Expression of MsLEC1- and MsLEC2-antisense genes in alfalfa plant lines causes severe embryogenic, developmental and reproductive abnormalities.

Although it has been proposed that plant lectins play a number of roles, the function of these proteins in normal plant growth and development has been unclear. To analyze the functions of putative alfalfa lectin genes, lines of transgenic alfalfa plants expressing approximately half of the open reading frame of MsLEC1 or MsLEC2, in the antisense or sense orientation, were established and analyzed. The antisense plants displayed severe abnormalities in embryogenesis, and both vegetative and reproductive development were perturbed. Some differences were observed between MsLEC1- and MsLEC2-antisense plants, and abnormalities were especially severe during the early stages of development in both the primary and secondary transgenic generations. In contrast, vector-control and sense-transgene plants exhibited normal growth and development. MsLEC1 and MsLEC2 mRNA accumulation levels were reduced in cognate antisense plants, especially during the later stages of embryogenesis, but also tended to be low in MsLEC1 sense-transgene plants. However, correlated with the phenotypic abnormalities observed in the MsLEC1-antisense plants was the specific reduction in the accumulation of a candidate MsLEC1 protein. Our results suggest that the MsLEC1 and MsLEC2 gene products, in addition to being important for embryogenesis, are required throughout alfalfa development.

Recombinant tobacco mosaic virus movement protein is an RNA-binding, alpha-helical membrane protein.

The 30-kDa movement protein (MP) is essential for cell-cell spread of tobacco mosaic virus in planta. To explore the structural properties of MP, the full-length recombinant MP gene was expressed in Escherichia coli, and one-step purification from solubilized inclusion bodies was accomplished by using anion exchange chromatography. Soluble MP was maintained at >4 mg/ml without aggregation and displayed approximately 70% alpha-helical conformation in the presence of urea and SDS. A trypsin-resistant core domain of the MP had tightly folded tertiary structure, whereas 18 aa at the C terminus of the monomer were rapidly removed by trypsin. Two hydrophobic regions within the core were highly resistant to proteolysis. Based on results of CD spectroscopy, trypsin treatment, and MS, we propose a topological model in which MP has two putative alpha-helical transmembrane domains and a protease-sensitive carboxyl terminus.