Gut-Expressed Vitellogenin Facilitates the Movement of a Plant Virus across the Midgut Wall in Its Insect Vector.

Many viral pathogens of global importance to plant and animal health are persistently transmitted by insect vectors. Midgut of insects forms the first major barrier that these viruses encounter during their entry into the vectors. However, the vector ligand(s) involved in the movement of plant viruses across the midgut barrier remains largely uncharacterized. Begomoviruses, many of which are disease agents of some major crops worldwide, are persistently transmitted by whiteflies (Bemisia tabaci). Here, in order to identify whitefly midgut proteins that interact with a devastating begomovirus, tomato yellow leaf curl virus (TYLCV), we performed midgut-specific TYLCV coat protein (CP) immunoprecipitation followed by high-throughput mass spectrometry proteomic analysis. We find that vitellogenin (Vg), a critical insect reproductive protein that has been considered to be synthesized by the fat body, is also synthesized by and interacts with TYLCV CP in the whitefly midgut. TYLCV appears to be internalized into midgut epithelial cells as a complex with Vg through endocytosis. Virus-containing vesicles then deliver the virus-Vg complexes to early endosomes for intracellular transport. Systematic silencing of Vg or midgut-specific immune blocking of Vg inhibited virus movement across the midgut wall and decreased viral acquisition and transmission by whitefly. Our findings show that a functional Vg protein is synthesized in the midgut of an insect and suggest a novel Vg mechanism that facilitates virus movement across the midgut barrier of its insect vector. IMPORTANCE An essential step in the life cycle of many viruses is transmission to a new host by insect vectors, and one critical step in the transmission of persistently transmitted viruses is overcoming the midgut barrier to enter vectors and complete their cycle. Most viruses enter vector midgut epithelial cells via specific interaction between viral structural proteins and vector cell surface receptor complexes. Tomato yellow leaf curl virus (TYLCV) is persistently transmitted by the whitefly Bemisia tabaci between host plants. Here, we find that TYLCV coat protein interacts with vitellogenin (Vg) in the whitefly midgut. This interaction is required for the movement of the virus crossing the midgut wall and thus facilitates viral acquisition and transmission by whitefly. This study reveals a novel mechanism of virus overcoming the insect midgut barrier and provides new insights into the function of Vg beyond serving as nutrition for developing embryos in insects.

A vector whitefly endocytic receptor facilitates the entry of begomoviruses into its midgut cells via binding to virion capsid proteins.

Many circulative plant viruses transmitted by insect vectors are devastating to agriculture worldwide. The midgut wall of vector insects represents a major barrier and at the same time the key gate a circulative plant virus must cross for productive transmission. However, how these viruses enter insect midgut cells remains poorly understood. Here, we identified an endocytic receptor complex for begomoviruses in the midgut cells of their whitefly vector. Our results show that two whitefly proteins, BtCUBN and BtAMN, compose a receptor complex BtCubam, for which BtCUBN contributes a viral-binding region and BtAMN contributes to membrane anchorage. Begomoviruses appear to be internalized together with BtCubam via its interaction with the 12-19 CUB domains of BtCUBN via clathrin-dependent endocytosis. Functional analysis indicates that interruption of BtCUBN and BtAMN lead to reduction of virus acquisition and transmission by whitefly. In contrast, CUBN-begomovirus interaction was not observed in two non-competent whitefly-begomovirus combinations. These observations suggest a major role of the specific endocytic receptor in facilitating viral entry into vector midgut cells.

A plant DNA virus replicates in the salivary glands of its insect vector via recruitment of host DNA synthesis machinery.

Whereas most of the arthropod-borne animal viruses replicate in their vectors, this is less common for plant viruses. So far, only some plant RNA viruses have been demonstrated to replicate in insect vectors and plant hosts. How plant viruses evolved to replicate in the animal kingdom remains largely unknown. Geminiviruses comprise a large family of plant-infecting, single-stranded DNA viruses that cause serious crop losses worldwide. Here, we report evidence and insight into the replication of the geminivirus tomato yellow leaf curl virus (TYLCV) in the whitefly (Bemisia tabaci) vector and that replication is mainly in the salivary glands. We found that TYLCV induces DNA synthesis machinery, proliferating cell nuclear antigen (PCNA) and DNA polymerase δ (Polδ), to establish a replication-competent environment in whiteflies. TYLCV replication-associated protein (Rep) interacts with whitefly PCNA, which recruits DNA Polδ for virus replication. In contrast, another geminivirus, papaya leaf curl China virus (PaLCuCNV), does not replicate in the whitefly vector. PaLCuCNV does not induce DNA-synthesis machinery, and the Rep does not interact with whitefly PCNA. Our findings reveal important mechanisms by which a plant DNA virus replicates across the kingdom barrier in an insect and may help to explain the global spread of this devastating pathogen.

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR.

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

Juvenile hormone regulates the differential expression of putative juvenile hormone esterases via methoprene-tolerant in non-diapause-destined and diapause-destined adult female beetle.

Juvenile hormone (JH) plays an essential role in regulating molting, metamorphosis, reproduction, and diapause (dormancy), in many insects and crustaceans. JH esterases (JHEs) can control JH titer by regulating JH degradation. Although the biochemistry and structure of JHEs have been well studied, regulation of their expression remains unclear. We identified three putative JHEs (JHE1, JHE2, JHE3) in the cabbage beetle Colaphellus bowringi, and investigated the regulation of their expression by JH signaling in non-diapause-destined (NDD, reproductive) and diapause-destined (DD) female adults. Sequence and phylogenetic tree analyses indicate that the three putative JHEs shared conserved motifs with the JHEs of other insects and one crustacean, and were similar to Coleopteran, Dipteran, Orthopteran, Hymenopteran, and Decapodan JHEs. They were, however, less closely related to Hemipteran and Lepidopteran JHEs. JHEs were more highly expressed in NDD female adults than in DD female adults. JH analog induction in DD female adults significantly upregulated the expression of JHE1 and JHE2, but had no effect on the expression of JHE3. Knockdown of the JH candidate receptor methoprene-tolerant (Met) in NDD female adults downregulated the expression of all three JHEs. These results suggest that JHE expression is positively correlated with JH signaling, and that Met may be involved in the JH-mediated differential expression of JHE in DD and NDD adult female C. bowringi.

[Differentiation of recombinant adenoviruses expressed MDV-1 VP22 subcellular localization pattern].

AIM: To study the transduction and localization mechanism of Marek's disease virus serotype 1 (MDV-1) CVI988 VP22 in different cells. METHODS: VP22 was expressed with recombinant adenovirus and identified by immunofluorescence assay (IFA) and Western blot. Lysates of recombinant virus infected 293 cells were added to normal MDBK cells to identify the transduction property of VP22. AD-293 cells infected with recombinant virus were fixed to investigate localization of VP22 at different time post infection. Transient expression of VP22 in AD-293 cells was carried out for control. RESULTS: The results showed that the VP22 expressed by recombinant adenovirus entered almost all the monolayer cells, which indicate the VP22 remains its transduction property. The VP22 first gather round the nucleus membrane, and then concentrated in particles in cytoplasm of 293 cells infected with recombinant adenovirus, compared with the nuclei localization pattern of VP22 in MDV infected CEF and transient expressed VP22 in 293 cells. CONCLUSION: The VP22 presented a different localization pattern in cells infected with different recombinant virus.