control non inoculated … TO THE MOLECULAR DIALOG ELUCIDATION A. Renier1*, S. Rapior2, B. Dreyfus1 and P. Jourand1 METHYLOTROPHY: 1Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), UMR 113 AgroM/CIRAD/INRA/IRD/UMII, TA 10/J, Campus International de Baillarguet, 34398 Montpellier cedex 5, France. 2Laboratoire de Botanique, Phytochimie et Mycologie, UMR 5175 CEFE, Faculté de Pharmacie, 15 Avenue Charles Flahault, BP 14491, 34093 Montpellier cedex 5, France. * adeline.renier@mpl.ird.fr FROM A GREAT ASSET IN SYMBIOSIS PROCESS … The bacterial genus Methylobacterium belongs to the -proteobacteria. The main feature of members of this genus is their ability to oxidize methanol, a methylotrophic property based on the presence of methanol dehydrogenase (MDH). Recently, strains isolated from three Crotalaria spp. were described as being a single novel methylotrophic Methylobacterium spp. This new species was named Methylobacterium nodulans for its ability to both nodulate and fix nitrogen specifically during symbiosis with Crotalaria spp. Methylotrophy of M. nodulans is expressed in root nodules formed during symbiosis with C. podocarpa Loss of methylotrophy strongly affects symbiosis The mxaF gene was expressed in apex root nodules suggesting that methanol is present inside the nodule and subsequently used by the bacteria. Mutant methylotroph - control non inoculated ORS 2060T wild type When compared to the wild type strain, inoculation of M. nodulans nonmethylotroph mutants in C. podocarpa decreased: Histochemical localization of the -galactosidase activity in 6-week-old C. podocarpa root nodules. the total root nodule number per plant up to 60%, the whole-plant nitrogen fixation capacity up to 42%, Methylotrophy appears to be essential in symbiosis between the bacteria M. nodulans and its host legume C. podocarpa. This function might be implicated at different levels of symbiosis. the total dry plant biomass up to 46%, During a first step of infection, methylotrophy could be involved in the bacterial ability to colonize or invade plant roots by detoxication of toxic methylated compounds produced by plant. Comparison of growth (5 weeks after inoculation) of C. podocarpa under 2 inoculation conditions. In a second step, during the symbiosis process, the methylotrophic function could contribute as supplementary energy source for the bacteria nitrogenase activity by recycling methanol produced through nodule tissue degradation. Lastly, the methanol being a key one-carbon metabolite regulating the expression of numerous genes, we cannot completely dismiss the possibillity that bacterial methylotrophy interfers in nodule cell-wall rearrangements or defence mechanisms by suppressing the methanol signal. … TO THE MOLECULAR DIALOG ELUCIDATION References Jourand P, Renier A, Rapior S, Mania de Faria S, Prin Y, Galiana A, Giraud E, Dreyfus B (2005) Role of methylotrophy during symbiosis between Methylobacterium nodulans and Crotalaria podocarpa. Molecular Plant-Microbe Interactions 18, 1061-1068. Sy A, Giraud E, Jourand P, Garcia N, Willems A, de Lajudie P, Prin Y, Neyra M, Gillis M, Boivin-Masson C, Dreyfus B (2001) Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes. Journal of Bacteriology 183, 214-220. FLAVONOIDS: During formation of legume-rhizobia symbioses flavonoids released from roots and seeds are well-known as inducers of the promoter of the bacterial nodulation genes. A crucial contribution to the infection process is highlighted when flavonoids interact with the constitutively expressed internal proteins of rhizobial regulatory nodD genes to form a transcriptional activators of other nod genes. Absorbance at 325 nm 15 20 Retention time (min) Separation of flavonoids from Crotalaria podocarpa root exsudate by reverse phase liquid chromatography (HPLC, C18). FLAVONOIDS The Nod protein products in response to transcriptional activation of nod genes, are responsible for the synthesis of reciprocal signal molecules to the host plant root - the chitolipooligosaccharides Nod factor. Nod D protein + Nod proteins Crotalaria podocarpa Effect of different signal molecules on nod gene expression of Methylobacterium nodulans was studied following beta-galactosidase activity. Preliminary data shown that unusual flavonoids act as nod gene activators while common flavonoid derivatives as genistein and daidzein are inefficient. nodD nod genes nod-box Methylobacterium nodulans NOD FACTOR H SH C18:1 NOD FACTOR: UNIVERSITE MONTPELLIER II Sciences et Techniques du Languedoc Chitolipooligosaccharides Nod factors are essential signals for rhizobial entry in legume roots, and the success or otherwise of the infection process is in large part determined by their structural features. Chemical structure of Nod factor from Methylobacterium nodulans (unpublished results). This compound was obtained after induction 48 hours by apigenin 5 µM.