Gene knockdown by intro-thoracic injection of double-stranded RNA in the brown planthopper, Nilaparvata lugens

Liu

et al. 2016

J Virol

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...

Section: Methodsmentioning

confidence: 99%

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus

Lan

Liu

et al. 2016

J Virol

“…The dsRNAs were microinjected into WBPHs following the method reported for the brown planthopper (23). Briefly, 50 thirdinstar nymphs of the WBPH were fed on SRBSDV-infected rice plants for 1 day.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, 50 thirdinstar nymphs of the WBPH were fed on SRBSDV-infected rice plants for 1 day. Then, individual nymphs were microinjected with 200 nl dsGFP or dsP7-1 (0.5 g/l) at the conjunction site between the prothorax and the mesothorax (23) and then kept on healthy rice plants. At 2 and 6 days post-first access to diseased plants (padp), we dissected the internal organs from 50 WBPHs that had received dsRNAs.…”

Section: Methodsmentioning

confidence: 99%

Virus-Induced Tubule: a Vehicle for Rapid Spread of Virions through Basal Lamina from Midgut Epithelium in the Insect Vector

Jia

Mao

et al. 2014

J Virol

The plant reoviruses, plant rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent propagative manner. These viruses induce the formation of viral inclusions to facilitate viral propagation in insect vectors. The intestines of insect vectors are formed by epithelial cells that lie on the noncellular basal lamina surrounded by visceral muscle tissue. Here, we demonstrate that a recently identified plant reovirus, southern rice black-streaked dwarf virus (SRBSDV), exploits virus-containing tubules composed of virus-encoded nonstructural protein P7-1 to directly cross the basal lamina from the initially infected epithelium toward visceral muscle tissues in the intestine of its vector, the white-backed planthopper (Sogatella furcifera). Furthermore, such tubules spread along visceral muscle tissues through a direct interaction of P7-1 and actin. The destruction of tubule assembly by RNA interference with synthesized double-stranded RNA targeting the P7-1 gene inhibited viral spread in the insect vector in vitro and in vivo. All these results show for the first time that a virus employs virus-induced tubule as a vehicle for viral spread from the initially infected midgut epithelium through the basal lamina, facilitating the rapid dissemination of virus from the intestine of the insect vector. IMPORTANCENumerous plant viruses are transmitted in a persistent manner by sap-sucking insects, including thrips, aphids, planthoppers, and leafhoppers. These viruses, ingested by the insects, establish their primary infection in the intestinal epithelium of the insect vector. Subsequently, the invading virus manages to transverse the basal lamina, a noncellular layer lining the intestine, a barrier that may theoretically hinder viral spread. The mechanism by which plant viruses cross the basal lamina is unknown. Here, we report that a plant virus has evolved to exploit virus-induced tubules to pass through the basal lamina from the initially infected midgut epithelium of the insect vector, thus revealing the previously undescribed pathway adapted by the virus for rapid dissemination of virions from the intestine of the insect vector.

“…To explore the function of NlEG1, we used RNAi as described by Liu et al (2010). Injection with doublestranded RNA (dsRNA) of NlEG1 decreased the transcript levels of NlEG1 in the whole body, salivary gland, midgut, fat body, and ovary of BPH by 81% to 95% over a period of 6 d (2-8 d post injection; Fig.…”

Section: Silencing Nleg1 Impairs Bph Feeding and Fecunditymentioning

confidence: 99%

A Salivary Endo-β-1,4-Glucanase Acts as an Effector That Enables the Brown Planthopper to Feed on Rice

et al. 2017

Plant Physiol.

100

ORCID IDs: 0000-0002-5714-7586 (J.Z.); 0000-0002-3262-6134 (Y.L.).The brown planthopper (BPH) Nilaparvata lugens is one of the most destructive insect pests on rice (Oryza sativa) in Asia. After landing on plants, BPH rapidly accesses plant phloem and sucks the phloem sap through unknown mechanisms. We discovered a salivary endo-b-1,4-glucanase (NlEG1) that has endoglucanase activity with a maximal activity at pH 6 at 37°C and is secreted into rice plants by BPH. NlEG1 is highly expressed in the salivary glands and midgut. Silencing NlEG1 decreases the capacity of BPH to reach the phloem and reduces its food intake, mass, survival, and fecundity on rice plants. By contrast, NlEG1 silencing had only a small effect on the survival rate of BPH raised on artificial diet. Moreover, NlEG1 secreted by BPH did not elicit the production of the defense-related signal molecules salicylic acid, jasmonic acid, and jasmonoyl-isoleucine in rice, although wounding plus the application of the recombination protein NlEG1 did slightly enhance the levels of jasmonic acid and jasmonoyl-isoleucine in plants compared with the corresponding controls. These data suggest that NlEG1 enables the BPH's stylet to reach the phloem by degrading celluloses in plant cell walls, thereby functioning as an effector that overcomes the plant cell wall defense in rice.