Genetic access to the obligate cyanobacterial endosymbiontwithin thefern host
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
Abstract Eukaryote-associated microbes are ubiquitous, but their essential roles in the development and ecology of their host is yet to be fully understood, partly because complex associations cannot be reconstituted and, in many instances, the genetic tools to elucidate those roles are not available. Here, we report the conjugative transfer of DNA intowithin threefern hosts.is a filamentous, N-fixing, heterocyst-forming cyanobacterium which is the predominant obligate endosymbiont of the complex microbial community associated with the floating ferns of the genus. The cyanobiont provides fixed nitrogen to its host, supporting maximum growth rates without any N-fertilizer and makingsymbioses both ecologically and agriculturally important. Triparental mating protocols and fluorescent reporter detection were optimized for the cyanobiont isolated from the fern, allowing the further demonstration of heterologous gene expression indriven by several promoters, including some of a CRISPR-associated transposon system.was then treated with a cytokinin hormone to render fern shoot apexes amenable toconjugation, permitting DNA transfer to, and gene expression in two distinct developmental stages ofwithin. These included (i) cells of filaments from the Shoot Apicalcolony, the only cyanobacterial stem-cell population vertically transmitted across fern generations, and (ii) cells from differentiated filaments in early formedleaf cavities. Our approach represents a technically groundbreaking
Abstract
Abstract Eukaryote-associated microbes are ubiquitous, but their essential roles in the development and ecology of their host is yet to be fully understood, partly because complex associations cannot be reconstituted and, in many instances, the genetic tools to elucidate those roles are not available. Here, we report the conjugative transfer of DNA intowithin threefern hosts.is a filamentous, N-fixing, heterocyst-forming cyanobacterium which is the predominant obligate endosymbiont of the complex microbial community associated with the floating ferns of the genus. The cyanobiont provides fixed nitrogen to its host, supporting maximum growth rates without any N-fertilizer and makingsymbioses both ecologically and agriculturally important. Triparental mating protocols and fluorescent reporter detection were optimized for the cyanobiont isolated from the fern, allowing the further demonstration of heterologous gene expression indriven by several promoters, including some of a CRISPR-associated transposon system.was then treated with a cytokinin hormone to render fern shoot apexes amenable toconjugation, permitting DNA transfer to, and gene expression in two distinct developmental stages ofwithin. These included (i) cells of filaments from the Shoot Apicalcolony, the only cyanobacterial stem-cell population vertically transmitted across fern generations, and (ii) cells from differentiated filaments in early formedleaf cavities. Our approach represents a technically groundbreaking advance for the genetic engineering of cyanobacterial endosymbioses that may be useful for other symbiotic systems, opening a way to investigate these important biological entities.