Stable Genetic Transformation and Heterologous Expression in the Nitrogen-fixing Plant Endosymbiont Frankia alni ACN14a

Abstract

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes, which are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. However the corresponding endosymbionts, Frankia and the rhizobia, are distantly related groups of bacteria, leading to questions of their symbiotic mechanisms and evolutionary history. To date, a stable system of genetic transformation has been lacking in Frankia. Here, we report the successful electrotransformation of Frankia alni ACN14a, by means of replicating plasmids expressing chloramphenicol-resistance for selection, and the use of GFP as a marker of gene expression. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation and circumvented this barrier by using unmethylated plasmids, which allowed the transformation of F. alni as well as the maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the plasmid transformation system was used with expression of egfp under the control of the nif gene cluster promoter, it was possible to demonstrate by fluorescence imaging the expression of nitrogen fixation in vesicles but not hyphae in nitrogen-limited culture.\n\nImportanceTo date, the study of Frankia-actinorhizal symbioses has been complicated by the lack of genetic tools for manipulation of Frankia, especially stable genetic transformation. The transformation system reported here, particularly coupled with marker genes, can be used to differentiate patterns of gene expression between Frankia hyphae and vesicles in symbiosis or in free-living conditions. This will enable deeper comparisons between Frankia-actinorhizal symbioses and rhizobia-legume symbioses in terms of molecular signaling and metabolic exchange that will broaden understanding of the evolution of these symbioses and potentially make possible their application in agriculture. The development of transformation methods will allow further down-stream applications including gene knock-outs and complementation that will, in turn, open up a much broader range of experiments into Frankia and its symbioses.