Rescue of Tomato spotted wilt tospovirus entirely from cDNA clones, establishment of the first reverse genetics system for a segmented (-)RNA plant virus

Abstract

The group of negative strand RNA viruses (NSVs) includes not only dangerous pathogens of medical importance but also serious plant pathogens of agronomical importance. Tomato spotted wilt tospovirus (TSWV) is one of those plant NSVs that cause severe diseases on agronomic crops and pose major threats to global food security. Its negative-strand segmented RNA genome has, however, always posed a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from cDNA clones, the first reverse genetics (RG) system for a segmented plant NSV. First, a replication and transcription competent mini-genome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by a 5’ Hammerhead and 3’ Ribozyme sequence of Hepatitis Delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA dependent RNA polymerase (RdRp) gene. Next, a movement competent mini-genome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. After further optimization, infectious TSWV and derivatives carrying eGFP reporters were successfully rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA and L(+)-agRNA. Viral rescue occurred in the additional presence of various viral suppressors of RNAi, but TSWV NSs interfered with the rescue of genomic RNA. The establishment of a RG system for TSWV now allows detailed molecular genetic analysis of all aspects of tospovirus life cycle and their pathogenicity.SignificanceFor many different animal-infecting segmented negative-strand viruses (NSVs), a reverse genetics system has been established that allows the generation of mutant viruses to study disease pathology and the role of cis- and trans-acting elements in the virus life cycle. In contrast to the relative ease to establish RG systems for animal-infecting NSVs, establishment of such system for the plant-infecting NSVs with a segmented RNA genome so far has not been successful. Here we report the first reverse genetics system for a segmented plant NSV, the Tomato spotted wilt tospovirus, a virus with a tripartite RNA genome. The establishment of this RG system now provides us with a new and powerful platform to study their disease pathology during a natural infection.

A Review of the Most Common and Economically Important Diseases That Undermine the Cultivation of Tomato Crop in the Mediterranean Basin

“…TSWV is a temperature-sensitive virus; in fact, depending on the isolate, the thermal inactivation points vary from 40 to 46 • C, with an exposure time of approximately 10 min [223]. TSWV infects a wide range of agricultural crops, including tomato [224,225], where it is responsible for the spotted wilt disease. TSWV is spread in all Mediterranean countries apart from Morocco [226] and may cause from 40-90% and up to 95% loss in yield and marketable value of tomato, respectively [46,47].…”

Piperazine Derivatives Containing the α-Ketoamide Moiety Discovered as Potential Anti-Tomato Spotted Wilt Virus Agents

“…The beginning of the infection cycle of TSWV requires at least RNP, which is the smallest infectious unit of the virus, and the N protein of TSWV is absolutely necessary for the remodeling of the infectious RNP complex. 33 When the data of three different activities (curative, protective, and inactivation) of the target compounds are compared against TSWV in vivo, it was found that the same compound was more effective in the inactivation activity; that is, the compound interacted with the virus and then inactivated the virus to inhibit the spread of the virus. Therefore, to investigate the potential action mode of the target compounds to inhibit TSWV, the real RNP composition was simulated by infecting N. benthamiana with L (+)opt + SR (+)eGFP + VSRs mediated by A. tumefaciens in vivo.…”

“…The fluorescence quantity and intensity of N. benthamiana were observed by a fluorescence microscope on the third and fifth days. 33 Expression of α-Enhanced Green Fluorescent Protein (α-eGFP) in Tobacco Was Detected by Western Blot. The sample was removed from −80 °C, and 1.0 g of sample was added to 1.0 mL of protein extraction buffer [50 mL; 5 mL of glycerin, 1.25 mL of 1 M Tris−HCl (pH 7.5), 18.6 mg of disodium ethylenediaminetetraacetic acid (EDTA), 438 mg of NaCl, 77 mg of dithiothreitol, 1 g of polyvinylpolypyrrolidone (PVPP), 0.25 mL of Triton X-100, and 1× protease inhibitor, white and muddy].…”

The Bunyavirales: The Plant-Infecting Counterparts

“…Only recently have the first reverse genetics systems been established for TSWV and RRV. The first one was established for TSWV [ 261 ], soon followed by the establishment of one for RRV [ 95 ]. For both viruses, particles and a systemic infection could be rescued entirely from cDNA clones.…”

“…There are no studies that address how microevolution affects any of the aforementioned taxa, largely because reverse genetics platforms have only recently become available [ 75 , 95 , 261 ]. On the other hand, there are investigations on how recombination, reassortment, and genome plasticity affect virus macroevolution.…”