes the loading of sRNAs into AGO2 by its interaction with AGO2, mRH3 with reduced interaction with AGO2 (Fig. 1d–g) was used. Coexpression of HA-AGO2 and pre-miR393b with wild-type RH3 or mRH3 in N. benthamiana plants showed that the accumulation of miR393b* bound to AGO2 was higher in plants coexpressing wild-type RH3 compared to those coexpressing mRH3 (Fig. 4e, f). This result was further confirmed in the in vitro loading assays (Fig. 4h, i). The mutant form of RH3 with impaired interaction indeed affected its ability to facilitate sRNA loading into AGO2.

Furthermore, the decreased loading of sRNAs into AGO2 in the rh3 mutant plants had functional consequences. The rh3-4 mutant plants exhibited higher accumulation of the MEMB12 protein, a target of miR393b* involved in the secretion of the PR1 protein¹² (Supplementary Fig. 15a). Additionally, upon treatment with a bacterial pathogen, the secretion of PR1 protein was decreased in the rh3-4 mutant plants compared to Col-0 plants (Supplementary Fig. 15b, c). Moreover, the transcripts of At1g28490 and At5g14180, which are predicted targets of miR5026⁵², were higher in rh3-4 mutant plants compared to Col-0 plants (Supplementary Fig. 15d). These findings further demonstrate the role of RH3 in promoting the functions of sRNAs bound to AGO2.

The RH3-AGO2 interaction at ER–chloroplast MCSs facilitates the loading of exogenous vsiRNAs into AGO2

Turnip yellow mosaic virus (TYMV), Turnip mosaic virus and other similar viruses, which are threats to crops and vegetables, have been shown to perform their replication processes at the chloroplast periphery^53,54,55. These viruses manipulate host membranes and modify the chloroplast membrane structure to facilitate their replication processes^56,57. During TYMV infection, there is a rearrangement of ER structures over viral vesicles, followed by the formation of chloroplast clumps surrounding these vesicles^53,58. These studies suggested that ER-chloroplast MCSs might be the active sites for the replication of TYMV and some other viruses. The 140K proteins of TYMV, which are required for the TYMV RNA replication, localizes to the chloroplast periphery^58,59,60. By coexpressing CFP-HDEL (ER) and dsRed-TYMV::140 K in A. thaliana seedling, we find that the replication complex of TYMV (dsRed-TYMV::140K) indeed associates with ER (Supplementary Fig. 16a). Additionally, the RH3-AGO2 interaction site co-localizes with 140K protein of TYMV at the chloroplast periphery (Supplementary Fig. 16b). We then infected A. thaliana seedling with dsRed-TYMV::140K and coexpressed nYFP-AGO2, RH3-cYFP, and CFP-HDEL. The results demonstrated the colocalization of the dsRed-TYMV::140K viral marker with the signals from CFP-HDEL (ER) and YFP (RH3-cYFP/nYFP-AGO2) (Fig. 5a). Moreover, RNA-fluorescence in situ hybridization assays revealed the localization of the replication intermediates of TYMV (TYMV dsRNA) at the chloroplast periphery, the majority of which co-localized with the RH3-AGO2 interaction site (Fig. 5b, Supplementary Fig. 16c). The accumulation of dsRNA at ER-chloroplast MCSs may activate RNA silencing-mediated plant resistances to viruses. Indeed, DCL4, responsible for processing viral dsRNAs⁶¹ (Supplementary Fig. 16d), and RNA-dependent RNA polymerase 6 (RDR6), which amplifies viral vsiRNAs⁶², were scattered around the chloroplast periphery (Supplementary Fig. 16d). These findings provide evidence that the TYMV replication may associate with RNA silencing at ER–chloroplast MCSs.

**Fig. 5: RH3-AGO2 interaction co-localizes with viral replication at ER–chloroplast MCSs.**

Roles of RH3 on sRNAs loading into AGO2 led us to investigate whether RH3 also facilities the loading of vsiRNAs into AGO2. The levels of vsiRNAs loaded into AGO2 in TYMV-infected Col-0, rh3-4, and pRH3::RH3-FLAG in rh3-4 plants were thus measured. Although the total vsiRNA levels in the rh3-4 mutant plants were higher than those in the Col-0 plants, the levels of vsiRNAs loaded into AGO2 were significantly reduced in the rh3-4 mutant plants (Fig. 5c). The levels in pRH3::RH3-FLAG in rh3-4 were comparable to those in Col-0 plants (Fig. 5c). These observations suggest that RH3 facilitates the loading of vsiRNAs into AGO2.

AGO2 has been shown to play a significant role in plant resistance to viruses^{13,14,15,16,17}. A previous study demonstrated that AGO2 contributes to limiting the TYMV infections⁶³. To investigate whether RH3 promotes plant antiviral immunity, the susceptibility of rh3-4 plants to TYMV was examined. Indeed, the viral genomic RNA and the RNA transcript of viral coat protein (CP) in systemically infected leaves of rh3-4 plants was increased compared to that in Col-0 or pRH3::RH3-FLAG in rh3-4 plants (Fig. 5d, e).

In response to viral infection, more RH3 is located to the chloroplast periphery to interact with AGO2. A BiFC assay conducted in TYMV-infected A. thaliana seedlings showed an increased YFP signal compared to the control plants (Fig. 5f, g), while the levels of AGO2 and RH3 protein were not significantly increased (Supplementary Fig. 16e). Additionally, an elevated RH3-CFP signal at the chloroplast periphery were observed in TYMV-infected samples (Fig. 5h, i). Immunofluorescence assays also showed increased colocalization between RH3 and AGO2 in TYMV-infected samples (Fig. 5j, k). These findings suggest that viral infection causes the relocation of RH3 to the chloroplast periphery, where it interacts with AGO2 and enhances the loading of sRNAs into AGO2.

To investigate the dependence of the antiviral effects of RH3 on AGO2, a rh3-4/ago2-1 double mutant was generated (Supplementary Fig. 17a, b) and infected with TYMV. Intriguingly, TYMV caused more severe disease symptoms in the rh3-4/ago2-1 plants (Supplementary Fig. 17c), and the accumulation of TYMV genomic RNAs was more abundant in the rh3-4/ago2-1 plants (Supplementary Fig. 17d). These results suggest that while RH3 promotes plant antiviral immunity, it does not solely depend on AGO2.

In addition to AGO2, other AGO proteins, such as AGO7, have been shown to participate in plant immune responses against viruses^3,64,65. RH3 may promote antiviral immunity by facilitating the loading of sRNA loading into AGO7 and other AGOs in addition to AGO2. Co-localization and interaction studies demonstrated that RH3 partially colocalized with AGO7 at the chloroplast periphery (Supplementary Fig. 18a) and interacted with AGO7 (Supplementary Fig. 18b). Moreover, RH3 was found to facilitate the loading of miR390 into AGO7 (Supplementary Fig. 18c). Interestingly, RH3 did not show a similar effect on the loading and regulation function of tested sRNAs associated with AGO1, but promoted the association of siRNA AtREP2 with AGO4 (Supplementary Fig. 18d–g). These findings indicate that RH3 may play a role in facilitating the loading of sRNAs into multiple AGO proteins.

Discussion

This study has revealed an important connection between viral replication and RNA silencing-mediated antiviral defense mechanisms in plants. It demonstrates that ER-chloroplast MCSs serve as sites for both the loading of sRNAs into AGO2 (Fig. 5l, middle panel) and viral replication (Fig. 5l, bottom panel). The RH3 protein is found to facilitate the loading of sRNAs into AGO2 at these sites. Furthermore, viral infections enhance the association of RH3 with AGO2, leading to increased loading of vsiRNAs into AGO2 and enhanced plant resistance to viruses.

Previous studies have highlighted the roles of the ER in RNA silencing, with AGO1, a peripheral membrane protein^66,67, being localized to the ER and the translation inhibitions of miRNA targets in ER-associated membrane-bound polysomes²⁶. In our study, we observed sRNAs and major RNA silencing components, including DCL, RDR, and AGOs, at the chloroplast periphery/ER-chloroplast MCSs. The loading of sRNAs into AGO2 was found to occur at these MCSs, and RH3 interacts with and facilitates the loading of AGO2 at these sites. AGO7 was found to associate with membranes that are coeluted with calnexin, a resident ER protein⁶⁸. Moreover, AGO7 tightly interacts with miR390 and stalls the MBPs bound to TAS3 transcripts^25,68,69, indicating that AGO7 localizes to the ER to cleave TAS3 transcripts. Additionally, approximately 60% of AGO7 colocalizes with the 6K2 protein of TEV in the cytoplasm⁶⁸. Notably, our results show that the TYMV 140K protein localizes at ER–chloroplast MCSs. RH3 also colocalizes with AGO7 at the chloroplast periphery and promotes the loading of miR390 into AGO7, suggesting that AGO7 may also load sRNAs at ER–chloroplast MCSs. Moreover, RH3 also promotes the loading of siRNA AtREP2 into AGO4. Therefore, ER-chloroplast MCSs may serve as important loading sites for sRNAs, and RH3 may has a broad role in facilitating their loading at these locations.

Chloroplast are sites for the synthesis of carbon, ATP, amino acids, purine, pyrimidine, salicylic acids, and jasmonic acids^54,55, which are required for the proliferation of viruses and host immune responses. Therefore, viruses target the chloroplast to eavesdrop cell compartments and membrane contents for their own proliferation⁵⁴, while simultaneously suppressing host immune responses. The ER is also a key site for the biosynthesis of proteins, lipids and other molecules that are also required for the proliferation of viruses⁷⁰. Many plant viruses remodel and use ER or ER-derived vesicles for viral proliferation^71,72. As MCSs contain molecules from two opposing organelles that may be required for viral proliferation, viruses may exploit host MCSs as centres to fulfill their proliferations^73,74. For example, the movement protein of Turnip vein clearing virus interacts with synaptotagmin SYTA, a protein enriched at ER–plasma MCSs, remodeling ER–plasma MCSs at plasmodesmata to form viral replication sites⁷⁵. Additionally, VAP27-1, another protein enriched at ER–plasma MCSs, also interacts with viral proteins and facilitates viral replication^76,77. Our study demonstrated that the replication of TYMV takes place at ER–chloroplast MCSs, emphasizing the importance of these sites in viral proliferation.

To efficiently inhibit viral replication, RNA silencing-mediated antiviral immunity may cytobiologically connect with viral replication sites such as ER-chloroplast MCSs. AGO2 and other AGO proteins, the central protein of RNA silencing, associate with vsiRNAs and play pivotal roles in plant resistances to viruses^{3,15,16,17,18,64,78}. To efficiently inhibit viral proliferation, AGOs need to be in the right place upon the infection of viruses¹⁹. Our study found that viral replications co-localize with RH3-AGO2 interaction sites at ER-chloroplast MCSs. AGO7 also plays important roles in plant resistance to viruses⁶⁵. As RH3 colocalizes with AGO7 at the chloroplast periphery and facilitates the loading of sRNAs into AGO7, AGO7 may also load vsiRNAs at these sites. Therefore, ER–chloroplast MCS provides a space not only for viral replication and loading of endogenous sRNAs but also for the loading and potential action of vsiRNA-RISCs, establishing a cytobiological link between viral replication and RNA silencing.

It is worth noting that same AGOs may load different types of sRNAs at different locations within the cell. For example, miRNAs are typically loaded into AGO1 in nuclei, while phasiRNAs are loaded into AGO1 in the cytoplasm⁸. Intriguingly, some unloaded mature miRNAs or miRNA/miRNA* duplexes have been found to accumulate in the cytoplasm, suggesting that the loading of the portion of specific miRNAs into AGO1 may also occur in the cytoplasm⁷⁹. Indeed, mobile miRNAs are reported to load into AGO1 in the cytoplasm⁸⁰. AGO2, on the other hand, not only loads miRNAs, miRNA*s, and vsiRNAs, but it also associates with diRNAs and participates in repairing double-strand breaks in the nucleus¹¹. Therefore, AGO2 may also have multiple subcellular localizations for sRNA loading.

In conclusion, this study uncovers the cytobiological connections between viral replication, RNA silencing, and plant antiviral defense mechanisms. ER tubules are present throughout the cytosol and form various MCSs with multiple organelles^{20,24,81,82,83}. Other organelles such as endosomes, lysosomes, peroxisomes, and mitochondria have also been suggested as replication sites for various plant viruses^46,56, and it is worth investigating roles of other ER–organelle MCSs in viral replications and RNA silencing.

Methods

Plant materials

Arabidopsis thaliana T-DNA insertion lines rh3-4 (SALK_005920)⁴⁹ were obtained from the Arabidopsis Biological Resource Center. The rh3-4 line was crossed with ago2-1 (SALK_003380) to generate homozygous rh3-4/ago2-1 double mutant. The ago2-1 mutant and pAGO2::HA-AGO2 in ago2-1 were previously described¹². Arabidopsis transgenic plant expressing RH3 under 35S promoter were generated by Agrobacterium tumefaciens (A. tumefaciens)-mediated flower dipping transformation in both Col-0 and rh3-4 backgrounds⁸⁴. Transformants were selected on 1/2 Murashige and Skoog (1/2 MS) agar plates supplemented with 0.1% basta to get p35S::RH3-CFP-HA in Col-0 and p35S::RH3-CFP-HA in rh3-4 plants. The p35S::amiR-RH3 construct was transformed into Col-0 to obtain amiR-RH3 transgenic plants. The pRH3::RH3-FLAG construct was transformed into rh3-4 to obtain pRH3::RH3-FLAG in rh3-4 transgenic plants. The p35S::3HA-GFP in Col-0 was previously described⁸⁵.

Typically, 4-week-old Arabidopsis plants were used for various assays. Due to the serious developmental defects, p35S::amiR-RH3 in Col-0 plant was not used in further experiment. The phenotype of p35S::amiR-RH3 in Col-0 T0 lines were shown. The T2 generations of stable transgenic lines were used for subsequent experiments. For viral infections, 4-week-old Arabidopsis plants were used for virus infection and the systemic leaves were harvested at 14 dpi for TYMV. 4-week-old N. benthamiana plants were used for viral infection and transient expression, and the inoculated leaves were harvested at 3 dpi. Protoplasts from 2-week-old Arabidopsis plants were used for transient expression in protoplasts. Adult plants were grown in a greenhouse at 22 °C ± 1 °C under a 12 h light/12 h dark cycle, while seedlings were grown on 1/2 MS plates in a growth chamber (22°C, 12 h light/12 h dark cycle). Additional details for individual experiments are provided in the figure legends. N. benthamiana wild-type plants were grown in a greenhouse at 22°C ± 1°C under 12 h light/12 h dark photoperiod. P19, an RNA silencing suppressor, was not included in transient expression experiments unless otherwise notified.

Plasmids and cloning procedures

To generate pENTR constructs, the coding sequences of RH3, AGO2, BFA1, Calnexin (CNX), AGO7, DCL4 and TOC64-III were amplified from cDNA derived from Col-0 plants. For Co-IP assays, the coding sequence of RH3, BFA1, AGO2, and AGO7 were recombined from pENTR vectors into pFH or pMDC32 vector using Gateway LR Clonase II Enzyme Mix (Invitrogen), resulting in the generation of p35S::RH3-FLAG, p35S::BFA1-FLAG, p35S::HA-AGO2, and p35S::HA-AGO7 constructs^86,87,88. For transient expression assay, the coding sequence of RH3, AGO2, AGO7, DCL4 and TOC64-III were recombined from pENTR vectors into pEarleyGate-102, pEarleyGate-104 or pBI121-RFP by Gateway LR Clonase II Enzyme Mix (Invitrogen), resulting in the generation of p35S::RH3-CFP-HA, p35S::YFP-AGO2, p35S::YFP-AGO7, p35S::DCL4-RFP, and p35S::TOC64-III-CFP-HA constructs. dsRed-TYMV-140K vector was constructed by recombinant PCR to ligate TYMV-140K into pGDR vector using SoSoo cloning kit (Tsingke). For the BiFC assay, the coding sequence of GST, RH3, BFA1, CNX and AGO2 were recombined from pENTR vector into pSITE-cEYFP-C1, pSITE-cEYFP-N1, pSITE-nEYFP-N1 or pSITE-nEYFP-C1, gateway-compatible BiFC vectors, using Gateway LR Clonase II Enzyme Mix (Invitrogen), resulting in the generation p35S::GUS-cEYFP-C1, p35S::RH3-cEYFP-N1, p35S::BFA1-cEYFP-N1, p35S::CNX-nEYFP-N1, and p35S::nEYFP-AGO2-C1 constructs⁸⁹. pENTR-GUS was provided by Gateway LR Clonase II Enzyme Mix (Invitrogen). p35S::nEYFP was constructed by inducing a stop codon in the region between nEYFP and AGO2 to p35S::nEYFP-AGO2-C1 construct. p35S::cEYFP was constructed by inducing a stop codon in the region between cEYFP and GUS to p35S::cEYFP-GUS-C1 construct. p35S::DCL1-YFP and pEarleyGate100-MIR393b were described previously^12,85. For the generation of pRH3::RH3-cEYFP, a 3026 bp RH3 promoter and RH3 genomic DNA fragment was amplified from Col-0 genomic DNA, and a cEYFP fragment with stop codon was recombined to the C terminal of RH3 by recombination PCR. The RH3 promotor-RH3-cEYFP fragment was cloned into pENTR vector with pENTR^TM/SD/D-TOPO^TM Cloning kit (Invitrogen) according to the manufacturer’s instructions, and further ligated to pV73 vector using Gateway LR Clonase II Enzyme Mix (Invitrogen). Similarly, pAGO2::HA-nEYFP-AGO2 was generated by recombining a 1576 bp native promotor with HA-nEYFP-AGO2 and ligating into pCAMBIA 1305 vector using using ClonExpress Ultra One Step Cloning Kit (Vazyme). p35S::RFP-HDEL, p35S::YFP-HDEL and p35S::CFP-HDEL⁴², p35S::RDR6-RFP⁹⁰ and Mit-GFP, Nuc-GFP, and Chl-GFP⁴⁰ were also described previously.

For prokaryotic expression, RH3-HA and GST-HA fragments were amplified with primers containing HA tag sequences using pENTR-RH3 and pENTR-GST as templates. The amplified RH3-HA or GST-HA fragments were inserted into pET-32a and digested with NcoI and XhoI, resulting in the generation of pET-32a-RH3-HA and pET-32a-GST-HA. Codon-optimized FLAG-AGO2 was synthesized (GenScript) and inserted into pET-28a digested with NdeI and SalI. RH3D70, RH3(1-250), RH3(251-500), and RH3(501-748) were amplified from pET-32a-RH3 using sets of primers: 5′ forward containing an NcoI digestion site and 3′ reverse primer containing HA tag sequence and XhoI digestion site, respectively. RH3D70Δ(251-350), RH3D70Δ(351-450), RH3D70Δ(451-550), RH3D70Δ(551-650), RH3D70Δ(651-748), RH3D70Δ(451-500), RH3D70Δ(501-550), RH3D70Δ(651-700), RH3D70Δ(701-748), RH3D70 K459A R460A R463A R467A, RH3D70 E466A D468A E474A, and RH3D70 D483A E486A D490A ligated pET-32a vectors were constructed by recombinant PCR followed by DpnI digestion with pET-32a-RH3D70 as template. pET-32a-/pFH-RH3K459A R460A R463A R467A (mRH3) were constructed by recombinant PCR followed by DpnI digestion with pET-32a-RH3 or pFH-RH3 as template.

The p35S::amiR-RH3 construct was generated by overlapping PCR using the pri-miR319a backbone and cloned into pEarleyGate-100 as described previously¹². A 6351 bp genomic DNA fragment containing the RH3 promoter and coding sequence was amplified from Col-0 genomic DNA and cloned into pENTR vector with pENTR^TM/SD/D-TOPO^TM Cloning kit (Invitrogen) according to the manufacturer’s instructions. The 6351 bp fragment was then cloned into pEarleyGate-302 using Gateway LR Clonase II Enzyme Mix (Invitrogen), resulting in the generation of pRH3::RH3-FLAG construct.

For in vivo protein expression-related clones, RH3-HA and GST-HA fragments were amplified with primers containing SP6-TMVΩ at 5′ end of RH3/GST as forward primer and 3′ RH3/GST with HA as reverse primer, and ligated to pEASY-Blunt vector. FLAG-AGO2 was amplified with primers containing TMVΩ and FLAG at 5′ end of AGO2 as forward primer and 3′ end of AGO2 as reverse primer, and ligate to pEASY-Blunt vector. Further mutant PCR was done to remove the sequence between T7 promoter and PCR product, and the vectors were ligated in E. coli. These vectors are digested by BstXI before performing transcription. All primers used for constructing these plasmids are listed in Supplementary Data 3.

Bacterial infection and PR1 protein extraction

Pseudomonas syringae pv. Tomato (Pst) DC3000 carrying the avrRpt2 effector was cultured at 28°C in KB medium supplemented with 50 µg/ml rifampicin and 50 µg/ml kanamycin. Bacteria treatments were performed as previously described¹². Briefly, 4-week-old plants were inoculated with Pst (avrRpt2) strain at a concentration of OD₆₀₀ = 0.02. The infiltrated leaves were harvested at 12 h post-inoculation (hpi). Before collecting intercellular wash fluid (IWF), equal amounts of leaves from different plants were vacuumed with 20 mM phosphate buffer (KH₂PO₄ and K₂HPO₄, pH 7.4) for 10 min, as previously described⁹¹. The leaves were then centrifuged at 1,500 g for 5 min to collect IWF. The PR1 protein in the total and intercellular fluid was examined using α-PR1 polyclonal rabbit antibody (1:4,000)¹², with Tubulin serving as a loading control.

Viral infection

The A. tumefaciens strain GV3101 carrying the genome of TYMV⁶⁰ was cultured at 28°C in LB medium supplemented with 25 µg/ml rifampicin, 50 µg/ml gentamicin, and 25 µg/ml kanamycin. The inoculations of TYMV were performed as described^53,92. Briefly, the A. tumefaciens GV3101 carrying TYMV constructs were resuspended with buffer [10 mM MgCl₂, 0.15 mM Acetosyringone (AS), and 10 mM 2-Morpholinoethanesulfonic Acid (MES)] to a final concentration of OD₆₀₀ = 0.5, respectively. For virus inoculation, 4-week-old A. thaliana plants were used, and systemic tissues were harvested at 14 dpi for protein and RNA analyses, as detailed in the “Plant material” section. For transient expression in A. thaliana seedling, 3-day-old Col-0 WT were used for virus inoculation and performed as previously described⁹³, and the inoculated leaves were collected at 4-5 dpi for fluorescence microscopy or Western blot analysis.

Transient expression in Arabidopsis seedling

The transient expression of p35S::CFP-HEDL, p35S::RH3-cYFP, p35S::nYFP-AGO2, pRH3::RH3-cEYFP, pAGO2::HA-nEYFP-AGO2 and p35S::dsRed-TYMV-140K in Arabidopsis seedling were performed as previously described⁹³. Briefly, the A. tumefaciens GV3101 carrying target construct were resuspended with buffer [5% sucrose, 0.2 mM AS, and 10 mM MES] to a final concentration of OD₆₀₀ = 2.0, respectively, and used to infiltrate 3-day-old Col-0 WT. The infiltrated plants were cultured for 3-5 days before being used for further experiments.

Protoplast isolation and transfection

Protoplasts were isolated as previously described⁹⁴. Briefly, leaves from 4-week-old plants were cut into 0.5-1 mm strips with razor blades. The leaf strips were then incubated in enzyme solution [1.5% cellulase R10, 0.4% macerozyme R10, 0.4 M mannitol, 20 mM KCl, 20 mM MES (pH 5.7), 10 mM CaCl₂, 0.1% BSA] in dark for 4 h with gentle shaking (40 rpm) at 37 °C. The resulting protoplasts were filtered and washed with the equal volume of pre-cold W5 solution [154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, and 2 mM MES (pH 5.7)] for three times. After centrifugation at 100 g for 2 min at 4 °C, the protoplasts were suspended in pre-cold W5 solution and incubated on ice for 30 min.

For PEG-mediated transfection, the protoplasts were resuspended in MMG solution [(0.4 M mannitol, 15 mM MgCl₂, 4 mM MES (pH 5.7)]. The transfection was performed by gently mixing 200 µl protoplasts with the desired construct (20 µg) and 220 µl PEG solution (40% PEG4000, 100 mM CaCl₂, 0.2 M mannitol) for 10 min at room temperature. The transfected protoplasts were then washed three times with W5 solution. After the washing steps, the transfected protoplasts were suspended with 1 ml W5 solution and cultured under white light at room temperature. CFP/GFP fluorescence in transfected protoplasts was observed 10 to 15 h after transfection using the confocal microscope. N. benthamiana protoplasts used for transient protein expression were imaged directly without transfection.

Microsome isolation

To isolate microsomes for determining the association of RH3 and AGO2 with the ER, the following procedure was performed with minor modifications²⁵. First, 2 g of seedlings were ground in 5 ml microsome isolation buffer [100 mM Tris-HCl (pH 7.5), 5 mM EGTA, 15 mM MgCl₂, 5 mM DTT, 0.3 M sucrose, and a tablet of cOmplete proteinase inhibitor per 50 ml] at 4 °C. The resulting cell lysate was filtered through two layers of miracloth, and a 100 µl aliquot was taken as the total proteins. Next, the cell lysate was centrifuged at 8000 g for 5 min twice to remove debris, resulting in the isolation of the cytosolic proteins. The soluble fraction resulting in the isolation was then performed ultracentrifugation for 30 min at 100,000 g, 4 °C. The resulting supernatant was collected for further Western blot assays and labeled as the “supernatant” fraction. The pellet obtained after ultracentrifugation was suspended in 5 ml isolation buffer, and applied onto a sucrose gradient (2.5 ml 20% sucrose/2.5 ml 60% sucrose). The sucrose gradient was then centrifuged at 100,000 g for 1 h at 4 °C. Microsomes were recovered from the interface between the two sucrose layers and precipitated by centrifugation at 100,000 g for 30 min at 4 °C.

For the isolation of microsomes for AGO2 immunoprecipitation, a similar procedure was followed with some modifications⁹⁵. The leaves were grounded in microsome isolation buffer at 4 °C. The cell lysate was filtered through two layers of miracloth, and subjected to centrifugation at 8000 g for 5 min twice to remove debris. The resulting soluble fraction was then performed ultracentrifugation for 30 min at 100,000 g at 4 °C. Both the supernatant and pellet (microsomes) were collected for further experiments.

Construction of sRNA library and data analysis

Total RNA (30 µg) was resolved on 15% urea-PAGE gel, and the 15-30 nt regions were cut from the gel. The sRNAs were recovered by soaking the smashed gel in 0.3 M NaCl overnight, followed by precipitation with ethanol⁹⁶. Then the total sRNAs and sRNAs extracted from AtAGO2 immunoprecipitation were used for sRNA libraries construction using the NEBNext® Small RNA library Prep Set for Illumina® (NEB, E7300S). Two biological replicates of sRNA libraries were constructed and sequenced using an Illumina Hiseq2500 SE50 V4 at Bionova Beijing.

The reads from the Illumina sRNA-seq were processed to remove the 3′ adaptor sequences and low-quality bases using fastp. Clean reads that aligned to rRNA, tRNA, and snoRNA were removed using Bowtie v1.3.0⁹⁷, and counts of reads mapped to 45S rRNA region were recorded as normalized background. sRNAs range in length from 18 to 30 nt with perfect matches to the Arabidopsis genome sequences (TAIR10 version) were used for further analysis and normalization. To compare sRNAs abundance in rh3-4 and Col-0 plants, the total sRNA library samples were normalized by calculating their expression levels (reads per million, RPM). sRNAs from the AtAGO2 IP sRNA library were normalized using the number of total 45S rRNA reads²⁵.

miRNA annotation file was downloaded from miRBase v21, and BEDtools⁹⁸ were used to quantify miRNAs, miRNA*s, phasiRNAs, and ta-siRNAs abundance. Adaptor-free reads within 21 and 22 nt were aligned for these regions, allowing for 1-nt shift on either the 5′ or 3′ end. The TAS gene regions (TAS1a, 1b, 1c, TAS2, TAS3a, 3b, 3c, and TAS4) were counted and summed separately for each size class (21 nt, 22 nt). A fold change of normalized counts between rh3-4 and Col-0 greater than 1 and a mean number of reads greater than 10 were considered as significant.

RT-qPCR analysis

Total RNA was extracted from leaves of 4-week-old plants using the RNAprep Pure Plant Kit (Tiangen) according to the manufacturer’s instructions. Purified total RNA (1 µg) was used for reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). Real-time PCR were performed using TB Green™Premix Ex Taq™ kit (Takara). Primers are listed in Supplementary Data 3. ACTIN2 was used as the internal control for normalization.

Northern blot

For low molecular weight RNAs, total RNAs or sRNAs extracted from AtAGO2 co-immunoprecipitation beads were separated on 14% urea-polyacrylamide denaturing gels. The gel was transformed to a Hybond membrane NX (GE healthcare) at 14 V overnight. The membrane was chemically crosslinked at 60 °C for 2 h and then incubated at 85 °C for 2 h⁹⁹. The membrane was pre-incubated with PerfectHyb™ Plus Hybridization Buffer liquid (Sigma-Aldrich) for 30 min, then hybridized overnight at 37 °C with ³²P-labeled DNA probes. For the detection of endogenous sRNAs, DNA probes reverse complementary to the sRNAs sequences were labeled with γ-³²P ATP by T4-polynucleotide kinase. For detection of vsiRNAs, PCR fragment of the CP in TYMV were used to synthesize the α-³²P-labeled probes randomly labeled by Prime-a-Gene® Labeling System (Promega) kit^100,101.

For high molecular weight RNAs, freshly infected systemic leaves infected with TYMV (0.1 g) were grounded in liquid nitrogen and RNAs were extracted with hot phenol, and precipitated with 4 M LiCl. 10 µg TYMV RNA was loaded onto a formaldehyde gel and transferred to Hybond membrane N⁺ (GE healthcare) overnight using 20 × SSC (3 M NaCl, 300 mM Sodium Citrate) transfer buffer. Methylene blue staining strip was used as a loading control. The membrane was then UV crosslinked at 1.2 × 10⁵ µJ/cm² for 2 min. After pre-incubation with PerfectHyb™ Plus Hybridization Buffer liquid (Sigma-Aldrich) for 30 min, the membrane was then hybridized overnight at 60 °C with α-³²P-labeled DNA probes. For virus gRNA detection, PCR fragment of the CP in TYMV were used to synthesize the α-³²P-labeled probes randomly labeled by Prime-a-Gene® Labeling System (Promega) kit^100,101. After hybridization, the membrane was washed with a buffer contains 2 × SSC and 0.025% SDS. Auto-radiography of the membrane was performed using a Typhoon Scanner. Images were quantified with ImageJ. Locked Nucleic Acid probes was used to enhance the sensitivity of Northern blot¹⁰². Sequences of probes and primers are listed in Supplementary Data 3.

Fluorescence in situ hybridization

In situ hybridization experiments were performed as previously described¹⁰³. Digoxin-labeled RNA probes reverse complementary to miR393b*, miR171 or U6, and biotin-labeled RNA probes reverse complementary to random fragment of chloroplast 16S rRNA were synthesized by BGI. Hypocotyl of 7-day-old Col-0 seedlings were collected and fixed by fixation buffer (120 mM NaCl, 2.7 mM KCl, 0.1% Tween-20, 80 mM EGTA, 5% formaldehyde, and 10% DMSO) under vacuum for 30 min at room temperature. The samples were then dehydrated in a series of solution: 100% methanol for 30 min, 100% ethanol for 30 min, 50% xylene in ethanol for 30 min, 100% ethanol for 30 min, and 100% methanol for 30 min. The samples were further digested with 125 µg/ml proteinase K for 30 min, and incubated with 0.16 M EDC at 60 °C for 2 h. Before hybridization, the samples were rinsed with fixation buffer without formaldehyde and pre-incubated with PerfectHyb™ Plus hybridization buffer for 1 h. Hybridization buffer containing 10 µl of 1 µM DIG-labeled miR393b*, miR171, or U6 probes and 5 µl of 1 µM biotin-labeled 16S rRNA probes were added to the samples. Hybridization was performed overnight at 50 °C. Immunodetection was carried out using Alexa FluorTM 594-conjugated streptavidin (1:200, Invitrogen) and FITC anti-digoxigenin antibody (1:200, Abcam) overnight at 4 °C. Finally, the samples were mounted in Vectashield mounting medium with DAPI and analyzed using a Zeiss LSM710 confocal laser-scanning microscope.

Immunofluorescence analysis of viral dsRNA

To detect TYMV dsRNA, A. thaliana seedling infected with TYMV were collected at 5 dpi for protoplast isolation. Immunofluorescence was performed as described previously¹⁰⁴. Briefly, protoplasts were incubated with 1 volume of fixing solution (4% paraformaldehyde, 0.25 M mannitol, and 50 mM PBS) for 15 min at room temperature. The fixed protoplasts were then washed three times with PBS (10 min each). The fixed protoplasts were transferred onto a cover slide and incubated with 5% BSA in PBS for 20 min at room temperature. The samples were further incubated with monoclonal antibody J2 (1:200, Scicons, 10010500) as the primary antibody and then treated with Alexa Fluor 594-conjugated secondary antibody (1:500, Invitrogen, A-11005). The samples were washed three times with PBS (10 min each) before processing to detect the fluorescence.

Immunoprecipitation and mass spectrometry

Four-week-old pAGO2::HA-AGO2 in ago2-1 and p35S::3HA-GFP in Col-0 plants were harvested and ground in liquid nitrogen. p35S::3HA-GFP in Col-0 was served as control. Total proteins were extracted from 3 g ground powder and suspended in 15 ml immunoprecipitation buffer [20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM MgCl₂, 5 mM DTT, 0.5% Tween-20, 1 tablet of cOmplete protease inhibitor per 50 ml] for 10 min at 4 °C. After centrifugation and filtration, the supernatant was immunoprecipitated with 20 μl anti-HA magnetic beads (Lablead) at 4 °C for 2 h. Followed by 4 times washing with washing buffer [250 mM NaCl, 5 mM MgCl₂, 5 mM DTT, 20 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 1 pellet of cOmplete protease inhibitor per 50 ml], proteins copurified with HA-AGO2 or HA-GFP were collected by boiling the beads at 95 °C for 10 min with SDS loading buffer. The pulldown proteins were resolved with 10% SDS-PAGE and the gel band pieces of three biological replicates were cut out and send to Analytical Instrumentation Center of Peking University for Mass spectrometry assay.

The mass spectrometry was done as described previously¹⁰⁵. Protein samples were digested using the endoproteinase trypsin enzyme. The digested peptides were extracted twice with 5% formic acid/50% acetonitrile. The extracted peptides were vacuum-centrifuged to dryness and resuspended in 0.1% Formic acid in water prior to LC-MS/MS analysis. For LC-MS/MS analysis, the samples were reconstituted in 0.2% formic acid, loaded onto a 100 μm × 2 cm pre-column and separated on a 75 μm × 15 cm capillary column with laser-pulled sprayer. The peptides were separated by the following HPLC gradient: 5-35% B in 60 min, 35-75% B in 4 min, then held at 75% B for 10 min (A = 0.1% formic acid in water, B = 0.1% formic acid in acetonitrile) at a flow rate of 300 nL/min. The eluted peptides were sprayed into a Velos Pro Orbitrap Elite mass spectrometer (Thermo Scientific, USA) equipped with a nano-ESI source. The mass spectrometer was operated in data-dependent mode with a full MS scan (375–1600 M/z) in FT mode at a resolution of 120000 followed by CID (Collision Induced Dissociation) MS/MS scans on the 10 most abundant ions in the initial MS scan.

The acquired mass spectrometry data was further processed using Thermo Proteome Discoverer 2.4 to align with a database downloaded from TAIR (https://www.arabidopsis.org/, version 2021-07-11). Protein differential analysis was processed with DEP pipeline¹⁰⁶. Briefly, protein abundance matrices from AGO2-IP and GFP-IP were combined and proteins that are only present in one repetition were removed. Variance-stabilized normalization (VSN) was performed as suggested by the DEP pipeline. Left-censored imputation method was used to impute missing values for protein with ‘0’ LFQ intensity. In details, gaussian distribution centered around a minimal value method integrated with DEP was used. Differentially proteins between AGO2-IP and GFP-IP were identified by protein-wise linear models and empirical Bayes statistics. Volcano plot were drawn by R package ggplot2. Proteins in AGO2-IP with p-value < 0.05 and fold change >2 were considered as AGO2 associated proteins, and highlighted in pink box (Supplementary Fig. 2a). The proteins that potentially interacts with AGO2 were listed in Supplementary Fig. 2b with proteins predicted to locate in chloroplast labeled in red.

Phylogenetic analysis

For the phylogenetic analysis of RH3 protein, the RH3 ortholog proteins in 24 plant species (identified or predicted) were used. These sequences were aligned using ClustalW as implemented within MEGA X. The maximum likelihood bootstrap consensus tree was generated using the JTT matrix-based model with discrete gamma distribution by MEGA X from 500 bootstraps^107,108.

Immunoprecipitation and Co-immunoprecipitation

Immunoprecipitation was performed as previously described¹². For immunoprecipitation with Arabidopsis, 3 g of leaf tissue collected from 4-week-old plants were grounded with liquid nitrogen and resuspended in 15 ml extraction buffer [20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM MgCl₂, 5 mM DTT, 0.5% Tween-20, 1 tablet of cOmplete protease inhibitor per 50 ml]. The protein extracts were then cleared by pre-incubating with 20 μl protein A beads (Invitrogen) for 1 h, and then incubated with 7 μl/g anti-AGO2 antibody (Agrisera, AS132682) overnight at 4 °C. After that, the samples were incubated with 50 µl protein A beads for 1 h and washed three times with washing buffer [20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM MgCl₂, 5 mM DTT, 0.5% Triton X-100, 1 tablet of cOmplete protease inhibitor per 50 ml]. The washed beads were resuspended for further analysis.

For AGO2 immunoprecipitation in supernatant and microsome fractions, purified microsomes were resuspended in 2 ml buffer (a mixture of extraction buffer and microsome isolation buffer in a 1:1 ratio), and 1 ml of supernatant was diluted in a 1:1 ratio with extraction buffer. The ratio of dilution was tested ahead to make sure the level of AGO2 protein in protein extracts is comparable in supernatant and microsome. For AGO2 immunoprecipitation in isolated microsome of Col-0 and rh3-4, purified microsomes were resuspended in 2 ml buffer (extraction buffer and microsome isolation buffer mixed with a ratio of 1:1). The protein extracts were cleared by pre-incubating with 5 μl protein A beads (Invitrogen) for 1 h and then incubated with 2 µl/g anti-AGO2 antibody (Agrisera, AS132682) overnight at 4 °C. After that, the products were incubated with 15 µl protein A beads for 1 h, followed with three washes. The washed beads were resuspended for further analysis.

Protein expression and purification

To overcome the difficulty of transient expressing AGO2 in the E. coli system, codon optimization was performed on the AGO2 nucleotide sequence. The optimized codon was shown in Supplementary Fig. 4. For the purification of His-tagged recombinant proteins in E. coli, the plasmids encoding 6 × His-FLAG-AGO2, 6 × His-TrxA-GST-HA were transformed into BL21 competent cells (DE3), while the plasmids encoding 6 × His-TrxA-RH3-HA and its variants were transformed into Rosetta competent cells (DE3). The transformed cells were grown in LB at 37 °C until reaching an optical density at OD₆₀₀ = 0.6. Protein expression was then induced by adding induced with 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 16 °C overnight. Subsequent protein purifications were carried out at 4 °C.

The bacterial cells were collected by centrifugation and resuspended in lysis buffer (50 mM phosphate buffer, pH 8.0, 300 mM NaCl, 1% Triton X-100, and 1 tablet of cOmplete EDTA-free protease inhibitor per 50 ml). The cells were then sonicated. After centrifugation, the cleared lysates were loaded onto a HisTrap column with Ni-nitrilotriacetic acid (NTA) resin and washed with 5 ml washing buffer (50 mM phosphate buffer, pH 8.0, 300 mM NaCl, 5 mM imidazole) for three times. The purified proteins were eluted with elution buffer (50 mM phosphate buffer, pH 8.0, 300 mM NaCl, and 300 mM imidazole). The purified AGO2, RH3 and its variants proteins were supplemented with 50% glycerol. The proteins were then frozen using liquid nitrogen and stored at -80 °C.

FLAG pull-down assay

10 µg purified 6 × His-FLAG-AGO2 protein in binding buffer (100 mM NaCl, 20 mM Tris-HCl, pH 7.5, and 0.05% NP-40) was incubated with anti-FLAG M2 agarose beads (Sigma-Aldrich) for 1 h at 4 °C. The beads were washed three times with binding buffer. After that, the washed beads were incubated with 30 µg 6 × His-GST-HA or 6 × His-RH3-HA, or its variants proteins, respectively. The mixture was rotated at 4 °C for 4 h. After the incubation, beads were washed six times with binding buffer. The beads were boiled at 95 °C for 10 min with SDS loading buffer [25 mM Tris-HCl (pH 6.8), 1% SDS (W/W), 1 mM DTT, 10% glycerol (V/V), 0.01% bromophenol blue]. The eluted proteins were separated on a 10% SDS-PAGE gel and stained with Coomassie brilliant blue R-250.

In vitro RISC assembly assay

An in vitro translation was performed using BYL as described previously with minor modifications^51,109. Briefly, RH3, mRH3, and GST mRNA was transcribed using SP6 High Yield Message Maker Kit and tailed by A-Plus™ Poly(A) Polymerase Tailing Kit. Subsequently, 1 µg of RH3-HA, mRH3-HA, and GST-HA mRNA were individually translated in a 10 µl BYL translation mixture at 25 °C for 1 h, respectively. Additionally, 1 µg of linearized AGO2 DNA was transcribed and translated using T_NT® Coupled Wheat Germ Extract System (Promega) according to the manufacturer’s protocol. After the translation of RH3, mRH3, and GST mRNA in BYL, the AGO2 translation mixture was mixed with the respective RH3, mRH3 or GST samples. sRNA duplexes (100 nM) with 5′-γ-³²P labeled passenger strands were added to the translation mixture and incubated at 25 °C for 1 h. 2 µl aliquots of the reaction mixture were transferred into new tubes, and used for Western blots analysis. Anti-FLAG antibody (1:2000, Easybio, BE7003) and anti-HA antibody (1:2000, Easybio, E2061) were used to detect the expression of AGO2 and RH3/mRH3/GST, respectively. 4 µl 5 × Stopping dye solution [500 mM Tris, 450 mM boric acid, 50 mM EDTA, 1 mg/ml bromophenol blue, 1 mg/ml xylene cyanol, 50% (v/v) glycerol] was added to the translation mix and run on native 6% PAGE gel. Radio labeled bands were detected using an image analyzer (Typhoon FLA7000).

Immunofluorescence analysis of protein localization

Immunofluorescence was performed as previously described⁸⁵. pRH3::RH3-FLAG in rh3-4, pAGO2::HA-AGO2 in ago2-1 and p35S::GFP-HDEL transgenic plants were used for this assay. 1-week-old seedlings grown on solid 1/2 MS medium were fixed using 4 % paraformaldehyde (Sigma) under vacuum for 60 min at room temperature. For TYMV infected samples, 3-week-old seedlings were used for TYMV inoculation, and systemic leaves were harvested at 7 dpi for fixation. For HA or AGO2 immunofluorescence, cell spreads were incubated with anti-HA (1:200, Abcam, ab18181) or anti-AGO2 (1:200, Agrisera, AS132682) antibody overnight. Goat anti-mouse antibody labeled with Dylight 488 (1:500, Abbkine, A23210) and Dylight 594 labeled goat anti-rabbit antibody (1:1000, Abbkine, A23420) was used as secondary antibody for HA and AGO2, respectively. For dual immunolocalization, cell spreads were first incubated with primary anti-FLAG antibody (1:500, Sigma-Aldrich, F1804) in blocking buffer (2% BSA in 1 × PBS, pH 7.4) at 4 °C overnight Subsequently, the spreads were incubated with either anti-Toc34 (1:200, PhytoAB, PHY1264S) or anti-AGO2 (1:200, Agrisera, AS132682) antibody for 7-8 h at 4 °C. Finally, the spreads were incubated with Dylight 488 labeled goat anti-mouse antibody (1:500, secondary antibody for FLAG) and Dylight 594 labeled goat anti-rabbit antibody (1:1000, secondary antibody for Toc34 and AGO2). Col-0 or ago2-1 fluorescence was used as a negative control. Images were captured using a Zeiss LSM 710 confocal microscope.

Antibodies

Proteins examined by Western blot were probed by α-AGO2 (1:1000, Agrisera, AS132682), a-Tubulin (1:5000, Easybio, BE0031), α-FLAG (1:2000, Easybio, BE7003), α-HA (1:2000, Easybio, BE2061), α-AGO1 (1:2000, Agrisera, AS09527), α-AGO4 (1:2000, Agrisera, AS09617), α-GFP (1:2000, Easybio, BE2001), α-Toc34 (1:2000, PhytoAB, HY1264S), α-H3 (1:2000, Easybio, BE7004), α-PEPC (1:2000, Agrisera, AS09458), α-Calnexin (1:2000, Agrisera, AS122365), α-Rbcl (1:2000, Agrisera, AS03037). Secondary antibodies were goat anti-rabbit IgG (1:5000, Easybio, BE0101) and goat anti-mouse IgG (1:5000, Easybio, BE0102). α-PR1 (1:4,000)¹², α-MEMB12 (1:2000)¹², α-CSD2 (1:4,000)²⁶ have been previously described, and their specificities were validated in paper listed above. Mouse monoclonal α-RH3 (1:2000) was produced by BGI, China, and its specificity was validated in Supplementary Fig. 9c.

Confocal fluorescence microscopy

Agro-infiltrated N. benthamiana leaf tissues, transfected protoplast, viral dsRNA, and immunolabelled samples were observed with a Zeiss LSM-710 confocal microscope equipped with Zeiss Zen 2012 software. The following excitation/emission wavelengths were used for specific fluorophores: 405 nm/454-490 nm for CFP, 488 nm/500-540 nm for GFP, 488 nm/510-530 nm for YFP, 561 nm/580-630 nm for RFP/mCherry, and 633 nm/665-721 nm for chlorophyll auto-fluorescence. For the colocalization of RH3-FLAG and AGO2 in immunofluorescence assay, Z stack sequential scanning was performed. Images were digitally captured with a Zeiss Axiocam camera at a 1024- by 1024-pixel resolution and processed with Zen 2.5 2018.

Immunoelectron microscopy

Immunoelectron microscopy was performed as described previously with minor modifications¹¹⁰. Briefly, 3-week-old p35S::RH3-CFP-HA in rh3-4 transgenic plants were cut into pieces and fixed in a fixation buffer (0.1% glutaraldehyde, 3% paraformaldehyde, 0.1 M phosphate, pH 7.2) for 3 h at 4 °C. Samples were then dehydrated in a gradient ethanol series (50%, 70%, 90%, and 100%). The tissue samples were then embedded in K4M resin and polymerized by UV light.

For the immuno-labeling of RH3-HA, a monoclonal α-HA antibody (1:100, Abcam, ab18181) was used as the primary antibody. A gold-coupled anti-mouse antibody (1:200, Sigma-Aldrich, G7652) was used as secondary antibody. The sections were post-stained with aqueous uranyl acetate/lead citrate and examined using a Hitachi H-7650 transmission electron microscope with a CCD camera operating at 80 kV (Hitachi High- Technologies Corporation).

Image intensity analysis

ImageJ version 1.8.0 was used to quantify the Northern blot and the western blot images (https://imagej.nih.gov/ij/index.html). Briefly, the background of images was subtracted with default threshold and integrated densities of the bands were determined. Zeiss Zen (blue edition) with auto threshold was used for the quantification of fluorescence intensity.

Statistics and reproducibility

Statistical parameters were shown in the figure legends. Two-tailed Student’s t tests were used to determine the difference between two groups. P-values were calculated and the cut off for significance was 0.05. P values were presented on the figure. ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.001, ****P < 0.0001. No statistical method was used to predetermine sample size. No data were excluded from the analyses.

Reporting summary

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

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD051119^111,112. The date set of sRNA deep sequencing have been deposited into NCBI under accession numbers PRJNA1096839. The analysis on the AGO2 IP-MS assay and sRNA deep sequencing are available in Supplementary Data 1 and 2. Oligos used in the study are reported in Supplementary Data 3. Source data are provided with this paper.

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Acknowledgements

We thank Dr. Hailing Jin, Dr. Guodong Ren, Dr. Yifan Lii, Dr. Manabu Yoshikawa, Dr. Masayuki Ishikawa and Dr. Shuzhen Men for helpful comments. We are grateful for Dr. James C. Carrington, Dr. Jean-François Laliberté, Dr. Fernando Ponz, Dr. Manabu Yoshikawa, Dr. Xiaofeng Cao, Dr. Yijun Qi, Dr. Lianwei Peng, Dr. Rongchen Lin, Dr. Fangfang Li, Dr. Xian-bing Wang, Dr. Mingmin Zhao, Dr. Taiyun Wei and Dr. Manling Shi for sharing materials. This work is supported by National Key Plan for Scientific Research and Development of China (2022YFD1400800 XZ), National Natural Science Foundation of China (32325042 XZ and 32090012 QL), Program of CAS (ZDBS-LY-SM027), National Natural Science Foundation of China (31700233 JH, 91954105 XZ and 91540116 XZ), Hainan Seed Industry Laboratory (B21HJ0104 XZ and B23CJ0105 XZ), Open Research Fund Program of State Key Laboratory of Integrated Pest Management (IPM2207 JW), Initiative Scientific Research Program, and Institute of Zoology, Chinese Academy of Sciences (2023IOZ0203 XZ and 2024IOZ0106 XZ).

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These authors contributed equally: Juan Huang, Juan Du.

Authors and Affiliations

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
Juan Huang, Juan Du, Yan Liu, Lu Lu, Yanzhuo Xu, Jianfei Shi, Qing Liu, Qi Li, Yang Liu, Yaqiu Chen, Meng Du, Yiming Zhao, Jinfeng Chen, Feng Cui & Xiaoming Zhang
College of Life Science, University of Chinese Academy of Sciences, Beijing, 100049, China
Juan Huang, Juan Du, Yanzhuo Xu, Jianfei Shi, Qing Liu, Yang Liu, Yiming Zhao, Feng Cui & Xiaoming Zhang
Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan, 250100, China
Yan Liu
College of Life Sciences, Hebei University, Baoding, Hebei, 071002, China
Liangxiao Huo, Weiran Wang, Chenxi Ding & Liya Wei
Vector-borne Virus Research Center, Fujian Province Key Laboratory of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
Jianguo Wu
Department of Ecology and Evolutionary Biology, University of Connecticut, 75 North Eagleville Road, Unit 3043, Storrs, CT, 06269, USA
Yao-Wu Yuan
SUSTech-PKU Institute of Plant and Food Science, Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
Ruixi Li
Hainan Seed Industry Laboratory, Sanya, 572025, China
Xiaoming Zhang

Authors

Juan Huang
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Juan Du
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Yan Liu
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Lu Lu
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Yanzhuo Xu
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Jianfei Shi
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Qing Liu
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Qi Li
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Yang Liu
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Yaqiu Chen
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Meng Du
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Yiming Zhao
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Liangxiao Huo
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Weiran Wang
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Chenxi Ding
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Liya Wei
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Jianguo Wu
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Yao-Wu Yuan
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Jinfeng Chen
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Ruixi Li
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Feng Cui
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Xiaoming Zhang
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Contributions

Conceptualization, X.Z.; Methodology, J.H., and J.D.; Investigation, J.H., J.D., Yan.L., Y.X., Y.C., Q.Liu., M.D., Yang.L., Y.Z., L.H., W.W., C.D.,; Formal Analysis, L.L, J.S.; Writing – Original Draft, X.Z., F.C., J.H., and J.D.; Writing –Review & Editing, X.Z., F.C., J.C., Y-W.Y., R.L., J.W., L.W., and Q.Li.; Funding Acquisition, X.Z., and Q.Li.; Supervision, X.Z., F.C., and L.W.

Corresponding authors

Correspondence to Feng Cui or Xiaoming Zhang.

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Nature Communications thanks Gaoyuan Song, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Huang, J., Du, J., Liu, Y. et al. RH3 enhances antiviral defense by facilitating small RNA loading into Argonaute 2 at endoplasmic reticulum–chloroplast membrane contact sites. Nat Commun 16, 1953 (2025). https://doi.org/10.1038/s41467-025-57296-6

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Received: 06 December 2022
Accepted: 18 February 2025
Published: 25 February 2025
DOI: https://doi.org/10.1038/s41467-025-57296-6