Subcellular dynamics and role of Arabidopsis ß-1,3-glucanases in cell-to-cell movement of tobamoviruses.

ß-1,3-Glucanases (BG) have been implicated in enhancing virus spread by degrading callose at plasmodesmata (Pd). Here, we investigate the role of Arabidopsis BG in tobamovirus spread. During Turnip vein clearing virus infection, the transcription of two pathogenesis-related (PR)-BG AtBG2 and AtBG3 increased but that of Pd-associated BG AtBG_pap did not change. In transgenic plants, AtBG2 was retained in the endoplasmic reticulum (ER) network and was not secreted. As a stress response mediated by salicylic acid, AtBG2 was secreted and appeared as a free extracellular protein localized in the entire apoplast but did not accumulate at Pd sites. At the leading edge of Tobacco mosaic virus spread, AtBG2 co-localized with the viral movement protein in the ER-derived bodies, similarly to other ER proteins, but was not secreted to the cell wall. In atbg2 mutants, callose levels at Pd and virus spread were unaffected. Likewise, AtBG2 overexpression had no effect on virus spread. However, in atbg_pap mutants, callose at Pd was increased and virus spread was reduced. Our results demonstrate that the constitutive Pd-associated BG but not the stress-regulated extracellular PR-BG are directly involved in regulation of callose at Pd and cell-to-cell transport in Arabidopsis, including the spread of viruses.

The constitutive expression of Arabidopsis plasmodesmal-associated class 1 reversibly glycosylated polypeptide impairs plant development and virus spread.

Arabidopsis class 1 reversibly glycosylated polypeptides (C1RGPs) were shown to be plasmodesmal-associated proteins. Transgenic tobacco (Nicotiana tabacum) plants constitutively expressing GFP tagged AtRGP2 under the control of the CaMV 35S promoter are stunted, have a rosette-like growth pattern, and in source leaves exhibit strong chlorosis, increased photoassimilate retention and starch accumulation that results in elevated leaf specific fresh and dry weights. Basal callose levels around plasmodesmata (Pd) of leaf epidermal cells in transgenic plants are higher than in WT. Such a phenotype is characteristic of virus-infected plants and some transgenic plants expressing Pd-associated viral movement proteins (MP). The local spread of Tobacco mosaic virus (TMV) is inhibited in AtRGP2:GFP transgenics compared to WT. Taken together these observations suggest that overexpression of the AtRGP2:GFP leads to a reduction in Pd permeability to photoassimilate, thus lowering the normal rate of translocation from source leaves to sink organs. Such a reduction may also inhibit the local cell-to-cell spread of viruses in transgenic plants. The observed reduction in Pd permeability could be due to a partial Pd occlusion caused either by the accumulation of AtRGP2:GFP fusion in Pd, and/or by constriction of Pd by the excessive callose accumulation.

Physical control of leafhoppers.

In 2000, a severe outbreak of phytoplasma-caused disease in Limonium spp. flowers devastated the industry in Israel; insecticides were not able to knock down and kill leafhopper vectors before they could transmit the pathogen. Nonchoice laboratory studies were conducted to determine the effect of UV-absorbing plastics on the movement of leafhoppers toward light; UV-absorbing plastic significantly reduced leafhopper movement. In choice trials conducted in sunlight, significantly more leafhoppers moved into the cage covered with regular plastic as opposed to the cage covered with UV-absorbing plastic. Field studies were conducted to determine at what height leafhoppers enter 2.5-3-m high walk-in tunnels; the majority enter the tunnels low to the ground, up to 1 m. Finally, field studies were conduced to compare leafhopper population levels in walk-in tunnels covered with UV-absorbing plastic or screening, and with ventilation holes at different heights above the ground. Elevated ventilation holes and UV-absorbing tunnel covering significantly reduced Orosius orientalis entrance into tunnels. Ramifications of these finding for leafhopper control are discussed.

Biological and molecular characterization of a novel carmovirus isolated from angelonia.

ABSTRACT A new carmovirus was isolated from Angelonia plants (Angelonia angustifolia), with flower break and mild foliar symptoms, grown in the United States and Israel. The virus, for which the name Angelonia flower break virus (AnFBV) is proposed, has isometric particles, approximately 30 nm in diameter. The experimental host range was limited to Nicotiana species, Schizanthus pinnatus, Myosotis sylvatica, Phlox drummondii, and Digitalis purpurea. Virions were isolated from systemically infected N. benthamiana leaves, and directly from naturally infected Angelonia leaves, using typical carmovirus protocols. Koch's postulates were completed by mechanical inoculation of uninfected Angelonia seedlings with purified virions. Isometric particles were observed in leaf dips and virion preparations from both Angelonia and N. benthamiana, and in thin sections of Angelonia flower tissue by electron microscopy. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dissociated purified virus preparations, a major protein component with a molecular mass of 38 kDa was observed. Virion preparations were used to produce virus-specific polyclonal antisera in both Israel and the United States. The antisera did not react with Pelargonium flower break virus (PFBV), Carnation mottle virus (CarMV), or Saguaro cactus virus (SgCV) by either enzyme-linked immunosorbent assay or immunoblotting. In reciprocal tests, antisera against PFBV, CarMV, and SgCV reacted only with the homologous viruses. The complete nucleotide sequence of a Florida isolate of AnFBV and the coat protein (CP) gene sequences of Israeli and Maryland isolates were determined. The genomic RNA is 3,964 nucleotides and contains four open reading frames arranged in a manner typical of carmoviruses. The AnFBV CP is most closely related to PFBV, whereas the AnFBV replicase is most closely related to PFBV, CarMV, and SgCV. Particle morphology, serological properties, genome organization, and phylogenetic analysis are all consistent with assignment of AnFBV to the genus Carmovirus.

Transgenic Gladiolus plants transformed with the bean yellow mosaic virus coat-protein gene in either sense or antisense orientation.

Transgenic Gladiolus plants transformed with the bean yellow mosaic virus (BYMV) coat-protein (CP) gene in either sense or antisense (AS) orientation were developed using biolistics. Four of the plants were confirmed to carry the CP gene in the sense orientation of the gene and seven plants in the AS orientation. Two of the CP plant lines and all of the AS lines showed DNA rearrangements of the transgene in addition to an intact copy of the transgene. The copy number ranged from one to nine. Of the 11 lines, eight had only one to four copies of the transgene. Transcription of the transgene occurred for three of the CP lines and five of the AS lines as determined by Northern hybridization. All 11 plant lines were challenged with BYMV using controlled aphid transmission. One month following aphid transmission, the transgenic plants were examined by immunoelectron microscopy for presence of the virus. Several transgenic plant lines containing either antiviral transgene showed a lower incidence of infection (percentage of plants infected as detected by immunoelectron microscopy) than the non-transformed plants. Most of the CP- and AS-transgenic plants that did not contain BYMV 1 month after challenge were found to contain BYMV the next season. It appeared that BYMV infection was delayed in the CP- and AS-transgenic lines but that the transgenes did not prevent eventual infection of BYMV. This is the first report of developing a floral bulb crop with antiviral genes to BYMV.

Molecular evolution of Turnip mosaic virus: evidence of host adaptation, genetic recombination and geographical spread.

Turnip mosaic virus (TuMV), a species of the genus Potyvirus, occurs worldwide. Seventy-six isolates of TuMV were collected from around the world, mostly from Brassica and Raphanus crops, but also from several non-brassica species. Host tests grouped the isolates into one or other of two pathotypes; Brassica (B) and Brassica-Raphanus (BR). The nucleotide sequences of the first protein (P1) and coat protein (CP) genes of the isolates were determined. One-tenth of the isolates were found to have anomalous and variable phylogenetic relationships as a result of recombination. The 5'-terminal 300 nt of the P1 gene of many isolates was also variable and phylogenetically anomalous, whereas the 380 nt 3' terminus of the CP gene was mostly conserved. Trees calculated from the remaining informative parts of the two genes of the non-recombinant sequences by neighbour-joining, maximum-likelihood and maximum-parsimony methods were closely similar, and so these parts of the sequences were concatenated and trees calculated from the resulting 1150 nt. The isolates fell into four consistent groups; only the relationships of these groups with one another and with the outgroup differed. The "basal-B" cluster of eight B-pathotype isolates was most variable, was not monophyletic, and came from both brassicas and non-brassicas from southwest and central Eurasia. Closest to it, and forming a monophyletic subgroup of it in most trees, and similarly variable, was the "basal-BR" group of eight BR pathotype Eurasian isolates. The third and least variable group, the "Asian-BR" group, was of 22 BR-pathotype isolates, all from brassicas, mostly Raphanus, and all from east Asia mostly Japan. The fourth group of 36 isolates, the "world-B" group, was from all continents, most were isolated from brassicas and most were of the B-pathotype. The simplest of several possible interpretations of the trees is that TuMV originated, like its brassica hosts, in Europe and spread to the other parts of the world, and that the BR pathotype has recently evolved in east Asia.