Systemic movement of an RNA plant virus determined by a point substitution in a 5' leader sequence.

Abstract

The ability of viruses to move through infected plants is an important determinant of host range and pathogenicity. We have investigated the genetic basis for the inability of the Type strain of barley stripe mosaic hordeivirus to undergo long-range systemic movement in the tobacco Nicotiana benthamiana. We show that, in this model system, a short open reading frame in the 5' leader of the smallest viral genomic RNA prevents long-range vascular movement. As predicted by the ribosome scanning model, the leader open reading frame decreases the efficiency with which the 5' proximal gene is translated in vitro. Thus, systemic pathogenicity in this system may be determined by the efficiency of translation of a viral gene in vivo and is not determined by the primary sequence of the encoded protein.The mechanisms by which plant viruses move from cell to cell and through the vasculature of infected plants are currently unknown. However, these processes generally require the interaction of specific viral gene products with the host and are an important constraint on viral host range and pathogenicity (1,2 Fig. 1) were prepared by digestion of full-length cloned cDNA with Kpn I and Hpa I, treatment with T4 DNA polymerase to render the ends flush, and recircularization of the large plasmid fragments according to standard procedures (9). Chimeric recombinants were constructed by exchanging 5' and 3' regions of y cDNA at the conserved HincIl and Pst I sites using yb deletion mutants of each strain. The 372-nucleotide (nt) direct repeat (4, 7) was removed from a full-length Type strain y cDNA clone by digestion with,Nru I and recircularization of the large plasmid fragment (see Fig. 1). This deletion mutant was then used to construct both the Nru I site cDNA chimeras and the oligonucleotide-directed mutations in the Type strain genetic background. In vitro transcription and plant inoculation with the mutant RNAs were carried out as described (4).A full-length cDNA clone of RNA y from the CV17 strain of BSMV was isolated in the transcription vector pBSMY(3'-T7) as described previously, except that the T7a Band-Aid oligonucleotide was used in place of T7y (for details, see ref.8). Sequencing of the cDNA clones using universal M13 primers was as described (8). Oligonucleotide-directed mutations were made by the procedure of Kunkel (10) as described (4) and confirmed by sequencing.$ Analysis of Systemic Movement. The systemic movement of BSMV in N. benthamiana was assessed visually by the appearance of disease symptoms and by detection of viral coat protein antigen in extracts from inoculated and systemically infected leaves (or their equivalent in the case of nonmoving strains and mutants) 12 days after inoculation. Protein extracts prepared from N. benthamiana leaves were fractionated by electrophoresis in SDS/15% polyacrylamide gels and blotted onto nitrocellulose, and viral coat protein was detected with anti-BSMV antiserum as described (4).In Vitro Translation. For in vitro translation, capped in vitro transcripts of...

Long-Distance Movement, Virulence, and RNA Silencing Suppression Controlled by a Single Protein in Hordei- and Potyviruses: Complementary Functions between Virus Families

“…In plants inoculated with BSMVdel␥b, Western blotting and RNA dot blot hybridization confirmed the absence of viral proteins and RNAs in the upper (noninoculated) leaves, and therefore the lack of systemic infection, at 6 weeks postinoculation (data not shown). These results differ from those reported previously (47) in which deletion of the ␥b gene delayed but did not fully abolish systemic infection. The different results could be explained by different genotypes of N. benthamiana used.…”

“…In previous studies, deletion of the BSMV gene encoding the cysteine-rich ␥b protein prevented systemic infection in barley (H. vulgare), a monocot host of the virus (46), and reduced the rate of systemic infection of BSMV in the dicot species N. benthamiana (47). We deleted the ␥b gene from the infectious cDNA of BSMV strain ND18 (BSMV ND18 ) (45) and tested its infectivity on our strain of N. benthamiana.…”

“…␥b is cysteine rich, has a putative zinc finger motif, possesses RNA-binding activity in vitro, and may influence hordeivirus gene expression (1,15,23,41,46,48). It is dispensable for virus replication and affects infection phenotypes in the natural host barley (Hordeum vulgare, a monocot) and the experimental host Chenopodium amaranticolor (a dicot) (45,47). Moreover, ␥b is implicated in seed transmissibility of hordeiviruses (19), and this protein also constitutes a virulence determinant (15,48).…”

Complete Nucleotide Sequence and Organization of the RNA Genome of Groundnut Rosette Umbravirus

“…There are examples in which more than one virus-coded protein is involved in movement of the virus within infected plants (e.g. Petty et at., 1990;Traynor et at., 1991;Nelson et al, 1993 ;Hilt & Dawson, I993 ;Taliansky & Garcia-Arenal, 1995) and the ORF3 proteins may play a role, for example, in long distance virus movement.…”

RNA-binding activities of barley stripe mosaic virus b fusion proteins

“…Plus-and minus-sense probes corresponding to the common 3' end (235nt) of BSMV RNAs were prepared from the vector pTZ18U and pTZI9U (Pharmacia) subclones constructed for hybridization analyses (Petty et al, 1990a). An RNAyspecific probe was obtained by subcloning a 210 bp EcoRV-KpnI 'blunt-ended' fragment containing the 7a-yb intergenic region of the genomic ND18 strain y42 cDNA clone (Petty et al, 1990b) into the HindIII site of pGEM-4Z (Promega), that bad been treated with Klenow polymerase. An additional 167 bp 'blunt ended' BstBI-StyI DNA fragment corresponding to the fla-flb intergenic region was subcloned in both orientations from the ND18 strain fl42 cDNA genomic clone.…”