Genetic diversity of RNA viruses infecting invertebrate pests of rice.

Invertebrate species are a natural reservoir of viral genetic diversity, and invertebrate pests are widely distributed in crop fields. However, information on viruses infecting invertebrate pests of crops is limited. In this report, we describe the deep metatranscriptomic sequencing of 88 invertebrate samples covering all major invertebrate pests in rice fields. We identified 296 new RNA viruses and 13 known RNA viruses. These viruses clustered within 31 families, with many highly divergent viruses constituting potentially new families and genera. Of the identified viruses, 13 RNA viruses clustered within the Fiersviridae family of bacteriophages, and 48 RNA viruses clustered within families and genera of mycoviruses. We detected known rice viruses in novel invertebrate hosts at high abundances. Furthermore, some novel RNA viruses have genome structures closely matching to known plant viruses and clustered within genera of several plant virus species. Forty-five potential insect pathogenic RNA viruses were detected in invertebrate species. Our analysis revealed that host taxonomy plays a major role and geographical location plays an important role in structuring viral diversity. Cross-species transmission of RNA viruses was detected between invertebrate hosts. Newly identified viral genomes showed extensive variation for invertebrate viral families or genera. Together, the large-scale metatranscriptomic analysis greatly expands our understanding of RNA viruses in rice invertebrate species, the results provide valuable information for developing efficient strategies to manage insect pests and virus-mediated crop diseases.

Bacillus velezensis ZN-S10 Reforms the Rhizosphere Microbial Community and Enhances Tomato Resistance to TPN.

Tomato pith necrosis (TPN) is a highly destructive disease caused by species of the Pseudomonas genus and other bacteria, resulting in a significant reduction in tomato yield. Members of the genus Bacillus are beneficial microorganisms extensively studied in the rhizosphere. However, in most cases, the potential of Bacillus members in controlling TPN and their impact on the rhizosphere microbial composition remain rarely studied. In this study, Bacillus velezensis ZN-S10 significantly inhibited the growth of Pseudomonas viridiflava ZJUP0398-2, and ZN-S10 controlled TPN with control efficacies of 60.31%. P. viridiflava ZJUP0398-2 significantly altered the richness and diversity of the tomato rhizobacterial community, but pre-inoculation with ZN-S10 mitigated these changes. The correlation analysis revealed that ZN-S10 maybe inhibits the growth of nitrogen-fixing bacteria and recruits beneficial bacterial communities associated with disease resistance, thereby suppressing the occurrence of diseases. In summary, the comparative analysis of the rhizosphere microbiome was conducted to explore the impact of ZN-S10 on the composition of rhizosphere microorganisms in the presence of pathogenic bacteria, aiming to provide insights for further research and the development of scientific and eco-friendly control strategies for this disease.

Novel RNA Viruses Discovered in Weeds in Rice Fields.

Weeds often grow alongside crop plants. In addition to competing with crops for nutrients, water and space, weeds host insect vectors or act as reservoirs for viral diversity. However, little is known about viruses infecting rice weeds. In this work, we used metatranscriptomic deep sequencing to identify RNA viruses from 29 weed samples representing 23 weed species. A total of 224 RNA viruses were identified: 39 newly identified viruses are sufficiently divergent to comprise new families and genera. The newly identified RNA viruses clustered within 18 viral families. Of the identified viruses, 196 are positive-sense single-stranded RNA viruses, 24 are negative-sense single-stranded RNA viruses and 4 are double-stranded RNA viruses. We found that some novel RNA viruses clustered within the families or genera of several plant virus species and have the potential to infect plants. Collectively, these results expand our understanding of viral diversity in rice weeds. Our work will contribute to developing effective strategies with which to manage the spread and epidemiology of plant viruses.

Metatranscriptomic Sequencing Suggests the Presence of Novel RNA Viruses in Rice Transmitted by Brown Planthopper.

Viral pathogens are a major threat to stable crop production. Using a backcross strategy, we find that integrating a dominant brown planthopper (BPH) resistance gene Bph3 into a high-yield and BPH-susceptible indica rice variety significantly enhances BPH resistance. However, when Bph3-carrying backcross lines are infested with BPH, these BPH-resistant lines exhibit sterile characteristics, displaying panicle enclosure and failure of seed production at their mature stage. As we suspected, BPH-mediated viral infections could cause the observed sterile symptoms, and we characterized rice-infecting viruses using deep metatranscriptomic sequencing. Our analyses revealed eight novel virus species and five known viruses, including a highly divergent virus clustered within a currently unclassified family. Additionally, we characterized rice plant antiviral responses using small RNA sequencing. The results revealed abundant virus-derived small interfering RNAs in sterile rice plants, providing evidence for Dicer-like and Argonaute-mediated immune responses in rice plants. Together, our results provide insights into the diversity of viruses in rice plants, and our findings suggest that multiple virus infections occur in rice plants.

Identification of Spodoptera frugiperda importin alphas that facilitate the nuclear import of Autographa californica multiple nucleopolyhedrovirus DNA polymerase.

Proteins containing nuclear localization signals (NLSs) are actively transported into the nucleus via the classic importin-α/ß-mediated pathway, and NLSs are recognized by members of the importin-α family. Most studies of insect importin-αs have focused on Drosophila to date, little is known about the importin-α proteins in Lepidoptera insects. In this study, we identified four putative importin-α homologues, Spodoptera frugiperda importin-α1 (SfIMA1), SfIMA2, SfIMA4 and SfIMA7, from Sf9 cells. Immunofluorescence analysis showed that SfIMA2, SfIMA4 and SfIMA7 localized to the nucleus, while SfIMA1 distributed in cytoplasm. Additionally, SfIMA4 and SfIMA7 were also detected in the nuclear membrane of Sf9 cells. SfIMA1, SfIMA4 and SfIMA7, but not SfIMA2, were found to associate with the C terminus of AcMNPV DNA polymerase (DNApol) that harbours a typical monopartite NLS and a classic bipartite NLS. Further analysis of protein-protein interactions revealed that SfIMA1 specifically recognizes the bipartite NLS, while SfIMA4 and SfIMA7 bind to both monopartite and bipartite NLSs. Together, our results suggested that SfIMA1, SfIMA4 and SfIMA7 play important roles in the nuclear import of AcMNPV DNApol C terminus in Sf9 cells.

Identification and Characterization of the Nucleolar Localization Signal of Autographa californica Multiple Nucleopolyhedrovirus LEF5.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 5 (LEF5) is highly conserved in all sequenced baculovirus genomes and plays an important role in production of infectious viral progeny. In this study, nucleolar localization of AcMNPV LEF5 was characterized. Through transcriptome analysis, we identified two putative nucleolar proteins, Spodoptera frugiperda nucleostemin (SfNS) and fibrillarin (SfFBL), from Sf9 cells. Immunofluorescence analysis demonstrated that SfNS and SfFBL were localized to the nucleolus. AcMNPV infection resulted in reorganization of the nucleoli of infected cells. Colocalization of LEF5 and SfNS showed that AcMNPV LEF5 was localized to the nucleolus in Sf9 cells. Bioinformatic analysis revealed that basic amino acids of LEF5 are enriched at residues 184 to 213 and may contain a nucleolar localization signal (NoLS). Green fluorescent protein (GFP) fused to NoLS of AcMNPV LEF5 localized to the nucleoli of transfected cells. Multiple-point mutation analysis demonstrated that amino acid residues 197 to 204 are important for nucleolar localization of LEF5. To identify whether the NoLS in AcMNPV LEF5 is important for production of viral progeny, a lef5-null AcMNPV bacmid was constructed; several NoLS-mutated LEF5 proteins were reinserted into the lef5-null AcMNPV bacmid with a GFP reporter. The constructs containing point mutations at residues 185 to 189 or 197 to 204 in AcMNPV LEF5 resulted in reduction in production of infectious viral progeny and occlusion body yield in bacmid-transfected cells. Together, these data suggested that AcMNPV LEF5 contains an NoLS, which is important for nucleolar localization of LEF5, progeny production, and occlusion body production.IMPORTANCE Many viruses, including human and plant viruses, target nucleolar functions as part of their infection strategy. However, nucleolar localization for baculovirus proteins has not yet been characterized. In this study, two nucleolar proteins, SfNS and SfFBL, were identified in Sf9 cells. Our results showed that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection resulted in redistribution of the nucleoli of infected cells. We demonstrated that AcMNPV late expression factor 5 (LEF5) could localize to the nucleolus and contains a nucleolar localization signal (NoLS), which is important for nucleolar localization of AcMNPV LEF5 and for production of viral progeny and yield of occlusion bodies.