In plants, the mevalonic acid (MVA) pathway provides precursors for the formation of triterpenes, sesquiterpenes, phytosterols and primary metabolites important for cell integrity. Here, we have cloned the cDNA encoding enzymes catalysing the final three steps of the MVA pathway from Madagascar periwinkle (Catharanthus roseus), mevalonate kinase (MVK), 5-phosphomevalonate kinase (PMK) and mevalonate 5-diphosphate decarboxylase (MVD). These cDNA were shown to functionally complement MVA pathway deletion mutants in the yeast Saccharomyces cerevisiae. Transient transformations of C. roseus cells with yellow fluorescent protein (YFP)-fused constructs reveal that PMK and MVD are localised to the peroxisomes, while MVK was cytosolic. These compartmentalisation results were confirmed using the Arabidopsis thaliana MVK, PMK and MVD sequences fused to YFP. Based on these observations and the arguments raised here we conclude that the final steps of the plant MVA pathway are localised to the peroxisome.

Catharanthus roseus synthesizes a wide range of valuable monoterpene indole alkaloids, some of which have recently been recognized as functioning in plant defence mechanisms. More specifically, in aerial organ epidermal cells, vacuole-accumulated strictosidine displays a dual fate, being either the precursor of all monoterpene indole alkaloids after export from the vacuole, or the substrate for a defence mechanism based on the massive protein cross-linking, which occurs subsequent to organelle membrane disruption during biotic attacks. Such a mechanism relies on a physical separation between the vacuolar strictosidine-synthesizing enzyme and the nucleus-targeted enzyme catalyzing its activation through deglucosylation. In the present study, we carried out the spatial characterization of this mechanism by a cellular and subcellular study of three enzymes catalyzing the synthesis of the two strictosidine precursors (i.e. tryptamine and secologanin). Using RNA in situ hybridization, we demonstrated that loganic acid O-methyltransferase transcript, catalysing the penultimate step of secologanin synthesis, is specifically localized in the epidermis. A combination of green fluorescent protein imaging, bimolecular fluorescence complementation assays and yeast two-hybrid analysis enabled us to establish that both loganic acid O-methyltransferase and the tryptamine-producing enzyme, tryptophan decarboxylase, form homodimers in the cytosol, thereby preventing their passive diffusion to the nucleus. We also showed that the cytochrome P450 secologanin synthase is anchored to the endoplasmic reticulum via a N-teminal helix, thus allowing the production of secologanin on the cytosolic side of the endoplasmic reticulum membrane. Consequently, secologanin and tryptamine must be transported to the vacuole to achieve strictosidine biosynthesis, demonstrating the importance of trans-tonoplast translocation events during these metabolic processes.Abbreviations BiFC, bimolecular fluorescence complementation; CFP, cyan fluorescent protein; ER, endoplasmic reticulum; G10H, geraniol 10-hydroxylase; GFP, green fluorescent protein; GUS, b-glucuronidase; IPAP, internal phloem-associated parenchyma; LAMT, loganic acid O-methyltransferase; -LW, leucine-trytophan lacking medium; -LWH, leucine-trytophan-histidine lacking medium; MEP, 2-C-methyl-D-erythritol 4-phosphate; MIA, monoterpene indole alkaloid(s); pGAD, GAL4 activation domain; pLex, LexA DNA-binding domain; SLS, secologanin synthase; SGD, strictosidine b-D-glucosidase; STR, strictosidine synthase; TDC, tryptophan decarboxylase; YFP, yellow fluorescent protein.

To gain insight into wasp factors that might be involved in the initial induction of galls on woody plants, we performed high throughput (454) transcriptome analysis of ovaries and venom glands of two cynipid gall wasps, Biorhiza pallida and Diplolepis rosae , inducing galls on oak and rose, respectively. De novo assembled and annotated contigs were compared to sequences from phylogenetically related parasitoid wasps. The relative expression levels of contigs were estimated to identify the most expressed gene sequences in each tissue. We identify for the first time a set of maternally expressed gall wasp proteins potentially involved in the interaction with the plant. Some genes highly expressed in venom glands and ovaries may act to suppress early plant defense signaling. We also identify gall wasp cellulases that could be involved in observed local lysis of plant tissue following oviposition, and which may have been acquired from bacteria by horizontal gene transfer. We find no evidence of virus-related gene expression, in contrast to many non-cynipid parasitoid wasps. By exploring gall wasp effectors, this study is a first step toward understanding the molecular mechanisms underlying cynipid gall induction in woody plants, and the recent sequencing of oak and rose genomes will enable study of plant responses to these factors.

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