bioRxiv preprint doi: https://doi.org/10.1101/496703; this version posted December 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Stable Genetic Transformation and Heterologous Expression in the Nitrogen-fixing 2 Plant Endosymbiont Frankia alni ACN14a 3 Isaac Gifforda#, Summer Vancea, Giang Nguyena, and Alison M Berrya 4 Author affiliations: aDepartment of Plant Sciences, University of California, Davis, 5 Davis, CA, United States 6 #Address correspondence to Isaac Gifford, [email protected] 7 Running Title: Genetic Transformation of Frankia alni 8 Word Counts: 9 Abstract: 221 words 10 Importance: 123 words 11 Text Body: 5315 words 12 Abstract 13 Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form 14 root nodule symbioses with a group of hosts known as the actinorhizal plants. These 15 plants are evolutionarily closely related to the legumes, which are nodulated by the 16 rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule 17 symbiosis, derived from common plant ancestors. However the corresponding 18 endosymbionts, Frankia and the rhizobia, are distantly related groups of bacteria, leading 19 to questions of their symbiotic mechanisms and evolutionary history. To date, a stable 20 system of genetic transformation has been lacking in Frankia. Here, we report the 1 bioRxiv preprint doi: https://doi.org/10.1101/496703; this version posted December 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 21 successful electrotransformation of Frankia alni ACN14a, by means of replicating 22 plasmids expressing chloramphenicol-resistance for selection, and the use of GFP as a 23 marker of gene expression. We have identified type IV methyl-directed restriction 24 systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia 25 transformation and circumvented this barrier by using unmethylated plasmids, which 26 allowed the transformation of F. alni as well as the maintenance of the plasmid. During 27 nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and 28 nitrogen-fixing vesicles. When the plasmid transformation system was used with 29 expression of egfp under the control of the nif gene cluster promoter, it was possible to 30 demonstrate by fluorescence imaging the expression of nitrogen fixation in vesicles but 31 not hyphae in nitrogen-limited culture. 32 Importance 33 To date, the study of Frankia-actinorhizal symbioses has been complicated by the 34 lack of genetic tools for manipulation of Frankia, especially stable genetic transformation. 35 The transformation system reported here, particularly coupled with marker genes, can be 36 used to differentiate patterns of gene expression between Frankia hyphae and vesicles in 37 symbiosis or in free-living conditions. This will enable deeper comparisons between 38 Frankia-actinorhizal symbioses and rhizobia-legume symbioses in terms of molecular 39 signaling and metabolic exchange that will broaden understanding of the evolution of 40 these symbioses and potentially make possible their application in agriculture. The 41 development of transformation methods will allow further down-stream applications 42 including gene knock-outs and complementation that will, in turn, open up a much 43 broader range of experiments into Frankia and its symbioses. 2 bioRxiv preprint doi: https://doi.org/10.1101/496703; this version posted December 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 44 Introduction 45 Bacteria in the genus Frankia form nitrogen-fixing root nodule symbioses with a 46 group of host plants, the actinorhizal plants. The actinorhizal plants are evolutionarily 47 closely related to the legumes within the Nitrogen-fixing Clade (NFC; Soltis et al., 1995). 48 The bacterial symbionts are only distantly related however: Frankia belongs to the 49 phylum Actinobacteria, comprised of high-GC-content gram-positive bacteria, whereas 50 the rhizobia, symbionts of the legumes, are gram-negative proteobacteria. Additionally, 51 Frankia is a multicellular, hyphal genus whereas the rhizobia are single-celled. In most of 52 the actinorhizal symbioses, and in nitrogen-limiting conditions in vitro, Frankia 53 differentiates into two cell types: vegetative hyphae and nitrogen-fixing vesicles. Vesicles 54 are surrounded by a lamellar hopanoid lipid envelope (Berry et al., 1993) that increases in 55 number of layers in response to oxygen tension thereby likely reducing the flow of 56 oxygen into the vesicle interior to protect the nitrogenase complex from deactivation 57 (Benson and Silvester, 1993). Thus, unlike the rhizobia which require their hosts to 58 regulate the flow of oxygen for nitrogen fixation in symbiosis (Appleby, 1984), Frankia 59 vesicles are capable of fixing nitrogen in atmospheric oxygen conditions (Benson and 60 Silvester, 1993). 61 Biological nitrogen fixation has potential applications in the development of 62 minimal input strategies for more sustainable agriculture globally and in nutrient- 63 deficient soils through genetic improvement of crops (Gutierrez, 2012). In efforts to 64 understand the mechanisms of root nodule symbioses, phylogenetic studies of the 65 Nitrogen-Fixing Clade have shown a shared predisposition underlying the evolution root 66 nodule symbioses between the actinorhizal plants and legumes (Werner et al., 2014; 3 bioRxiv preprint doi: https://doi.org/10.1101/496703; this version posted December 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 67 Battenberg et al., 2018). The actinorhizal and legume hosts share the Common Symbiotic 68 Pathway, a signaling pathway that leads to the development of the nodule in response to 69 the symbiont (reviewed in Oldroyd 2013) as well as several key gene orthologs in the 70 process of nodule development (Battenberg et al., 2018; Griesmann et al., 2018). Clear 71 differences also exist in the genetics of nodulation in the two bacterial groups, leaving 72 major questions concerning the evolution and development of root-nodule symbioses in 73 the bacterial partners. The rhizobial signaling molecules that trigger nodulation in many 74 of the legume hosts, called Nod factors, are synthesized by nod genes and have been 75 extensively studied (Oldroyd, 2013). The majority of Frankia genomes do not contain 76 clear sets of nod gene homologs with the exception of several of the cluster II Frankia: 77 genomes of these Frankia contain homologs of the rhizobial nodABC genes that are 78 expressed in symbiosis (Persson et al., 2015). Other mechanisms have been found to 79 trigger legume nodulation by rhizobia, including an effector protein injected into the host 80 by a type III secretion system (Okazaki et al., 2013); however genes responsible for these 81 pathways lack homologs in Frankia genomes as well. Signals from Frankia in clusters I 82 and III that elicit host root responses have been detected experimentally, but the chemical 83 composition has not been defined (Ceremonie et al., 1999; Cissoko et al., 2018). 84 The major advances in knowledge of rhizobial symbioses have been made 85 possible by the development of genetic tools for dissecting the metabolic and regulatory 86 pathways in the microsymbiont-host interactions. Long et al. (1982) originally 87 demonstrated the role of the nod genes in symbiosis by complementing nodulation- 88 deficient mutants of Sinorhizobium meliloti with nod genes encoded on a replicating, 89 broad host-range, plasmid. More recently, expression of marker genes in rhizobia has 4 bioRxiv preprint doi: https://doi.org/10.1101/496703; this version posted December 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 90 enabled a wider-range of experiments including the tracking of microsymbionts during 91 the host infection process (Gage, 2002) and the identification of regulatory networks 92 involved in symbiotic interactions (Spaepen et al., 2009). However, to date, there has 93 been no stable genetic transformation system for Frankia (Kucho et al., 2009). As 94 reported here, a transformation system in Frankia will thus enable functional inquiries 95 into the diverse mechanisms involved in establishing root-nodule symbiosis and 96 maintaining biological nitrogen fixation in actinorhizal symbioses. These inquiries will, 97 in turn, contribute to a wider understanding of the origins of root nodule symbioses and 98 mechanisms of Frankia and rhizobia interaction with hosts in the NFC. 99 Frankia has several barriers to transformation. Actinobacteria in general have low 100 rates of