The open reading frame 5A of foxtail mosaic virus is expressed in vivo and is dispensable for systemic infection

Abstract

Summary. Infectious transcripts were successfully derived from full-length cDNA clones of foxtail mosaic potexvirus (FoMV). Full-length clones were constructed by RT-PCR whereby 5 and 3 genomic segments of 2.7 and 3.4 kb, respectively, were ligated into Bluescript II KS. The in vitro RNA transcripts were infectious to moncotyledonous (barley) and dicotyledonous (Chenopodium amaranticolor) plant species. Individual mutation studies on clones of each of the five major ORFs confirmed predicted gene function for the polymerase, TGB (triple gene block), and coat protein (CP) genes. Protoplast studies on expression of a unique open reading frame, ORF 5A, which initiates 143 nts upstream of the CP before it "reads through" the CP, revealed that the 5A protein was produced in vivo. Mutation analysis of the 5A ORF indicated, however, that it was not required for either replication or for productive infection of plants. However, the nucleic acid sequences encoding the extended CP segment were shown to be important for CP expression. Additional mutations in 5A had no effect on FoMV replication in protoplasts but rendered the virus noninfectious to plants. A correlation with diminished CP production from both mutant clones implies that synthesis of subgenomic CP mRNA was compromised, and this limited systemic infection.

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants

“…A VIGS vector-based single viral genome is easy to handle and it usually possesses high efficiency to cause infection of plants, a prerequisite for effective VIGS. Second, FoMV produces only mild symptoms or is latent in many native and adaptive hosts (Robertson et al, 2000). Indeed, our pFoMV-sg VIGS vector generally induces very mild mosaic symptoms in emerging leaves of barley, wheat, and foxtail millet plants.…”

“…The full-length Foxtail mosaic virus (FoMV) cDNA was PCR-amplified using FoMV cDNA clone P9 (Robertson et al, 2000) as a template with primer pairs P1-F/P1-R, and cloned into StuI/XbaI-digested pYL44, a pBIN19-based T-vector containing a duplicated CaMV 35S promoter and nitric oxide synthase (NOS) terminator (Liu et al, 2002b), to generate pFoMVwt. Further, DNA fragment 1 containing FoMV nt 4258-5371 (GenBank accession: EF630359) and DNA fragment 2 containing FoMV nt 5202-6008 were PCR-amplified using primer pairs P2-F/P2-R and P3-F/P3-R, respectively.…”

“…FoMV is similar to PVX and other potexviruses in their genomic organization. It consists of a positive-sense single-stranded RNA genome with a 59-methylguanosine cap, a 39-polyadenylated tail, and five major open reading frames (ORFs) and a unique 5A gene (Robertson et al, 2000;Bruun-Rasmussen et al, 2008). Each of the five major ORFs encodes a functional protein as in PVX (Bruun-Rasmussen et al, 2008).…”

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize

“…Later, an RNAbased full-length infectious clone was constructed and a revised genome was published (Robertson et al, 2000;Bruun-Rasmussen et al, 2008). Similar to PVX, the FoMV genome contains five ORFs: ORF1 expressed from the genomic RNA; ORF2, ORF3, and ORF4 expressed from sgRNA1 and sgRNA2; and ORF5 expressed from sgRNA3.…”

“…One significant difference in the genome organization of the two viruses is that FoMV has a unique ORF5A that initiates 143 nucleotides upstream of the CP. The 5A protein is produced in vivo, but it is not required for replication or for the systemic infection of plants (Robertson et al, 2000). Previously, the FoMV infectious clone was adapted as a transient gene expression vector by substitution of the TGB or CP gene with target genes (Liu and Kearney, 2010).…”