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B G McLean

Publications and source records attributed to B G McLean.

13 recordsLinked to original sources
PubMed1997

TOUSLED is a nuclear serine/threonine protein kinase that requires a coiled-coil region for oligomerization and catalytic activity.

The TOUSLED (TSL) gene is essential for the proper morphogenesis of leaves and flowers in Arabidopsis thaliana. Protein sequence analysis predicts TSL is composed of a carboxyl-terminal protein kinase catalytic domain and a large amino-terminal regulatory domain. TSL fusion proteins, expressed in and purified from yeast, were used to demonstrate TSL protein kinase activity in vitro. TSL trans-autophosphorylates on serine and threonine residues, and phosphorylates exogenous substrates. Using the yeast two-hybrid system, TSL was found to oligomerize via its NH2-terminal domain. A deletion series indicates that a region containing two alpha-helical segments predicted to participate in a coiled-coil structure is essential for oligomerization. TSL localizes to the nucleus in plant cells through an essential NH2-terminal nuclear localization signal; however, this signal is not necessary for protein kinase activity. Finally, deletion mutants demonstrate a strict correlation between catalytic activity and the ability to oligomerize, arguing that activation of the protein kinase requires interaction between TSL molecules.

Amino Acid Sequence↗
PubMed1995

Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells.

Tobacco mosaic virus movement protein P30 complexes with genomic viral RNA for transport through plasmodesmata, the plant intercellular connections. Although most research with P30 focuses on its targeting to and gating of plasmodesmata, the mechanisms of P30 intracellular movement to plasmodesmata have not been defined. To examine P30 intracellular localization, we used tobacco protoplasts, which lack plasmodesmata, for transfection with plasmids carrying P30 coding sequences under a constitutive promoter and for infection with tobacco mosaic virus particles. In both systems, P30 appears as filaments that colocalize primarily with microtubules. To a lesser extent, P30 filaments colocalize with actin filaments, and in vitro experiments suggested that P30 can bind directly to actin and tubulin. This association of P30 with cytoskeletal elements may play a critical role in intracellular transport of the P30-viral RNA complex through the cytoplasm to and possibly through plasmodesmata.

Actins↗
PubMed1994

Mutants of Agrobacterium VirA that activate vir gene expression in the absence of the inducer acetosyringone.

In the presence of inducer molecules produced by wounded plants, the VirA/VirG two-component positive regulatory system of Agrobacterium tumefaciens initiates transcription of virulence genes required for crown gall tumor formation. Exactly how this system enables the bacterium to respond to an environmental signal is not known, but phosphorylation of VirA and VirG plays a role. To analyze further the function of VirA, we chemically mutagenized the virA gene. Two mutants that activate vir transcription without the plant inducer acetosyringone were found; these mutants alter VirA function by distinct mechanisms. One mutant functions entirely independently of acetosyringone, whereas the activity of the second mutant is enhanced by acetosyringone. Both mutants function best at acid pH, but respond differently to specific monosaccharides that stimulate induction by wild-type VirA. Both mutant phenotypes are dominant over wild-type VirA, and both need the conserved histidine at the autophosphorylation site for strong inducer-independent vir transcription.

Acetophenones↗
PubMed1993

Cell-to-cell movement of plant viruses.

To establish an infection, most plant viruses move from cell to cell in the plant. Virus-encoded movement proteins mediate this process and appear to use two mechanisms for transport. Both mechanisms involve interaction with and potential modification of plant intercellular connections, the plasmodesmata. Thus, although viral movement proteins are a diverse group, they share an ability to interact with specific plant components.

Movement↗
PubMed1993

Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall-associated protein kinase.

In host plants, cell-to-cell spread of tobacco mosaic virus (TMV) presumably occurs through intercellular connections, the plasmodesmata. TMV movement is mediated by a specific virus-encoded single-strand nucleic acid-binding protein, P30. The mechanism by which P30 operates is largely unknown. Here, we demonstrate that P30 expressed in transgenic plants is a phosphoprotein. We have developed an assay for in vitro phosphorylation of purified P30 by plant cell wall fractions and have localized the phosphorylation sites to amino acid residues Ser-258, Thr-261, and Ser-265. Interestingly, the P30 phosphorylation sites do not correspond to any known consensus phosphorylation sites for protein kinases. While P30 binding to single-stranded DNA (ssDNA) was shown to involve Thr-261, phosphorylation of this residue does not appear to play a role in binding activity. The protein kinase activity contained in the cell wall fractions was developmentally regulated, expressed predominantly in leaves. Within a leaf, this protein kinase activity increased with leaf maturation and correlated with the reported development of secondary plasmodesmata, sites of P30 accumulation. We suggest that phosphorylation may represent a mechanism for the host plant to sequester P30 following its localization to cell walls.

Amino Acid Sequence↗
PubMed1990

Plants contain highly divergent actin isovariants.

Actin protein isovariants have been identified in animals with distinct cytoplasmic or muscle specific patterns of expression. Analysis of vascular plant actin gene sequences suggests that an even greater diversity should exist within the plant actin protein families, but previous studies on plant proteins have not demonstrated the presence of multiple actin isovariants. Antibodies recognizing a conserved amino-terminal plant actin peptide, a family of plant actin peptides from a variable region, and two monoclonal antibodies to conserved epitopes within animal actins were used to identify isovariants of soybean actin resolved by two-dimensional isoelectric focusing (IEF) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Approximately six to eight actin isovariants with pI values ranging from 5.1 to 5.8 have been identified from soybean hypocotyls, stems, leaves, and roots with varying amounts of most isovariants present in all four organs. Acidic isovariants were present in much higher levels in leaves and stems. Antisera with lambda-class actin specificity detected a subset of three isovariants in all organs examined. One monoclonal and one antipeptide antisera are shown to react well with a wide variety of plant actin isovariants. Similar patterns of actin isovariants were detected in the distant angiosperms, Arabidopsis, petunia, and maize. It is likely that many of these diverse classes of isovariants have been preserved throughout vascular plant evolution and reflect the ancient diversity within plant actin gene families. The extreme difference among isovariants implies the presence of a complex actin-based cytoskeletal system in plants.

Actins↗
PubMed1990

Characterization of the expression of the petunia glycine-rich protein-1 gene product.

We have examined the expression of the petunia (Petunia hybrida) glycine-rich protein-1 (ptGRP1) gene product using an antibody raised against a synthetic peptide comprising amino acids 22 through 36 of the mature ptGRP1 protein. This antibody recognizes a single protein of 23 kilodaltons. Cell fractionation studies showed that, as predicted (CM Condit, RB Meagher [1986] Nature 323: 178-181), ptGRP1 is most likely localized in the cell wall. In addition, it was found that (extractable) ptGRP1 is present in much higher abundance in unexpanded than in fully expanded tissue, with highest levels of accumulation in the bud. This same developmentally regulated pattern of protein expression was found in all varieties of petunia tested. In addition, tissue blots of petunia stem sections showed that ptGRP1 is localized to within the vascular tissue (to at least the phloem or cambium) and to either the epidermal cells or to a layer of collenchyma cells directly below the epidermis. Localization of ptGRP1 antigen in these cell types is shown to occur at different times in the overall development of the plant and at different quantitative levels.

Journal Article↗
PubMed1990

Tissue-specific expression of divergent actins in soybean root.

It has been proposed that the evolution of distinct classes of genes encoding the kappa-, lambda-, and mu-actins in soybean is the result of an ancient divergence in patterns of actin gene expression. In this study, antisera against a family of synthetic actin peptides from a divergent region within the predicted actin polypeptide sequences have been used to explore the differential expression of plant actins. Antiserum elicited against a 16-residue synthetic lambda-actin peptide SAc4:257 reacted with a 46-kilodalton protein in soybean extracts, showed specificity for the lambda-peptide over the divergent kappa- and mu-actin peptides in enzyme-linked immunosorbent assays, and reacted strongly and preferentially with root protoderm in apical roots and in lateral root primordia. Antiserum elicited against the synthetic kappa-actin peptide SAc1:257 reacted with 46-kilodalton protein on protein gel blots, showed partial specificity toward the immunogenic kappa-peptide over the divergent lambda- and mu-peptides, and reacted strongly with all root tissues with the exception of root cap. These data support the hypothesis that ancient classes of plant actin genes may have been preserved because of their role in developmentally controlled differences in tissue-specific actin expression and/or function. The possibility that other diverse actin classes have unique patterns of regulation is discussed.

Actins↗