Plants pre-infected with a mild variant of a virus frequently become protected against more severe variants of the same virus through the cross protection phenomenon first discovered in 1929. Despite its widespread use in managing important plant virus diseases, the mechanism of cross protection remains poorly understood. Recent investigations in our labs, by analyzing the whole-plant dynamics of a turnip crinkle virus (TCV) population, coupled with cell biological interrogation of individual TCV variants, revealed possible novel mechanisms for cross protection and the closely related process of superinfection exclusion (SIE). Our new mechanistic model postulates that, for RNA viruses like TCV, SIE manifests a viral function that denies progeny viruses the chance of re-replicating their genomes in the cells of their “parents,” and it collaterally targets highly homologous superinfecting viruses that are indistinguishable from progeny viruses. We further propose that SIE may be evolutionarily selected to maintain an optimal error frequency in progeny genomes. Although primarily based on observations made with TCV, this new model could be broadly applicable to other viruses as it provides a molecular basis for maintaining virus genome fidelity in the face of the error-prone nature of virus replication process.

Insect melanization triggered by the conversion of prophenoloxidase to active phenoloxidase via serine proteases (SPs) is an important immediate immune response. However, how phytoplasmas evade this immune response to promote their propagation in insect vectors remains unknown. Here, we demonstrate that infection of leafhopper vectors with rice orange leaf phytoplasma (ROLP) activates the mild melanization response in hemolymph. ROLP-encoded effector protein SRP1 is highly expressed in leafhopper hemolymph, where it competitively binds to SP2, thereby inhibiting SP2-mediated cleavage of prophenoloxidase into active phenoloxidase. Consequently, microinjection of SRP1 effectively suppresses the melanization response and enhances ROLP propagation. The histidine residue at position 23 of SRP1 is essential for SRP1-SP2 interaction, and the mutation of this position abolishes its ability to inhibit such SP2-meidated cleavage, ultimately promoting melanization response and inhibiting ROLP propagation. Our findings provide insights into how phytoplasmas antagonize insect melanization response to facilitate their persistent transmission.

Persistently transmitted plant viruses encounter multiple membrane and tissue barriers in the process of completing their infection routes within their insect vectors. Some of these viruses have been reported to overcome the elaborate barriers of the central nervous system (CNS) to travel through the nervous tissues, but the specific mechanisms of this process remain unknown. Here, we report the axonal transport mechanism of rice yellow stunt virus (RYSV), a nucleorhabdovirus, in the CNS of the green rice leafhopper (Nephotettix cincticeps). The infection route of RYSV in the internal organs of its insect vector after ingestion of the virus was investigated by immunofluorescence microscopy. RYSV was first detected in the epithelial cells of midgut regions, from where it proceeded to the nervous system, and finally into the salivary glands. We then utilized immunofluorescence and electron microscopy to investigate the distribution of RYSV particles within the leafhopper CNS, demonstrating that non-enveloped viral particles distributed along the microtubule-based neurofilaments in the axon cytoplasm following the direct interaction of leafhopper α-tubulin with the RYSV M protein. Tubulin inhibitors inhibited the dissemination of RYSV to the CNS, then into the salivary glands in leafhoppers. We therefore describe a mechanism of plant virus transport through CNS axons as an alternative means of rapid viral dissemination in an insect vector.

In China, the rice pathogen Rice yellow stunt virus (RYSV), a member of the genus Nucleorhabdovirus in the family Rhabdoviridae, was a severe threat to rice production during the1960s and1970s. Fundamental aspects of the biology of this virus such as protein localization and formation of the RYSV viroplasm during infection of insect vector cells are largely unexplored. The specific role(s) of the structural proteins nucleoprotein (N) and phosphoprotein (P) in the assembly of the viroplasm during RYSV infection in insect vector is also unclear. In present study, we used continuous leafhopper cell culture, immunocytochemical techniques, and transmission electron microscopy to investigate the subcellular distributions of N and P during RYSV infection. Both GST pull-down assay and yeast two-hybrid assay were used to assess the in vitro interaction of N and P. The dsRNA interference assay was performed to study the functional roles of N and P in the assembly of RYSV viroplasm. Here we demonstrated that N and P colocalized in the nucleus of RYSV-infected Nephotettix cincticeps cell and formed viroplasm-like structures (VpLSs). The transiently expressed N and P are sufficient to form VpLSs in the Sf9 cells. In addition, the interactions of N/P, N/N and P/P were confirmed in vitro. More interestingly, the accumulation of RYSV was significantly reduced when the transcription of N gene or P gene was knocked down by dsRNA treatment. In summary, our results suggest that N and P are the main viral factors responsible for the formation of viroplasm in RYSV-infected insect cells. Early during RYSV infection in the insect vector, N and P interacted with each other in the nucleus to form viroplasm-like structures, which are essential for the infection of RYSV.

In the field, 80% of plant viruses are transmitted by insect vectors. When ingested by a sap-sucking insect such as Recilia dorsalis, persistently transmitted viruses such as rice stripe mosaic virus (RSMV) infect the gut epithelium and eventually pass to the salivary glands where they will be transmitted to the next rice (Oryza sativa) plant. To efficiently exploit insect vectors for transmission, plant viruses must overcome various immune mechanisms within the vectors, including autophagy. However, understanding how plant viruses overcome insect autophagic defenses remains limited. In this study, we provide evidence that infection with RSMV triggers an autophagic antiviral response in leafhopper cells. In this response, the G protein of RSMV binds to a leafhopper AMP-activated protein kinase (AMPK), leading to enhanced phosphorylation of Beclin-1 (BECN1), thereby inducing autophagy. Knockdown of AMPK and genes encoding members of the phosphoinositide 3-kinase (PI3K) complex composed of the autophagy-related protein 14 (ATG14), BECN1, and vacuolar protein sorting 34 (VPS34) facilitated viral infection in leafhoppers. To suppress leafhopper-induced autophagy, RSMV M protein specifically interacts with ATG14, resulting in the disintegration of PI3K complexes. This leads to reduced phosphatidylinositol-3-phosphate content and thus inhibits the G-protein- induced autophagy. Our study sheds light on the mechanism by which this rice virus evades insect autophagy antiviral defenses.

IThe plastome of Manilkara zapota is found to be 158,386 bp long with the typical quadripartite structure of angiosperms, contains two inverted repeats (IRs) of 26,099 bp each, a large single-copy (LSC) region of 87,745 bp, and a small single-copy (SSC) region of 18,443 bp. The plastome contains 114 genes, consisting of 80 unique protein-coding genes, 30 unique tRNA genes, and 4 unique rRNA genes. The overall A/T content in the plastome of M. zapota is 63.00%. The phylogenetic analysis indicated that M. zapota is close to Sideroxylon wightianum within Sapotaceae in this study.