Here we report on a simple and reproducible system of Agrobacterium-mediated transient gene expression assay that utilizes infiltration of young Nicotiana benthamiana leaves. Although some of the phenomena described in this paper have been already reported by other researchers, here we have further developed them. The highest level of transient gfp gene expression was detected in the youngest leaves of N. benthamiana infiltrated with A. tumefaciens strains AGL0 and EHA105 precultured in the presence of 450-600 microM acetosyringone. Although the maximum level of transient gfp gene expression was restricted presumably by RNA silencing, it was completely suppressed in the presence of the viral protein HC-Pro. The transient expression system described here can be used to identify new viral suppressors of RNA silencing, for detailed analysis of unidentified genes and for industrial production of proteins in plants as well.
Abstrad: Hippeastrum x hortorum bulbs produce red pigments upon wounding. Analysis by nc, HPLC, MS and NMR revealed that this pigment, which is absent in unwounded tissue, is a mixture composed of an orange-coloured chalcone (3,2',4'-trihydroxy-4-methoxychalcone) and 3 f1avans (7,4'-dihydroxy-8-methylflavan, 7,3'-dihydroxy-4'-methoxyflavan and 7-hydroxy-3'-4'-methylenedioxyflavan). The colourless f1avans can be oxidised to red-coloured dimers or polymers. The induction of these "phytoalexins" is achieved by wounding and can be further amplified by biotic and abiotic elicitors; e.g. cell walls of yeast appeared to be a potent elicitor. Immediately after wounding or elicitation the apparent activities of phenylalanine ammonium lyase (PAL) and peroxidase, which are probably involved in the biosynthesis of the red pigments increase substantially (both enzymes are hardly measurable in unwounded tissue) and reach a transient maximum after 2-3d whereas pigment formation becomes visible after 2 d and reaches a maximum after 9 to 12 d. Since cycloheximide inhibits the formation of the phytoalexins, a de novo synthesis of the corresponding enzymes is likely. Total phytoalexin extracts showed an inhibition of bacterial growth (e.g. of Bacillus subtilis, B. megaterium) and of feeding by polyphagous larvae of the moth Syntomis mogadorensis. These data imply that the wounding-and elicitor-induced phytoalexins appear to function as defence compounds against microorganisms and herbivores.
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