1. Microbiology and Infectious Disease
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Exosomes mediate horizontal transmission of viral pathogens from insect vectors to plant phloem

  1. Qian Chen
  2. Yuyan Liu
  3. Jiping Ren
  4. Panpan Zhong
  5. Manni Chen
  6. Dongsheng Jia
  7. Hongyan Chen
  8. Taiyun Wei  Is a corresponding author
  1. Vector-borne Virus Research Center, Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, China
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Cite this article as: eLife 2021;10:e64603 doi: 10.7554/eLife.64603

Abstract

Numerous piercing-sucking insects can horizontally transmit viral pathogens together with saliva to plant phloem, but the mechanism remains elusive. Here, we report that an important rice reovirus has hijacked small vesicles, referred to as exosomes, to traverse the apical plasmalemma into saliva-stored cavities in the salivary glands of leafhopper vectors. Thus, virions were horizontally transmitted with exosomes into rice phloem to establish the initial plant infection during vector feeding. The purified exosomes secreted from cultured leafhopper cells were enriched with virions. Silencing the exosomal secretion-related small GTPase Rab27a or treatment with the exosomal biogenesis inhibitor GW4869 strongly prevented viral exosomal release in vivo and in vitro. Furthermore, the specific interaction of the 15-nm-long domain of the viral outer capsid protein with Rab5 induced the packaging of virions in exosomes, ultimately activating the Rab27a-dependent exosomal release pathway. We thus anticipate that exosome-mediated viral horizontal transmission is the conserved strategy hijacked by vector-borne viruses.

Introduction

Many devastating plant, animal, and human viral pathogens are horizontally transmitted by arthropod insects (Eigenbrode et al., 2018; Mayer et al., 2017). For example, rice stripe virus transmitted by planthoppers is a serious agricultural threat in rice-growing countries throughout Asia (He et al., 2017), and Zika virus (ZIKV) transmitted by mosquitoes has been a recent public health threat in the Americas (Liu et al., 2017). In general, arthropod-borne viruses (arboviruses) establish their initial infection in the insect midgut, which is disseminated to the hemolymph and ultimately spread into the salivary glands, from which virions are introduced into susceptible hosts together with saliva (Hogenhout et al., 2008; Wei and Li, 2016). Insect salivary gland cells are filled with abundant apical plasmalemma-lined cavities, where saliva is stored (Mao et al., 2017; Wei and Li, 2016). Arboviruses have to pass through the apical plasmalemma into insect salivary cavities, thereby moving with salivary flow to establish the initial infection in hosts (Mayer et al., 2017; Wei and Li, 2016). The secretory cells in the central region of salivary glands of the whitefly have been demonstrated to determine the transmissibility of begomoviruses (Wei et al., 2014). However, how arboviruses overcome cavity plasmalemma barriers for successful viral transmission is still poorly understood.

Frequently, arboviruses are sequestered into multivesicular bodies (MVBs) in the salivary glands of insect vectors (Ammar and Nault, 1985; Gray et al., 2014; Gray and Gildow, 2003; Janzen et al., 1970; Mao et al., 2017; Shikata and Maramorosch, 1965). MVBs are spherical endosomal organelles containing small vesicles formed by the inward budding of the limiting membrane into the endosomal lumen (Chahar et al., 2015). MVBs fuse with the plasma membrane to release the internal vesicles into the extracellular space in an exocytic manner (Chahar et al., 2015). These released vesicles are known as exosomes, which can transport various cellular regulatory RNAs from cell to cell, thereby receiving extensive research attentions (Chahar et al., 2015; Geisler and Coller, 2013; Janas et al., 2015; Kim et al., 2017). Thus, exosome biogenesis starts with the maturation from the early endosomes into late endosomes (Chahar et al., 2015). Several proteins are implicated in exosome biogenesis, including Rab GTPases and the tetraspanin CD63 (Kalluri and LeBleu, 2020). Endosomal Rab GTPases, such as Rab27a, Rab5, and Rab11, can regulate exosome biogenesis, trafficking, and/or release from cultured animal cells (Akers et al., 2013; Alenquer and Amorim, 2015; Zeigerer et al., 2015). Notably, early endosomal Rab5 is also detected within the MVBs and exosomes (Logozzi et al., 2009; Pisitkun et al., 2004; Vidal and Stahl, 1993). For example, the purified exosomes from hepatitis C virus-infected and uninfected hepatoma cells contain Rab5 (Ramakrishnaiah et al., 2013). In Arabidopsis, a model plant system, purified exosomes from leaf apoplasts also contain the Rab5 GTPase homologue ARA7 (Rutter and Innes, 2018). Indeed, the depletion of Rab5 strongly inhibits exosomal formation and secretion (Ostrowski et al., 2010). Furthermore, Rab27a functions in the docking of MVBs in the plasma membrane, thereby regulating exosome secretion (Ostrowski et al., 2010). For example, Herpes simplex virus-1 glycoprotein can interact with Rab27a to mediate the exosomal release pathway (Temme et al., 2010). Several arboviruses, such as dengue virus (DENV) and ZIKV, hijack exosomes to release virions, as observed in cultured mosquito cells (Martínez-Rojas et al., 2020; Reyes-Ruiz et al., 2019; Vora et al., 2018). Moreover, the secreted exosomes from arbovirus-infected mosquito cells can transmit viruses to cultured mammalian cells (Martínez-Rojas et al., 2020; Reyes-Ruiz et al., 2019; Vora et al., 2018). However, whether exosome-mediated viral spread occurs in infected host tissues or organs in vivo remains unknown. The final bottleneck in viral infection via insect vectors, that is, the cavity plasmalemma of salivary glands, is a principal determinant of the ability of an insect species to transmit a virus (Hogenhout et al., 2008; Wei and Li, 2016). Accordingly, we can reasonably regard the cavity plasmalemma of insect salivary glands as the plasma membrane of cultured infected cells. Thus, how arboviruses exploit exosomes to overcome the cavity plasmalemma barriers of vector salivary glands is a unique system for us to more easily address the role of viral release via the exosomal pathway in vivo.

Numerous plant viral pathogens of agricultural importance are persistently transmitted by piercing-sucking insect vectors, such as leafhoppers, planthoppers, and whiteflies, to plant phloem (Hogenhout et al., 2008; Wei and Li, 2016). Rice dwarf virus (RDV), the first recorded plant arbovirus transmitted by leafhopper vectors in 1895, causes a severe rice disease in Asia (Wei and Li, 2016). We have previously demonstrated that the plant reovirus RDV can be secreted by cultured leafhopper cells via infectious exosomes (Wei et al., 2008; Wei et al., 2009). Here, we report that RDV hijacks the exosomal release pathway to traverse the apical plasmalemma into saliva-stored salivary cavities, ultimately moving with exosomes into the phloem of rice plants to establish initial viral infection via leafhopper vectors.

Results

RDV traverses the apical plasmalemma into leafhopper salivary cavities via the exosomal release pathway

The salivary glands of the leafhopper Nephotettix cincticeps consist of a pair of principal and accessory salivary glands (Figure 1—figure supplement 1A). The principal salivary gland (PSG) contains six types of cells (I–VI) (Figure 1—figure supplement 1A), which are filled with apical plasmalemma-lined cavities (Figure 1A and Figure 1—figure supplement 1A). Immunofluorescence microscopy showed that RDV disseminated into the salivary cavities by traversing the apical plasmalemma in all types of PSG cells (Figure 1—figure supplement 1). We further used electron microscopy to visualize how RDV disseminated into the salivary cavities. In the cytoplasm of virus-infected type III cells, double-layered RDV particles (approximately 65 nm in diameter) were often engulfed into small vesicles within the MVBs at the periphery of salivary cavities (Figure 1B and C). The attachment of MVBs to the cavity plasmalemma drove the enation of the apical plasmalemma to form a membrane extrusion toward the cavity (Figure 1D and E). This extrusion was followed by the fusion of the apical plasmalemma of cavities with the plasma membrane of MVBs (Figure 1F–H). Finally, virus-containing small vesicles (exosomes) were released into the salivary cavities (Figure 1I and J). RDV-specific antibody specifically recognized viral particles within the MVBs or exosomes (Figure 1—figure supplement 2A–C). Virus-free exosomes measured 53–135 nm in diameter, but the packaging of viral particles enlarged exosomes to 110–302 nm in diameter in the salivary cavities (Figure 1K). Together, our observations suggest that RDV exploits an exosomal release pathway to mediate viral entry into the salivary cavities via passage through the apical plasmalemma.

Figure 1 with 2 supplements see all
Transmission electron micrographs showing that RDV traverses the apical plasmalemma into the salivary cavities via the exosomal release pathway.

At 14 days padp, virus-infected salivary glands of N. cincticeps were examined by transmission electron microscopy. (A) Salivary cavities in type III cells. (B–C) Virus-containing MVBs at the periphery of salivary cavities attached to the apical plasmalemma. Panel C showing an enlarged image of the boxed area in panel B. (D–E) The propulsion of MVBs led to the enation of the apical plasmalemma toward the cavity. (F–H) Virus-containing MVBs fused with the apical plasmalemma of cavities. (I–J) Virus-containing exosomes were released into the cavities. SC, salivary cytoplasm; APL, apical plasmalemma; Cv, cavity; Ex, exosome; VEx, virus-containing exosome; MVB, multivesicular body; Mv, microvilli. Bars, 2 μm (A), 500 nm (B, F), 200 nm (C, D, G, I and J), and 100 nm (E and H). (K) The mean diameters of exosomes within the salivary cavities, as measured from more than 30 virus-free or virus-containing exosomes. The diameters of exosomes are shown in a dot plot, with the middle line representing the mean value and the top and bottom lines representing the SD. ****p<0.0001.

Viral infection activates the formation of exosomes for the delivery of viral particles to the salivary cavities

Rab27a is an important regulator of exosome release, and the tetraspanin protein CD63 has been described as a key factor in exosomes production (Kalluri and LeBleu, 2020; Ostrowski et al., 2010). Immunoelectron microscopy indicated that the antibodies against Drosophila Rab27a and N. cincticeps CD63 can be used to label virus-containing MVBs and exosomes in virus-infected salivary glands (Figure 2A and B). Furthermore, these two antibodies also labeled some vesicle-like structures in uninfected salivary glands (Figure 2A and B). Immunofluorescence microscopy confirmed that Rab27a or CD63 was colocalized with RDV antigens to form puncta along the cavity plasmalemma or within cavities in virus-infected salivary glands (Figure 2C and D). Viral infection induced a significant increase in the average number of Rab27a- or CD63-specific punctate structures in type III cells (Figure 2E). RT-qPCR and western blot assays indicated that RDV infection significantly increased the relative expression of Rab27a and CD63 in the salivary glands (Figure 2F and G). Thus, RDV infection activated the formation of exosomes for carrying of viral particles. Taken together, these results suggest that viral release from salivary glands into the cavities potentially involves secretory exosomes derived from MVBs.

RDV dissemination into the salivary cavities via Rab27a-involved exosomal release pathway.

(A–B) Immunogold labeling of Rab27a (A) or CD63 (B) on virus-containing MVBs or exosomes in the salivary glands of N. cincticeps. Virus-infected (panels I and II) or uninfected (panels III) salivary glands of N. cincticeps were immunolabeled with Rab27a-specific IgG or CD63-specific IgG as primary antibody, followed by treatment with 15 nm gold particle-conjugated goat antibody against rabbit IgG as secondary antibody. SC, salivary cytoplasm; MVB, multivesicular body; Cv, cavity; Ex, exosome; VEx, virus-containing exosome. Arrows indicate gold particles. Bars, 100 nm. (C–D) Immunofluorescence assay of the distribution of Rab27a (C) or CD63 (D) during viral infection of the salivary glands of N. cincticeps. Salivary glands of nonviruliferous or viruliferous N. cincticeps were fixed, immunostained with virus-FITC (green), Rab27a- rhodamine (red) or CD63-rhodamine (red), and actin dye phalloidin-Alexa Fluor 647 carboxylic acid (blue). Then, immunostained salivary glands were processed for immunofluorescence microscopy. Panels I and II (merged) are the enlarged images of the boxed areas in C and D. Arrows show the colocalization puncta in the salivary cavities. APL, apical plasmalemma; Cv, cavity. Bars, 5 μm. (E) The mean number of Rab27a- or CD63-positive puncta in infected or uninfected salivary glands. Twenty random 70 × 70 μm fields of type III cell samples from infected or uninfected salivary glands were examined. (F) RT-qPCR assay showing the transcript levels of Rab27a and CD63 in the salivary glands of viruliferous or nonviruliferous leafhoppers. RT-qPCR results were normalized against the actin expression level, and the transcript levels of Rab27a or CD63 in the salivary glands of nonviruliferous leafhopper were normalized as 1. (G) Western blot assay showing the expression levels of Rab27a and CD63 in the salivary glands of viruliferous or nonviruliferous leafhoppers. The relative intensities of bands in the analyses of Rab27a and CD63 are shown below. Coomassie-blue-stained gels demonstrate the loading of equal amounts of proteins. Data shown here are representative of three biological replicates. Means (± SD) from three biological replicates are shown in E and F. *p<0.05, **p<0.01. V-, nonviruliferous; V+, viruliferous.

We then knocked down the expression of Rab27a by RNA interference (RNAi) to confirm the role of exosomes in RDV dissemination from salivary glands. Viruliferous leafhoppers were microinjected with double-stranded RNAs targeting Rab27a or green fluorescence protein (dsRab27a or dsGFP) and then transferred to rice seedlings. RT-qPCR and western blot assays showed that the accumulation levels of the major outer capsid protein P8 of RDV and Rab27a in the salivary glands of dsRab27a-treated insects were significantly decreased compared with in dsGFP controls (Figure 3A and B). Immunofluorescence microscopy confirmed that the knockdown of Rab27a expression substantially reduced the accumulation of virus- and Rab27a-positive puncta in the salivary glands, especially within the salivary cavities (Figure 3C and D). As expected, dsRab27a treatment significantly decreased the ability of viruliferous leafhoppers to transmit viruses to rice plants (Figure 3E). Taken together, these results indicate that the exosomal release of viruses into salivary cavities is greatly impaired by the knockdown of Rab27a expression in leafhoppers, which significantly suppresses the horizontal transmission of RDV from insect salivary glands to rice plants.

Knockdown of Rab27a expression reduces viral dissemination from salivary glands via the exosomal release pathway.

(A) RT-qPCR assay showing the transcript levels of RDV P8 and Rab27a expression in the salivary glands of dsRab27a- or dsGFP-treated insects. Results were normalized against the actin expression level. Means (± SD) from three biological replicates are shown. The relative expression levels of the genes in individual insects are shown in a dot plot, with the middle line representing the mean value and the top and bottom lines representing the SD. *p<0.05, **p<0.01. (B) Western blot assay showing the expression levels of RDV P8 and Rab27a in the salivary glands of dsRab27a- or dsGFP-treated insects. The relative intensities of bands in the analyses of Rab27a or RDV P8 are shown below. Coomassie-blue-stained gels demonstrated the loading of equal amounts of protein. Data shown here are representative of three biological replicates. (C) Immunofluorescence microscopy showing the distribution of Rab27a during viral infection in the salivary glands of dsRab27a- or dsGFP-treated insects. Salivary glands were fixed, immunolabeled with virus-FITC (green), Rab27a-rhodamine (red) and actin dye phalloidin-Alexa Fluor 647 carboxylic acid (blue), and processed for immunofluorescence microscopy. APL, apical plasmalemma; Cv, cavity. Bars, 5 μm. (D) The mean number of Rab27a–RDV antigen colocalization puncta in the cavities of dsGFP- and dsRab27a-treated salivary glands. Twenty random 70 × 70 μm fields in type III cell samples from infected or uninfected salivary glands were examined, and means (± SD) from three biological replicates are shown. **p<0.01. (E) The transmission rates by viruliferous dsGFP- or dsRab27a-treated leafhoppers. Means (± SD) from three biological replicates are shown. **p<0.01.

RDV is secreted from cultured insect vector cells via the exosomal release pathway

Previously, we demonstrated that MVBs were involved in the exosomal release of RDV particles from infected cultured leafhopper cells (Wei et al., 2009). At 48 hr post-inoculation (hpi) with RDV at a multiplicity of infection (MOI) of 1, electron microscopy showed that double-layered RDV particles were sequestered in the endosomal compartments (Figure 4A–I), which then formed MVBs in the cytoplasm of cultured N. cincticeps cells (Figure 4A–II). Finally, virus-containing exosomes were released from infected cultured cells after the fusion of MVBs with the plasma membrane (Figure 4A–III and IV), as described previously (Wei et al., 2008; Wei et al., 2009). Immunoelectron microscopy confirmed that RDV-specific antibody recognized viral particles within exosomes (Figure 1—figure supplement 2D). Furthermore, Rab27a and CD63 antibodies reacted with virus-containing exosomes in virus-infected cells and some vesicle-like structures in uninfected cells (Figure 4B). Next, we purified exosomes from the extracellular medium of infected or uninfected cultured leafhopper cells. Negative staining electron microscopy showed that virus-containing exosomes (114–307 nm in diameter) can be isolated from the extracellular medium of infected cells, which were thereby larger than virus-free exosomes (58–138 nm in diameter) secreted from uninfected cells (Figure 4C and D).

Figure 4 with 1 supplement see all
The secretion of RDV from cultured insect vector cells via the exosomal release pathway.

(A) At 48 hpi, virus-infected cultured leafhopper cells were examined by transmission electron microscopy. RDV virions were sequestered in the endosomal compartments (I), which then developed into MVBs (II). Finally, virus-containing exosomes were secreted from cultured cells (III-IV). Bars, 100 nm (I, III, IV) and 200 nm (II). (B) Immunogold labeling of Rab27a or CD63 in virus-free (I, III) or virus-containing (II, IV) exosomes. Arrows indicate gold particles. At 48 hpi, cultured cells were immunostained with Rab27a-specific IgG or CD63-specific IgG as primary antibody, followed by treatment with 15 nm gold particle-conjugated goat antibody against rabbit IgG as secondary antibody. Bars, 100 nm. (C) Negative staining electron microscopy showing the purified exosomes from the extracellular media of uninfected (I) or virus-infected (II) cultured cells. Bars, 100 nm. (D) The diameters of purified exosomes are shown in a dot plot, with the middle line representing the mean value and the top and bottom lines representing the SD. ****p<0.0001. (E–F) Virus-infected cultured cells treated with dsRab27a (E) or 20 μM GW4869 (F) were immunostained with virus-rhodamine (red) and Rab27a-FITC (green) and examined by immunofluorescence microscopy. Treatments with dsGFP (E) or DMSO (F) served as the controls. Arrows indicate the colocalization puncta of virus-rhodamine and Rab27a-FITC on the cell surface or outside of cells. Panels I show the enlarged images of the boxed areas in panels E or F. Bars, 5 μm. (G) Negative staining electron microscopy showing the purified exosomes from the extracellular media of dsGFP-, dsRab27a-, DMSO-, or 20 μM GW4869-treated virus-infected cultured cells. Panel I, dsGFP- and dsRab27a-treated cells; Panel II, DMSO- and GW4869-treated cells. Bars, 100 nm. (H) RT-qPCR assay showing the transcript levels of RDV P8 and Rab27a in dsRab27a- or dsGFP-treated cells. Results were normalized against the actin transcript level; the expression levels of Rab27a or P8 in dsGFP-treated cells were normalized as 1. Means (± SD) from three biological replicates are shown. *p<0.05. (I) Western blot assay showing the expression levels of RDV P8 and Rab27a in dsRab27a- or dsGFP-treated cells. The relative intensities of bands in the analyses of Rab27a and P8 are shown below. Coomassie-blue-stained gels demonstrated the loading of equal amounts of protein. Data shown here are representative of three replicates. (J) RT-qPCR assay showing the transcript levels of RDV P8 in GW4869 (10 or 20 μM)- or DMSO-treated cells. Means (± SD) from three biological replicates are shown. *p<0.05. Ex in A–C, exosome; VEx in A–C and G, virus-containing exosome; MVB in A, multivesicular body.

GW4869 is a cell-permeable but selective inhibitor for exosome production and release (Vora et al., 2018). Immunofluorescence microscopy showed that treatment with dsRab27a or 20 μM GW4869 followed by RDV infection (MOI of 1) efficiently inhibited virus-containing exosome formation and release (Figure 4E and F). Negative staining electron microscopy of purified exosomes secreted from virus-infected cells further confirmed that treatment with dsRab27a or GW4869 efficiently abolished exosome formation (Figure 4G). RT-qPCR or western blot assays showed that the accumulation levels of RDV P8 and Rab27a in dsRab27a-treated cultured cells were substantially decreased compared with that in dsGFP-treated controls (Figure 4H and I). Treatment of leafhopper cells with 20 μM GW4869 also significantly reduced RDV P8 transcript accumulation, compared with the DMSO control (Figure 4J).

Our previous study showed the RDV particles were assembled at the periphery of the viroplasm, the site of viral replication and assembly, and then engulfed by MVBs in virus-infected cultured leafhopper cells (Wei et al., 2006b; Wei et al., 2008; Wei et al., 2009). We examined whether the treatment with dsRab27a or GW4869 had any effects on the replication of RDV in cultured cells. The cultured cells treated with dsRNAs, DMSO, or 20 μM GW4869 were inoculated with purified RDV at a MOI of 10 which guaranteed viral infection rate was 100%. At 48 hpi, the extracellular medium and the cells were collected. Treatment with dsRab27a or GW4869 significantly reduced RDV P8 transcript loads in the extracellular medium, but did not significantly reduce RDV P8 transcript loads in cell-associated viruses (Figure 4—figure supplement 1). These results demonstrated that RDV normally proliferated in the infected cells, but viral release from the cells was impeded by the inhibition of exosome formation. Taken together, these results indicate that RDV can be released from cultured insect vector cells via the Rab27a-dependent exosomal release pathway.

RDV P2 inherently interacts with Rab5 to mediate the exosomal release of virions

We then investigated how RDV virions were sequestered by exosomes. The outer layer of RDV virion contains the major outer capsid protein P8 and minor outer capsid protein P2 (Miyazaki et al., 2016). Structural analysis shows that P2 has an L-shaped flexible structure with a 10-nm-long domain anchored on the viral surface (P2C) and a 15-nm-long domain oriented toward the exterior (P2N), which recognizes cellular receptors to mediate viral entry into insect vector cells (Miyazaki et al., 2016Figure 5A). Rab5 is localized on the membrane surface of both early and mature endosomal MVBs (Gorvel et al., 1991; Mercer et al., 2010). The open-reading frames (ORFs) of N. cincticeps Rab5 and Rab27a were 645 and 672 bp long, respectively, and encoded approximately 23 kDa and 25 kDa predicted proteins, respectively, with the typical Rab family motifs (Figure 5—figure supplement 1A and Figure 5—figure supplement 2A). Each gene sequence was deposited in GenBank (accession numbers MW266984 and MW266985, respectively). Phylogenic analysis showed that the amino acid sequences of N. cincticeps Rab5 and Rab27a clustered with homologs in the order Hemiptera (Figure 5—figure supplement 1B and Figure 5—figure supplement 2B). Yeast two-hybrid assays showed that RDV P2N, but not RDV P2C or RDV P8, specifically interacted with Rab5 (Figure 5B). However, P2N did not interact with Rab27a from N. cincticeps (Figure 5—figure supplement 2C). Glutathione S-transferase (GST) pull-down assays confirmed these interactions (Figure 5C). Immunoelectron microscopy indicated that the antibody against Drosophila Rab5 can be used to label virus-containing exosomes in virus-infected salivary glands (Figure 5D). Notably, Rab5 antibody could react with viral particles within exosomes (Figure 5D). Furthermore, this antibody can also label some vesicle-like structures in uninfected salivary glands (Figure 5D). Immunofluorescence microscopy confirmed that Rab5 was colocalized with RDV antigens to form puncta within the salivary cavities (Figure 5E). RDV infection induced the accumulation of virus- and Rab5-positive puncta in the salivary glands, especially within the salivary cavities (Figure 5F). RT-qPCR and western blot assays indicated that RDV infection significantly increased the expression of Rab5 in the salivary glands (Figure G and H). Furthermore, RT-qPCR and western blot assays showed that the accumulation levels of RDV P8 and Rab5 in the salivary glands of dsRab5-treated insects were significantly decreased compared with that in dsGFP controls (Figure 5I and J). RT-qPCR assay also showed that the knockdown of Rab5 expression did not significantly affect Rab27a expression and that the knockdown of Rab27a expression also did not significantly affect Rab5 expression (Figure 5—figure supplement 2D). However, immunofluorescence microscopy showed that some Rab5-labeled punctate structures were colocalized with Rab27a-labeled punctate structures in the salivary glands (Figure 5—figure supplement 2E). Taken together, we deduce that the packaging of RDV into exosomes is caused by the direct interaction of RDV P2N with Rab5.

Figure 5 with 3 supplements see all
RDV P2 interacts with endosomal Rab5 of leafhopper.

(A) Schematic representation of the structures of RDV particle and P2 protein. P2 is composed of an L-shaped flexible structure with a 15 nm domain (aa 1–688, P2N) toward the exterior, and a 10 nm domain (aa 689–1149, P2C) on the viral envelope. (B) RDV P2N, but not RDV P2C or P8, specifically interacted with Rab5 in a yeast two-hybrid assay. Transformants on SD/-Trp-Leu-Ade-His plates are labeled as follows: +, positive control, i.e., pGBKT7-53/pGADT7-T; –, negative control, i.e., pGBKT7-Lam/pGADT7-T; Rab5 +P8, pGADT7-Rab5/pGBKT7-P8; Rab5 +P2C, pGADT7-Rab5/pGBKT7-P2C; Rab5 +P2N, pGADT7-Rab5/pGBKT7-P2N. Yeast cultures appeared blue in the β-galactosidase assay. (C) GST pull-down assay demonstrating the interaction of P2N with Rab5. Rab5 or RDV P8 fused with GST served as the bait, and GST alone served as the control. P2N or Rab5 fused with His served as the prey. The bait protein or the GST control were incubated with cell lysate expressing the His-fused protein. Input and pull-down samples were probed with antibodies against GST or His for western blot assays. (D) Immunogold labeling of Rab5 on exosomes in the salivary cavities. The salivary glands from nonviruliferous (panel I) or viruliferous (panel II) leafhoppers were immunostained with Rab5-specific IgG as primary antibody, followed by treatment with 15 nm gold particle-conjugated goat antibody against rabbit IgG as secondary antibody. Arrows indicate gold particles. Ex, exosome; VEx, virus-containing exosome. Bars, 100 nm. (E) Immunofluorescence assay showing the distribution of Rab5 during virus infection in the salivary glands of N. cincticeps. Virus-infected or uninfected salivary glands of N. cincticeps were fixed, immunostained with virus-FITC (green), Rab5-rhodamine (red), and actin dye phalloidin-Alexa Fluor 647 carboxylic acid (blue). Immunostained salivary glands were then processed for immunofluorescence microscopy. Arrows show the colocalization puncta in the salivary cavities. APL, apical plasmalemma; Cv, cavity. Bars, 10 μm. (F) The mean number of Rab5-positive puncta in infected or uninfected salivary glands. Twenty random 70 × 70 μm fields of type III cell samples from infected or uninfected salivary glands were examined. (G) RT-qPCR assay showing the transcript levels of Rab5 in the salivary glands of viruliferous or nonviruliferous leafhoppers. RT-qPCR results were normalized against the actin expression level, and the transcript levels of Rab5 in the salivary glands of nonviruliferous leafhopper were normalized as 1. Means (± SD) from three biological replicates are shown in F and G. *p<0.05; **p<0.01. (H) Western blot assay showing the expression levels of Rab5 in the salivary glands of viruliferous or nonviruliferous leafhoppers. The relative intensities of bands in the analyses of Rab5 and RDV P8 are shown below. Coomassie-blue-stained gels demonstrated the loading of equal amounts of proteins. Data shown here are representative of three biological replicates. (I) RT-qPCR assay showing the transcript levels of RDV P8 and Rab5 in the salivary glands of dsRab5- or dsGFP-treated insects. Results are normalized against the actin expression level. The relative expression levels of the genes in individual insects are shown in a dot plot, with the middle line representing the mean value and the top and bottom lines representing the SD. *p<0.05. (J) Western blot assay showing the expression levels of RDV P8 and Rab5 in the salivary glands of dsRab5- or dsGFP-treated insects. The relative intensities of bands are shown below. Coomassie-blue-stained gels demonstrated the loading of equal amounts of protein. Data shown here are representative of three replicates. V-, nonviruliferous insects; V+, viruliferous insects.

To further determine how RDV P2N recognizes endosomal MVBs, we infected cultured Spodoptera frugiperda (Sf9) cells with recombinant baculoviruses overexpressing His-fused P2N or Myc-fused Rab5. When expressed alone, P2N formed punctate structures, while the overexpressed Rab5 was diffusely distributed in the cytoplasm of Sf9 cells (Figure 6A). The coinfection led to the redistribution of Rab5 into the punctate structures of P2N (Figure 6A). Notably, antibodies against Drosophila Rab5 or Rab27a could also be used to label the endosome-like compartments in the cytoplasm of Sf9 cells (Figure 6B), as previously reported for cells of other species (Nielsen et al., 1999; Simonsen et al., 1998; Wucherpfennig et al., 2003). We found that the expressed P2N was colocalized with Rab5- or Rab27a-labeled endosome-like compartments (Figure 6B). In particular, P2N-specific endosome-like compartments can release into the extracellular medium of Sf9 cells (Figure 6B). Treatment with GW4869 (20 μM) or chloroquine (280 μM) significantly prevented the formation of P2N-specific endosome-like compartments (Figure 6C and Figure 6—figure supplement 1). Furthermore, the GFP-fused P2N (P2N-GFP) in Sf9 cells also appeared as punctuate inclusions (Figure 6—figure supplement 2A–B). Treatment with chloroquine (280 μM) or GW4869 (20 μM) also significantly reduced the mean of fluorescence intensity and accumulation of P2N-GFP (Figure 6—figure supplement 2C to E). Thus, RDV P2N had the inherent ability to recognize endosomes, even in the absence of viral infection. Taking together, these results indicate that the specific and inherent interaction of RDV P2 with Rab5 mediates the packaging of RDV particles into exosomes.

Figure 6 with 2 supplements see all
RDV P2N is associated with the endosomal compartments in Sf9 cells.

(A) Immunofluorescence assay showing the colocalization of RDV P2N with Rab5 of N. cincticeps in Sf9 cells. RDV P2N-His and Rab5-Myc were expressed alone or co-expressed. Sf9 cells were fixed at 48 hpi and immunolabeled with His-Alexa Fluor 488 (green) and Myc-Alexa Fluor 555 (red). (B) The association of RDV P2N with the endosomal compartments labeled by Rab5- or Rab27a-specific IgG in Sf9 cells. The control or P2N-expressing cells were immunostained with Rab5- or Rab27a-rhodamine and His-Alexa Fluor 488. Arrows indicate the release of P2N-specific endosomal compartments into the extracellular medium. (C) Endosomal inhibitors inhibited the association of P2N with the endosomal compartments. Sf9 cells were infected with recombinant baculovirus encoding P2N-His. At 48 hpi, cells were treated with GW4869 (20 μM) or chloroquine (280 μM). Cells were immunostained with His-Alexa Fluor 488 prior to examination by immunofluorescence microscopy. Bars, 5 μm.

RDV is released from the salivary glands into rice phloem with the exosomes

Generally, viruses in the salivary cavities move with salivary flow to the canal in the stylets and ultimately are injected into the sieve cells of plant phloem as piercing-sucking insect vectors feed on susceptible hosts (Hogenhout et al., 2008; Wei and Li, 2016). We then tested whether RDV-containing exosomes in the salivary cavities could be ejected with saliva into rice plants. Immunofluorescence assays showed that the antibodies against Rab5, Rab27a, or CD63 did not react with any specific structures in rice phloem (Figure 7A). However, Rab5, Rab27a, or CD63 appeared in the sieve tube cells of rice phloem after being fed on by nonviruliferous leafhoppers for 2 days (Figure 7B), confirming that the exosomes were released with salivary flow into plant phloem via leafhopper feeding. Notably, RDV was accompanied by Rab5-, Rab27a-, or CD63-involved exosomes into the sieve tube cells of rice phloem after viruliferous leafhoppers feeding and then spread through rice phloem to establish initial infection (Figure 7C). Western blot assay further showed that more Rab5, Rab27a, and CD63 proteins were detected in rice seedlings that had been fed on by viruliferous leafhoppers for 2 days compared to those observed after feeding by nonviruliferous leafhoppers (Figure 7D). More importantly, western blot assay confirmed that the antibodies against Rab5, Rab27a, or CD63 did not react with the components in rice phloem (Figure 7D). Together, these results suggest that RDV is disseminated together with secretory exosomes from insect vector salivary glands into rice phloem, ultimately accomplishing viral transmission to the plant host.

Figure 7 with 1 supplement see all
Exosome-mediated RDV delivery to the phloem of rice plants via leafhopper feeding.

(A–C) Immunofluorescence microscopy showing the distribution of RDV antigens, Rab27a, CD63 or Rab5 in the phloem of rice plants after 2 day feeding of viruliferous or nonviruliferous leafhoppers. Rice plants without leafhopper feeding (A) and fed on by nonviruliferous (B) or viruliferous (C) leafhoppers were immunolabeled with virus-FITC (green) and Rab27a, CD63, or Rab5-rhodamine (red) and then examined by immunofluorescence microscopy. Panels i to iii are enlarged images of the boxed areas in panels I to III, respectively. The green and red panels show the separate green (RDV antigens) or red fluorescence (Rab27a, CD63, or Rab5 antigens) of the merged images in panels i to iii. Areas enclosed with a dotted line indicate phloem. AS, air space; P, phloem; PV, pitted vessel; AV, annular vessel; ST, sieve tube; VBS, vascular bundle sheath. Bars, 10 μm. (D) Western blot assay of RDV P8, Rab27a, CD63, or Rab5 in rice plants that had been fed on by viruliferous or nonviruliferous leafhoppers. Samples were separated by SDS-PAGE and detected with P8-, Rab27a-, CD63-, or Rab5-specific antibody. V-, rice plants that had been fed on by nonviruliferous leafhoppers; V+, rice plants that had been fed on by viruliferous leafhoppers; Control, rice plants without leafhopper feeding. The relative intensities of bands in analyses of Rab5, Rab27a, and CD63 are shown below. Coomassie-blue-stained gels demonstrated the loading of equal amounts of protein. Data shown here are representative of three biological replicates.

Discussion

The horizontal transmission of arboviruses from the salivary glands of insect vectors to susceptible hosts is an essential process for viral survival in nature. Through horizontal transmission, infectious virions are ejected into hosts by insect stylets and then robustly establish the initial infection in hosts. Exosome-mediated spread of virions and various cellular or viral regulatory RNAs from cell to cell has gained considerable interest among researchers (Chahar et al., 2015; Geisler and Coller, 2013; Janas et al., 2015; Kim et al., 2017; Mori et al., 2008; Tanggis et al., 2017; Nguyen et al., 2003); however, these studies have been limited to in vitro systems of cultured animal cells. Here, we show that the specific interaction of the 15-nm-long domain of RDV P2 on viral surface with Rab5 on the cytoplasmic face of endosomes induces the packaging of intact virions into endosomal MVBs in salivary glands. Such MVBs attach to the apical plasmalemma and induce an exocytosis process promoting the release of virus-containing exosomes into salivary cavities. The average diameters of the salivary canal of leafhoppers in Cicadellidae range from 3 μm to 500 nm (Brozek and Herczek, 2000; Leopold et al., 2003; Zhao et al., 2010), greatly exceeding the maximum diameter of virus-containing exosomes. Therefore, such virus-containing exosomes can move easily through the salivary canal. Finally, the virus can be horizontally transmitted with exosomes into rice phloem to establish the initial infection. Inhibition of exosome secretion by silencing Rab5 or Rab27a can prevent such viral exosomal release into salivary cavities, ultimately suppressing viral transmission by insect vectors. More importantly, viral infection upregulates the expression of Rab5 or Rab27a, thereby activating the formation of exosomes for delivery of viral particles to the salivary cavities. Accordingly, the packaging of virions enlarges the normal sizes of exosomes secreted by infected salivary glands cells. Thus, the virus has evolved strategies to activate and exploit the exosomal release pathway into the salivary cavity, facilitating viral transmission by leafhopper vectors to rice phloem together with saliva (Figure 8).

Proposed model of RDV hijacking the exosomal release pathway to be released from salivary glands of leafhopper vectors into rice phloem.

In the proposed model, the specific interaction of RDV P2N and Rab5 causes the internalization and delivery of viruses to endosomes. Then, these virus-containing endosomes are engulfed in the MVBs in the cytoplasm. The attachment of MVBs with the apical plasmalemma drives the enation of the apical plasmalemma toward the cavity. The fusion of MVBs with apical plasmalemma leads to the release of virus-containing exosomes to the cavities. This exosomal release pathway is mediated by Rab27a. Exosomes are secreted with the salivary flow from the salivary cavities into the stylets and are then injected into the plant phloem as leafhoppers feed on rice plants. The virus released into the phloem establishes the initial replication within plant cells. APL, apical plasmalemma; MVB, multivesicular body; Cv, cavity; SD, salivary duct; En, endosome; Ex, exosome; P, phloem; AS, air space; PV, pitted vessel.

We have shown that the 15-nm-long domain of RDV P2 can interact with cellular receptors on the plasma membrane of cultured leafhopper cells to facilitate viral entry, or with the outer membrane protein of the obligate bacterial symbiont Sulcia of leafhoppers to facilitate viral transovarial transmission (Jia et al., 2017; Miyazaki et al., 2016; Wei et al., 2007; Zhou et al., 2007). Thus, RDV P2 has evolved to interact with distinct receptors in insect bodies to enable the virus to overcome various tissue or organ barriers. RDV enters insect vector cells through receptor-mediated, clathrin-dependent endocytosis and is sequestered in endosomal compartments (Wei et al., 2007). Furthermore, RDV P2 can induce membrane fusion in insect vector cells (Zhou et al., 2007). We also show that the 15-nm-long domain of RDV P2 has the inherent ability to recognize endosomal compartments even in the absence of viral infection. Thus, RDV P2–Rab5 interaction leads to the recruitment of Rab5 on the surface of virions within the exosomes. These observations are fully consistent with the role of RDV P2 in the viral package within MVBs via interaction with Rab5 and subsequent fusion of MVBs with the cell membrane for exosome secretion. Indeed, exosomes isolated from mammalian or plant cells have also been reported to contain the early endosome marker Rab5 (Logozzi et al., 2009; Ramakrishnaiah et al., 2013; Song et al., 2019). Purified exosomes secreted from infected leafhopper cells were larger and contained virions. The inhibition of exosomal secretion by silencing Rab27a or treatment with GW4869 suppresses such virus-mediated exosomal release, confirming the functional relevance of infectious exosomes with RDV dissemination (Wei et al., 2008; Wei et al., 2009). Thus, the use of the continuous insect cell culture system provides more conclusive proof that RDV can be released from insect vector cells via a Rab27a-dependent exosomal release pathway. Similarly, numerous arboviruses, such as DENV and ZIKV, are packaged in MVBs and secreted from cultured mosquito vector cells via the exosomal release pathway (Martínez-Rojas et al., 2020; Reyes-Ruiz et al., 2019; Vora et al., 2018). Thus, the exosomes play a conserved role in arbovirus dissemination.

Generally, in the secretory cells of insect salivary glands, arboviruses are frequently packaged in MVBs (Ammar and Nault, 2002; Gray et al., 2014; Gray and Gildow, 2003; Janzen et al., 1970; Mao et al., 2017; Shikata and Maramorosch, 1965). We thus anticipate that arboviruses may have evolved a conserved strategy to exploit the exosomal release pathway to ensure viral horizontal transmission from insect vectors into plant or mammalian hosts. In particular, exosome-mediated viral release from the apical plasmalemma into cavities does not cause substantial damage to salivary glands, conferring an evolved advantage for RDV to be persistently transmitted into plant phloem. Furthermore, exosomes potentially operate as an immune evasion strategy to ensure the initial infection of viruses in plant phloem (Chahar et al., 2015). Identification of the molecular modes or determinants of the transmission of arboviruses from vectors to hosts is a basic but very fundamental research objective. For the first time, we show the role of virus-induced exosomes in viral horizontal transmission from the salivary glands of an insect vector to a plant host in vivo.

Information concerning plant-cell–derived extracellular vesicles is rather limited. Most of the available information about plant exosomes highlights the implication of the SNARE SYP121 protein and comes from studies of plant–fungi interactions and plant–bacteria interactions (Nielsen et al., 2012; Cai et al., 2018). Rab11 is involved in exosome secretion and the recycling of cell wall components (Nielsen et al., 2008). The purified exosomes from Arabidopsis leaf apoplasts contained the Rab5 GTPase homologue ARA7 (Rutter and Innes, 2018). Turnip mosaic virus (TuMV) infection induces the formation of extracellular vesicles containing viral RNAs in infected leaves (Movahed et al., 2019). Rab proteins, such as RabG3E, RabD2B, and Rab7, were detected in TuMV-induced extracellular vesicles (Movahed et al., 2019). It is well known that various cellular or viral regulatory RNAs or proteins can be packaged into exosomes (Chahar et al., 2015), and thus, exosome-mediated viral horizontal transmission may modulate phloem–insect–virus interactions, an exciting frontier of plant science. Hence, our study shows that exosomes contribute to plant–insect interactions.

Materials and methods

Insects, viruses, and antibodies

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Nonviruliferous individuals of leafhopper N. cincticeps were collected from Yunnan Province in southwestern China and propagated for several generations in the laboratory. Rice plants infected with RDV were also initially collected from Yunnan Province and propagated via transmission by N. cincticeps under greenhouse conditions. Rabbit polyclonal antisera against RDV antigens and P8 were provided by Dr. Toshihiro Omura (National Agricultural Research Center, Japan). Polyclonal antibodies against Rab27a and Rab5 from Drosophila endosomes were obtained from Beyotime Biotechnology, Nantong, China. There were 57% and 79% amino acid sequence similarities between Drosophila and N. cincticeps Rab27a and between Drosophila and N. cincticeps Rab5, respectively (Figure 5—figure supplement 3). Preliminary tests also showed the specificity of these two antibodies in reacting with Rab27a or Rab5 of N. cincticeps. Polyclonal antibody against N. cincticeps CD63 (GenBank accession MW978787) was prepared by Genscript Biotech Corporation, Nanjing, China, as approved by the Science Technology Department of Jiangsu Province of China. IgGs were isolated from polyclonal antisera by using a protein A-Sepharose affinity column (Pierce Biotechnology, Waltham, MA, USA). IgGs against RDV antigens, Rab27a, CD63, or Rab5 were directly conjugated to FITC or rhodamine, respectively, according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). The actin dyes phalloidin-rhodamine and phalloidin-Alexa Fluor 647 carboxylic acid, His tag antibody conjugated to Alexa Fluor 488 (His-Alexa Fluor 488), and Myc tag antibody conjugated to Alexa Fluor 555 (Myc-Alexa Fluor 555) were obtained from Thermo Fisher Scientific. Actin was detected with actin-specific IgG (Sigma).

Immunofluorescence microscopy for examination of viral infection and release in salivary glands

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Nonviruliferous second-instar nymphs of N. cincticeps were fed on RDV-infected rice plants for 2 days and then maintained on healthy rice seedlings. At different days padp, the salivary glands of leafhoppers were dissected, fixed in 4% (v/v) paraformaldehyde in PBS for 2 hr, and then permeabilized in 0.2% Triton-X for 1 hr. The salivary glands were then immunolabeled with virus-specific IgG conjugated to FITC (virus-FITC), Rab27a-, CD63-, or Rab5-specific IgGs conjugated to rhodamine (Rab27a-rhodamine, CD63-rhodamine, or Rab5-rhodamine), and the actin dyes (phalloidin-rhodamine or phalloidin-Alexa Fluor 647 carboxylic acid). Immunostained salivary glands were then processed for immunofluorescence microscopy.

Electron microscopy for examination of viral release into salivary cavities

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At 14 days padp, salivary glands dissected from leafhoppers were fixed, dehydrated and embedded as previously described (Mao et al., 2017). The ultrathin sections were examined under a transmission electron microscope (H-7650; Hitachi, Tokyo, Japan). For immunoelectron microscopy, ultrathin sections were immunolabeled with virus-, Rab27a-, CD63-, or Rab5-specific IgG as the primary antibody and were then treated with goat anti-rabbit IgG conjugated with 15-nm-diameter gold particles as the secondary antibody (Abcam, Cambridge, UK).

RT-qPCR and western blot assays to detect the effects of viral infection on exosomal production and release

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At 14 days padp, total RNAs or proteins were extracted from the salivary glands of 30 viruliferous or nonviruliferous leafhoppers for to detect the relative transcript or protein levels of Rab27a and Rab5 during viral infection. RT-qPCR was performed using the SYBR Green PCR Master Mix kit (Promega, Madison, WI, USA). The actin transcript of N. cincticeps served as the internal reference, and the relative gene expression levels were calculated using the 2-ΔΔCT method. For western blot assay, RDV P8-, Rab27a-, CD63-, or Rab5-specific IgG served as the primary antibody and goat anti-rabbit IgG-peroxidase served as the secondary antibody. The band intensities of western blot assay were quantified with ImageJ software.

To examine the release of exosomes from salivary glands, more than 80 viruliferous or nonviruliferous leafhoppers prepared as described above were fed on two healthy rice seedlings (approximately 10 cm in height) for 2 days. The tested plants were then collected separately and subjected to protein extraction in equal quantities. Equal amounts of proteins from each group were loaded for western blot assay.

Effect of synthesized dsRNAs on viral release from salivary glands

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The dsRNAs targeting 500 bp regions of Rab27a or Rab5 were synthesized in vitro using the T7 RiboMAX Express RNAi System (Promega). Nonviruliferous second-instar nymphs were fed on diseased rice plants. At 8 days padp, the insects were microinjected with dsRab27a, dsRab5, or dsGFP (about 0.05 μg/insect) at the intersegment region of the thorax using a Nanoject II Auto-Nanoliter Injector (Spring). Thereafter, they were transferred to healthy rice seedling for recovery. At 14 days padp, the salivary glands of approximately 100 of these leafhoppers were first collected, and the corresponding bodies were individually tested using RT-PCR assay to confirm they were viruliferous. Then the salivary glands of dsRNAs-treated leafhoppers, of which bodies were viruliferous, were tested using RT-qPCR, western blot, or immunofluorescence assays to examine the effects of synthesized dsRNAs on the expression of RDV P8, Rab5, or Rab27a, as described above.

To investigate the transmission rates of RDV by N. cincticeps treated with dsRab27a, more than 300 s instar individuals were fed on RDV-infected rice plants for 2 days. At 8 days padp, groups of 100 insects were each microinjected with dsRab27a or dsGFP. At 14 days padp, the individual insects were transferred into glass tubes that each contained a single rice seedling for 2 days. Then the insects were individually tested using RT-PCR to determine whether they were viruliferous. Thirty days later, the plants inoculated with viruliferous N. cincticeps were subjected to RT-PCR detection of RDV P8 gene. The transmission rates of RDV by N. cincticeps were calculated as the percentage of RT-PCR-positive plants out of the total number of plants.

Examination of RDV release from cultured leafhopper cells

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Synchronous infection of continuous cultured cells of N. cincticeps with RDV was initiated as described by Wei et al., 2006a. At 48 hpi, infected cultured cells were fixed, dehydrated, and embedded. Then, ultrathin sections were cut and processed for electron microscopy, as previously described (Wei et al., 2006a). For immunoelectron microscopy, thin sections were immunolabeled with Rab27a- or CD63-specific IgG as the primary antibody and then treated with goat anti-rabbit IgG conjugated with 15-nm-diameter gold particles as the secondary antibody (Abcam).

To inhibit exosomal secretion from cultured cells via RNAi, 16 μg of dsRNAs and 12 μL of Cellfectin II reagent (Thermo Fisher Scientific) were diluted individually in 100 μL of LBM medium without antibiotics and fetal bovine serum, mixed gently together at room temperature for 20 min, and incubated with cultured cells for 8 hr. To chemically inhibit exosomal secretion, cultured cells were incubated with GW4869 (10 or 20 μM) or DMSO for 6 hr. Thereafter, cultured cells treated with dsRNAs, DMSO, or GW4869 were inoculated with purified RDV at a MOI of 1 in a solution of 0.1 M histidine that contained 0.01 M MgCl2 (pH 6.2; His-Mg) at 25°C for 2 hr, and then recovered with LBM medium. At 48 hpi, cultured cells were fixed, permeabilized, and immunolabeled with virus-specific IgG conjugated to rhodamine (virus-rhodamine), virus-FITC, or Rab27a-specific IgG conjugated to FITC (Rab27a-FITC) for immunofluorescence microscopy. Additionally, the treated cells were also harvested for RT-qPCR and western blot assays, as described above.

To investigate whether the inhibition of exosome formation affected viral replication, the cultured cells treated with dsRNAs, DMSO, or 20 μM GW4869 were inoculated with purified RDV at a MOI of 10. At 48 hpi, the extracellular medium and the cells were collected. The viral accumulation of each sample was determined by RT-qPCR assay, as described above.

Exosome isolation

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To isolate exosomes from the cellular supernatant, a Total Exosome Isolation kit (from cell culture media) (Thermo Fisher Scientific) was used. In brief, the media from infected or uninfected cultured leafhopper cells as well as dsRNAs-treated cells with 80% confluence were harvested and centrifuged at 2000 × g for 30 min to remove cells and debris. The samples were incubated with Exosome Isolation reagent at one half volume of the medium at 4°C overnight. After centrifugation at 10,000 × g for 1 hr at 4°C, the pellets were resuspended. The exosomes were negatively stained with aqueous 2% uranyl acetate and then examined with an electron microscope.

Yeast two-hybrid and GST pull-down assays

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We used a Matchmaker Gold Yeast Two-hybrid system (Clontech, Mountain View, CA, USA) to examine the interaction of Rab27a or Rab5 with the outer capsid proteins of RDV. The Rab27a and Rab5 genes were separately inserted into the pGADT7 vector to construct the prey plasmids. The full-length ORFs of P8, P2N (aa 1–688), and P2C (aa 689–1149) from RDV were separately cloned into the pGBKT7 vector as the bait plasmids and then used to transform yeast strain AH109 to confirm the absence of self-activation and toxicity. The prey and bait plasmids were then co-transformed into AH109, and β-galactosidase activity was detected on SD/-Ade-His-Leu-Trp/X-α-Gal culture medium. The interaction of pGBKT7-53 with pGADT7-T served as a positive control and that of pGBKT7-Lam with pGADT7-T served as a negative control.

A GST pull-down assay was conducted as described previously (Chen et al., 2019). The full-length ORFs of Rab5 and P8 were separately cloned into the pGEX-3x vector to construct plasmids expressing the GST fusion protein as baits (GST-Rab5 and GST-P8, respectively). Rab5 and P2N were separately cloned into the pEASY-Blunt E1 Expression Vector (Transgen Biotech, Beijing, China) to construct plasmids expressing the His fusion protein as preys (His-Rab5 and His-P2N). The recombinant proteins GST-Rab5 and GST-P8, as well as GST, were separately expressed in Escherichia coli stain BL21. Lysates were then incubated with glutathione-Sepharose beads (Amersham, Stafford, UK) and subsequently with the recombinant proteins His-P2N and His-Rab5, respectively. Finally, the eluates were analyzed by western blot assay using GST-tag and His-tag antibodies (Sigma-Aldrich), respectively.

Recombinant baculovirus expressing P2N

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The coding region of RDV P2N was amplified with a reverse primer that contained the coding sequences of the His-tag or GFP and a forward primer to construct P2N-His or P2N-GFP. The coding region of Rab5 was amplified with a reverse primer that contained the coding sequence of the Myc tag and a forward primer. These purified PCR products were cloned into the pDEST8 vector (Thermo Fisher Scientific), using an In-Fusion HD Cloning Kit (Clontech), to construct recombinant baculoviruses containing P2N or Rab5. Recombinant bacmids were generated by transforming E. coli DH10Bac (Thermo Fisher Scientific) with the recombinant baculoviruses.

Next, Sf9 cells were transfected with purified recombinant bacmid in the presence of Cellfectin II (Thermo Fisher Scientific), according to the manufacturer’s instructions. At 48 hpi, the cells were processed for immunofluorescence microscopy with His-Alexa Fluor 488 or Rab5-rhodamine. Furthermore, cells expressing P2N-GFP were directly observed under a fluorescence microscope.

To chemically inhibit the exosomal pathway, Sf9 cells were incubated with GW4869 at a final concentration of 10 μM or 20 μM or chloroquine at a final concentration of 140 μM or 280 μM. At 48 hpi, treated Sf9 cells were fixed, permeabilized and immunolabeled with His-Alexa Fluor 488 for immunofluorescence microscopy. Then, the flow cytometry was performed to examine the effect of the inhibitory chemicals on the GFP fluorescence intensity of P2N-GFP. Briefly, approximately 1 × 106 cells were collected and washed once with PBS, resuspended in PBS, and immediately examined with a flow cytometer (Apogee, Hemel Hempstead, UK). Data from three independent biological experiments were analyzed using Histogram software and displayed as a plot of fluorescence intensity of GFP (x-axis) against cell number (y-axis).

Frozen sections of rice plants

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Then, approximately 15 viruliferous or nonviruliferous leafhoppers were reared in small cages to inoculate the two rice seedlings for 2 days, respectively (Figure 7—figure supplement 1). The feeding areas of the tested plants were embedded with O.C.T. Compound (Sakura, Torrance, CA, USA) and then sectioned with a Shandon Cryotome FSE (Thermo Fisher Scientific). The sections were ultimately immunolabeled with virus-FITC and Rab5-, CD63-, or Rab27a-rhodamine, and processed for immunofluorescence microscopy, as shown above. Rice without leafhopper feeding was treated exactly as the same.

Statistical analyses

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All quantitative data presented in the text and figures were analyzed with two-tailed t-tests in GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA).

Data availability

Sequencing data have been deposited in GenBank under accession numbers MW266984, MW266985 and MW978787.

The following data sets were generated
    1. Chen Q
    2. Liu Y
    3. Ren J
    4. Zhong P
    5. Chen M
    6. Jia D
    7. Chen H
    8. Wei T
    (2021) NCBI Nucleotide
    ID MW978787. Exosomes mediate horizontal transmission of viral pathogens from insect vectors to plant phloem.
    1. Chen Q
    2. Liu Y
    3. Ren J
    4. Zhong P
    5. Chen M
    6. Jia D
    7. Chen H
    8. Wei T
    (2020) NCBI Nucleotide
    ID MW266984. Exosomes mediate horizontal transmission of viral pathogens from insect vectors to plant phloem.
    1. Chen Q
    2. Liu Y
    3. Ren J
    4. Zhong P
    5. Chen M
    6. Jia D
    7. Chen H
    8. Wei T
    (2020) NCBI Nucleotide
    ID MW266985. Exosomes mediate horizontal transmission of viral pathogens from insect vectors to plant phloem.

References

  1. Conference
    1. Brozek J
    2. Herczek A
    (2000)
    Modifications in the mouth parts structure in selected species of Cicadellidae (Hemiptera: cicadomorpha)
    IEEE. pp. 19–25.
    1. Vidal MJ
    2. Stahl PD
    (1993)
    The small GTP-binding proteins Rab4 and ARF are associated with released exosomes during reticulocyte maturation
    European Journal of Cell Biology 60:261–267.

Decision letter

  1. Jacqueline Monaghan
    Reviewing Editor; Queen's University, Canada
  2. Detlef Weigel
    Senior Editor; Max Planck Institute for Developmental Biology, Germany

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Although the transmission of viruses by insects is well established, relatively little is known about what form they take when delivered to the plant phloem from insect stylets. This report demonstrates that rice dwarf virus is packaged into exosomes in its insect vector and that these exosomes are subsequently delivered to the plant phloem. These results have important implications for understanding virus transmission and movement with the plant. We think you have addressed the concerns raised previously and commend you on submitting a revised version that is significantly improved.

Decision letter after peer review:

Thank you for submitting your article "Exosomes mediate horizontal transmission of viral pathogens from insect vectors to plant phloem" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Detlef Weigel as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Verify exosome identify:

Some concerns were raised regarding the identity/origin of exosomes. These points are especially important to address as your main conclusion is that the virus is shuttled from the insect to the plant via insect-derived exosomes.

2) Antibody specificity:

Some concerns were raised regarding the specificity of antibodies used in immunofluorescence and western blot experiments. It is not clear that these antibodies have been properly vetted for use in this heterologous system. It is important to have controls without antibodies in some immunofluorescence assays to rule out any plant autofluorescence, as well as in western blots to rule out any cross-reaction with plant proteins.

3) Justify use of Rab markers for exosomes:

Some concerns were raised around the use of Rab GTPases as optimal markers for exosomes. The authors need to justify their use. Our suggestion is to conduct a few comparative assays alongside better-established exosomal markers (such as tetraspanins; orthologs of mammalian DC63).

4) Specificity of knock-downs in miRNA experiments:

Due to sequence similarity among Rab proteins, concerns were raised regarding the specificity of the miRNA-mediated knock down assays. Please verify that the intended transcripts have been specifically knocked down.

5) Size of exosomes:

Please provide exosome measurements to back up any claims of size differences.

Reviewer #1:

The authors used an arsenal of methods and experiments to show that the leafhopper transmitted RDV is associated with exosomes in the insect vector salivary system and it is secreted in the plant vascular system with the insect exosomes. the authors managed to show using electron and confocal microscopy that RDV is exosome-associated, however, controls in some experiments are missing, especially those showing the association of the insect exosomes with the plant vascular system.

This manuscript investigated the transmission of Rice Dwarf Virus (RDV) by leafhoppers and suggest that this virus uses the exosome pathway in salivary glands to be transmitted to host plant phloem by employing exosomal bodies which shuttle viral particles into the plant phloem where it starts infection. The manuscript presents an evidence of how vector-borne viruses use their relationships with their arthropod vectors to be well-transmitted to subsequent plant hosts and initiate infection. The use of exosome is a unique strategy by which membrane-associated structure are secreted into plant sieve elements for further infection and their formation is regulated by the expression of associated genes with the exosomal pathway. The authors used both electron and fluorescent microscopy as well as molecular biology tools to track the mode of transmission of RDV and the involved pathways. Some points require clarification and potentially additional experiments to fully support the conclusions.

1. In figure 1 the authors indicate the involvement of exosomes for shuttling RDV virions, however they did not use immunogold labeling as they did in figure 2 to track the virions. To support those images I would also perform immunogold labeling as in the size range of RDV and ribosomes and maybe other organelle structures are close and the images can be misleading without using specific antibody labeling and subsequent gold detection.

2. In figure 2 the authors indeed performed immunogold labeling, however, the amount of presented results in this regards are insufficient, and this kind of experiments needs to be quantified in order to make sure the labeling observed is not random as many times the gold particles attach non specifically. Although the subsequent results with regards to Rab27a, Rab5 and P8 show that those exosome-related genes are indeed induced upon virus infection and the silencing of two of them reduced their levels and colocalization with the virus.

3. I think better labeling of figure 4 panels, especially figure 4D and G is needed, to show the released virions which need to be marked.

4. Figure 7 shows that RDV is secreted into plant sieve elements with exosomes derived in the salivary glands of its leafhopper vector. The authors mainly used immunofluorescence and western blot. Control immunofluorescence images without antibodies or other controls are not provided. It is known that plant tissues strongly auto fluoresce at some wavelengths and thus it necessary to show controls for all these images.

5. The hypothesis is that exosome containing RDV virions are secreted into phloem. Since the authors can measure the diameter of such exosomes, I wonder if they can show measurements of exosomes and then compare this to the diameter of the salivary canal in the insect stylet. I assume the diameter of the salivary canal is way thinner than the exosome, and i wonder how such exosomes can move through the salivary canal if they are bigger. More information needs to be provided as this is the only route such exosomes would leave the insect and enter the plant.

Reviewer #2:

The authors present in this manuscript compelling data that the Rice Dwarf Virus (RDV) might be transmitted from the grasshopper vector into the phloem of rice host plant via insect exosomes. The authors support this hypothesis with their findings of i) co-localization of the vector MVB marker proteins Rab27a/Rab5 and virion particles and ii) physical interaction of the N-terminal viral capsid protein P2 (P2N) with vector Rab5. Moreover, transient knockdown of vector Rab27a/Rab5 via dsRNA treatment inhibits viral packaging into MVBs and rice transmission.

In general, the text is well written and experiments are well designed and explained. However, in order to state that exosomes are the means of virus transmission into rice phloem (see title) examination of exosomes with additional biomarkers and nanoparticle track analysis is necessary. Further, regarding the provided experimental data, some experiments would benefit from additional controls to ensure specificity of the experimental outcome and their interpretation.

Comments for the authors:

Rab27a and Rab5 dsRNA knockdown experiments. Please, provide control experiments of the specificity of targeted knockdown. Since Rab GTPases contain highly conserved domains, co-suppression might have occurred. For example, this could be tested by dsRNA Rab27a and Rab5 cross-check.

It was unclear whether Rab GTPases are optimal exosome biomarkers, and a suggestion is to include tetraspanins (orthologs of the mammalian CD63) as most commonly used exosome biomarkers.

It was unclear whether the specificity of the used antibodies has been vetted. Both anti-Rab antibodies are raised against Drosophila proteins and are polyclonal.

Quantification of difference in protein levels (Western blot) requires at least three biological replicates (Figure 2G, 3B, 4F, 5G, 7b).

In Figure 2A, the authors state that "Rab27a antibodies specifically labelled virus-containing exosomes", but Rab27a signals appear also in non-viral loaded exosomes in Figure 2A-II.

The authors state in lines 137/138"… KD Rab27a significantly reduced the accumulation of virus and Rab27a positive puncta in salivary glands". Such statement requires quantitative analysis and proper statistics in order to claim such significance.

Knockdown of Rab27a as well as chemical inhibition of endosome formation leads to reduced viral P8 expression (virus quantity) and cellular foci in fluorescence microscopy images. However, these data do not present exosomal release of virus, as interpreted by the authors.

The cell culture system was used to isolate exosomes and IGL via Rab27a antibody by electron microscopy. The authors claim that exosomes with viral particles are enlarged compared to non-virus containing exosomes. Here, size measurement and relative quantities by nanoparticle track analysis should be performed to support this statement. Additional biomarkers such as tetraspanins would support the identity of exosomes. Again, like in Figure 2, Rab27a labeled exosomes free of virus, thus Rab27a is not specific to virus-containing exosomes.

The authors state that "treatments of dsRNA Rab27a significantly abolished the formation of exosomes", similar to GW54869. Such statement must be verified by exosome quantification using nanoparticle track analysis.

No explanation was provided why Rab5 localization via antibody or Rab5-cMyc expression look so different, see Figure 6A versus B.

In Figure 6C, it seems that pictures for mock and DMSO have a longer exposure time than GW4869 and chloroquine. In order to claim any GFP signal intensity difference, quantitative analysis on biological replicates must be provided.

In Figure 7A, there seems to be cross-reaction of the Rab5 antibody with rice protein(s) leading to background signals in non-feeding samples. This makes it difficult to compare Rab5 translocation during feeding.

Figure 7D: Label WB with viruliferous or non- viruliferous instead of 1 and 2.

Here, I would suggest a complementary experiment, in which extracted exosomes, e.g. from infected grasshopper cell culture, to be injected in the rice phloem, to reproduce effects of viral spread, as observed in infected rice. Would such exosome injection lead to disease symptoms in rice?

Figure S1: Please clarify what Roman numbering indicates.

Figure S4B: What was the way to normalize fluorescence intensity (considering background noise)?

Figure S5E: axis labels of graphs are not readable.

Reviewer #3:

The manuscript under review addresses a timely and interesting question regarding how viruses are transmitted by insects and suggests that a plant virus, RDV, upon replicating in the cells of the insect vector, is secreted in exosomes and delivered to the insect saliva, and subsequently to the plant phloem. Although many studies have shown the involvement of exosomes and related machinery in animal viruses, very little is known about this phenomenon with plant viruses. The authors present very high-quality data in support of their conclusions. Indeed, the microscopy images are very well presented and convincing. At the same time, the authors present important data that suggest a very plausible mechanism for how the virus is packaged into multi-vesicular endosomes (MVEs) before being secreted. They show that the viral coat protein (CP) interacts with Rab5, a well-established identity marker for early endosomes. This mechanistic work is validated using several lines of evidence using protein-protein interaction methods and cell biology. Furthermore, they show that the CP and RDV particles associate with Rab27a, a marker for several secretion pathways, including exosomes. In addition, the association with the Rabs is shown to be biologically relevant as knock downs affect virus transmission. As such, there is a convincing story here regarding the packaging of RDV into exosomes in the insect vector.

There is only one major issue, regarding the putative detection of insect-derived exosomes in the plant that should either be addressed or be toned down (see point 9 below). Although the authors make a case for the insect proteins Rab5 and Rab27a being important for vectoring the virus, there are controls missing regarding the specificity of detection of these proteins in the plant. This point needs to be addressed.

The flow of the manuscript and presentation of data in the figures is good and relatively easy to follow. However, the writing needs to be significantly improved before publication in terms of form and grammar. The manuscript would also be improved by more discussion of how this data fits with the current state of knowledge on exosomes in plants. For example, Rab5 is associated with early endosomes. As such, is it logical, or consistent with the literature, to detect Rab5 in exosomes? This report will be of interest to a broad range of researchers and it is important to introduce and discuss the dynamics of the endomembrane system to this broad audience.

Comments for the authors:

1. Figure 3b. The RNAi-mediated knock-down of Rab27b does not result in a dramatic reduction of the Rab27b protein (1 vs. 1.41). Is this result representative? That is, the qRT-PCR results suggest that the efficacy of knock down varies widely between individual insects.

2. At several points, the authors indicate that virus-containing exosomes as being larger than normal exosomes, but is there some quantification for this?

3. Figure legends: generally, the figure legends are not acceptable for a scientific publication. Legends need to contain all the information required to understand what was done in the experiments, without interpretation of the results. For example, multiple figure legends talk about immunostaining with virus-rhodamine red and Rab27a-FITC. Presumably, the virus was detected with anti virus antibodies labelled with rhodamine, not with actual virus. Likewise, in Figure 5C it is simply indicated "GST pulldown" or "proteins incubated with cell lysate". While one can figure out what is meant, this is not the way to formally write a figure legend. These are just two small examples and all legends should be revisited.

4. Figure 4e. KD of Rab27a is again not that remarkable by western. At the same time neither is reduction of virus P8 – 1.39 in dsGFP vs. 1 in dsRab27a (would it not make more sense to normalize to dsGFP as 1).

However, would it not actually make sense that virus levels are not that different upon KD of Rab27a? According to the authors, Rab27a is important for virus transmission, but it should not affect replication of the virus; only its ability to infect other cells. Presumably, replication is not taking place in the MVBs.

Same point for results using GW4869.

5. Same point as above for Figure 5 and KD or Rab5. It is perhaps not feasible, but would it be more informative to investigate difference in total virus accumulation versus extracellular virus accumulation (in cell culture – maybe in saliva?).

6. Figure 5D (and elsewhere): The images indicate exosomes and virus-containing exosomes. The latter are from virus-infected cells, but in this case, how do you know that there are virus particles in these exosomes? Is there a method used to differentiate the "bodies" in regular exosomes versus viruses"

7. Figure 6B: P2N shows colocalization with Rab5 and, in a separate set of experiments, with Rab27a. Thus, P2N colocalizes with both proteins, but are all three colocalized. In vertebrate systems invagination of exosomes is thought to be concomitant to replacement of Rab5 with Rab7, presumably prior to recruitment of Rab27a, which is associated with later stages related to secretion. Given what is known about these Rab GTPases, is there precedent for them to be present on the same vesicles.

Do you have direct data showing colocalization of Rab5 and Rab27a?

8. Please discuss more what is known about exosomes in plants and if possible their known associations with different Rabs.

9. Figure 7D. Western blots show Rab5 and Rab27a accumulation in plants exposed to leafhoppers, with a moderate difference in accumulation of both when exposed to viruliferous insects. These blots should include samples from plants not exposed to leafhoppers to demonstrate a lack of cross-reaction to any plant protein. This is particularly important given that there is a non-negligible background in the immunofluorescence microscopy of non-feeding plants. The antibodies used in this study were raised against Drosophila proteins and quick BLAST of Drosophila Rab5, and to a lesser extent Rab27a, against rice shows significant blocks of exact or near exact homology.

It is thus important to determine if the signals seen are Rab GTPases from the insect or from the plant. It is not implausible that feeding from viruliferous and non-viruliferous could induce the expression of Rab GTPases. Indeed, given the amount Rab proteins putatively transferred from insect to plant, these proteins would presumably be diluted throughout the entire plant. Is it plausible that the insect would secrete enough of these proteins to detectable in all parts of the phloem?

10. Supplementary figure 4 (and related to figure 6): data is shown on the quantification of fluorescence as a measure of P2N. It is indicated that there is less in the presence of the two inhibitors. However, if there is no longer a concentration of P2N in specific vesicles, then one would expect the diffuse protein to be much less detectable. As such, and quantification based on fluorescence is probably not accurate and should rather be assayed by western blot.

11. Movahed et al., have reported Turnip Mosaic Virus Components Are Released into the Extracellular Space by Vesicles in Infected Leaves. These vesicles also appeared to contain several Rab proteins, albeit not the as those investigated here (and these are plant derived exosomes). Nonetheless, this study should be discussed in the context of the current manuscript.

https://doi.org/10.7554/eLife.64603.sa1

Author response

Essential revisions:

1) Verify exosome identify:

Some concerns were raised regarding the identity/origin of exosomes. These points are especially important to address as your main conclusion is that the virus is shuttled from the insect to the plant via insect-derived exosomes.

We prepared for the antibody against CD63 of N. cincticeps, which can be used for labelling the MVB and exosomes in the salivary glands and cultured leafhopper cells. The related contents appeared in Figures 2, 4 and 7. Furthermore, we have added the necessary plant or antibody controls to prove the specificity for Rab5, Rab27a and CD63 antibodies to react with the exosome components secreted from insect vectors, but not with rice components. Related results were shown in Figure 7.

2) Antibody specificity:

Some concerns were raised regarding the specificity of antibodies used in immunofluorescence and western blot experiments. It is not clear that these antibodies have been properly vetted for use in this heterologous system. It is important to have controls without antibodies in some immunofluorescence assays to rule out any plant autofluorescence, as well as in western blots to rule out any cross-reaction with plant proteins.

The amino acid sequences of Rab27a and Rab5 of N. cincticeps had no significant similarity by using Blast N, although they showed the conserved domain of Rab GTPases. The relative expression of Rab27a and Rab5 genes of N. cincticeps in the salivary glands after the treatment with dsRab27a or Rab5 were tested. The absence of co-suppression in the treatments of dsRab27a or dsRab5 was concluded. Furthermore, there were 57% and 79% amino acid sequence similarities between Drosophila and N. cincticeps Rab27a and between Drosophila and N. cincticeps Rab5, respectively. Although both Rab antibodies were prepared against the antigen derived from Drosophila, these antibodies showed the specificity of N. cincticeps samples in western blots with correct molecular weights, and in the immunofluorescence and immunoelectron microscopy with the localization of MVBs and exosomes. These results confirmed that both Rab antibodies in this study were useful for recognizing exosomes in N. cincticeps. The related contents were shown on lines 241-246, 389-391, Figures 2-5 and Figure 5—figure supplement 2.

We have added the necessary plant or antibody controls to prove the specificity for Rab5, Rab27a and CD63 antibodies to react with the exosome components secreted from insect vectors, but not with rice components. The controls without antibodies were added to exclude the plant autofluorescence. Western blot assay showed that the utilization of Rab5, Rab27a and CD63 antibodies did not cause the cross-reaction with plant proteins. The related contents were shown in Figures 7.

3) Justify use of Rab markers for exosomes:

Some concerns were raised around the use of Rab GTPases as optimal markers for exosomes. The authors need to justify their use. Our suggestion is to conduct a few comparative assays alongside better-established exosomal markers (such as tetraspanins; orthologs of mammalian DC63).

We prepared for the antibody against CD63 of N. cincticeps, which can be used for labelling the MVB and exosomes in the salivary glands and leafhopper cultured cells. The related contents were shown in Figures 2, 4 and 7.

4) Specificity of knock-downs in miRNA experiments:

Due to sequence similarity among Rab proteins, concerns were raised regarding the specificity of the miRNA-mediated knock down assays. Please verify that the intended transcripts have been specifically knocked down.

The amino acid sequences of Rab27a and Rab5 of N. cincticeps had no significant similarity by using Blast N, although they showed the conserved domain of Rab GTPases. The relative expression of Rab27a and Rab5 genes of N. cincticeps in the salivary glands after the treatment with dsRab27a or Rab5 was tested. RT-qPCR assay also showed that the knockdown of Rab5 expression did not significantly affect Rab27a expression, and that the knockdown of Rab27a expression also did not significantly affect Rab5 expression. The related contents were shown in Figure 5—figure supplement 2D.

5) Size of exosomes:

Please provide exosome measurements to back up any claims of size differences.

We have measured the sizes of virus-free and virus-containing exosomes by negative staining electron microscopy, and showed that the virus-containing exosomes are larger than virus-free exosomes. The related contents were shown in Figure 1K and Figure 4D.

Reviewer #1:

[…] 1. In figure 1 the authors indicate the involvement of exosomes for shuttling RDV virions, however they did not use immunogold labeling as they did in figure 2 to track the virions. To support those images I would also perform immunogold labeling as in the size range of RDV and ribosomes and maybe other organelle structures are close and the images can be misleading without using specific antibody labeling and subsequent gold detection.

We characterized double-layered RDV particles of approximately 65 nm in diameter, which were bigger than ribosomes with a diameter of 25-30 nm. We have performed immunogold labeling to track the virions. The related results were shown on lines 119-121 and 172-173, as well as Figure 1—figure supplement 2.

2. In figure 2 the authors indeed performed immunogold labeling, however, the amount of presented results in this regards are insufficient, and this kind of experiments needs to be quantified in order to make sure the labeling observed is not random as many times the gold particles attach non-specifically. Although the subsequent results with regards to Rab27a, Rab5 and P8 show that those exosome-related genes are indeed induced upon virus infection and the silencing of two of them reduced their levels and colocalization wit the virus.

We have presented the new images in Figure 2A-B to show the immunogold labelling of Rab27a and CD63 on the MVBs or exosomes. For these immunoelectron microscopic images, the gold particles on MVBs or exosomes are specific and not random.

3. I think better labeling of figure 4 panels, especially figure 4D and G is needed, to show the released virions which need to be marked.

The arrows in the enlarge images and the new images in Figure 4E and F indicated the released virions.

4. Figure 7 shows that RDV is secreted into plant sieve elements with exosomes derived in the salivary glands of it's leafhopper vector. The authors mainly used immunofluorescence and western blot. Control immunofluorescence images without antibodies or other controls are not provided. It is known that plant tissues strongly auto fluoresce at some wavelengths and thus it necessary to show controls for all these images.

The controls without antibodies were added to Figure 7A to show the weak background caused by autofluorescence of plant.

5. The hypothesis is that exosome containing RDV virions are secreted into phloem. Since the authors can measure the diameter of such exosomes, I wonder if they can show measurements of exosomes and then compare this to the diameter of the salivary canal in the insect stylet. I assume the diameter of the salivary canal is way thinner than the exosome, and i wonder how such exosomes can move through the salivary canal if they are bigger. More information needs to be provided as this is the only route such exosomes would leave the insect and enter the plant.

The average diameters of salivary canal of most leafhoppers in Cicadellidae range from 3 μm to 500 nm (Brozek and Herczek, 2000; Zhao et al., 2010; Leopold et al., 2003), which are bigger than the virus-containing exosome. Therefore, such virus-containing exosomes can move through the salivary canal. We also added this discussion on lines 306-309.

Reviewer #2:

[…] Comments for the authors:

Rab27a and Rab5 dsRNA knockdown experiments. Please, provide control experiments of the specificity of targeted knockdown. Since Rab GTPases contain highly conserved domains, co-suppression might have occurred. For example, this could be tested by dsRNA Rab27a and Rab5 cross-check.

The amino acid sequences of Rab27a and Rab5 of N. cincticeps had no significant similarity according to Blast N, although they showed the conserved domain of Rab GTPases. The relative expression of Rab27a and Rab5 genes of N. cincticeps in the salivary glands after the treatment with dsRab27a or Rab5 was tested. RT-qPCR assay also showed that the knockdown of Rab5 expression did not significantly affect Rab27a expression and that the knockdown of Rab27a expression also did not significantly affect Rab5 expression. The related contents were shown in Figure 5—figure supplement 2D.

It was unclear whether Rab GTPases are optimal exosome biomarkers, and a suggestion is to include tetraspanins (orthologs of the mammalian CD63) as most commonly used exosome biomarkers.

We have prepared for the antibody against CD63 of N. cincticeps, which can be used for labelling the MVB and exosomes in the salivary glands and leafhopper cultured cells. The related contents were shown in Figures 2, 4 and 7.

It was unclear whether the specificity of the used antibodies has been vetted. Both anti-Rab antibodies are raised against Drosophila proteins and are polyclonal.

Rab27a or Rab5 from N. cincticeps and Drosophila had 57% or 79% amino acid similarity, respectively. Although both Rab antibodies were prepared against the antigen derived from Drosophila, these antibodies showed the specificity of N. cincticeps samples in western blots with correct molecular weights, and in the immunofluorescence and immunoelectron microscopy with the localization of MVBs and exosomes. These results confirmed that both Rab antibodies in this study were useful for recognizing exosomes in N. cincticeps. The related contents were shown on lines 244-246, 389-391, and on Figures 2-5 and Figure 5—figure supplement 2E.

Quantification of difference in protein levels (Western blot) requires at least three biological replicates (Figure 2G, 3B, 4F, 5G, 7b).

The results of western blot represent three biological replicates. The descriptions were shown in the Figure Legends of Figures 2G, 3B, 4I, 5H, 5J, 7D and Figure 6—figure supplement 1C.

In Figure 2A, the authors state that "Rab27a antibodies specifically labelled virus-containing exosomes", but Rab27a signals appear also in non-viral loaded exosomes in Figure 2A-II.

We have revised the similar sentences in the full text.

The authors state in lines 137/138"… KD Rab27a significantly reduced the accumulation of virus and Rab27a positive puncta in salivary glands". Such statement requires quantitative analysis and proper statistics in order to claim such significance.

We have added the results of quantitative analysis to show the change of virus, Rab5, Rab27a and CD63, as detected by immunofluorescence microscopy in Figures 2E, 3D, and 5F.

Knockdown of Rab27a as well as chemical inhibition of endosome formation leads to reduced viral P8 expression (virus quantity) and cellular foci in fluorescence microscopy images. However, these data do not present exosomal release of virus, as interpreted by the authors.

To further confirm that the treatment of dsRab27a and GW4869 would inhibit viral exosomal release from infected cultured cells without significant effect on viral propagation, we perform one new experiment. The cultured cells treated with dsRNAs (dsGFP and dsRab27a), DMSO or GW4869 were inoculated with purified RDV at a MOI of 10 which guaranteed viral infection rate was 100%. At 48 hpi, the extracellulaar medium and the cells were collected. We showed that the treatment of dsRab27a or GW4869 significantly reduced RDV P8 transcript loads in the extracellular medium, but did not significantly reduce RDV P8 transcript loads in cell-associated viruses. These results demonstrated that RDV had proliferated in the infected cells but viral release from the cells had been impeded by the inhibition of exosomes formation. The related contents were shown on lines 192-203 and Figure 4—figure supplement 1.

The cell culture system was used to isolate exosomes and IGL via Rab27a antibody by electron microscopy. The authors claim that exosomes with viral particles are enlarged compared to non-virus containing exosomes. Here, size measurement and relative quantities by nanoparticle track analysis should be performed to support this statement. Additional biomarkers such as tetraspanins would support the identity of exosomes. Again, like in Figure 2, Rab27a labeled exosomes free of virus, thus Rab27a is not specific to virus-containing exosomes.

For the size measurement and relative quantities by nanoparticle track analysis, we have contacted the expert of this method, and known that the quantity of purified exosomes from the salivary glands or medium of infected monolayer of cultured leafhopper cells are not enough for this analysis. Thus, we have measured the sizes of virus-free and virus-containing exosomes in salivary glands, and proved the virus-containing exosomes were larger compared to virus-free exosomes. We also used negative staining electron microscopy to observe the purified exosomes from cultured cells and analyzed the size. The related contents were shown on lines 121-123 and 177-180, as well as Figures 1K and 4D.

We prepared for the antibody against CD63 of N. cincticeps, which can be used for labelling the MVB and exosomes in the salivary glands and leafhopper cultured cells. The related contents were shown in Figures 2, 4 and 7. For the comment “Again, like in Figure 2, Rab27a labeled exosomes free of virus, thus Rab27a is not specific to virus-containing exosomes”, we have checked the full text and revised the similar sentences for Rab27a, CD63 and Rab5.

The authors state that "treatments of dsRNA Rab27a significantly abolished the formation of exosomes", similar to GW54869. Such statement must be verified by exosome quantification using nanoparticle track analysis.

For the relative quantities by nanoparticle track analysis, we have contacted the expert of this method, and known that the quantity of purified exosomes from the medium of infected monolayer of N. cincticeps cultured cells are not enough for this analysis. Instead, we performed the negative staining electron microscopy to analyze the impaired purified exosomes secreted from dsRNAs- and GW4869-treated cells. The related contents were shown on lines 184-187, and Figure 4G.

No explanation was provided why Rab5 localization via antibody or Rab5-cMyc expression look so different, see Figure 6A versus B.

We used the baculovirus expression system in Sf9 cells to overexpress N. cincticeps Rab5 fused with cMyc tag (Rab5-cMyc). The results showed that the expressed N. cincticeps Rab5 colocalized with RDV P2N fused with His (Figure 6A). Furthermore, we also used antibody against Drosophila Rab5 to label Rab5 of Sf9 cells. The results showed RDV P2N could colocalize with Rab5 of Sf9 cells (Figure 6B). The explanation was on lines 248-258.

In Figure 6C, it seems that pictures for mock and DMSO have a longer exposure time than GW4869 and chloroquine. In order to claim any GFP signal intensity difference, quantitative analysis on biological replicates must be provided.

We have provided the new images for mock and DMSO, which did not show a longer exposure time than GW4869 and chloroquine in Figure 6C. Furthermore, we have quantified the GFP signal intensity of mock, DMSO, GW4869 and chloroquine treatments, and did three biological replicates. The results were demonstrated in Figure 6—figure supplement 1 and 2.

In Figure 7A, there seems to be cross-reaction of the Rab5 antibody with rice protein(s) leading to background signals in non-feeding samples. This makes it difficult to compare Rab5 translocation during feeding.

Rab5 antibody did cause weak background signal in non-feeding samples in some degree. However, with the feeding time increasing, the immunofluorescence of Rab5 antibody in samples of nonviruliferous leafhopper feeding or viruliferous leafhopper feeding strengthened, and showed obvious differences. These results showed that Rab5 translocation to plant phloem during vector feeding. We also added a control without antibodies to show the autofluorescence of plant tissues. We hope these data could prove the release of Rab5 from leafhopper to rice plants. The results were shown in Figure 7A.

I am wondering, if it would be possible to isolate exosomes from salivary glands from leafhoppers either non-viruliferous, viruliferous, or viruliferous upon injection of dsRNA Rab27a. Such data would significantly support the engulfment of virus into exosomes complementing the here provided EM images.

It seems impossible for us to isolate exosomes from salivary glands from leafhoppers, because the leafhoppers are so tiny that the exosome isolated from salivary glands using the general method are very limited.

Figure 7D: Label WB with viruliferous or non- viruliferous instead of 1 and 2.

We have revised this.

Here, I would suggest a complementary experiment, in which extracted exosomes, e.g. from infected grasshopper cell culture, to be injected in the rice phloem, to reproduce effects of viral spread, as observed in infected rice. Would such exosome injection lead to disease symptoms in rice?

This suggestion is good for the inoculation of animal cultured cells with exosomes purified from insect cultured cells. However, as a persistent-propagative plant virus (RDV), the purified virus-containing exosomes from infected cultured cells of leafhopper must be firstly acquired by insect vectors during feeding, and then the viruses can transmit into plants from insect salivary glands. Thus, it is impossible for the direct inoculation of purified virus-containing exosomes from infected cultured cells into rice plants.

Figure S1: Please clarify what Roman numbering indicates.

We have explained this in Figure legend.

Figure S4B: What was the way to normalize fluorescence intensity (considering background noise)?

We further used western blot assay to test the expression levels of P2N-His in different treatments in Figure 6—figure supplement 1C.

Figure S5E: axis labels of graphs are not readable.

We are sorry for making you puzzled. We added the explanations to the Figure legend.

Reviewer #3:

The manuscript under review addresses a timely and interesting question regarding how viruses are transmitted by insects and suggests that a plant virus, RDV, upon replicating in the cells of the insect vector, is secreted in exosomes and delivered to the insect saliva, and subsequently to the plant phloem. Although many studies have shown the involvement of exosomes and related machinery in animal viruses, very little is known about this phenomenon with plant viruses. The authors present very high-quality data in support of their conclusions. Indeed, the microscopy images are very well presented and convincing. At the same time, the authors present important data that suggest a very plausible mechanism for how the virus is packaged into multi-vesicular endosomes (MVEs) before being secreted. They show that the viral coat protein (CP) interacts with Rab5, a well-established identity marker for early endosomes. This mechanistic work is validated using several lines of evidence using protein-protein interaction methods and cell biology. Furthermore, they show that the CP and RDV particles associate with Rab27a, a marker for several secretion pathways, including exosomes. In addition, the association with the Rabs is shown to be biologically relevant as knock downs affect virus transmission. As such, there is a convincing story here regarding the packaging of RDV into exosomes in the insect vector.

There is only one major issue, regarding the putative detection of insect-derived exosomes in the plant that should either be addressed or be toned down (see point 9 below). Although the authors make a case for the insect proteins Rab5 and Rab27a being important for vectoring the virus, there are controls missing regarding the specificity of detection of these proteins in the plant. This point needs to be addressed.

We have added the necessary plant or antibody controls to prove the specificity for antibodies against Rab5, Rab27a and CD63 to react with exosome components secreted from insect vectors, but not with rice components. Related results were shown in Figure 7.

The flow of the manuscript and presentation of data in the figures is good and relatively easy to follow. However, the writing needs to be significantly improved before publication in terms of form and grammar. The manuscript would also be improved by more discussion of how this data fits with the current state of knowledge on exosomes in plants. For example, Rab5 is associated with early endosomes. As such, is it logical, or consistent with the literature, to detect Rab5 in exosomes? This report will be of interest to a broad range of researchers and it is important to introduce and discuss the dynamics of the endomembrane system to this broad audience.

In the Introduction and Discussion, we have added the sentences to induce and discuss the dynamics of the exosomes biogenesis, including plant system. The published literatures indicated that Rab5 was also detected in the exosomes or extracellular vesicles isolated from mammalian or plant cells, especially in virus-infected systems. Furthermore, our data of RDV P2-Rab5 interaction would lead to the recruitment of Rab5 on the surface of virions within the exosomes. These observations (Figure 5) are fully consistent with the role of RDV P2 in viral package within MVB via interaction with Rab5. Thus, our finding is logical and consistent with the published literatures. The related contents appear on lines 65-72 in the Introduction, and on lines 302-306, 332-338 in the Discussion.

Comments for the authors:

1. Figure 3b. The RNAi-mediated knock-down of Rab27b does not result in a dramatic reduction of the Rab27b protein (1 vs. 1.41). Is this result representative? That is, the qRT-PCR results suggest that the efficacy of knock down varies widely between individual insects.

We have performed the western blot again, and analyzed the relative intensities of bands. The new data was shown in Figure 3B.

2. At several points, the authors indicate that virus-containing exosomes as being larger than normal exosomes, but is there some quantification for this?

We have measured the sizes of virus-free and virus-containing exosomes, and proved that virus-containing exosomes were larger compared to virus-free exosomes. The related sentences were added on lines 121-123 and 177-180. The images were added to Figures 1K and 4D.

3. Figure legends: generally, the figure legends are not acceptable for a scientific publication. Legends need to contain all the information required to understand what was done in the experiments, without interpretation of the results. For example, multiple figure legends talk about immunostaining with virus-rhodamine red and Rab27a-FITC. Presumably, the virus was detected with anti virus antibodies labelled with rhodamine, not with actual virus. Likewise, in Figure 5C it is simply indicated "GST pulldown" or "proteins incubated with cell lysate". While one can figure out what is meant, this is not the way to formally write a figure legend. These are just two small examples and all legends should be revisited.

We have carefully revised the Figure legends in the full text.

4. Figure 4e. KD of Rab27a is again not that remarkable by western. At the same time neither is reduction of virus P8 – 1.39 in dsGFP vs. 1 in dsRab27a (would it not make more sense to normalize to dsGFP as 1).

Thank you very much. We analyzed the intensities of bands again, and normalize to dsGFP as 1. The reduction of Rab27a is remarkable: 0.43 in dsRab27a vs. 1.00 in dsGFP. The reduction of P8 0.54 in dsRab27a vs. 1.00 in dsGFP. The relative data was also renewed in Figure 4I.

However, would it not actually make sense that virus levels are not that different upon KD of Rab27a? According to the authors, Rab27a is important for virus transmission, but it should not affect replication of the virus; only its ability to infect other cells. Presumably, replication is not taking place in the MVBs. Same point for results using GW4869.

To further confirm that the treatment of dsRab27a and GW4869 would inhibit viral exosomal release from infected cultured cells without significant effect on viral propagation, we perform one new experiment. The cultured cells treated with dsRNAs (dsGFP and dsRab27a), DMSO or GW4869 were inoculated with purified RDV at a MOI of 10 which guaranteed viral infection rate was 100%. At 48 hpi, the extracellular medium and the cells were collected. We showed that the treatment of dsRab27a or GW4869 significantly reduced RDV P8 transcript loads in the extracellular medium, but did not significantly reduce RDV P8 transcript loads in cell-associated viruses. These results demonstrated that RDV had proliferated in the infected cells but viral release from the cells had been impeded by the inhibition of exosomes formation. The related contents were shown on lines 192-203 and Figure 4—figure supplement 1.

5. Same point as above for Figure 5 and KD or Rab5. It is perhaps not feasible, but would it be more informative to investigate difference in total virus accumulation versus extracellular virus accumulation (in cell culture – maybe in saliva?).

We have analyzed the accumulation levels of cell-associated viruses and extracellular viruses in leafhopper cultured cells after different treatments for inhibiting exosomal formation. However, such experiments are not available for insect saliva due to the limited harvest.

6. Figure 5D (and elsewhere): The images indicate exosomes and virus-containing exosomes. The latter are from virus-infected cells, but in this case, how do you know that there are virus particles in these exosomes? Is there a method used to differentiate the "bodies" in regular exosomes versus viruses"

We added the evidence of immunoelectron microscopy to prove the double-layered particles in exosomes were viruses. RDV particles, confirmed by immunoelectron microscopy, were easily distinguished by their spherical appearance and diameter (65 nm), and they were not found in uninfected controls. The related contents were shown on lines 111-114, 119-121, 172-173 and Figure 1—figure supplement 2.

7. Figure 6B: P2N shows colocalization with Rab5 and, in a separate set of experiments, with Rab27a. Thus, P2N colocalizes with both proteins, but are all three colocalized. In vertebrate systems invagination of exosomes is thought to be concomitant to replacement of Rab5 with Rab7, presumably prior to recruitment of Rab27a, which is associated with later stages related to secretion. Given what is known about these Rab GTPases, is there precedent for them to be present on the same vesicles. Do you have direct data showing colocalization of Rab5 and Rab27a?

We have added Figure 5—figure supplement 2E to show that some puncta labeled by Rab27a-FITC could colocalize with the puncta labeled by Rab5-rhodamine in the salivary glands from nonviruliferous N. cincticeps.

Furthermore, our data of RDV P2-Rab5 interaction would lead to the recruitment of Rab5 on the surface of virions within the exosomes. These observations (Figure 5) are fully consistent with the role of RDV P2 in viral package within MVB via interaction with Rab5. In the Introduction and Discussion, we have added the sentences to discuss the dynamics of the exosomes biogenesis, including plant system. The published literatures indicated that Rab5 was also detected in the exosomes or extracellular vesicles isolated from mammalian or plant cells, especially in virus-infected systems. Thus, our finding is logical and consistent with the published literatures. The related contents appear on lines 65-72 in the Introduction, and on lines 302-306, 332-338 in the Discussion.

8. Please discuss more what is known about exosomes in plants and if possible their known associations with different Rabs.

We have discussed the exosomes in plants and their known associations with Rabs on lines 364-373.

9. Figure 7D. Western blots show Rab5 and Rab27a accumulation in plants exposed to leafhoppers, with a moderate difference in accumulation of both when exposed to viruliferous insects. These blots should include samples from plants not exposed to leafhoppers to demonstrate a lack of crossreaction to any plant protein. This is particularly important given that there is a non-negligible background in the immunofluorescence microscopy of non-feeding plants. The antibodies used in this study were raised against Drosophila proteins and quick BLAST of Drosophila Rab5, and to a lesser extent Rab27a, against rice shows significant blocks of exact or near exact homology.

We reconducted this biological experiment for 3 replicates. Western blot assay proved the obvious difference of Rab5, CD63 or Rab27a accumulation between viruliferous and non-viruliferous feeding samples. Furthermore, the utilization of Rab5, CD63 or Rab27a antibody did not cause the cross-reaction with plant proteins. Related results were shown in Figure 7D.

The amino acid sequences of Rab27a and Rab5 of N. cincticeps had no significant similarity according to Blast N, although they showed the conserved domain of Rab GTPases. The relative expression of Rab27a and Rab5 genes of N. cincticeps in the salivary glands treated with dsRab27a or Rab5 were tested. The absence of co-suppression with the treatments of dsRab27a or dsRab5 was concluded. The related results were shown in Figure 5—figure supplement 2D.

Furthermore, there were 57% and 79% amino acid sequence similarities between Drosophila and N. cincticeps Rab27a and between Drosophila and N. cincticeps Rab5, respectively. Although both Rab antibodies were prepared against the antigen derived from Drosophila, these antibodies showed the specificity of N. cincticeps samples in western blot with correct molecular weights, and in the immunofluorescence and immunoelectron microscopy with the localization of MVBs and exosomes. These results confirmed that both Rab antibodies in this study were useful for recognizing exosomes in N. cincticeps. The related contents were shown on lines 241-246, 389-391, and on Figures 2-5 and Figure 5—figure supplement 3.

It is thus important to determine if the signals seen are Rab GTPases from the insect or from the plant. It is not implausible that feeding from viruliferous and non-viruliferous could induce the expression of Rab GTPases. Indeed, given the amount Rab proteins putatively transferred from insect to plant, these proteins would presumably be diluted throughout the entire plant. Is it plausible that the insect would secrete enough of these proteins to detectable in all parts of the phloem?

We have added the plant controls which were not fed on by leafhoppers. The immunofluorescence result showed the weak background signals of Rab27a and Rab5 in the non-feeding control, and showed strong and specific fluorescence signals in leafhopper-feeding samples. It was suggested that the signals were from insect but not plant. Furthermore, western blot assay confirmed that Rab27a and Rab5 antibodies did not react with the plant samples. These results were shown in Figure 7A and 7D.

Theoretically, Rab proteins would be diluted throughout the entire plant. However, as shown in Figure 7—figure supplement 1, in our study, approximate 15 viruliferous or nonviruliferous leafhoppers were reared in small cages to inoculate rice seedlings for 2 days. We even fed 80 viruliferous or nonviruliferous leafhoppers on 2 rice seedlings to enrich the proteins for western blot assay. Such methods are useful for accumulation of enough amount Rab proteins putatively transferred from insect to plant phloem. The related results were shown in Figure 7.

10. Supplementary figure 4 (and related to figure 6): data is shown on the quantification of fluorescence as a measure of P2N. It is indicated that there is less in the presence of the two inhibitors. However, if there is no longer a concentration of P2N in specific vesicles, then one would expect the diffuse protein to be much less detectable. As such, and quantification based on fluorescence is probably not accurate and should rather be assayed by western blot.

We have added western blot to show the difference of P2N expression level among the treatments in Figure 6—figure supplement 1C. Furthermore, in Figure 6—figure supplement 2, we carefully compared the quantification of fluorescence as a measure of P2N-GFP after the treatment of inhibitors.

11. Movahed et al., have reported Turnip Mosaic Virus Components Are Released into the Extracellular Space by Vesicles in Infected Leaves. These vesicles also appeared to contain several Rab proteins, albeit not the as those investigated here (and these are plant derived exosomes). Nonetheless, this study should be discussed in the context of the current manuscript.

We have discussed the related contents of TuMV extracellular vesicles and their associated Rab proteins on lines 370-373.

https://doi.org/10.7554/eLife.64603.sa2

Article and author information

Author details

  1. Qian Chen

    Vector-borne Virus Research Center, Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8442-6089
  2. Yuyan Liu

    Vector-borne Virus Research Center, Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China
    Contribution
    Investigation, Visualization
    Competing interests
    No competing interests declared
  3. Jiping Ren

    Vector-borne Virus Research Center, Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  4. Panpan Zhong

    Vector-borne Virus Research Center, Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Manni Chen

    Vector-borne Virus Research Center, Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  6. Dongsheng Jia

    Vector-borne Virus Research Center, Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  7. Hongyan Chen

    Vector-borne Virus Research Center, Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  8. Taiyun Wei

    Vector-borne Virus Research Center, Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China
    Contribution
    Conceptualization, Resources, Supervision, Visualization, Methodology, Project administration, Writing - review and editing
    For correspondence
    weitaiyun@fafu.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0732-9752

Funding

National Natural Science Foundation of China (31772124)

  • Qian Chen

National Natural Science Foundation of China (31920103014)

  • Taiyun Wei

National Natural Science Foundation of China (31972239)

  • Qian Chen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are grateful to Dr. Toshihiro Omura (National Agricultural Research Center, Tsukuba, Japan) for providing the antibodies against intact viral particles and P8. This work was supported by grants from the National Natural Science Foundation of China (Grants 31772124, 31920103014, and 31972239).

Senior Editor

  1. Detlef Weigel, Max Planck Institute for Developmental Biology, Germany

Reviewing Editor

  1. Jacqueline Monaghan, Queen's University, Canada

Publication history

  1. Received: November 4, 2020
  2. Accepted: June 21, 2021
  3. Version of Record published: July 2, 2021 (version 1)

Copyright

© 2021, Chen et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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