Overexpression of arginase in Arabidopsis thaliana influences defence responses against Botrytis cinerea

Arabidopsis possesses two arginase-encoding genes, ARGAH1 and ARGAH2, catalysing the catabolism of arginine into ornithine and urea. Arginine and ornithine are both precursors for polyamine biosynthetic pathways. We observed an accumulation of ARGAH2 mRNA in Arabidopsis upon inoculation with the necrotrophic pathogen Botrytis cinerea. Transgenic lines displaying either overexpression of ARGAH2 or simultaneous silencing of both Arabidopsis arginase-encoding genes were created and their resistance to B. cinerea infection evaluated. Overexpression of arginase resulted in changes in amino acid accumulation, while polyamine levels remained largely unaffected. Silencing lines were affected in both amino acid and putrescine accumulation. Arabidopsis plants overexpressing the arginase gene were less susceptible to B. cinerea, whereas silencing lines remained as susceptible as the wild type. We discuss how arginase might interact with plant defence mechanisms. These results provide new insights into amino acid metabolic changes under stress

A reproducible genetic transformation system for cultivated Phaseolus acutifolius (tepary bean) and its use to assess the role of arcelins in resistance to the Mexican bean weevil

A reproducible Agrobacterium tumefaciens-mediated genetic transformation method that delivers fertile and morphologically normal transgenic plants was developed for cultivated tepary bean (Phaseolus acutifolius L. Gray). Factors contributing to higher transformation efficiencies include (1) a low initial concentration of bacteria coupled with a longer cocultivation period with callus, (2) an initial selection of callus on a medium containing low levels of the selectable agent, (3) omission of the selectable agent from the medium during callus differentiation to shoots and (4) the efficient conversion of transgenic shoots into fertile plants. All plants regenerated with this procedure (T0) were stably transformed, and the introduced foreign genes were inherited in a Mendelian fashion in most of the 33 independent transformants. Integration, stable transmission and high expression levels of the transgenes were observed in the T1 and/or T3 progenies of the transgenic lines. The binary transformation vectors contained the ?-glucuronidase reporter gene, the neomycin phosphotransferase II selectable marker gene and either an arcelin 1 or an arcelin 5 gene. Arcelins are seed proteins that are very abundant in some wild P. vulgaris L. genotypes showing resistance to the storage insect Zabrotes subfasciatus (Boheman) (Coleoptera, Bruchidae). Transgenic beans from two different cultivated P. acutifolius genotypes with high arcelin levels were infested with Z. subfasciatus, but they were only marginally less susceptible to infestation than the non-transgenic P. acutifolius. Hence, the arcelin genes tested here are not major determinants of resistance against Z. subfasciatus

Expression of dicistronic transcriptional units in transgenic tobacco.

We investigated whether the two cistrons of a dicistronic mRNA can be translated in plants to yield both gene products. The coding sequences of various reporter genes were combined in dicistronic units, and their expression was analyzed in stably transformed tobacco plants at the RNA and protein levels. The presence of an upstream cistron resulted in all cases in a drastically reduced expression of the downstream cistron. The translational efficiency of the gene located downstream in the dicistronic units was 500- to 1,500-fold lower than that in a monocistronic control; a 500-fold lower value was obtained with a dicistronic unit in which both cistrons were separated by 30 nucleotides, whereas a 1,500-fold lower value was obtained with a dicistronic unit in which the stop codon of the upstream cistron and the start codon of the downstream cistron overlapped. As a strategy to select indirectly for transformants with enhanced levels of expression of a gene which is by itself nonselectable, the gene of interest can be cloned upstream from a selectable marker in a dicistronic configuration. This strategy can be used provided that the amount of dicistronic mRNA is high. If, on the other hand, the expression of the dicistronic unit is too low, selection of the downstream cistron will primarily give clones with rearranged dicistronic units

Fouilles archéologiques de Pessinonte: la campagne de 2001

Devreker John, Devos Geert, Bauters Luc, Braeckman Kurt, De Clercq Wim, Monsieur Patrick, Van Peteghem André. Fouilles archéologiques de Pessinonte : la campagne de 2002. In: Anatolia Antiqua, Tome 12, 2004. pp. 83-95

Agrobacterium-mediated transformation of Phaseolus vulgaris L. using indirect organogenesis

A protocol for genetic transformation of Phaseolus vulgaris L. cv. CIAP7247F via Agrobacterium tumefaciens was established. Primary green nodular calli and proliferative calli were used as target explants. Several factors such as Agrobacterium strain, plasmid, light conditions, bacterial concentration, co-cultivation period and type of callus were studied for optimization of the Agrobacterium-mediated transformation. The highest DNA transfer occurred when proliferative calli were inoculated with strain EHA105 harbouring pCAMBIA3301 plasmid at a density of OD600 = 0.5, and co-cultivated under 16 h light/8 h dark photoperiod for 6 days. The transformation system integrates Agrobacterium-mediated DNA transfer with efficient regeneration via indirect organogenesis, and using the bar gene as selectable marker and glufosinate ammonium (herbicide finale) for callus selection. The proposed system allowed obtaining transgenic lines with Mendelian inheritance of the transgenes, as demonstrated by PCR analyses. These results also validated the effectiveness of a regeneration protocol via indirect organogenesis for regeneration of transformed bean cells. This system constitutes a major initial step for common bean transformation using A. tumefaciens