Reliable Transformation Method Research Articles

Agrobacterium tumefaciens-mediated transformation of Nigrospora sp. isolated from switchgrass leaves and antagonistic toward plant pathogens

Nigrospora is a diverse genus of fungi colonizing plants through endophytic, pathogenic, or saprobic interactions. Endophytic isolates can improve growth and development of host plants, as well as their resistance to microbial pathogens, but exactly how they do so remains poorly understood. Developing a reliable transformation method is crucial to investigate these mechanisms, in particular to identify pivotal genes for specific functions that correlate with specific traits. In this study, we identified eight isolates of Nigrospora sp. internally colonizing the leaves of switchgrass plants cultivated in North Carolina. Using an Agrobacterium tumefaciens-mediated transformation approach with control and GFP-expressing vectors, we report the first successful transformation of two Nigrospora isolates. Finally, we demonstrate that wild-type and transgenic isolates both negatively impact the growth of two plant pathogens in co-culture conditions, Bipolaris maydis and Parastagonospora nodorum, responsible for the Southern Leaf Blight and Septoria Nodorum Blotch diseases, respectively. The GFP-transformed strains developed here can therefore serve as accurate reporters of spatial interactions in future studies of Nigrospora and pathogens in the plant. Finally, the transformation method we describe lays the foundation for further genetic research on the Nigrospora genus to expand our mechanistic understanding of plant-endophyte interactions.

Random transposon insertion in the Mycoplasma hominis minimal genome

Mycoplasma hominis is an opportunistic human pathogen associated with genital and neonatal infections. Until this study, the lack of a reliable transformation method for the genetic manipulation of M. hominis hindered the investigation of the pathogenicity and the peculiar arginine-based metabolism of this bacterium. A genomic analysis of 20 different M. hominis strains revealed a number of putative restriction-modification systems in this species. Despite the presence of these systems, a reproducible polyethylene glycol (PEG)-mediated transformation protocol was successfully developed in this study for three different strains: two clinical isolates and the M132 reference strain. Transformants were generated by transposon mutagenesis with an efficiency of approximately 10−9 transformants/cell/µg plasmid and were shown to carry single or multiple mini-transposons randomly inserted within their genomes. One M132-mutant was observed to carry a single-copy transposon inserted within the gene encoding P75, a protein potentially involved in adhesion. However, no difference in adhesion was observed in cell-assays between this mutant and the M132 parent strain. Whole genome sequencing of mutants carrying multiple copies of the transposon further revealed the occurrence of genomic rearrangements. Overall, this is the first time that genetically modified strains of M. hominis have been obtained by random mutagenesis using a mini-transposon conferring resistance to tetracycline.

Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species.

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species.

A simple and reliable multi-gene transformation method for switchgrass

A simple and reliable Agrobacterium -mediated transformation method was developed for switchgrass. Using this method, many transgenic plants carrying multiple genes-of-interest could be produced without untransformed escape. Switchgrass (Panicum virgatum L.) is a promising biomass crop for bioenergy. To obtain transgenic switchgrass plants carrying a multi-gene trait in a simple manner, an Agrobacterium-mediated transformation method was established by constructing a Gateway-based binary vector, optimizing transformation conditions and developing a novel selection method. A MultiRound Gateway-compatible destination binary vector carrying the bar selectable marker gene, pHKGB110, was constructed to introduce multiple genes of interest in a single transformation. Two reporter gene expression cassettes, GUSPlus and gfp, were constructed independently on two entry vectors and then introduced into a single T-DNA region of pHKGB110 via sequential LR reactions. Agrobacterium tumefaciens EHA101 carrying the resultant binary vector pHKGB112 and caryopsis-derived compact embryogenic calli were used for transformation experiments. Prolonged cocultivation for 7 days followed by cultivation on media containing meropenem improved transformation efficiency without overgrowth of Agrobacterium, which was, however, not inhibited by cefotaxime or Timentin. In addition, untransformed escape shoots were completely eliminated during the rooting stage by direct dipping the putatively transformed shoots into the herbicide Basta solution for a few seconds, designated as the 'herbicide dipping method'. It was also demonstrated that more than 90 % of the bar-positive transformants carried both reporters delivered from pHKGB112. This simple and reliable transformation method, which incorporates a new selection technique and the use of a MultiRound Gateway-based binary vector, would be suitable for producing a large number of transgenic lines carrying multiple genes.

Functional integration of multiple genes into the genome of the oleaginous yeastRhodosporidium toruloides

The basidiomycetous yeast Rhodosporidium toruloides represents an excellent producer for microbial lipids and carotenoids. However, further rational engineering of this unconventional yeast remains challenging partially because of the absence of efficient and reliable transformation method. In this study, we developed an Agrobacterium-mediated transformation (ATMT) protocol for effective gene integration into the R.toruloides genome. Both haploid and diploid strains were successfully modified, and the integration was confirmed by colony PCR, Western blot analysis and genome walking. We further demonstrated that multiple genes could be integrated by consecutive ATMT, leading to engineered strains simultaneously resistant to multiple antibiotics. Our results provided a practical method for functional integration and expression of exogenous genes in R.toruloides, which should facilitate the development of genetic tools and the construction of superior strains to produce biofuel molecules and biochemicals.

A reliable approach to the solution of Navier–Stokes equations

In this paper we will demonstrate an affective approach of solving Navier–Stokes equations by using a very reliable transformation method known as the Cole–Hopf transformation, which reduces the problem from nonlinear into a linear differential equation which, in turn, can be solved effectively.

A reliable transformation method and heterologous expression of β-glucuronidase in Lentinula edodes

A simple and reliable mushroom transformation procedure based on electroporation of basidiospores or mycelial fragments was developed. This method eliminated the problem of protoplast preparation, the transformation efficiency were 30–150 transformants per μg DNA and the hygromycin resistant marker gene and gus were expressed in Lentinula edodes successfully. No false positive antibiotic-resistant cultures were detected by PCR amplification and the β-glucuronidase (GUS) expression was maintained stable during mitotic cell division without selection pressure for more than 6 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. Using the glyceraldehyde-3-phosphate dehydrogenase ( gpd) promoter with the first intron of gpd gene, the average GUS activity in L. edodes reached 144.6 ± 3.9 U mg − 1 soluble protein, while only 30.1 ± 0.7 U mg − 1 soluble protein was detected for those without the intron. The percentage of GUS in total soluble protein was 5.67 × 10 − 4 (0.06%) for the transformant with the highest GUS activity. This rapid and convenient electroporation procedure offers a new approach for the genetic manipulation and tool to tag genes of important edible mushroom species.