A novel virus transmitted through pollination causes ring-spot disease on gentian (Gentiana triflora) ovaries

Abstract

In this study, we identified a novel virus from gentian (Gentiana triflora) that causes ring-spots on ovaries. Furthermore, the virus causes unusual symptoms, ring-spots that appear specifically on the outer surface of the ovarian wall after pollination. Pollen grains carrying the virus were used to infect host plants by hand-pollination. RNA extracted from purified virions indicated that the virus had two segments, RNA1 and RNA2. The full-length cDNA sequence indicated that RNA1 had two ORFs: ORF1 had methyltransferase and helicase motifs, and ORF2 had an RNA-dependent RNA polymerase motif. RNA2 had five ORFs encoding a coat protein, triple gene block proteins 1-3 and a cysteine-rich protein. The length of RNA1 was 5519 bases and that of RNA2 was 3810 bases not including a polyU/polyA region between the first and second ORFs. Viral RNA does not have a polyA tail at the 39 end. Sequence similarity and phylogenetic analysis suggested that the virus is closely related to members of the genera Pecluvirus and Hordeivirus but distinct from them. These combined results suggest that the causal agent inducing ring-spot symptoms on gentian ovaries is a new virus belonging to the family Virgaviridae but not to any presently known genus. We tentatively name the virus gentian ovary ring-spot virus.

P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas

“…To make Cl-90-1 Br2-P3B No. 30 without GFP (Fig. 1B), the SalI-BamHI fragment of Cl-SB with GFP was used to replace that of Cl-P1HC with GFP, thus producing Cl-P1HCϩSB without GFP, and the NheI-SalI fragment of Cl-NS with GFP was then used to replace that of Cl-P1HCϩSB without GFP to produce the final vector.…”

“…In this study, we constructed pWCl/P3-No. 30 (7). We cloned P3N-PIPO of Cl-No.30 as a fragment from the 5= end of P3 to the position corresponding to the stop codon of Cl-RB PIPO (located downstream of the stop codon of the PIPO frame of Cl-No.30).…”

“…Agrobacterium-mediated transient expression. Agrobacterium-mediated transient expression was conducted as described previously (30). Agrobacterium tumefaciens LBA4404 cells transformed with each construct were suspended in MES buffer [10 mM 2-(N-morpholino)ethanesulfonic acid, 10 mM MgCl 2 (pH 5.7)], and the suspensions were adjusted to an optical density at 600 nm (OD 600 ) of 1.0.…”

“…Western blots were conducted as described previously (30,32). Proteins were resolved in 12% NuPAGE Bis-Tris gels (Thermo Fisher Scientific) using MES-SDS buffer (FLAG-tagged protein detection) or in 10% SDS-PAGE using Tris-glycine buffer (coat protein [CP] detection [33]), followed by electrotransfer onto a polyvinylidene difluoride (PVDF) membrane.…”

“…Cyn1 has been suggested to be an NB-LRR gene whose product recognizes ClNo. 30 and induces an HR associated with cell death (20,21).…”