Degradation of tobacco mosaic virus movement protein by the 26S proteasome

Cell-to-cell spread of tobacco mosaic virus is facilitated by the virus-encoded 30-kDa movement protein (MP). This process involves interaction of viral proteins with host components, including the cytoskeleton and the endoplasmic reticulum (ER). During virus infection, high-molecular-weight forms of MP were detected in tobacco BY-2 protoplasts. Inhibition of the 26S proteasome by MG115 and clasto-lactacystin-beta-lactone enhanced the accumulation of high-molecular-weight forms of MP and led to increased stability of the MP. Such treatment also increased the apparent accumulation of polyubiquitinated host proteins. By fusion of MP with the jellyfish green fluorescent protein (GFP), we demonstrated that inhibition of the 26S proteasome led to accumulation of the MP-GFP fusion preferentially on the ER, particularly the perinuclear ER. We suggest that polyubiquitination of MP and subsequent degradation by the 26S proteasome may play a substantial role in regulation of virus spread by reducing the damage caused by the MP on the structure of cortical ER.     

Regulation by ABA of beta-Conglycinin Expression in Cultured Developing Soybean Cotyledons

The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the β-subunit of β-conglycinin, one of the major storage proteins of soybean seeds. The amount of the α′-and α-subunits of β-conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the β-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and β-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against β-conglycinin, there was an increased amount of pre-β-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-β-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of β-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the β-subunit of β-conglycinin in developing cultured soybean cotyledons.     

Developmental Regulation of beta-Conglycinin in Soybean Axes and Cotyledons

Analysis of the expression of genes encoding the β-conglycinin seed storage proteins in soybean has been used to extend our understanding of developmental gene expression in plants. The α, α′, and β subunits of β-conglycinin are encoded by a multigene family which is organ-specific in its expression. In this study we report the differentially programmed accumulation of the α, α′, and β subunits of β-conglycinin. Multiple isomeric forms of each subunit are present in the dry seed, but the timing of their accumulation is unique for each subunit. The previously reported variation in amount of α′ and α subunits in axis and cotyledons is also reflected in the amount of subunit specific mRNA which is present in each tissue. The β subunit, previously undetected in soybean axes, is found to be synthesized but rapidly degraded. These differences in β-conglycinin protein accumulation may be reflected by the morphological differences observed in protein bodies between these two tissues.     

Expression of a soybean β-conclycinin gene under the control of the Cauliflower Mosaic Virus 35S and 19S promoters in transformed petunia tissues

A gene encoding the -subunit of -conglycinin was ligated to the 19S and 35S promoters of Cauliflower Mosaic Virus and introduced into petunia plants on a disarmed Ti-plasmid using Agrobacterium tumefaciens. Transformed cells were regenerated into whole plants and ummunoreactive polypeptides and hybridizable, polyadenylated mRNA were detected in transformed tissues. Expression from the 35S promoter was 10 to 50 times greater than expression from the 19S promoter. The level of immunodetectable polypeptides was greater in seeds than in leaves or callus tissue. In addition, the pattern of -polypeptide breakdown products was distinctive in seeds and leaves. We conclude that in seeds the higher levels of the -polypeptide reflect enhanced stability of this protein.     

Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants

A chimeric gene encoding the alfalfa mosaic virus (AlMV) coat protein was constructed and introduced into tobacco and tomato plants using Ti plasmid-derived plant transformation vectors. The progeny of the self-fertilized transgenic plants were significantly delayed in symptom development and in some cases completely escaped infection after inoculated with AlMV. The inoculated leaves of the transgenic plants had significantly reduced numbers of lesions and accumulated substantially lower amounts of coat protein due to virus replication than the control plants. These results show that high level expression of the chimeric viral coat protein gene confers protection against AlMV, which differs from other plant viruses in morphology, genome structure, gene expression strategy and early steps in viral replication. Based on our results with AlMV and those reported earlier for tobacco mosaic virus, it appears that genetically engineered cross-protection may be a general method for preventing viral disease in plants.