J. A. Dodds

A defective movement protein of TMV in transgenic plants confers resistance to multipleviruses whereas the functional analog increases susceptibility

Transgenic tobacco plants that express a gene encoding a defective mutant of the tobacco mosaic virus (TMV) movement protein which are known to be resistant to several tobamoviruses were inoculated with viruses from different taxonomic groups to determine the breadth of resistance. There were significant delays in the time of appearance of disease symptoms and/or there was reduced systemic accumulation of virus in upper leaves of plants inoculated with tobacco rattle tobravirus, tobacco ringspot nepovirus, alfalfa mosaic alfamovirus, peanut chlorotic streak caulimovirus, and cucumber mosaic cucumovirus. Conversely, tobacco plants that express a gene encoding the functional tobacco mosaic virus wild-type movement protein accelerated symptom development, enhanced the severity of symptom formation, and/or increased the accumulation of these viruses and, additionally, TMV. Our results indicate that there are similar functions among the movement proteins of a number of plant viruses despite the apparent lack of sequence similarity between them.

Cross protection between strains of cucumber mosaic virus: effect of host and type of inoculum on accumulation of virions and double-stranded RNA of the challenge strain.

Two strains of cucumber mosaic virus (CMV) differed in three characteristics of value for cross-protection experiments. Their virions and also two of their double-stranded RNAs could be separated and distinguished by electrophoresis on polyacrylamide gels, and the symptoms of one strain were milder than the other in tobacco, tomato, and squash. The mild strain, CMV-S, protected plants of these three hosts from the effects of the second strain, CMV-P, and also prevented the accumulation of virions and ds RNAs of the challenge strain. Protection was detected in leaves inoculated with the challenge strain and also in later formed leaves. The only exception to this result was the accumulation of ds RNAs and to a lesser extent virions of the challenge strain when infectious viral RNA was used as the challenge inoculum instead of virus particles. This breakdown of cross protection occurred only in those leaves inoculated with the challenge strain RNA. No accumulation of challenge ds RNAs or virions occurred in later formed leaves. Tomato and tobacco plants infected with CMV-S were not protected from infection by tobacco mosaic virus.

Comparison of Detection Methods for Citrus Tristeza Virus in Field Trees During Months of Nonoptimal Titer

Enzyme-linked immunosorbent assay (ELISA) can reliably detect citrus tristeza virus (CTV) in samples collected during approximately 6 months of a typical year. Two reverse transcriptase polymerase chain reaction (RT-PCR) methods (total nucleic acid extract and immunocapture based) were evaluated and compared to ELISA in order to develop a more sensitive assay for CTV. From May 1994 to October 1995, 6 sweet orange trees infected with CTV from each of 2 geographic areas (Riverside and the San Joaquin Valley) were tested monthly by each method. In the months of August (San Joaquin Valley samples) and September (Riverside and San Joaquin Valley samples) several of the trees had a significant loss of virus titer such that CTV was not reliably detected by ELISA. By contrast, the 2 PCR methods gave definitive positive results for CTV in samples collected during these months. Different tissue types were analyzed by each of the above assays. Petioles and midribs, both phloem-rich tissues, were each satisfactory for ELISA, while distal leaf tips did not always produce a positive result. All tissue types were equally efficient in producing a positive result in both PCR-based assays. The results of this study provide a basis for CTV testing by PCR in months when virus titer drops to a level generally unacceptable for using ELISA.

Assembly and characterization of hybrid virus-inorganic nanotubes

Recently, rod-shaped viruses have attracted attention as biological templates for assembly of nanostructures. Tobamoviruses such as the type strain of Tobacco mosaic virus (TMV-U1, or -common) have a cylindrical shape and dimensions suitable for nanoelectronic applications: 300nm long and 18nm in diameter with a 4nm axial channel. TMV particles can be coated with metals, silica, or semiconductor materials and may also form end-to-end assemblies to be used as interconnects or device channels. In this letter, we report the preparation of TMV-U1 templated organic-metal nanotubes, and their structural characterization using transmission electron microscopy and micro-Raman spectroscopy. Reproducible phonon signatures different from that of native TMV-U1 were observed from the metal-coated TMVs. Our results indicate that Raman spectroscopy can be used for monitoring of the bio-assisted nanostructure assembly and for analyzing the vibrational modes of the resulting bio-inorganic junctions.

Segregation of distinct variants from Citrus tristeza virus isolate SY568 using aphid transmission.

A well-studied severe isolate of Citrus tristeza virus (CTV) known as SY568 has previously been shown to contain multiple variants of the virus which differ in their genetic and biological characters. Aphid transmission was used in an attempt to segregate some of these variants for further characterization. Resulting infections gave symptoms which varied from asymptomatic to more severe than the inoculum source. RNase protection assays (RPAs) were used to compare nine regions of the CTV genome and determine whether unique strains could be identified. Five aphid-transmitted subcultures, with fingerprints that were different from those of the inoculum sources in at least one genomic area, were then cloned, sequenced, and compared with known isolates. An asymptomatic strain was shown to be different in every area of the CTV genome when examined by RPA and sequencing of selected regions. Mixed-infection studies using graft transmission of the asymptomatic subculture and two of the more severe aphid-transmitted subcultures showed that the mild strain was not able to compete well when in the presence of any of the severe variants tested, and its titer was significantly reduced from that seen in single infection. The mild strain and a selected severe strain were singly graft inoculated into five different citrus hosts (sweet orange, grapefruit, sour orange, lemon, and lime), where they maintained their distinct biological and genetic characteristics.

First Report of Angelonia flower break virus in Nemesia spp. and Other Ornamental Plants in California

Between June 2006 and July 2007, ornamental plant samples were collected from four counties in California (Riverside, Sacramento, San Diego, and Santa Barbara) and tested for the presence of Angelonia flower break virus (AnFBV) using ELISA (Agdia, Inc., Elkhart, IN). Tissue samples were from propagation facilities or wholesale outlets except those from Riverside County, which were from retail stores. Thirteen positive samples were found in three varieties each of Angelonia and Nemesia spp. and seven varieties of Verbena spp., with at least one positive from each county. Foliar symptoms ranged from asymptomatic to a mild mosaic with distinct flower breaking in the Angelonia spp. Results were confirmed by reverse transcription (RT)-PCR of the coat protein gene (1) and the 1,172-bp amplicons were sequenced. Viral isolates from the three varieties of the Angelonia spp. had 98 to 99% nucleotide similarity and 99 to 100% amino acid identity to the Maryland strain of AnFBV (1; GenBank Accession No. DQ221212), with 91 to 92% nucleotide similarity and 96 to 97% amino acid identity to the Israel and Florida strains (GenBank Accession Nos. DQ223771 and DQ219415). All viral isolates from the Nemesia and Verbena spp. plants had nucleotide similarities of 96 to 98% and 98% amino acid identity to the Israel and Florida strains, with 91 to 92% nucleotide similarity to the Maryland strain. AnFBV has been previously reported in Angelonia and Verbena spp. among other hosts (1,2), but not in Nemesia spp. and not in California. This recently described carmovirus appears to be well established in the state in a variety of ornamental plant species. References: (1) S. Adkins et al. Phytopathology 96:460, 2006. (2) F. Assis-Filho et al. Plant Dis. 90:1115, 2006.

First report of Nemesia ring necrosis virus in North America in ornamental plants from California.

During the last several years, two California propagators have detected what was believed to be the tymovirus Scrophularia mottle virus (ScrMV) in several ornamental plant species on the basis of enzyme-linked immunosorbent assay (ELISA) using a ScrMV antibody system. Symptoms were generally mild, ranging from nonsymptomatic to a mild mosaic. Our laboratory confirmed the presence of a tymovirus in one Verbena sp. and two Diascia spp. cultivars on the basis of dsRNA analysis that showed bands of approximately 6,400 and 300 nucleotides representing the genomic and coat protein subgenomic RNAs, respectively. While these plants and those that were experimentally infected (Nicotiana benthamiana and N. clevelandii) also tested positive for ScrMV by ELISA, the host range did not match that published for ScrMV, notably the lack of symptoms in Chenopodium quinoa and the lack of systemic infection in Datura stramonium. A similar host range result was reported in Europe for another tymovirus that cross reacts with ScrMV antiserum, Nemesia ring necrosis virus (NeRNV) (2). Using NeRNV specific primers (1), we used reverse transcription-polymerase chain reaction (RT-PCR) to test plants that had previously tested positive for ScrMV by ELISA and had dsRNAs typical of a tymovirus. An amplicon of the appropriate size (960 bp) for NeRNV was obtained from each of five samples. Using ScrMV specific primers, the same samples failed to amplify the expected product. We have found NeRNV in three Diascia spp., one Verbena sp., and one Nemesia sp. plants in two counties in California (Riverside and San Diego). When the RT-PCR products were sequenced, they all had 99% sequence identities to NeRNV with 4 to 7 single nucleotide changes (GenBank Accession Nos. DQ648150 to DQ648154). Notably, each of the five amplicons had changes at nucleotides 5134 (G to C) and 5549 (G to T) when compared with the European isolates of NeRNV, which did not result in any amino acid changes. To our knowledge, this is the first report of NeRNV in North America and more specifically, in California. References: (1) R. Koenig et al. J. Gen. Virol. 86:1827, 2005. (2) A. L. Skelton et al. Plant Pathol. 53:798, 2004.

Naturally Occurring Variants of Satellite Tobacco Mosaic Virus

ABSTRACT Four natural variants of satellite tobacco mosaic virus (STMV) were compared with each other and with the type strain. Differences were detected in double-stranded RNA, single-stranded RNA, and virion electrophoretic mobility patterns, while the size and antigenicity of the coat protein were similar for all. RNase protection assays detected differences in the genomes of each of the four new variants, which differed not only from each other, but also from that of type STMV. Infectious RNA transcripts were made from complementary DNA clones of one variant (STMV 10) with a genome apparently smaller than that of type STMV. A 71-base deletion in the region that contains the 6.8-kDa protein in type STMV was detected by sequence analysis of the STMV 10 clones, a result that is confirmed by the lack of a 6.8-kDa in vitro translation product for STMV 10. Only minor sequence differences exist elsewhere in the genome compared with that of type STMV. Type STMV and STMV 10 each successfully cross-protected against the other when tobacco plants were inoculated 10 days apart.