A CRM1-dependent nuclear export signal in Autographa californica multiple nucleopolyhedrovirus Ac93 is important for the formation of intranuclear microvesicles.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is highly conserved in all sequenced baculovirus genomes, and it plays important roles in both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles. In this study, we characterized a cellular CRM1-dependent nuclear export signal (NES) of AcMNPV Ac93. Bioinformatic analysis revealed that AcMNPV Ac93 may contain an NES at amino acids 115-125. Green fluorescent protein (GFP) fused to the NES (GFP:NES) of AcMNPV Ac93 is localized to the cytoplasm of transfected cells. Multiple point mutation analysis demonstrated that NES is important for the nuclear export of GFP:NES. Bimolecular fluorescence complementation experiments and co-immunoprecipitation assays confirmed that Ac93 interacts with Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits cellular CRM1-dependent nuclear export of GFP:NES. To determine whether the NES in AcMNPV Ac93 is important for the formation of intranuclear microvesicles, an ac93-null AcMNPV bacmid was constructed; the wild-type and NES-mutated Ac93 were reinserted into the ac93-null AcMNPV bacmid. Immunofluorescence analysis showed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in infected cells, while the construct containing point mutations at residues 123 and 125 of Ac93 resulted in a defect in budded virus production and the abolishment of intranuclear microvesicles. Together, these data demonstrate that Ac93 contains a functional NES, which is required for the production of progeny viruses and the formation of intranuclear microvesicles.IMPORTANCEAutographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is important for the formation of intranuclear microvesicles. However, how the baculovirus manipulates Ac93 for the formation of intranuclear microvesicles is unclear. In this study, we identified a nuclear export signal (NES) at amino acids 115-125 of AcMNPV Ac93. Our results showed that the NES is required for the interaction between Ac93 and Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits the nuclear export of green fluorescent protein fused to the NES. Our analysis revealed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in AcMNPV-infected cells. Together, our results indicate that Ac93 participates in the formation of intranuclear microvesicles via the Ac93 NES-mediated CRM1 pathway.

Multifaceted interactions between host ESCRT-III and budded virus-related proteins involved in entry and egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus.

The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.

Annual (2023) taxonomic update of RNA-directed RNA polymerase-encoding negative-sense RNA viruses (realm Riboviria: kingdom Orthornavirae: phylum Negarnaviricota).

In April 2023, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Impacts of maize hybrids with different nitrogen use efficiency on root-associated microbiota based on distinct rhizosphere soil metabolites.

Plant genotypes shape root-associated microbiota that affect plant nutrient acquisition and productivity. It is unclear how maize hybrids modify root-associated microbiota and their functions and relationship with nitrogen use efficiency (NUE) by regulating rhizosphere soil metabolites. Here, two N-efficient (NE) (ZD958, DMY3) and two N-inefficient (NIE) maize hybrids (YD9953, LY99) were used to investigate this issue under low N (60 kg N ha-1 , LN) and high N (180 kg N ha-1 , HN) field conditions. NE hybrids had higher yield than NIE hybrids under LN but not HN. NE and NIE hybrids recruited only distinct root-associated bacterial microbiota in LN. The bacterial network stability was stronger in NE than NIE hybrids. Compared with NIE hybrids, NE hybrids recruited more bacterial taxa that have been described as plant growth-promoting rhizobacteria (PGPR), and less related to denitrification and N competition; this resulted in low N2 O emission and high rhizosphere NO3 - -N accumulation. NE and NIE hybrids had distinct rhizosphere soil metabolite patterns, and their specific metabolites were closely related to microbiota and specific genera under LN. Our findings reveal the relationships among plant NUE, rhizosphere soil metabolites, root-associated microbiota, and soil nutrient cycling, and this information is informative for breeding NE crops.

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.

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.

Induction of Low Temperature Tolerance in Wheat by Pre-Soaking and Parental Treatment with Melatonin.

Low temperatures seriously depress germination and seedling establishment in wheat and it is of great significance to explore approaches to improve wheat tolerance to low temperatures. In this study, the effects of seed pre-soaking and parental treatment with melatonin on seed germination and low temperature tolerance during the early growing stage in wheat were studied. The results showed that pre-soaking with melatonin increased the germination rate, improved antioxidant capacity and accelerated starch degradation under low temperature, which alleviated low temperature-induced damage to the chloroplasts in coleoptiles of wheat seedlings. Parental melatonin treatment during grain filling stage significantly decreased the grain weight. Seeds from parental melatonin-treated plants showed higher germination rates and higher antioxidant enzyme activity than the control seeds under low temperature. In addition, parental treatment with melatonin modulated the activities of carbohydrate metabolism enzymes, which contributes to enhanced low temperature tolerance in wheat offspring. It was suggested that both seed pre-soaking and parental treatment with melatonin could be the effective approaches for low temperature tolerance induction in wheat.

The N Terminus of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase Is Required for Efficient Viral DNA Replication and Virus and Occlusion Body Production.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) plays a crucial role in viral DNA synthesis, and the N terminus (residues 1 to 186) is highly conserved in the baculovirus DNApol family. However, the functional role of the N terminus of DNApol has not yet been characterized. Here we report a functional analysis of the AcMNPV DNApol N terminus. We truncated the DNApol N terminus to construct truncation mutants Bac-GFP-PolΔ64, Bac-GFP-PolΔ110, and Bac-GFP-PolΔ186, which lack 64, 110, and 186 N-terminal residues, respectively. Although the truncation mutants rescued viral DNA synthesis and infectious virus production, the level of DNA replication decreased, and Bac-GFP-PolΔ64, Bac-GFP-PolΔ110, and Bac-GFP-PolΔ186 showed 10-fold, 89-fold, and 891-fold reductions in infectious viral yield compared to that of the wild-type repair virus, respectively. Production of occlusion bodies was compromised for all truncation mutants. Further bioinformatic analysis showed that the first 64 amino acids (aa) at the extreme N terminus contains a conserved α(-helix)-ß(-sheet)-ß-ß secondary-structure region, and further downstream sequence from aa 67 to 186 is comprised of four conserved sequence motifs. Multiple alanine point substitutions in the α-ß-ß-ß structure region or the four sequence motifs in the N terminus impaired viral DNA replication and resulted in reduction of virus yield and occlusion body production. Together, our results suggested that the secondary structure and four conserved motifs within the N terminus of AcMNPV DNApol are important for viral DNA synthesis, infectious virus yield, and production of occlusion bodies.IMPORTANCE DNA polymerase (DNApol) is highly conserved in all baculoviruses and is required for viral DNA replication. The N terminus is one of the highly conserved regions of baculovirus DNApols. Our results showed that the N terminus of baculovirus DNA polymerase plays an important role in efficient viral DNA synthesis and infectious virus yield and production of occlusion bodies. We identified five features, including a highly conserved secondary structure and four conserved amino acid motifs, in the AcMNPV DNApol N terminus, all of which are important for efficient viral DNA synthesis, infectious virus yield, and production of occlusion bodies.

A betabaculovirus DNA polymerase cannot substitute for the DNA polymerase of the alphabaculovirus Autographa californica nucleopolyhedrovirus.

DNA polymerase (DNApol) is present in all baculoviruses and plays a crucial role in viral DNA replication. Previously we showed that the DNApol of the alphabaculovirus group II Spodoptera litura nucleopolyhedrovirus (SpltNPV) could partially substitute for the DNApol of a group I alphabaculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, it is not known if a betabaculovirus DNApol could subsititute for the alphabaculovirus DNApol in AcMNPV. In this report, DNApol of the betabaculovirus Pieris rapae granulovirus (PiraGV) was inserted into a dnapol-null AcMNPV bacmid, creating Bac-AcΔpol:PrPol. The repair virus did not spread to neighboring cells; virus growth curve and real-time PCR revealed that the PiraGV dnapol substitution abrogated AcMNPV DNA replication and virus production. Immunofluorescence microscopy showed that PiraGV DNApol could be expressed and localized to the nucleus. Collectively, our results suggested that the alphabaculovirus AcMNPV DNApol could not be replaced by a DNApol from the betabaculovirus, PiraGV.

Rescue of dnapol-null Autographa californica multiple nucleopolyhedrovirus with DNA polymerase (DNApol) of Spodoptera litura nucleopolyhedrovirus (SpltNPV) and identification of a nuclear localization signal in SpltNPV DNApol.

DNA polymerase (DNApol) is highly conserved in all baculoviruses and plays an essential role in viral DNA replication. It determines the fidelity of baculovirus DNA replication by inserting the correct nucleotides into the primer terminus and proofreading any mispaired nucleotides. DNApols of groups I and II of the genus Alphabaculovirus in the family Baculoviridae share many common structural features. However, it is not clear whether a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNApol can be substituted by a group II NPV DNApol. Here we report the successful generation of AcMNPV dnapol-null virus being rescued by a group II Spodoptera litura NPV (SpltNPV) dnapol (Bac-AcΔPol : Slpol). Viral growth curves and quantitative real-time PCR showed that the dnapol replacement reduced the level of viral production and DNA replication of Bac-AcΔPol : SlPol compared with WTrep, a native dnapol insertion in an AcMNPV dnapol-null virus. Light microscopy showed that production of occlusion bodies for Bac-AcΔPol : Slpol was reduced. We also identified a nuclear localization signal (NLS) for the SpltNPV DNApol C terminus at residues 827-838 by mutational analysis and confocal microscopy. Multiple point substitution of SpltNPV DNApol NLS abrogated virus production and viral DNA replication. Overall, these data suggested that the NLS plays an important role in SpltNPV DNApol nuclear localization and that SpltNPV DNApol cannot efficiently substitute the AcMNPV DNApol in AcMNPV.

Autographa californica multiple nucleopolyhedrovirus DNA polymerase C terminus is required for nuclear localization and viral DNA replication.

UNLABELLED: The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production. IMPORTANCE: The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are insufficient for viral DNA synthesis and virus replication. Rather, we identified three features, including two nuclear localization signals and a highly conserved 10-amino-acid sequence in the AcMNPV DNApol C terminus, all three of which are important for both nuclear localization of DNApol and for DNApol activity, as measured by viral DNA synthesis and virus replication.

Characterization of heavy metal, antibiotic pollution, and their resistance genes in paddy with secondary municipal-treated wastewater irrigation.

Secondary municipal-treated wastewater irrigation may introduce residual antibiotics into the agricultural systems contaminated with certain heavy metals, ultimately leading to the coexistence of antibiotics and heavy metals. The coexistence may induce synergistic resistance to both in the microbial community. Here, we investigated the effects of long-term municipal-treated irrigation for rice on the microbiome and resistome. The results showed that the target antibiotics were undetectable in edible grains, and the heavy metal concentrations did not exceed the standard in edible rice grains. Heavy metal resistance genes (MRGs) ruvB and acn antibiotic resistance genes (ARGs) sul1 and sul2 were the dominating resistant genes. The coexistence of antibiotics and heavy metals affected the microbial community and promoted metal and antibiotic resistance. Network analysis revealed that Proteobacteria were the most influential hosts for MRGs, ARGs, and integrons, and co-selection may serve as a potential mechanism for resistance maintenance. MRG czcA and ARG sul1 can be recommended as model genes to study the co-selection of ARGs and MRGs in environments. The obtained results highlight the importance of considering the co-occurrence of heavy metals and antibiotics while developing effective methods to prevent the transmission of ARGs. These findings are critical for assessing the possible human health concerns associated with secondary municipal-treated wastewater irrigation for agriculture and improving the understanding of the coexistence of heavy metals and antibiotics.

Municipal-treated wastewater as a practical alternative to conventional rice irrigation: Effects on antibiotic resistance genes, virulence factors and human bacterial pathogens in soil, and responses of rice grain quality.

Reuse of municipal-treated wastewater for agricultural irrigation is becoming increasingly prevalent due to growing demand and decline in freshwater supplies. However, the microbial contamination profile, including antibiotic resistance genes (ARGs), virulence factors (VFs), and human bacterial pathogens (HBPs) in agricultural soil irrigated with municipal-treated wastewater for paddy cultivation, was unknown. Here, metagenomic analysis was applied to provide a systematic insight into the resistome, VFs and HBPs in paddy soils irrigated with municipal-treated wastewater. The obtained results revealed that the residual antibiotics in municipal-treated wastewater has an impact on the antibiotic resistome by increasing both the total number and abundance of ARGs. Furthermore, it was found that sul1 could serve as a potential risk indicator for assessing ARG contamination. VFs, core HBP abundance, and dangerous pathogens remain unaffected by municipal-treated wastewater irrigation for paddy. The good coexistence patterns of ARGs-HBPs and ARGs-VFs demonstrated the presence of resistant pathogenic bacteria. The network analysis revealed that ARGs-bearing Legionella pneumophila, Mycobacterium marinum, Bordetella pertussis, Staphylococcus aureus, and Pseudomonas aeruginosa might be ranked as high-risk HBPs. Additionally, our investigation also demonstrated that reuse of municipal-treated wastewater for agricultural irrigation had no detrimental effects on rice plant growth and grain quality. This study was the first to investigate the response of VFs and HBPs in paddy soil under long-term municipal-treated wastewater irrigation. The obtained results provide a scientific basis for the safe application of municipal-treated wastewater.

Conservative strip tillage system in maize maintains high yield and mitigates GHG emissions but promotes N2O emissions.

Optimizing N application under straw-covered strip tillage is of great significance to the rational utilization of stover resources as well as ensure food and ecosystem security, and especially N2O emissions from agricultural systems. Quantifying N2O emissions and even the carbon footprint (CF) from agricultural systems is crucial for future protecting agricultural production systems. A two-year field experiment was conducted on black soil in Northeast China, which set up two tillage systems: strip tillage with straw returning (ST) and conventional tillage (control: CT) without straw and three nitrogen rates: 0, farmers' practice (Nfp 240 kg hm-2), and optimized nitrogen fertilizer (Nopt 180 kg hm-2). We examined the characteristics of N2O emissions and CF under the ST and CT systems. Among them, we indirectly calculated GHG emissions using the LCA method. Compared with CT, the ST system significantly reduces indirect GHG emissions, but did significantly increase direct cumulative N2O emissions by 20.7 %, most likely because the higher soil residual nitrate nitrogen content, WFPS, and soil temperature under ST was 13.0 %, 2 % and 5.7 % higher than that under CT. Nopt treatment markedly reduced cumulative N2O emissions by 36.0 %, CFarea, CFyield, and CFNPV by 22.4 %, 23.1 %, and 23.5 % in ST, respectively, compared to Nfp. The reduction in energy use of machinery in ST results in lower fuel consumption and thus generating less CF. What's more, the decrease of CFyield and CFNPV between nitrogen application treatments under ST was 5.2 % and 7.7 % higher than CT, respectively. ST system can effectively achieve higher grain yield and mitigate GHG emissions on black soil in Northeast China compared with CT, but attention should be paid to N2O emissions in the soil during the maize growth period. The sustainability of balancing GHG emissions, and economic and environmental benefits can be achieved by optimizing nitrogen fertilizer manage.

Identification of an Endogenous Strong Promoter in Burkholderia sp. JP2-270.

Burkholderia is the second largest source of natural product bacteria after Actinomyces and can produce many secondary metabolites including pyrrolnitrin (PRN). Natural products of microbial origin are usually found in trace amounts, so in metabolic engineering, promoter engineering is often used to regulate gene expression to increase yield. In this study, an endogenous strong promoter was identified based on RNA-seq to overexpress biosynthetic genes to increase the production of PRN. By analyzing the transcriptomic data of the antagonistic bacterium Burkholderia sp. JP2-270 in three different development periods, we screened 50 endogenous promoters with high transcriptional activity, nine of which were verified by an obvious fluorescent signal via fluorescence observation. Then, combined with RT-qPCR analysis, Php, the promoter of a hypothetical protein, was found to be significantly expressed in all three periods. In order to increase the suitability of endogenous promoters, the promoter Php was shortened at different lengths, and the results show that a sequence length of 173 bp was necessary for its activity. Moreover, this promoter was used to overexpress the PRN biosynthesis genes (prnA, prnB, prnC and prnD) in JP2-270, resulting in a successful increase in gene expression levels by 40-80 times. Only the overexpression of the prnB gene successfully increased PRN production to 1.46 times that of the wild type. Overall, the endogenous strong promoters screened in this study can improve gene expression and increase the production of secondary metabolites in JP2-270 and other strains.

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.

Selection and characterization of Autographa californica multiple nucleopolyhedrovirus DNA polymerase mutations.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) is essential for viral DNA replication. AcMNPV mutants resistant to aphidicolin, a selective inhibitor of viral DNA replication, and abacavir, an efficacious nucleoside analogue with inhibitory activity against reverse transcriptase, were selected by the serial passage of the parental AcMNPV in the presence of increasing concentrations of aphidicolin or abacavir. These drug-resistant mutants had either a single (C543R) (aphidicolin) or a double (C543R and S611T) (abacavir) point mutation within conserved regions II and III. To confirm the role of these point mutations in AcMNPV DNA polymerase, a dnapol knockout virus was first generated, and several repair viruses were constructed by transposing the dnapol wild-type gene or ones containing a single or double point mutation into the polyhedrin locus of the dnapol knockout bacmid. The single C543R or double C543R/S611T mutation showed increased resistance to both aphidicolin and abacavir and, even in the absence of drug, decreased levels of virus and viral DNA replication compared to the wild-type repair virus. Surprisingly, the dnapol mutant repair viruses led to the generation of occlusion-derived viruses with mostly single and only a few multiple nucleocapsids in the ring zone and within polyhedra. Thus, these point mutations in AcMNPV DNA polymerase increased drug resistance, slightly compromised virus and viral DNA replication, and influenced the viral morphogenesis of occlusion-derived virus.

Insights into antibiotic resistance-related changes in microbial communities, resistome and mobilome in paddy irrigated with reclaimed wastewater.

Reclaimed wastewater (reclaimed wastewater, RWW) from municipal wastewater treatment plants for paddy irrigation is a well-established practice to alleviate water scarcity. However, the reuse may result in the persistent exposure of the paddy to residual antibiotics in RWW. Continuous presence of even low-level antibiotics can exert selective pressure on microbiota, resulting in the proliferation and dissemination of antibiotic resistance genes (ARGs) in paddy. In this study, metagenomic analysis was applied to firstly deciphered the effects of residual antibiotics on microbiome and resistome in constructed mesocosm-scale paddy soils. The diversity and abundance of ARG have remarkably risen with the increasing antibiotic concentration in RWW. Network analysis revealed that 28 genera belonging to six phyla were considered as the potential ARG hosts, and their abundances were enhanced with increasing antibiotic concentrations. A partial least-squares path model indicated that the microbial community was the principal direct driver of the ARG abundance and the resistome alteration in paddy soil under long-term RWW irrigation. Microbes may acquire ARGs via horizontal gene transfer. IntI1 could play an essential role in the propagation and spread of ARGs. Functional analysis suggested that enhanced SOS response and T4SSs (Type IV secretion systems) modules could stimulate horizontal transfer potential and promote the ARG abundance. The obtained results provide a scientific decision for assessing the ecological risk of RWW application.

2-Oxoglutarate contributes to the effect of foliar nitrogen on enhancing drought tolerance during flowering and grain yield of soybean.

Drought severely affects the growth and yield of soybean plants especially during the flowering period. To investigate the effect of 2-oxoglutarate (2OG) in combination with foliar nitrogen (N) at flowering stage on drought resistance and seed yield of soybean under drought stress. This experiment was conducted in 2021 and 2022 on drought-resistant variety (Hefeng 50) and drought-sensitive variety (Hefeng 43) soybean plants treated with foliar N (DS + N) and 2-oxoglutarate (DS + 2OG) at flowering stage under drought stress. The results showed that drought stress at flowering stage significantly increased leaf malonaldehyde (MDA) content and reduced soybean yield per plant. However, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were significantly increased by foliar N treatment, and 2-oxoglutarate synergistically with foliar N treatment (DS + N + 2OG) was more beneficial to plant photosynthesis. 2-oxoglutarate significantly enhanced plant N content, glutamine synthetase (GS) and glutamate synthase (GOGAT) activity. Furthermore, 2-oxoglutarate increased the accumulation of proline and soluble sugars under drought stress. Under drought stress, soybean seed yield was increased by DS + N + 2OG treatment by 16.48-17.10% and 14.96-18.84% in 2021 and 2022, respectively. Thus, the combination of foliar N and 2-oxoglutarate better mitigated the adverse effects of drought stress and could better compensate for the yield loss of soybean under drought stress.

Salt priming induces low-temperature tolerance in sugar beet via xanthine metabolism.

To understand the physiological mechanisms involved in xanthine metabolism during salt priming for improving low-temperature tolerance, salt priming (SP), xanthine dehydrogenase inhibitor (XOI), exogenous allantoin (EA), and back-supplemented EA (XOI + EA) treatments were given and the low-temperature tolerance of sugar beet was tested. Under low-temperature stress, salt priming promoted the growth of sugar beet leaves and increased the maximum quantum efficiency of PS II (Fv/Fm). However, during salt priming, either XOI or EA treatment alone increased the content of reactive oxygen species (ROS), such as superoxide anion and hydrogen peroxide, in the leaves under low-temperature stress. XOI treatment increased allantoinase activity with its gene (BvallB) expression under low-temperature stress. Compared to the XOI treatment, the EA treatment alone and the XOI + EA treatment increased the activities of antioxidant enzymes. At low temperatures, the sucrose content and the activity of key carbohydrate enzymes (AGPase, Cylnv, and FK) were significantly reduced by XOI compared to the changes under salt priming. XOI also stimulated the expression of protein phosphatase 2C and sucrose non-fermenting1-related protein kinase (BvSNRK2). The results of a correlation network analysis showed that BvallB was positively correlated with malondialdehyde, D-Fructose-6-phosphate, and D-Glucose-6-phosphate, and negatively correlated with BvPOX42, BvSNRK2, dehydroascorbate reductase, and catalase. These results suggested that salt-induced xanthine metabolism modulated ROS metabolism, photosynthetic carbon assimilation, and carbohydrate metabolism, thus enhancing low-temperature tolerance in sugar beet. Additionally, xanthine and allantoin were found to play key roles in plant stress resistance.