The Frankia alni Symbiotic Transcriptome

Abstract

The actinobacteria Frankia spp. are able to induce the formation of nodules on the roots of a large spectrum of actinorhizal plants, where they convert dinitrogen to ammonia in exchange for plant photosynthates. In the present study, transcriptional analyses were performed on nitrogen-replete free-living Frankia alni cells and on Alnus glutinosa nodule bacteria, using whole-genome microarrays. Distribution of nodule-induced genes on the genome was found to be mostly over regions with high synteny between three Frankia spp. genomes, while nodule-repressed genes, which were mostly hypothetical and not conserved, were spread around the genome. Genes known to be related to nitrogen fixation were highly induced, nif (nitrogenase), hup2 (hydrogenase uptake), suf (sulfur-iron cluster), and shc (hopanoids synthesis). The expression of genes involved in ammonium assimilation and transport was strongly modified, suggesting that bacteria ammonium assimilation was limited. Genes involved in particular in transcriptional regulation, signaling processes, protein drug export, protein secretion, lipopolysaccharide, and peptidoglycan biosynthesis that may play a role in symbiosis were also identified. We also showed that this Frankia symbiotic transcriptome was highly similar among phylogenetically distant plant families Betulaceae and Myricaceae. Finally, comparison with rhizobia transcriptome suggested that F. alni is metabolically more active in symbiosis than rhizobia.

Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia

“…Bacterial strains and growth conditions Frankia alni ACN14a (Normand and Lalonde, 1982) was grown according to Schwencke (1991) at 28 1C in defined (BAP) medium containing 5 mM ammonium chloride (N-replete condition, BAP þ ) or not (N-fixing condition, BAP À ) (Murry et al, 1984) with some minor modifications (Alloisio et al, 2010). The 5-day-old cultures were used for in vitro analyses.…”

“…Plant growth conditions and mRNA extraction A. glutinosa seeds were harvested from an A. glutinosa specimen growing on the Rhô ne River banks in Lyon, France, as described previously (Alloisio et al, 2010;Hocher et al, 2011). Seeds were grown and inoculated with the compatible F. alni strain ACN14a (Normand and Lalonde, 1982) as described previously (Alloisio et al, 2010).…”

“…Seeds were grown and inoculated with the compatible F. alni strain ACN14a (Normand and Lalonde, 1982) as described previously (Alloisio et al, 2010). For RNA extraction, non-inoculated roots at 0 and 7 days post inoculation (d.p.i.…”

“…Five biological replicates were used for each condition. Total RNA was purified from roots and nodules using the RNeasy plant mini kit (Qiagen, Courtaboeuf, France) (Alloisio et al, 2010). Residual DNA was removed from RNA samples using the Turbo DNA free kit (Ambion, Aix-en-Provence, France).…”

“…However, key steps in the symbiotic process, including how fixed nitrogen is transferred from symbionts to host plants, are still only partially understood despite efforts made in this area (Benson and Silvester, 1993;Benson et al, 2011;Oldroyd et al, 2011;Pawlowski et al, 2011). High-throughput sequencing of expressed sequence tags (ESTs) and DNA arrays are powerful tools to understand biological processes and have been widely used recently to study nitrogen-fixing symbiotic interactions (Ampe et al, 2003;Alloisio et al, 2010;Hocher et al, 2011;Heath et al, 2012). Nevertheless, there is presently no obvious candidate gene identified in either rhizobia or Frankia to account for release of nitrogen compounds to plants.…”