An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration.

A method for producing large numbers of transgenic wheat plants has been developed. With this approach, an average of 9.7% of immature embryo explants were transformed and generated multiple self-fertile, independently transformed plants. No untransformed plants, or escapes, were regenerated. This transformation procedure uses morphogenic calli derived from scutellum tissue of immature embryos of Triticum aestivum cv. Bobwhite co-bombarded with separate plasmids carrying a selectable marker gene (bar) and a gene of interest, respectively. Transformed wheat calli with a vigorous growth phenotype were obtained by extended culture on media containing 5.0 mg/l bialaphos. These calli retained morphogenic potential and were competent for plant regeneration for as long as 11 months. The bar gene and the gene of interest were co-expressed in T0 progeny plants. This wheat transformation protocol may facilitate quantitative production of multiple transgenic plants and significantly reduce the cost and labor otherwise required for screening out untransformed escapes.

Cylindrical inclusion bodies of wheat streak mosaic virus and three other potyviruses only self-assemble in mixed infections.

Potyviruses produce cylindrical inclusions (CIs) in the cytoplasm of infected cells. Immunogold labelling and electron microscopy of embedded and sectioned wheat and maize cells doubly infected by different potyviruses revealed no mixing of inclusion proteins in CIs. The viruses were wheat streak mosaic virus (WSMV), agropyron mosaic virus and hordeum mosaic virus, in wheat, and WSMV and maize dwarf mosaic virus in maize. The three viruses in wheat were indistinguishable morphologically and in ultrastructural features but can be separated by serology and host range. The absence of phenotypic mixing in CIs showed that in the presence of CI proteins of other potyviruses, assembly was either highly virus-specific, or that no opportunity existed for CI proteins to assemble into hybrid CIs in mixed infections.

Structural proteins of three viruses in the Potyviridae adhere only to their homologous cylindrical inclusions in mixed infections.

Immunoelectron microscopy of serial sections of leaf cells of wheat doubly infected by wheat streak mosaic virus and Agropyron mosaic virus or wheat spindle streak mosaic virus showed that structural proteins or virions associated only with homologous cylindrical inclusions (CI) or with self in aggregates. CI protein of WSMV was seen firmly attached to, and extended through, a plasmodesma. Based on CI protein similarities with transport protein families, membrane-associated secretion proteins, other transport proteins in the GTPase superfamily, and the here reported findings, it is suggested that CI proteins could serve as cell-to-cell movement proteins in the Potyviridae.

Immunohistochemical localization of barley stripe mosaic virions in infected wheat cells.

Barley stripe mosaic virus particles were localized in ultrathin sections with colloidal gold-labeled specific IgG or antiserum followed by gold-labeled goat anti-rabbit IgG. On the average, 1.5 gold particles were attached per virus rod. A statistical analysis of counts of gold and virus particles showed that the staining procedure was highly reproducible from experiment to experiment and after several independently prepared colloidal gold solutions. The procedure should be useful for the intracellular localization of any protein to which an antibody can be prepared.

Peripheral vesicles in proplastids of barley stripe mosaic virus-infected wheat cells contain double-stranded RNA.

Cytochemical and immunoelectron microscopy localized dsRNA in root tips systemically infected with barley stripe mosaic virus (BSMV). Anti-polyinosinic:polycytidylic acid (Anti-poly(I):poly(C)) serum was used as a primary stain followed by gold-labeled goat anti-rabbit IgG complexes. Only vesicles of vesiculated proplastids of early-infected cells but not proplastids of cells from noninoculated plants, contained dsRNA. This supports the notion of a role for proplastids in BSMV replication.

Immunocytology shows the presence of tobacco etch virus P3 protein in nuclear inclusions.

Intracellular localization studies of various potyvirus proteins have been made in hope of finding clues to their function(s). Immunocytological studies localized many of the tobacco etch virus (TEV)-encoded proteins in infected cells. We used antiserum against the nonstructural P3 protein of TEV to determine the subcellular location of the P3 protein in ultrathin sections of virus-infected cells. Immunogold labeling with the antiserum showed labels associated with nucleoli, nuclei, or NIs, Absorption of antiserum with purified NIs or P3 protein resulted in no labeling. TEV NIs are known to contain a bifunctional genome-linked protein-viral proteinase (NIa-VPg) and RNA-dependent RNA polymerase (NIb). It appeared that the TEV P3 protein was a third nonstructural viral protein of NIs of TEV if the NIa-VPg is considered one protein. The presence of P3 in NIs was also supported by Western blot assays. P3 protein in the nucleolus and nucleus could indicate that it, too, is involved in early stages of viral replication.

Chromic-acid formaldehyde fixation of nucleic acids of bacteriophage phi6 and infectious bovine rhinotracheitis virus.

Bacteriophage phi6 nucleic acid was present as a torus after chromic acid-formaldehyde-OSO4 fixation and acetone and propylene oxide dehydration. A herpes virus, infectious bovine rhinotracheitis virus, had its DNA mostly as a torus, collapsed in the centre, or as a network, after glutaraldehyde-OSO4 fixation, but in an uncollapsed torus or network formation after chromic acid-formaldehyde-OSO4. This fixative stabilized nucleic acids, allowing acetone dehydration and plastic embedding without collapse of nucleic acid to the centre of the virion.

Accumulation of barley stripe mosaic virus is significantly reduced in transgenic wheat plants expressing a bacterial ribonuclease.

An rnc70 gene encoding a mutant bacterial ribonuclease III (RNase III) was introduced into wheat (Triticum aestivum cv. Bobwhite) by microprojectile bombardment. T1, T2, and T3 plants regenerated from three transgenic callus lines were challenged with barley stripe mosaic virus. Plants expressing RNase III exhibited a high level of resistance to the virus infection. This resistance was evidenced by the absence of virus symptoms and reduced accumulation of virions in these plants. The result demonstrates that this pathogen-targeted resistance strategy can be effectively employed in conferring resistance to viral diseases of cereal crops.

Molecular cloning and sequencing of the coat protein gene of a Nebraskan isolate of tobacco necrosis virus: the deduced coat protein sequence has only moderate homology with those of strain A and strain D.

A partial nucleotide sequence spanning the coat protein (CP) gene of a Nebraskan isolate of tobacco necrosis virus (TNV-NE) has been determined. The sequence contains at least four open reading frames (ORFs). The 5'-terminal ORF encodes a protein that has 86% and 38% homology with the polymerases of strains A (TNV-A) and D (TNV-D), respectively. The second and third ORFs probably encode 10.7 kDa and 6.2 kDa proteins (p 10.7 and p 6.2). These are respectively 90% and 96% amino acid homologous encoded by similar ORFs in TNV-A but only 26% and 20% homologous with those in TNV-D. The fourth 3'-proximal ORF encodes the 30.3 kDa CP. The amino acid sequence of TNV-NE CP is only 51% and 44% homologous to those of TNV-A and TNV-D, respectively. Thus, the CP genes of TNV-NE, TNV-A, and TNV-D are quite different. Like the sequences to the 5' side of the CP gene, that of TNV-NE is more closely related to TNV-A than to TNV-D.

Ultrastructure of bacteriophage phi 6: arrangement of the double-stranded RNA and envelope.

Pseudomonas phaseolicola cells synchronously infected with phi 6 were fixed and embedded by several procedures. The diameter of phi 6 measured 75 nm and its nucleocapsid 60 nm. Virus nucleocapsids were icosahedral and surrounded by a double membrane. In planar sections the nucleic acid appeared as a hexagonal ring and in cross section as two angular structures.

A mammalian 2-5A system functions as an antiviral pathway in transgenic plants.

Resistance to virus infections in higher vertebrates is mediated in part through catalysis of RNA decay by the, interferon-regulated 2-5A system. A functional 2-5A system requires two enzymes, a 2-5A synthetase that produces 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) in response to double-stranded RNA, and the 2-5A-dependent RNase L. We have coexpressed these human enzymes in transgenic tobacco plants by using a single plasmid containing the cDNAs for both human RNase L and a low molecular weight form of human 2-5A synthetase under control of different, constitutive promoters. Expression of the human cDNAs in the transgenic plants was demonstrated from Northern blots, by specific enzyme assays, and by immunodetection (for RNase L). Infection of leaves, detached or in planta, of the coexpressing transgenic plants by tobacco mosaic virus, alfalfa [correction of alfafa] mosaic virus, or tobacco etch virus resulted in necrotic lesions. In contrast, leaves expressing 2-5A synthetase or RNase L alone and leaves containing the plasmid vector alone produced typical systemic infections. While alfalfa mosaic virus produced lesions only in the inoculated leaves regardless of the concentration of virus in the inoculum, high, but not low, levels of tobacco etch virus inoculum resulted in escape of virus to uninoculated leaves. Nevertheless, there was a substantial reduction of tobacco etch virus yield as measured by ELISA assay in the coexpressing transgenic plants. These results indicate that expression of a mammalian 2-5A system in plants provides resistance to virus infections.