Diverse nucleotide substitutions in rice base editing mediated by novel TadA variants.

CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops. The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes. Compared with the well-developed adenine base editors (ABEs), cytosine base editors (CBEs) and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice, significantly limiting their application. Here, we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice. We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID∗Δ, and TadA-CDd outperformed TadA-E27R/N46L. A C-to-G base editor (CGBE) engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P. In addition, a dual base editor constructed with a single protein, TadDE, enabled simultaneous, highly efficient C-to-T and A-to-G editing in rice. Collectively, our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice, providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.

Identification and genomic insights into a strain of Bacillus velezensis with phytopathogen-inhibiting and plant growth-promoting properties.

The use of biological agents offers a sustainable alternative to chemical control in managing plant diseases. In this study, Bacillus velezensis IFST-221 was isolated from the rhizosphere of a healthy maize plant amidst a population showing severe disease symptoms. The investigation demonstrated a broad-spectrum antagonistic activity of IFST-221 against eight species of pathogenic ascomycetes and oomycetes, suggesting its potential utility in combating plant diseases like maize ear rot and cotton Verticillium wilt. Additionally, our study unveiled that IFST-221 has demonstrated significant plant growth-promoting properties, particularly in maize, cotton, tomato, and broccoli seedlings. This growth promotion was linked to its ability to produce indole-3-acetic acid, nitrogen fixation, phosphate and potassium solubilization, and biofilm formation in laboratory conditions. A complete genome sequencing of IFST-221 yielded a genome size of 3.858 M bp and a GC content of 46.71%. The genome analysis identified 3659 protein-coding genes, among which were nine secondary metabolite clusters with known antimicrobial properties. Additionally, three unknown compounds with potentially novel properties were also predicted from the genomic data. Genome mining also identified several key genes associated with plant growth regulation, colonization, and biofilm formation. These findings provide a compelling case for the application of B. velezensis IFST-221 in agricultural practices. The isolate's combined capabilities of plant growth promotion and antagonistic activity against common plant pathogens suggest its promise as an integrated biological agent in disease management and plant productivity enhancement.

Analyzing the bibliometrics of brain-gut axis and Parkinson's disease.

Background: Parkinson's disease (PD), characterized by the loss of dopaminergic neurons, is a progressive neurodegenerative disorder. Recent research has revealed a significant connection between gut microbiota and PD. To gain insight into research interests, disciplinary contexts, and potential future directions, a comprehensive bibliometric analysis was conducted on the brain-gut axis and PD literature published between 2014 and 2023. Methods: Relevant literature records were gathered from the Web of Science Core Collection on August 11, 2023. The data were then analyzed by Biblioshiny R packages and VOSviewer (version 1.6.19). Results: The dataset revealed an upward trend in annual scientific publications on the brain-gut axis and PD, with an annual growth rate of 50.24%. China, the United States, and Italy were the top three most productive countries/regions. The journal "International Journal Of Molecular Sciences" published the most articles, while "Movement Disorders" received the highest number of citations. Professor Keshavarzian A emerged as the most prolific author, while Professor Scheperjans F held the highest h-index. Keyword analysis highlighted "alpha-synuclein" as the most frequent term, with "mouse model," "inflammation," and "risk" as emerging research topics. Additionally, "central nervous system" and "intestinal bacterial overgrowth" attracted increasing attention. Conclusion: This study examined current trends and hotspots in the bibliometric landscape of the brain-gut axis and PD research. Future research directions should explore the functional and metabolic activities of gut microbiota. Additionally, transitioning from observational to interventional study designs offers the potential for personalized interventions and disease prediction. These findings can guide researchers in navigating the latest developments and shaping the future directions of this field.

Molecular characterization and pathogenicity of a novel monopartite geminivirus infecting tobacco in China.

The occurrence of geminiviruses causes significant economic losses in many economically important crops. In this study, a novel geminivirus isolated from tobacco in Sichuan province of China, named tomato leaf curl Chuxiong virus (TLCCxV), was characterized by small RNA-based deep sequencing. The full-length of TLCCxV genome was determined to be 2744 nucleotides (nt) encoding six open reading frames. Phylogenetic and genome-wide pairwise identity analysis revealed that TLCCxV shared less than 91% identities with reported geminiviruses. A TLCCxV infectious clone was constructed and successfully infected Nicotiana benthamiana, N. tabacum, N. glutinosa, Solanum lycopersicum and Petunia hybrida plants. Furthermore, expression of the V2, C1 and C4 proteins through a potato virus X vector caused severe chlorosis or necrosis symptom in N. benthamiana. Taken together, we identified a new geminivirus in tobacco plants, and found that V2, C1 and C4 contribute to symptom development.

First report of Hibiscus latent Singapore virus and Hibiscus latent Fort Pierce virus infecting Lantana camara in China.

Hibiscus latent Singapore virus (HLSV) and Hibiscus latent Fort Pierce virus (HLFPV) both belong to the genus Tobamovirus in the family Virgaviridae. The genomes of both HLSV and HLFPV consist of a linear positive sense single-stranded RNA of about 6.3 kb. HLSV is the causal agent of hibiscus leaf crinkle disease. Infections of HLSV in hibiscus (Hibiscus rosa-sinensis) have so far only been reported in Singapore, Japan and Malaysia (Srinivasan et al., 2002; Yoshida et al., 2018; Yusop et al., 2021). In 2017, leaf curling and chlorosis symptoms of lantana (Lantana camara) plants were found in Chenshan Botanical Garden, Shanghai, China. To detect potential virus(es) in these lantana samples, leaves from one lantana plant were collected and total RNA was extracted with RNAiso Plus (TaKaRa). A cDNA library was prepared by TruSeq RNA Sample Prep Kit (Illumina) after removing ribosomal RNA by Ribo-ZeroTM rRNA Removal Kit (Epicentre). The paired-end sequencing was then performed on an Illumina NovaSeq 6000. A total of 61,085,018 high quality reads were obtained and de novo assembly by StringTie revealed 124,516 contigs (greater than 50 bp, N50=719 bp) with an average length of 537 bp. BLASTx analyses in the National Center for Biotechnology Information (NCBI) database showed that 1 long contig of 6,305 bp, assembled of 1794 clean reads, shared significant nucleotide similarities with the genomic sequence of HLSV, and 1 contig of 6,271 bp, assembled of 3174 clean reads, shared significant similarities with the genomic sequence of HLFPV, yielding an average coverage of the whole genome at 42.65 and 75.83 per million reads, respectively. To obtain the complete genome of the viral RNA in this lantana sample, eleven overlapping regions covering the entire HLSV viral genome, and nine overlapping regions covering the entire HLFPV viral genome were amplified by reverse transcription-PCR (RT-PCR) and sequenced. In addition, the exact 5' and 3' ends of the genomic RNA of each virus were determined by rapid amplification of the cDNA ends (RACE) (Wang et al. 2020). The complete genome of the identified HLSV, deposited in GenBank: MZ020960, is 6,486 nt in length and shows 98.4% nucleotide sequence identity with HLSV Singapore isolate (GenBank: AF395898). Similar to other HLSV isolates, this virus isolate possesses an internal poly(A) tract of 87 nucleotides, which is crucial to virus replication (Niu et al., 2015). The complete genome of the Lantana HLFPV isolate is 6,463 nt (GenBank MZ020961) including a 73 nt internal poly(A) tract, and has 98.4% nt identity to HLFPV-Japan (AB917427). In two other lantana plants from the same site, the presence of HLSV and HLFPV was confirmed by RT-PCR using the primer pairs (5'-GCATCTGCATAACACGGTTG-3'/5'-ACGTTGTAGTAGACGTTGTTGTAG-3' and 5'-GGACCTTGCTAATCCGCTAAAGTTG-3'/5'-GGTCCATGTCCATCCAGATGCAATC-3'). In addition to the HLSV and HLFPV genomes, BLASTx analysis of three contigs of 3,006 bp, 2,845 bp and 2,200 bp, assembled of 1328, 352 and 2280 clean reads respectively, showed high identity to RNAs 1 (MG182148), 2 (DQ412731) and 3 (KY794710) of cucumber mosaic virus. To the best of our knowledge, this is the first report of L. camara as a new natural host of HLSV and HLFPV, and first identification of a mixed infection of HLSV and HLFPV.

The C4 Protein of TbLCYnV Promotes SnRK1 ß2 Degradation Via the Autophagy Pathway to Enhance Viral Infection in N. benthamiana.

Geminiviruses are a group of single-stranded DNA viruses that have developed multiple strategies to overcome host defenses and establish viral infections. Sucrose nonfermenting-1-related kinase 1 (SnRK1) is a key regulator of energy balance in plants and plays an important role in plant development and immune defenses. As a heterotrimeric complex, SnRK1 is composed of a catalytic subunit α (SnRK1 α) and two regulatory subunits, ß and γ. Previous studies on SnRK1 in plant defenses against microbial pathogens have mainly focused on SnRK1 α. In this study, we validated the interaction between the C4 protein encoded by tobacco leaf curl Yunnan virus (TbLCYnV) and the regulatory subunit ß of Nicotiana benthamiana SnRK1, i.e., NbSnRK1 ß2, and identified that the Asp22 of C4 is critical for TbLCYnV C4-NbSnRK1 ß2 interactions. NbSnRK1 ß2 silencing in N. benthamiana enhances susceptibility to TbLCYnV infection. Plants infected with viral mutant TbLCYnV (C4D22A), which contains the mutant version C4 (D22A) that is incapable of interacting with NbSnRK1 ß2, display milder symptoms and lower viral accumulation. Furthermore, we discovered that C4 promotes NbSnRK1 ß2 degradation via the autophagy pathway. We herein propose a model by which the geminivirus C4 protein causes NbSnRK1 ß2 degradation via the TbLCYnV C4-NbSnRK1 ß2 interaction to antagonize host antiviral defenses and facilitates viral infection and symptom development in N. benthamiana.

Genomic characterization of a novel cytorhabdovirus infecting Ixeris denticulata in China.

Ixeris denticulata is a perennial herbal plant with important medical and economic value. In this study, a novel rhabdovirus from I. denticulata with leaf curling and mottle symptoms was identified through next-generation sequencing and molecular cloning approaches. Based on the host species and properties of this virus, it was tentatively named "Ixeris denticulata-associated rhabdovirus" (IdaRV). IdaRV has a negative-sense RNA genome that is 12,705 nucleotides in length and has five open reading frames (ORFs) in the order 3'-nucleoprotein -phosphoprotein -movement protein -matrix protein -large RNA-dependent RNA polymerase-5'. Pairwise sequence comparisons showed that IdaRV had 42.2-53.0% sequence identity to members of the genera Cytorhabdovirus, Varicosavirus, Betanucleorhabdovirus, Gammanucleorhabdovirus, Dichorhavirus, and Alphanucleorhabdovirus in the subfamily Betarhabdovirinae. BLASTp searches indicated that putative products of ORF1, ORF2, ORF3, ORF4, and ORF5 of IdaRV are most closely related to those of rudbeckia virus 1 (RudV1, GenBank accession number ON185810), with 32.1%, 21.3%, 52.4%, 37.6%, and 57.1% amino acid sequence identity, respectively, at the protein level. Phylogenetic analysis showed that IdaRV forms a smaller branch with RudV1, which belongs to the genus Cytorhabdovirus. These results establish IdaRV as a novel rhabdovirus in the genus Cytorhabdovirus of the family Rhabdoviridae.

Plant-virus arms race beyond RNA interference.

Plants use RNA interference for basal antiviral immunity, but emerging evidence suggests that additional RNA-targeting defense mechanisms also defend against invading viruses. Recent advancements in the understanding of RNA decay, RNA quality control, and N6-methyladenosine (m6A) RNA modifications have unveiled new insights into the molecular arms race between plants and viruses.

m6A modification of plant virus enables host recognition by NMD factors in plants.

N6-methyladenosine (m6A) is the most abundant eukaryotic mRNA modification and is involved in various biological processes. Increasing evidence has implicated that m6A modification is an important anti-viral defense mechanism in mammals and plants, but it is largely unknown how m6A regulates viral infection in plants. Here we report the dynamic changes and functional anatomy of m6A in Nicotiana benthamiana and Solanum lycopersicum during Pepino mosaic virus (PepMV) infection. m6A modification in the PepMV RNA genome is conserved in these two species. Overexpression of the m6A writers, mRNA adenosine methylase A (MTA), and HAKAI inhibit the PepMV RNA accumulation accompanied by increased viral m6A modifications, whereas deficiency of these writers decreases the viral RNA m6A levels but enhances virus infection. Further study reveals that the cytoplasmic YTH-domain family protein NbECT2A/2B/2C as m6A readers are involved in anti-viral immunity. Protein-protein interactions indicate that NbECT2A/2B/2C interact with nonsense-mediated mRNA decay (NMD)-related proteins, including NbUPF3 and NbSMG7, but not with NbUPF1. m6A modification-mediated restriction to PepMV infection is dependent on NMD-related factors. These findings provide new insights into the functionality of m6A anti-viral activity and reveal a distinct immune response that NMD factors recognize the m6A readers-viral m6A RNA complex for viral RNA degradation to limit virus infection in plants.

Plant virology in the 21st century in China: Recent advances and future directions.

Plant viruses are a group of intracellular pathogens that persistently threaten global food security. Significant advances in plant virology have been achieved by Chinese scientists over the last 20 years, including basic research and technologies for preventing and controlling plant viral diseases. Here, we review these milestones and advances, including the identification of new crop-infecting viruses, dissection of pathogenic mechanisms of multiple viruses, examination of multilayered interactions among viruses, their host plants, and virus-transmitting arthropod vectors, and in-depth interrogation of plant-encoded resistance and susceptibility determinants. Notably, various plant virus-based vectors have also been successfully developed for gene function studies and target gene expression in plants. We also recommend future plant virology studies in China.

Development and application of monoclonal antibody-based dot-ELISA and colloidal gold immunochromatographic strip for rapid, specific, and sensitive detection of tomato brown rugose fruit virus.

Tomato brown rugose fruit virus (ToBRFV) is an emerging tobamovirus that has become a great concern to tomato production industry. Due to the lack of resistant cultivars, precise detection of ToBRFV is essential to prevent the spread of ToBRFV. In this study, we produced highly sensitive and specific monoclonal antibodies against ToBRFV and established dot-enzyme-linked immunosorbent assay (dot-ELISA) and colloidal gold immunochromatographic strip (CGICS)-based methods for ToBRFV detection. These two methods could specifically detect ToBRFV without cross-reaction with seven tested tobamoviruses and three frequently occurring tomato-infecting viruses. Sensitivity analysis showed that the limit of detection of the established dot-ELISA and CGICS methods reached up to 1:6400 and 1:10,000 (w/v, g/mL) dilution of ToBRFV-infected tomato tissue, respectively. Further analyses using field-collected tomato foliar and fruit samples showed that the results obtained by dot-ELISA and CGICS were consistent with those obtained by reverse transcription polymerase chain reaction. The established methods here allow for specific, sensitive, and robust detection of ToBRFV, and will be helpful for precise monitoring and early warning of ToBRFV.

Two viral proteins translated from one open reading frame target different layers of plant defense.

Multilayered defense responses are activated upon pathogen attack. Viruses utilize a number of strategies to maximize the coding capacity of their small genomes and produce viral proteins for infection, including suppression of host defense. Here, we reveal translation leakage as one of these strategies: two viral effectors encoded by tomato golden mosaic virus, chloroplast-localized C4 (cC4) and membrane-associated C4 (mC4), are translated from two in-frame start codons and function cooperatively to suppress defense. cC4 localizes in chloroplasts, to which it recruits NbPUB4 to induce ubiquitination of the outer membrane; as a result, this organelle is degraded, and chloroplast-mediated defenses are abrogated. However, chloroplast-localized cC4 induces the production of singlet oxygen (1O2), which in turn promotes translocation of the 1O2 sensor NbMBS1 from the cytosol to the nucleus, where it activates expression of the CERK1 gene. Importantly, an antiviral effect exerted by CERK1 is countered by mC4, localized at the plasma membrane. mC4, like cC4, recruits NbPUB4 and promotes the ubiquitination and subsequent degradation of CERK1, suppressing membrane-based, receptor-like kinase-dependent defenses. Importantly, this translation leakage strategy seems to be conserved in multiple viral species and is related to host range. This finding suggests that stacking of different cellular antiviral responses could be an effective way to abrogate viral infection and engineer sustainable resistance to major crop viral diseases in the field.

Resistance to Planthoppers and Southern Rice Black-streaked Dwarf Virus in Rice Germplasms.

The damage caused by white-back planthopper (WBPH, Sogatella furcifera) and brown planthopper (BPH, Nilaparvata lugens), as well as southern rice black-streaked dwarf virus (SRBSDV), considerably decreases grain yield of rice. Identification of rice germplasms with sufficient resistance to planthoppers and SRBSDV is essential to the breeding and deployment of resistant varieties and hence the control of the pests and disease. In this study, 318 rice accessions were evaluated for their reactions to the infestation of both BPH and WBPH at the seedling stage using the standard seed-box screening test (SSST) method, insect quantification was further conducted at the end of tillering and grain-filling stages in field trials. Accessions HN12-239 and HN12-328 were resistant to both BPH and WBPH at all tested stages. Field trials were conducted to identify resistance in the collection to SRBSDV based on the virus infection rate under artificial inoculation. RHT and HN12-239 were moderately resistant to SRBSDV. In addition, we found that WBPH did not penetrate stems with stylets, but did more probing bouts and then xylem sap ingestion when feeding on HN12-239 than the susceptible control rice TN1. The resistance of rice accessions HN12-239, HN12-328 and RHT to BPH, WBPH and/or SRBSDV, should be valuable to the development of resistant rice varieties.