Cuiping Mo

As a tropical fruit of considerable economic importance, passionfruit (Passiflora edulis Sims) is extensively cultivated in the tropical and subtropical regions of China; however, the widespread incidence of viral diseases has significantly hampered the safety of its production. Rapid, sensitive, and visual detection of plant viruses is essential to effectively prevent and manage these viral diseases. In this study, we developed a visual detection system (displaying under blue light or UV) utilizing reverse transcription recombinase-aided amplification (RT-RAA) in conjunction with CRISPR/Cas12a to detect three viruses harmful to passionfruit production: telosma mosaic virus (TelMV), East Asian passiflora virus (EAPV), and passiflora mottle virus (PaMoV). Within this system, the optimal primer concentration for the RT-RAA reaction was determined to be 0.4 μM for all three viruses, with an optimal reaction temperature of 37 °C. The optimal reaction times were established as 20 min for TelMV, 15 min for EAPV, and 30 min for PaMoV. The entire detection process could be completed within 30 min without the need for sophisticated equipment or instruments. For TelMV and EAPV, the detection system demonstrated the capability to detect samples at a dilution of 10⁶, representing an approximately 10⁴-fold improvement over RT-PCR, while for PaMoV, it could identify samples at a dilution of 10⁶, representing an approximately 10²-fold improvement over traditional RT-PCR methods. These results confirm the successful development of the CRISPR/Cas12a-based detection systems. Subsequently, the system was applied for in situ detection of the three target viruses in field settings, yielding results that were fully consistent with laboratory-based RT-PCR assays, a consistency which underscores the system’s strong potential for field application in detecting important crop viruses.

Southem rice black-streaked dwarf virus (SRBSDV), a fijivirus with a double-stranded RNA genome of 10 segments and 13 proteins, severely impairs rice growth and development. The functions of P8, one of the major coat proteins of SRBSDV, are still poorly understood. In this study, the SRBSDV-encoded P8 protein was used as bait in a yeast two-hybrid screen of a rice cDNA library, which identified an interaction between P8 and auxin response factor 15 (OsARF15). The interaction was then confirmed by GST pull-down (in vitro) and co-immunoprecipitation (Co-IP) (in vivo) assays. Truncated protein analysis further revealed that P8 primarily binds to the C-terminal region of OsARF15. Over-expression and knockout of OsARF15 rice plants were constructed and then inoculated by SRBSDV. The results showed that over-expression of OsARF15 promoted SRBSDV infection whereas its knockout enhanced virus resistance. Meanwhile, the effect of OsARF15 on root development was observed and found that knocking out OsARF15 significantly promoted the growth of rice roots at the seeding stage. These results demonstrate that the SRBSDV P8 protein facilitates viral infection by interacting with the C-terminal region of OsARF15. Our findings have further strengthened the role of P8 in the viral infection process.

Sonchus (Sonchus oleraceus) was originated from Europe and now is cultivated worldwide. The wild resources of sonchus are very abundant, it has rich nutritional value and medicinal value. In this s...

Banana (Musa spp.) is an important fruit in tropical and subtropical regions and an essential food crop in some developing countries. China has a long history of banana cultivation and ranks second in global banana production, with a planting area exceeding 11 million hectares (FAOSTAT, 2023). Banana mild mosaic virus (BanMMV) is a flexuous filamentous virus infecting bananas and a banmivirus in the Betaflexiviridae family. Its infection often results in symptomless plants of Musa spp., and the virus has a worldwide distribution, which can explain its high prevalence (Kumar et al., 2015). BanMMV infection often causes transitory symptoms, such as mild chlorotic streaks and mosaics, on young leaves (Thomas, 2015). The mixed infection of BanMMV with other banana-infecting viruses such as banana streak viruses (BSV) and cucumber mosaic virus (CMV), can exacerbate the mosaic symptoms of BanMMV (Fidan et al., 2019). In October 2021, we collected twenty-six leaf samples of suspected viral disease of bananas from four cities (Huizhou, Qingyuan, Zhanjiang, and Yangjiang) in Guangdong province, two cities (Hekou and Jinghong) in Yunnan province, two cities (Yulin and Wuming) in Guangxi Zhuang autonomous region. After fully mixing these infected samples, we divided them into two pools and sent them to Shanghai Biotechnology Corporation (China) for metatranscriptome sequencing. Each sample contained about 5 g of leaves in total. Zymo-Seq RiboFree Total RNA Library Prep Kit (Zymo Research, USA) was used for ribosomal RNA depletion and library preparations. Illumina sequencing (Illumina NovaSeq 6000) was carried out by Shanghai Biotechnology Corporation (China). Paired-end (150 bp) sequencing of the RNA library was performed on an Illumina HiSeq 2000/2500 platform. Clean reads were assembled by a metagenomic de novo assembly using the CLC Genomics Workbench (version: 6.0.4). Then the non-redundant protein database in the National Center for Biotechnology Information (NCBI) was used for BLASTx annotation. A total of 79,528 contigs were generated from the clean reads (68,878,162) through de novo assembly. A contig of 7265 nucleotides (nts) showed the highest nucleotide sequence identity (90.08%) to the genome of BanMMV isolate EM4-2 (GenBank accession no. OL826745.1). We designed specific primers according to the BanMMV CP gene (Table S1), tested the twenty-six leaf samples collected from the above-mentioned eight cities, and found that only one sample of Fenjiao (Musa ABB Pisang Awak) in Guangzhou city was infected with this virus. The symptoms of banana leaves containing BanMMV were slight chlorosis and yellowing of leaf edges (Fig. S1). We failed to detect other banana viruses, such as BSV, CMV, and banana bunchy top virus (BBTV) in the BanMMV-infected banana leaves. RNA from the infected leaves was extracted, and the assembled contig was confirmed by overlapping PCR amplification across the whole sequence (Table S1). All ambiguous regions were amplified by PCR and RACE, and the products were subjected to Sanger sequencing. The complete genome of the virus candidate was 7310 nts in length, excluding the poly (A) tail. The sequence was deposited in GenBank under accession number ON227268 (isolate BanMMV-GZ from Guangzhou). A schematic representation of the genome organization of BanMMV-GZ is shown in Fig. S2. Its genome has five open reading frames (ORF) encoding RNA-dependent RNA polymerase (RdRp), three triple gene block proteins necessary for cell-to-cell movement (TGBp1 to TGBp3) and a coat protein (CP), similar to other BanMMV isolates (Kondo et al., 2021). Phylogenetic analyses of the complete nt sequence of the full genome and RdRp gene using the neighbor-joining (NJ) method also clearly placed the BanMMV-GZ firmly within all isolates of BanMMV (Fig. S3). To our knowledge, this is the first report of BanMMV infecting bananas in China, extending the geographical range of this viral disease around the world. Accordingly, larger-scale BanMMV investigations must be conducted to determine the distribution and prevalence of BanMMV in China.