Folklore has many stories about the healing power of the elderberry and elder flower

Due to the barrier created by type IV enzymes in the free-living condition , horizontal transfer involving Frankia in the soil is likely limited to other actinobacteria whose genomes similarly lack methylation , many of whom are endophytes . Inside the host, however, Frankia is likely more receptive to genes from other endophytic taxa as well. Combined, these factors could result in the preferential acquisition of genes involved in growth and host-microbe interactions in planta. In legume symbioses, exudates from hosts into their rhizospheres have been proposed to promote the conjugative transfer of symbiosis genes from a donor group of rhizobia to others, broadening the range of symbionts available to the host . Down-regulation of type IV restriction genes in actinorhizal nodule symbioses could be another mechanism that enhances horizontal transfer of genes related to interactions with plants by making the recipient Frankia more susceptible in the endophytic environment. M. tuberculosis showed much lower transcription of its annotated type IV methyladenine targeting restriction enzyme than other actinobacteria. M. tuberculosis expresses an adenine methyltransferase in hypoxic conditions that regulates the expression of genes likely involved with survival during macrophage infection . For this reason it is likely that M. tuberculosis responds to methylated DNA differently than other actinobacteria; indeed electrotransformation of M. tuberculosis can be readily achieved with methylated plasmids replicated in E. coli DH5α , suggesting that methylated DNA is not digested in M. tuberculosis. In this study derivatives of broad host-range plasmid pSA3 were capable of replication in F. alni. This shows that the broad host-range origin is capable of replication in Frankia and supports its use as a vector for the manipulation of Frankia spp. The parent plasmid of pSA3, pIP501,grow bucket replicates in a very broad range of bacteria including Streptomyces lividans and E. coli indicating the potential for transformation of additional actinobacteria with these plasmids.

The expression of the egfp gene of plasmid pIGSAFnif was up-regulated in N media compared with expression in N media, at proportional levels to the expression of the nifH nitrogenase gene , demonstrating for the first time that expression of reporter genes can be manipulated in Frankia. This transformation system resulted in the ability to visualize the expression of nitrogen fixation genes in vitro by fluorescence microscopy . Interestingly, fluorescence was detected in both the spherical portion of the vesicle as well as in the stalk that connects to the hyphae, suggesting that nitrogen fixation genes are expressed in both parts of the vesicle. Previous studies have shown that the vesicle envelope is deposited around the stalk as well as the spherical part of the vesicle , supporting the observation that nitrogen fixation can occur in the stalk. Although the fluorescence observed when egfp was expressed under the control of the F. alni nif cluster promoter was predominantly in the vesicles, some fluorescence was occasionally observed in hyphae under nitrogen-fixing conditions whereas in N media there was no observable fluorescence . This suggests that there can be condition-dependent expression of nif genes in the hyphae as well as the vesicles induced by nitrogen limitation. Frankia spp. in symbiosis with members of the Casuarinaceae have been reported to fix nitrogen in hyphae, since no vesicles are differentiated ; this pattern correlated with the formation of a lignified host cell wall in the symbiotic tissue that likely reduces oxygen partial pressure . In liquid culture, there may be zones of low pO2 that develop in portions of a Frankia hyphal colony where nitrogen fixation could be induced. Frankia spp. in symbiosis have been suggested to be more autonomous than rhizobial microsymbionts due to their ability to control the flow of oxygen with the formation of vesicles, and due as well to the expression of more metabolic pathways in the micro-symbiont in symbiosis. These factors potentially allow Frankia to be more metabolically independent from their hosts .

The development of genetic tools for the manipulation of Frankia will allow further exploration into these and other distinctive molecular aspects of actinorhizal symbioses, which will, in turn, further inform analyses of the evolution and diversity of root nodule symbiosis. The transformation methods presented here should be applicable for genetic experiments in other Frankia strains. Because plasmid pIGSAF has a broad host-range origin of replication and expresses egfp under the control of a constitutive promoter, the plasmid is likely to be usable in other strains as well. Even in the absence of selection, plasmid pIGSAF was found to be stable in F. alni cultures for at least 3 weeks and cultures continued to show fluorescence after at least 4 weeks . Three to 4 weeks is a time period that sufficiently spans the stages of nodulation and early nitrogen fixation in a broad-spectrum of hosts. This includes Alnus glutinosa with F. alni ACN14a , and Casuarina cunninghamiana , Discaria trinervis , Shepherdia argentaea , and Datisca glomerata . This suggests that transformants can be used to inoculate plants to study of the role of Frankia and its interactions with hosts during nodule establishment and symbiosis. In future this system could be modified using recombination or site-specific integrases to anchor genes within the genome. Differential regulation of reporter genes such as egfp can be used to localize the expression of genes identified by genomics and transcriptomics in specific Frankia cell types, in different growth conditions, and in symbiosis. Replicating plasmids may also enable the study of gene function by constitutive expression of selected genomic genes, by promoter switching or by knock-down experiments expressing anti-RNAs to genes of interest . Circumventing the natural restriction systems of Frankia will also increase the transformation rate of non-replicating plasmids and enable higher efficiency recombination, which can be combined with CRISPR systems, for gene knock-out experiments as attempted by Kucho et al. . While this manuscript was in review a separate method for the transformation of Frankia spp. utilizing conjugation with a methylation-positive E. coli was reported by Pesce et al. . Conjugative transfer has been shown to evade recipient restriction systems , thus presumably allowing Frankia spp. to circumvent the type IV restriction barrier identified in this study.

Nevertheless, the transformation efficiency of Streptomyces spp. by conjugation has been shown to increase over 104 -fold with unmethylated DNA . Thus, in addition to enabling the transformation of Frankia spp. by electroporation, our analysis of restriction systems in Frankia spp. can be further utilized to improve the transformation efficiency of conjugative transfer to Frankia spp. through the use of methylation-deficient E. coli donors. Optimized transformation efficiency will be crucial for future studies involving the generation of recombinants including gene knock-outs. Elderberry is a part of the Viburnaceae family and grows all over the world, including Europe, North America, and Asia.Due to the vast geographic and morphological variety within Sambucus, there have historically been many species within the genus. However, a reorganization by Bolli reclassified some of the most common species under Sambucus into subspecies of S. nigra. More recently, elderberry was moved out of the Adoxoaceae family, which had already been changed before when elderberry was taken out of the Caprifoliaceae family. These changes have impacted the three subspecies most of interest in this work: the European elderberry S. nigra ssp. nigra; the American elderberry S. nigra ssp. canadensis; and the blue elderberry S. nigra ssp. cerulea .However, due to wide acceptance of this naming scheme for the subspecies, it will be used through this work to align with the current naming, but previous works cited may use the former species names. Furthermore, some sources refer to the entire plant as an “elder”,dutch bucket for tomatoes while others refer to the plant as “elderberry”, which is also used to denote the fruit of the plant. In this work, “elderberry” is used to discuss the plant as well as the fruit. “Elderflower” is used to refer to the blossoms of the plant. European elderberry is the most well-studied and widely used subspecies of elderberry in the market. This subspecies grows throughout the European continent, including countries such as Slovenia, Portugal, and Austria. The fruit and flower have been studied for decades for their composition and bio-activity, and while elderberry and elder flower are not new ingredients to the market, they have garnered more attention in the last several years as consumers look for more natural remedies and supplements to support their health. This has been especially true during the COVID-19 pandemic, in which elderberry became a popular ingredient in immunity-supporting supplements. Thus, investigating other elderberry subspecies like the blue elderberry, the focus of future chapters, allows for farmers in the United States to capitalize on this demand, but more information is needed on this particular plant if it is going to be used in consumer products. There is a long, rich history of the use of different parts of the elderberry plant by many cultures. For example, the wood has been used for kindling and musical instruments. Indeed, the name of the plant is derived from various ancient words related to instruments. The flowers and berries have been used in a variety of beverages, foods, and other herbal supplements.\

The plant itself has been revered by many cultures, with a story about the “Elder Mother” living within the plant would protect those near the plant. It was even expected to ask the Elder Mother for the berries or flowers before taking them; without permission, she may seek revenge. The leaves, branches, flowers, and berries were believed to have protective powers for a home and the leaves were also used during burial rituals by some Celtic people. The personification and deep reverence for the elderberry show the importance of the plant through generations. Hippocrates and Pliny the Elder both wrote about elderberry and its medicinal properties. Tribes indigenous to North America used flowers and fruit for medicinal and beverages. Berries were also used as a natural dye for baskets and branches were used to make musical instruments. Elderberry is a perennial, deciduous plant native to many regions of the northern hemisphere. Elderberry plants are neither tree nor bush, as the plant sends new canes up each season, which without pruning, can lead to a large, shrub-like plant that can be several meters tall and wide. They prefer to grow in sunny, riparian climates with moist, well-drained soil, though subspecies in North America can be drought-tolerant. While pruning even down to the ground level of the elderberry can improve yield and accessibility for harvesting, there is a limitation on pruning of the blue elderberry in the Central Valley of California. Due to the threatened status of the Valley Elderberry Longhorn Beetle , which lives only in the elderberry, branches larger than one inch in protected areas should not be pruned or removed from a growing site.Elderberry shrubs typically produce small white flowers with five petals in the spring, though the elder flowers of the blue elderberry are a creamy yellow color. Small, dark blue-purple berries ripen in the summer in large clusters called umbels or cymes, though there are examples of subspecies that have some variation to these morphologies, such as the blue elderberry that has a white bloom on the berries, causing the berries to look blue, and S. racemosa, which are red.Variation can also occur within a subspecies due to growing conditions, such as soil type, precipitation, and temperatures, as well as a key differentiation tool: cultivars or genotypes. There are established cultivars or genotypes of the European subspecies , such as Sambu or Haschberg, as well as of the American subspecies , like Bob Gordon or Wylewood. Cultivars can have more consistent growing patterns, such as blooming or ripening all at once, and desired chemical compositions, such as increased anthocyanins, thus are more desirable to use in large scale growing of elderberry for commercial use. Blue elderberry does not have any established genotypes to date. If commercial interest in this subspecies continues to expand, effort should be made to develop cultivars with consistent quality and improved harvest ability, which is hampered right now due to flowers and berries ripening throughout a season, instead of a smaller window of time like the American and European subspecies. Indeed, starting this work can help increase the commercial interest viability of the blue elderberry. A primary driver in interest in the composition of elderberry and elder flower is for their potential health benefits. Several reviews have recently been published on this topic; thus, it will not be explored in depth here.