A SYNERGISM ASSOCIATED WITH MIXED INFECTIONS OF PANICUM MOSAIC VIRUS AND ITS SATELLITE VIRUS
K-BG SCHOLTHOF and M TURINA
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, 77843, USA
Background and objectives
Panicum mosaic virus (PMV) forms 30-nm icosahedral particles composed of a positive-sense single-stranded RNA of ca 4300 nucleotides (nt) and capsid protein (CP) subunits of 26 kDa. Comparative sequence analysis of PMV indicates that it should be provisionally assigned to a new genus, Panicovirus , within the Tombusviridae. The two 5' proximal open reading frames (ORFs), p48 and p112, comprise the replicase proteins. The 3' proximal ORFs, p8, p6.6/p8-FS, p15, and a 26-kDa CP, are expressed from a single subgenomic RNA (sgRNA), most likely by a cap-independent leaky scanning mechanism. Disruption of one or more of the ORFs encoded on the PMV sgRNA abolishes systemic infection, but all of the mutants are competent for replication in pearl millet (Pennisetum glaucum) protoplasts. PMV also supports the replication of a satellite virus (SPMV). Sequence analyses of cDNA clones derived from the 826-nt single-stranded SPMV RNA revealed four putative ORFs, two on the genomic RNA and two on the complementary RNA . SPMV ORF1 encodes a 17-kDa CP which forms 16-nm icosahedral particles containing the satellite virus RNA. Pearl millet plants systemically infected with PMV alone display mild mosaic, but co-infection with SPMV causes severe mosaic, stunting, and a lack of seed set. The availability of full-length infectious cDNA clones of PMV and SPMV  permit molecular analyses of the interaction between these genomes, in order to understand the basis for the severe symptom phenotype.
Results and conclusions
Recent experiments with PMV or mixed infections of PMV+SPMV suggest that SPMV induces a synergism, resulting in severe chlorosis and stunting of pearl millet plants, an increased titre of PMV, and more rapid distribution of the virus throughout the plant. The accumulation of PMV RNA, CP and p8 protein, and the SPMV CP and RNA, on inoculated and upper-inoculated pearl millet leaves, was monitored daily from 3 to 10 days post-inoculation (d.p.i.). The mixed infections of PMV+SPMV resulted in the accumulation of PMV in the upper uninoculated leaves approximately 1 day ahead of an infection of PMV alone. In addition, there was a 2- to 10-fold increase in the concentration of PMV RNA, CP, and p8 in the mixed PMV+SPMV infections when compared to infections of PMV alone. At 7 d.p.i., analyses of total RNA on sucrose density gradients indicated that PMV RNA increased ca 4-fold in the upper uninoculated leaves in the mixed infection of PMV+SPMV compared to the single infection of PMV. The rRNA concentrations in pearl millet plants were reduced significantly in both PMV and PMV+SPMV infections when compared to uninoculated plants at 7 d.p.i. To evaluate the severe symptom phenotype, site-directed oligonucleotide mutagenesis was used to individually disrupt the four putative SPMV ORFs. Pearl millet plants co-inoculated with transcripts of PMV and the SPMV mutants exhibited symptoms that were similar to the PMV+SPMV wild-type infection. These data suggest that SPMV RNA may affect symptom development. Further evaluation of the SPMV capsid protein may reveal its potential role in other aspects of the synergism, including enhancement of the movement and/or replication of PMV.
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