1984). 2019;223:2120–33. Proc Natl Acad Sci U S A. 2019;164:2417–29. The successful SYNV MR experiments provided the opportunity to develop the full-length SYNV infectious cDNA clones for use in N. benthamiana plants. 2011;156:587–95. 2019; Gao et al. Plant Biotechnol J. Institute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi 43600, Malaysia. Developments in plant negative-strand RNA virus reverse genetics. Google Scholar. 2019). 2012; Jarugula et al. 2008; Liu and Kearney 2010; Hefferon 2017). PubMed Central  The CCR4 proteins were recruited by P into the N-RNA complexes and induced turnover of cellular mRNAs that nonspecifically bind to the BYSMV N protein (Zhang et al. 2008;26:3395–403. Furthermore, although NSR viruses can replicate in insect vectors, appropriate insect cell lines are lacking for recovery of plant NSR viruses from their respective infectious clones (Hogenhout et al. 2015; Cao et al. Since plant positive-strand RNA virus genomes are messenger RNAs (mRNAs), reverse genetics systems of these viruses can be achieved by simply introducing in vitro synthesized RNA transcripts into susceptible plant cells. The polymerase chain reaction: an improved method for the analysis of nucleic acids. All authors read and approved the final manuscript. Rhabdoviruses have well-defined transcription units and modular genome structure, as well as polar transcription effects (Jackson et al. Nat Biotechnol. 2012;160:582–90. Pattnaik AK, Ball LA, Legrone A, Wertz GW. Pp 1-20. Yamada K, Terahara T, Kurata S, Yokomaku T, Tsuneda S, Harayama S. Environ Microbiol. 2018;9:1419. It is a member of the Emaravirus genus that includes nine species of virus with four to eight negative-sense RNAs (Di Bello et al. BMC Genet. Effect of Exogenous Transcription Factors Integration Sites on Safety and Pluripotency of Induced Pluripotent Stem Cells. To improve accumulation of these core proteins and minireplicon derivatives, suppressors of RNA silencing (VSR) proteins, the barley stripe mosaic virus (BSMV) γb, tomato bushy stunt virus (TBSV) p19, and tobacco etch virus (TEV) HC-Pro, were combined into a binary vector to express together in single cells (Ganesan et al. 2019; Feng et al. We also highlighted important applications of these reverse genetics of NSR viruses in viral gene function analyses, investigation of virus-insect-plant interactions, and genomic studies of insect vectors and host plants. As obligate parasites, most of plant viruses employ host cytoskeletons for intracellular movement. Consequently, the codon-optimized RdRp, rather than the wild-type RdRp protein, supported transcription and replication of the TSWV S gRNA-based MR system when co-expressed with N protein and four VSRs. Our studies clearly provide new protocols for developing reverse genetics systems of plant NSR viruses infecting monocot plants and arthropod vectors. Plant Biotechnol J. 2013; Wang et al. PubMed Central  2013;25:2429–43. The rhabdovirus NC complexes, the minimal infectious units, are composed of the genomic RNA (gRNA) and the viral N protein, the P protein and the L protein. 2007;18:134–41. CCR4, a RNA decay factor, is hijacked by a plant cytorhabdovirus phosphoprotein to facilitate virus replication. 2020). Ganesan U, Bragg JN, Deng M, Marr S, Lee MY, Qian S, et al. Arch Virol. 2020). Moreover, insect vectors play an important role in the life cycles of some viruses and obligate virus transmission. Front Microbiol. Gao Q, Xu W-Y, Yan T, Fang X-D, Cao Q, Zhang Z-J, et al. 2011). 2011; Dugdale et al. Finally, we injected crude extracts obtained from recombinant BYSMV-RFP-infected N. benthaminia leaves into thoraxes of healthy SBPHs. 2019b). Since numerous intron splicing sites were predicted to exist in the RdRp gene sequence, they then constructed a codon-optimized viral RdRp gene by removing potential splicing sites and used it to stably express the large RdRp protein (Fig. Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. The BYSMV genome encodes ten proteins including five structural proteins and five accessory proteins in the order of 3′–N–P–P3–P4/P5–P6–M–G–P9–L–5′ (Yan et al. 2015). developed an effective mechanical method by using an airbrush to deliver agrobacterium cultures (Pang et al. Negative-stranded RNA (NSR) viruses cause serious diseases in a broad range of hosts, including human, vertebrate, invertebrate, and plant organisms. 2020 Aug 26;23(1):5-13. doi: 10.2478/bjmg-2020-0003. The authors declare that they have no competing interests. Science. Since then, reverse genetics systems of numerous plant positive-strand RNA viruses have been developed and exploited as vectors for gene expression and virus-induced RNA silencing (Hefferon 2014; Cody and Scholthof 2019). Recently, Pang et al. 2019a;93:e00296–19. Ishibashi K, Matsumoto-Yokoyama E, Ishikawa M. A tomato spotted wilt virus S RNA-based replicon system in yeast. 1d). J Exp Bot. 2020). Drug Discov Today. Intriguingly, the CK1-mediated phosphorylation resulted in transformation of BYSMV P from 42 kDa (P42) to 44 kDa, thereby facilitating the transition from virus replication to transcription (Gao et al. The recombinant SYNV vector carrying a tgtRNA (tRNA-gRNA-tRNA) and a Cas9 was successfully delivered into plants, which resulted in single or multiplex mutagenesis in systemically infected tissues of tobacco plants (Ma et al. Thus, significant advances in understanding how plant positive-strand RNA viruses interact with their hosts have been achieved in recent few years. Tomato spotted wilt virus is a segmented NSR virus and belongs to the genus Orthotospovirus in the family Tospoviridae (Kormelink et al. eCollection 2020 Jun. EMBO J. 2019). The termini of VSV DI particle RNAs are sufficient to signal RNA encapsidation, replication, and budding to generate infectious particles. Zaidi SS-e-A, Mansoor S. Viral vectors for plant genome engineering. 2011). 2020 Jul 23;11(8):839. doi: 10.3390/genes11080839. 2019). We further modified the BYSMV vectors into versatile expression platforms for co-expression of three foreign proteins in cereal plants and SBPHs. Front Plant Sci. Kormelink R, Garcia ML, Goodin M, Sasaya T, Haenni AL. Briefly, these difficulties have severely affected the development of reverse genetics system of TSWV in plants. 2009;141:219–36. Recently, our group have constructed the infectious cDNA clones of a plant cytorhabdovirus, barley yellow striate mosaic virus (BYSMV), which can be rescued in monocot plants and insect vectors (Fang et al. 2017). Important discoveries in regard to pathogenicity mechanisms, the analysis of genome packaging and studies of the entry mechanism of viruses into their host cells are a few examples of the power of reverse genetics. Vaccine. a Agrobacterium tumefaciens-mediated expression of genes in N. benthamiana leaves was utilized for simultaneous delivery of plant NSR viruses core protein genes and genomic RNAs in single cells.

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