Tongyan Tian

Lettuce infectious yellows virus: in vitro acquisition analysis using partially purified virions and the whitefly Bemisia tabaci

Virions of lettuce infectious yellows virus (LIYV; genus Crinivirus) were purified from LIYV-infected plants and their protein composition was analysed by SDS-PAGE and immunoblotting. Virion preparations contained the major capsid protein (CP), but the minor capsid protein (CPm), p59 and the HSP70 homologue were also identified by immunoblot analysis. Immunogold labelling analysis showed that CP constituted the majority of the LIYV virion capsid, but CPm was also part of the capsid and localized to one end of the virion, similar to the polar morphology seen for viruses in the genus Closterovirus. p59 and the HSP70 homologue were not detected on virions by immunogold labelling, but were always detected in virion preparations by immunoblot analysis. Purified LIYV virions were used for in vitro acquisition analysis with Bemisia tabaci whiteflies and were efficiently transmitted to plants. Infectivity neutralization analyses were done using antisera to the LIYV-encoded CP, CPm, p59 and HSP70 homologue. Only antiserum to the CPm effectively neutralized LIYV transmission by B. tabaci. These data suggest that the LIYV-B. tabaci transmission determinants are associated with purified virions, and that the LIYV virion structural protein CPm is involved in transmission by B. tobaci.

Asynchronous Accumulation of Lettuce Infectious Yellows Virus RNAs 1 and 2 and Identification of an RNA 1 trans Enhancer of RNA 2 Accumulation

ABSTRACT Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartiteCrinivirus, Lettuce infectious yellow virus(LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation.

First report of Cucurbit yellow stunting disorder virus (genus Crinivirus) in North America.

In late summer 1999, field- and greenhouse-grown melon plants (Cucumis melo) showing severe stunting and yellowing symptoms were observed near Donna in southern Texas and near the town of Reynosa in northern Mexico. Symptoms were typical of those caused by viruses in the genus Crinivirus, family Closteroviridae. High populations of Bemisia spp. whiteflies were associated with these plantings, with many plants showing heavy infestation. Laboratory analyses showed that melon plants from both locations were infected by the whitefly-transmitted Cucurbit yellow stunting disorder virus (CYSDV). Positive hybridization reactions with digoxigenin-UTP-labeled transcript probes corresponding to the CYSDV heat shock protein 70 (HSP70) homolog coding region (1) were obtained for RNAs extracted from symptomatic plants. Similar probes for the related Lettuce infectious yellows virus (LIYV) and Beet pseudo-yellows virus (BPYV), two whitefly-transmitted viruses previously reported from North America (2), did not hybridize with the RNAs. Definitive confirmation of CYSDV was obtained by performing reverse-transcription polymerase chain reaction (RT-PCR) analyses for two distinct CYSDV coding regions. RT-PCR with primers corresponding to CYSDV, but not LIYV or BPYV HSP70 homolog coding regions, gave specific (≈500 bp) products from corresponding test plants. RNAs from healthy control plants gave no RT-PCR product. Because the HSP70 coding region is highly conserved (2), we also performed RT-PCR with primers designed for the Spanish CYSDV capsid protein gene (GenBank accession AJ243000). Positive RT-PCR products of ≈700 bp were obtained only from the Texas and Mexico melon plants. CYSDV is a widespread and damaging virus of cucurbits in southern Europe and the Middle East (2). This is the first report of this important virus in North America. References: (1) Tian et al. Phytopathology 86:1167, 1996. (2) Rubio et al. Phytopathology 89:707, 1999.

A Mutation in the Lettuce Infectious Yellows Virus Minor Coat Protein Disrupts Whitefly Transmission but Not In Planta Systemic Movement

ABSTRACT The Lettuce infectious yellows virus (LIYV) RNA 2 mutant p1-5b was previously isolated from Bemisia tabaci-transmitted virus maintained in Chenopodium murale plants. p1-5b RNA 2 contains a single-nucleotide deletion in the minor coat protein (CPm) open reading frame (ORF) that is predicted to result in a frameshift and premature termination of the protein. Using the recently developed agroinoculation system for LIYV, we tested RNA 2 containing the p1-5b CPm mutant genotype (agro-pR6-5b) in Nicotiana benthamiana plants. We showed that plant infection triggered by agro-pR6-5b spread systemically and resulted in the formation of virions similar to those produced in p1-5b-inoculated protoplasts. However, virions derived from these mutant CPm genotypes were not transmitted by whiteflies, even though virion concentrations were above the typical transmission thresholds. In contrast, and as demonstrated for the first time, an engineered restoration mutant (agro-pR6-5bM1) was capable of both systemic movement in plants and whitefly transmission. These results provide strong molecular evidence that the full-length LIYV-encoded CPm is dispensable for systemic plant movement but is required for whitefly transmission.

Specific inclusion bodies are associated with replication of lettuce infectious yellows virus RNAs in Nicotiana benthamiana protoplasts

Nicotiana benthamiana mesophyll protoplasts, either mock-inoculated or inoculated using in vitro transcripts derived from lettuce infectious yellows virus (LIYV) RNA 1- and/or RNA 2-cloned cDNAs were analysed by transmission electron microscopy (TEM) and, in some cases, also by immunogold labelling. TEM revealed the main cytopathological effects of LIYV infections in N. benthamiana protoplasts infected with RNAs 1 and 2: (a) typical closterovirus-induced (beet yellows virus-type) accumulations of vesiculated cytoplasmic membranes as inclusion bodies, sometimes with associated virions; (b) scattered aggregations of virions within the cytoplasm; and (c) electron-dense plasmalemma deposits. These were not seen in mock-inoculated protoplasts. Protoplasts inoculated only with LIYV RNA 1 contained vesiculated cytoplasmic inclusion bodies, but not virions or plasmalemma deposits. Thus, infection by only LIYV RNA 1 is sufficient to induce characteristic closterovirus vesiculated cytoplasmic inclusion bodies. However, both LIYV RNAs 1 and 2 are needed for production of virions and plasmalemma deposits.