植物负链RNA病毒反向遗传学体系前沿研究进展

Advances in reverse genetics system of plant negative-strand RNA viruses

负链RNA病毒是一类动植物上的重要病原,不仅危害人类、哺乳动物的健康,而且严重威胁家禽和农作物的安全生产.病毒反向遗传学体系是一种从cDNA克隆拯救病毒的现代病毒学分子操作技术,对于研究病毒侵染循环、致病机制和寄主相互作用至关重要.尽管动物负链RNA病毒反向遗传学体系的构建在20多年前就已取得成功,但植物负链RNA病毒的反向遗传学体系直到近年才有所突破.我国和美国科学家通过各种创新手段,相继成功构建了多种不分节段和分节段植物负链RNA病毒的反向遗传学体系,为植物负链RNA病毒的进一步深入研究提供了关键的技术体系,开启了植物负链RNA病毒研究的新时代.本文系统地回顾了植物负链RNA病毒反向遗传学体系的发展现状和最新研究进展,并对该领域未来发展方向和应用前景进行了探讨.

Negative-strand RNA(NSR) viruses contain not only medical important animal pathogens but also agricultural important plant pathogens. These NSR viruses cause deadliest diseases in humans, livestocks and agronomic crops and pose great threat to the health of human being and food security worldwide. The virus reverse genetics system is an important molecular genetic technology to study viral gene functions in virus infection cycle, disease pathology and virus-host interactions. For animal-infecting NSR viruses, the reverse genetics system has been established for more than twenty years. In contrast to the well-established reverse genetics systems for animal-infecting NSR viruses, establishment of such system for the plant-infecting NSR viruses turned out to be an extremely challenge work. Many research groups throughout the world have tried to use the similar strategies used for animal NSR viruses to establish reverse genetics systems for plant-infecting NSR viruses in the last twenty years but none of groups have succeeded. The absence of the reverse genetics system posed a major obstacle to molecular genetic manipulation and subsequent investigation of gene function and disease pathology for plant-infecting NSR viruses. Recently, big breakthroughs have been made in the establishment of the reverse genetics systems for plant NSR viruses by scientists from China and America, respectively. The systems were recently established for both nonsegmented and segmented plant NSR viruses. The reverse genetics system for a nonsegmented plant NSR virus was firstly developed for sonchus yellow net virus(SYNV), a non segmented nucleorhabdovirus NSR.Using the similar strategies as SYNV, barley yellow striate mosaic virus(BYSMV), a nonsegmented cytorhabdovirus NSR,was then succeeded in the establishment of reverse genetics system. The first reverse genetics system for a segmented plant NSR virus was developed using tomato spotted wilt virus(TSWV), a tospovirus with tripartite negative-stranded/ambisense RNA genomes. The reverse genetics system was also recently developed for rose rosette virus(RRV), an emaravirus with seven negative-sense mono-cistronic RNA genomes. The establishment of the reverse genetics system for plant NSR viruses typically involves two steps. The first step requires the construction of a mini-genome replication system. The viral genomic or antigenomic RNAwas flanked by hammerhead and ribozyme to generate exact viral 5′ and 3′end, and driven by 35S promoter. The mini-replicon construct was co-expressed with the constructs expressing nucleocapsid(N) protein, phosphoprotein(P;essential for rhabdovirus) and viral RNA-dependent RNA polymerase(Rd Rp). For the segmented plant NSR viruses such as TSWV, codon optimization and removal of intron-splicing sites of Rd Rp sequence are critical for the expression of a functional Rd Rp from 35S-driven constructs in planta. The second step involves the rescue of virus entirely from full-length infectious c DNA clones in plant. The recombinant virus can carry green fluorescence protein(GFP) gene reporter and infect Nicotiana benthamiana plant systemically. The establishment of reverse genetics for different plant-infecting NSR viruses now provided powerful systems to investigate all aspects of virus infection cycle and disease pathology. The recent breakthroughs of reverse genetics systems have opened a new era for the investigation of plant-infecting NSR viruses in the future.