Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae.

Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from Escherichia coli to Saccharomyces cerevisiae. We demonstrated that there was no significant change in conjugation frequency when plasmid size increased from 56.5 to 138.6 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids.

Designer Sinorhizobium meliloti strains and multi-functional vectors enable direct inter-kingdom DNA transfer.

Storage, manipulation and delivery of DNA fragments is crucial for synthetic biology applications, subsequently allowing organisms of interest to be engineered with genes or pathways to produce desirable phenotypes such as disease or drought resistance in plants, or for synthesis of a specific chemical product. However, DNA with high G+C content can be unstable in many host organisms including Saccharomyces cerevisiae. Here, we report the development of Sinorhizobium meliloti, a nitrogen-fixing plant symbioticα-Proteobacterium, as a novel host that can store DNA, and mobilize DNA to E. coli, S. cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. To achieve this, we deleted the hsdR restriction-system in multiple reduced genome strains of S. meliloti that enable DNA transformation with up to 1.4 x 105 and 2.1 x 103 CFU µg-1 of DNA efficiency using electroporation and a newly developed polyethylene glycol transformation method, respectively. Multi-host and multi-functional shuttle vectors (MHS) were constructed and stably propagated in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum. We also developed protocols and demonstrated direct transfer of these MHS vectors via conjugation from S. meliloti to E. coli, S. cerevisiae, and P. tricornutum. The development of S. meliloti as a new host for inter-kingdom DNA transfer will be invaluable for synthetic biology research and applications, including the installation and study of genes and biosynthetic pathways into organisms of interest in industry and agriculture.

Development of a Transformation Method for Metschnikowia borealis and other CUG-Serine Yeasts.

Yeasts belonging to the Metschnikowia genus are particularly interesting for the unusual formation of only two needle-shaped ascospores during their mating cycle. Presently, the meiotic process that can lead to only two spores from a diploid zygote is poorly understood. The expression of fluorescent nuclear proteins should allow the meiotic process to be visualized in vivo; however, no large-spored species of Metschnikowia has ever been transformed. Accordingly, we aimed to develop a transformation method for Metschnikowiaborealis, a particularly large-spored species of Metschnikowia, with the goal of enabling the genetic manipulations required to study biological processes in detail. Genetic analyses confirmed that M. borealis, and many other Metschnikowia species, are CUG-Ser yeasts. Codon-optimized selectable markers lacking CUG codons were used to successfully transform M. borealis by electroporation and lithium acetate, and transformants appeared to be the result of random integration. Mating experiments confirmed that transformed-strains were capable of generating large asci and undergoing recombination. Finally, random integration was used to transform an additional 21 yeast strains, and all attempts successfully generated transformants. The results provide a simple method to transform many yeasts from an array of different clades and can be used to study or develop many species for various applications.

Inside out: root cortex-localized LHK1 cytokinin receptor limits epidermal infection of Lotus japonicus roots by Mesorhizobium loti.

During Lotus japonicus-Mesorhizobium loti symbiosis, the LOTUS HISTIDINE KINASE1 (LHK1) cytokinin receptor regulates both the initiation of nodule formation and the scope of root infection. However, the exact spatiotemporal mechanism by which this receptor exerts its symbiotic functions has remained elusive. In this study, we performed cell type-specific complementation experiments in the hyperinfected lhk1-1 mutant background, targeting LHK1 to either the root epidermis or the root cortex. We also utilized various genetic backgrounds to characterize expression of several genes regulating symbiotic infection. We show here that expression of LHK1 in the root cortex is required and sufficient to regulate both nodule formation and epidermal infections. The LHK1-dependent signalling that restricts subsequent infection events is triggered before initial cell divisions for nodule primordium formation. We also demonstrate that AHK4, the Arabidopsis orthologue of LHK1, is able to regulate M. loti infection in L. japonicus, suggesting that an endogenous cytokinin receptor could be sufficient for engineering nitrogen-fixing root nodule symbiosis in nonlegumes. Our data provide experimental evidence for the existence of an LHK1-dependent root cortex-to-epidermis feedback mechanism regulating rhizobial infection. This root-localized regulatory module functionally links with the systemic autoregulation of nodulation (AON) to maintain the homeostasis of symbiotic infection.

Into the Root: How Cytokinin Controls Rhizobial Infection.

Leguminous plants selectively initiate primary responses to rhizobial nodulation factors (NF) that ultimately lead to symbiotic root nodule formation. Functioning downstream, cytokinin has emerged as the key endogenous plant signal for nodule differentiation, but its role in mediating rhizobial entry into the root remains obscure. Nonetheless, such a role is suggested by aberrant infection phenotypes of plant mutants with defects in cytokinin signaling. We postulate that cytokinin participates in orchestrating signaling events that promote rhizobial colonization of the root cortex and limit the extent of subsequent infection at the root epidermis, thus maintaining homeostasis of the symbiotic interaction. We further argue that cytokinin signaling must have been crucial during the evolution of plant cell predisposition for rhizobial colonization.

A plant-specific HUA2-LIKE (HULK) gene family in Arabidopsis thaliana is essential for development.

In Arabidopsis thaliana, the HUA2 gene is required for proper expression of FLOWERING LOCUS C (FLC) and AGAMOUS, key regulators of flowering time and reproductive development, respectively. Although HUA2 is broadly expressed, plants lacking HUA2 function have only moderately reduced plant stature, leaf initiation rate and flowering time. To better understand HUA2 activity, and to test whether redundancy with similar genes underlies the absence of strong phenotypes in HUA2 mutant plants, we identified and subsequently characterized three additional HUA2-LIKE (HULK) genes in Arabidopsis. These genes form two clades (HUA2/HULK1 and HULK2/HULK3), with members broadly conserved in both vascular and non-vascular plants, but not present outside the plant kingdom. Plants with progressively reduced HULK activity had increasingly severe developmental defects, and plants homozygous for loss-of-function mutations in all four HULK genes were not recovered. Multiple mutants displayed reproductive, embryonic and post-embryonic abnormalities, and provide detailed insights into the overlapping and unique functions of individual HULK genes. With regard to flowering time, opposing influences were apparent: hua2 hulk1 plants were early-flowering, while hulk2 hulk3 mutants were late-flowering, and hua2 acted epistatically to cause early flowering in all combinations. Genome-wide expression profiling of mutant combinations using RNA-Seq revealed complex transcriptional changes in seedlings, with FLC, a known target of HUA2, among the most affected. Our studies, which include characterization of HULK expression patterns and subcellular localization, suggest that the HULK genes encode conserved nuclear factors with partially redundant but essential functions associated with diverse genetic pathways in plants.