Plant reoviruses, rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent-propagative manner. How such persistent infection of plant viruses in insect vectors is established and maintained remains poorly understood. In this study, we used rice gall dwarf virus (RGDV), a plant reovirus, and its main vector leafhopper Recilia dorsalis as a virus–insect system to determine how the small interference (siRNA) pathway modulates persistent infection of a plant virus in its insect vector. We showed that a conserved siRNA antiviral response was triggered by the persistent replication of RGDV in cultured leafhopper cells and in intact insects, by appearance of virus-specific siRNAs, primarily 21-nt long, and the increased expression of siRNA pathway core components Dicer-2 and Argonaute-2. Silencing of Dicer-2 using RNA interference strongly suppressed production of virus-specific siRNAs, promoted viral accumulation, and caused cytopathological changes in vitro and in vivo. When the viral accumulation level rose above a certain threshold of viral genome copy (1.32 × 1014 copies/μg insect RNA), the infection of the leafhopper by RGDV was lethal rather than persistent. Taken together, our results revealed a new finding that the siRNA pathway in insect vector can modulate persistent infection of plant viruses.

IMPORTANCE Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH).Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 ؋ 10 9 copies of the SRBSDV P10 gene/g of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector. Many viral pathogens that cause significant global health and agricultural problems are arthropod borne and transmitted via an insect vector. Many plant viruses are transmitted in a persistent manner by sap-sucking insects, including planthoppers, leafhoppers, thrips, and aphids (1-3). Plant viruses, when ingested by their insect vectors, establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles (1-3). The viruses then move from the midgut muscles into the hemolymph and finally into the salivary glands, from where they can be introduced into plant hosts (1-3). Efficient viral infection of the midgut epithelium, where initial infection is established, is thus a determinative factor in the ability of an insect species to transmit a certain plant virus.This ability of an insect species to transmit a virus, which has been described as vector competence, is common in nature (4, 5). For many years, the midgut barrier has been believed to be the principal determinant of vector competence (1-9). For example, infection of the incompetent insect vectors of Rice dwarf virus and SRBSDV, two plant reoviruses, and Tomato spotted wilt virus (TSWV), a tospovirus, is restricted to the midgut epithelium (7-10). The inability of incompetent insect vectors to transmit these viruses may be caused by a failure of the virus to efficiently replicate in or disseminate from the midgut epithelium (1-5). It is possible that viral titers in the midgut epithelium of incompetent vectors are unable to reach the threshold needed for viral dissemination. The innate immune response in the midgut epithelium may effectively control viral accumulation, thus blocking viral transmission by incompetent vectors. However, whether the initial antiviral immune pathway(s) in the insect midgut can modulate the competence of plant virus vectors is not unknown.Several mosquito and Drosophila innate immune responses have been reported to have an antiviral effect. These responses include RNA interfere...

Rice stripe virus (RSV) infects rice and causes great yield reduction in some Asian countries. In this study, rice cDNA library was screened by a Gal4-based yeast two-hybrid system using pc4, a putative movement protein of RSV, as the bait. A number of positive colonies were identified and sequence analysis revealed that they might correspond to ten independent proteins. Two of them were selected and further characterized. The two proteins were a J protein and a small Hsp, respectively. Interactions between Pc4 and the two proteins were confirmed using coimmunoprecipitation. Implications of the findings that pc4 interacted with two chaperone proteins were discussed.

BackgroundViral disease has become the most severe constraint for the cultivation and production of Passiflora edulis in China. The infection of Telosma mosaic virus (TeMV), a potyvirus, and its effects on the phytochemical components of P. edulis remain largely unknown in China.MethodsP. edulis plants showing distorted leaves and severe mosaic skin on green fruit were identified with TeMV infection through traditional transmission electron microscopy, RT-PCR and modern small RNA sequencing (sRNA-seq) platform. The contents of phytochemical components and the activities of antioxidative enzymes were compared between virus-infected and virus-free P. edulis to confirm the effects of TeMV infection on host plant.ResultsFirstly, approximately 700 nm linear virus particles, representing TeMV, were detected in infected P. edulis fruits and leaves with Electron microscopy. Partial coat protein genes of TeMV were successfully amplified by RT-PCR in infected P. edulis leaves and fruits but not in healthy plants. Abundant small interference RNAs (siRNAs) sequences, showing several characterizations, were specifically generated from the TeMV genome in infected plant fruits by sRNA-seq platform. Furthermore, fruit length, fruit thickness (wideness) and fruit weight decreased significantly due to TeMV infection. The levels of total protein and total sugar increased significantly; however, the level of total fat, total acid and vitamin C decreased obviously after TeMV infection. The level of total phenols, a secondary metabolite, was obviously higher in TeMV-infected than TeMV-free P. edulis fruit. The activities of superoxide dismutases (SOD) and catalases (CAT) obviously increased in TeMV-infected in comparison with healthy P. edulis fruit.ConclusionsTeMV infection adversely affected the development of P. edulis fruits, differently and selectively modulated the phytochemical components of P. edulis fruits. In turn, P. edulis plants enhanced their tolerance to the stress of TeMV infection by increasing the secondary metabolite level and the antioxidative capacity. This is of significant importance to understand the effects of TeMV infection on the biochemical changes and the antioxidant defense mechanism in P. edulis.

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The green rice leafhopper, Nephotettix cincticeps, is a major rice pest in Southeast Asia and Southern China. Novel control strategies must be explored to control the rice pest. Behavior or fitness regulation of insect by modulating the Troponin C (TnC) may be a novel strategy in the comprehensive management of the insect pest. However, characterizations and functions of TnC, especially regarding effect of its RNA interference-mediated gene knockdown on the behavior or fitness of N. cincticeps remain unknown. Here, we successfully cloned and characterized TnC gene from N. cincticeps (Nc-TnC). We demonstrated that Nc-TnC ubiquitously transcribed at all development stages and special tissues in adult insects, with relative higher levels at the adult stage and in the intestinal canal. Microinjection- or oral membrane feeding-based transient knockdown of Nc-TnC adversely affected the performance or fitness, such as the decreased survival, feeding capacity, weight, and fecundity of N. cincticeps. Furthermore, we revealed that the expression of Nc-TnC was suppressed by its interaction with rice dwarf virus-encoded nonstructural protein 10, which ultimately affected detrimentally the corresponding parameters of the performance or fitness of N. cincticeps. In conclusion, our data deepen understanding of Nc-TnC functions during the development of and viral infection in N. cincticeps. It imply Nc-TnC may serve as a potential target for N. cincticeps control in future.

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