Apple latent spherical virus (ALSV)-induced gene silencing in a medicinal plant, Lithospermum erythrorhizon.

Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have been shown advantageous in introducing iRNA genes into non-model plants. This study describes the development of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRNA was detected in infected plants but not in escape and mock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the VIGS system silence target genes in soil-grown L. erythrorhizon.

ALSV-Based Virus-Induced Gene Silencing in Apple Tree (Malus × domestica L.).

Virus-induced gene silencing (VIGS) is a fast and efficient tool to investigate gene function in plant as an alternative to knock down/out transgenic lines, especially in plant species difficult to transform and challenging to regenerate such as perennial woody plants. In apple tree, a VIGS vector has been previously developed based on the Apple latent spherical virus (ALSV) and an efficient inoculation method has been optimized using biolistics. This report described detailed step-by-step procedure to design and silence a gene of interest (GOI) in apple tree tissues using the ALSV-based vector.

Contribution of Tomato torrado virus Vp26 coat protein subunit to systemic necrosis induction and virus infectivity in Solanum lycopersicum.

BACKGROUND: Tomato torrado virus (ToTV) infection manifests with burn-like symptoms on leaves, leaflets and upper stem parts of susceptible infected plants. The symptoms caused by ToTV may be considered as one of the most severe virus-induced forms of systemic necrosis, which spreads within the whole plant and leads to a lethal phenotype. However, to date there are no data revealing which viral genes encode for a specific pathogenicity determinant that triggers the plant necrotic response for any torradovirus. In this study we evaluated the influence of three coat protein subunits of ToTV: Vp23, Vp26 and Vp35, transiently expressed from a PVX-based vector, and checked their association with the induction of systemic necrosis in infected Solanum lycopersicum L. (cv. Beta Lux), a natural host of ToTV. METHODS: To estimate how ToTV coat protein subunits might contribute in plant response to virus infection we over-expressed the proteins from PVX-based vector in tomato and analyzed enzymatic activities related with plant defense response. By doing protein qualitative analysis performed by mass spectrometry we indicated the PR10 in protein fraction with induced ribonuclease activity. RESULTS: We observed that only the Vp26 enhanced PVX pathogenicity causing severe necrosis of the infected plant. Moreover, we indicated increased RNase and oxidative activities in plants infected with PVX-Vp26 chimeras only. Importantly, we suspected that this increased RNase activity is associated with increased accumulation of PR10 mRNA and products of its translation. CONCLUSIONS: On the basis of the obtained results, we indicated that Vp26 acts as the elicitor of hypersensitive response-like reactions of PVX-Vp26 manifesting with enhanced pathogenicity of the recombined PVX. This might be the first described suspected necrosis determinant of torradoviruses infecting tomatoes.

Aphid transmission of Lettuce necrotic leaf curl virus, a member of a tentative new subgroup within the genus Torradovirus.

Lettuce necrotic leaf curl virus (LNLCV) was described as the first non-tomato-infecting member of the genus Torradovirus. Until today, the virus was found only in The Netherlands in two different areas in open field crops of lettuce. In 2015, LNLCV was accepted by the ICTV as a new member of the genus Torradovirus. The tomato-infecting (TI) torradoviruses Tomato torrado virus (ToTV), Tomato marchitez virus (ToMarV) and Tomato chocolàte virus (ToChV) are transmitted by at least three whitefly species in a semi-persistent and stylet-borne manner. As LNLCV was transmitted in open fields in The Netherlands, where whiteflies are present only in low incidence, transmission studies were set up to identify the natural vector of LNLCV. Whitefly species which survive Dutch open field conditions during summer, as well as lettuce colonizing aphid species, were tested for their ability to transmit LNLCV. Lengths of acquisition and inoculation periods were chosen in accordance with the conditions for TI torradoviruses. Transmission experiments involving whiteflies were never successful. Transmission with aphids was only successful in case of the lettuce-currant aphid, Nasonovia ribisnigri. Localization of LNLCV virions in N. ribisnigri with a nested RT-PCR indicated the stylets as possible retention sites. The willow-carrot aphid Cavariella aegopodii did not transmit LNLCV in our transmission experiment but the virus could be detected in the stylets of this aphid, leaving C. aegopodii as a possible vector for LNLCV.

Evidence for participation of RNA 1-encoded elicitor in Cowpea mosaic virus-mediated concurrent protection.

The cowpea (Vigna unguiculata) line Arlington, inoculated with Cowpea mosaic virus (CPMV), showed no symptoms, and no infectivity or accumulation of capsid antigen was detected at several days after inoculation. Coinoculation, but not sequential inoculation, of CPMV with similar concentrations of another Comovirus; Cowpea severe mosaic virus (CPSMV), resulted in reduced numbers of CPSMV-induced lesions. This apparent, CPMV-mediated reduction in number of CPSMV-induced infection centers was termed concurrent protection. We report results obtained by inoculating two nearly isogenic cowpea lines derived from a CPMV-susceptible cowpea crossed to Arlington, one line CPMV-susceptible and the other resistant. The CPMV virions B and M, encapsidating genomic RNAs 1 and 2, respectively, were extensively purified by gradient centrifugation. In the CPMV-resistant cowpea, either CPMV or CPMV B affected concurrent protection against CPSMV and against two distinct non-Comoviruses: Cherry leafroll virus and Southern bean mosaic virus. Adding CPMV M to the inoculum did not enhance CPMV-B-mediated protection. CPMV B was ineffective in protecting CPMV-susceptible cowpea. We postulate that CPMV-mediated concurrent protection is elicited in CPMV-resistant cowpea by a CPMV RNA-1-encoded factor and acts to reduce accumulation or spread of CPMV and certain coinoculated challenging viruses in or from the inoculated cell. Coinoculated CPMV did not protect CPMV-resistant cowpea against Tomato bushy stunt virus or Cucumber mosaic virus.