A GREAT ASSET IN SYMBIOSIS PROCESS

1. A. Renier1, S. Rapior2, Y. Prin1, S. Miana de Faria1 , A.Galliana1, E. Giraud, 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. A GREAT ASSET IN SYMBIOSIS PROCESS A B 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). C Recently, strains isolated from three Crotalaria spp. were described as being a single novel methylotrophic Methylobacteriumspp. (Sy et al. 2001). This new species was named Methylobacterium nodulans for its ability to both nodulate Fig 1. A: Crotalaria podocarpa; B: 6-week-old nodules of C. podocarpa inoculated wtih Methylobacterium nodulans ORS 2060T wild type; C: Ultrastructure of M. nodulans bacteroids in 6-week-old nitrogen-fixing C. podocarpa nodules. and fix nitrogen specifically during symbiosis with Crotalaria spp. (Jourand et al. 2004; Sy et al. 2001). What’s role of methylotrophy during symbiosis between M. nodulans and the host legume C. podocarpa ? Methylotrophy of M. nodulans is expressed in root nodules formed during symbiosis with C. podocarpa Loss of methylotrophy strongly affects symbiosis Construction of M. nodulans ORS 2060T negative methylotroph mutants by insertional mutagenesis of transposon mTn5-GGm. Construction of M. nodulans ORS 2060T recombinant strain containing mxaF-LacZ transcriptional fusion promoter. The mxaF geneencodes the largest -polypeptide subunit of MDH and was use to monitor the expression of methylotroph genes in planta. A A’ control non inoculated ORS 2060T wild type mutant type 1 mutant type 2 ORS 2060 pCM132 mxaF-LacZ ORS2060 pCM132 mxaF-LacZ B B’ ORS 2060T wild type ORS 2060T wild type Fig 3. Comparaison of growth (5 weeks after inoculation) of C. podocarpa under four inoculaion conditions. Inoculation of M. nodulans nonmethylotroph mutants in C. podocarpa decreased : the total root nodule number per plant up to 60%, Fig 2. Histochemical localization of the -galactosidase activity in 6-week-old C. podocarpa root nodules. The mxaF gene was expressed in apex root nodules suggesting that methanol is present inside the nodule and subsequently used by the bacteria. the whole-plant nitrogen fixation capacity up to 42%, the total dry plant biomass up to 46%, During symbiosis, we hypothesize that methanol could be produced from pectin cell wall demethylation. compared to the wild-type strain. Methylotrophy appears to be essential in symbiosis between the bacteria M. nodulans and it host legume C. podocarpa. This function might be implicated at different levels of symbiosis. 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. 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 poduced 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. References Jourand P, Giraud E, Bena G, Sy A, Willems A, Gillis M, Dreyfus B, de Lajudie P (2004)Methylobacterium nodulans sp. nov., for a group of aerobic, facultatively methylotrophic, legume root-nodule-forming and nitrogen-fixing bacteria. International Journal of Systematic and Evolutionary Microbiology54, 2269-2273. 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 Interactions18, 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 Bacteriology183, 214-220.