Abstract
In the terrestrial ecosystem, plant–microbe symbiotic associations are ecologically and economically important processes. To better understand these associations at structural and functional levels, different molecular and biochemical tools are applied. In this study, we have constructed a suite of vectors that incorporates several new elements into the rhizosphere stable, broad-host vector pME6031. The new vectors are useful for studies requiring multi-color tagging and visualization of plant-associated, Gram-negative bacterial strains such as Pseudomonas plant growth promotion and biocontrol strains. A number of genetic elements, including constitutive promoters and signal peptides that target secretion to the periplasm, have been evaluated. Several next generation fluorescent proteins, namely mTurquoise2, mNeonGreen, mRuby2, DsRed-Express2 and E2-Crimson have been incorporated into the vectors for whole cell labeling or protein tagging. Secretion of mTurquoise2 and mNeonGreen into the periplasm of Pseudomonas fluorescens SBW25 has also been demonstrated, providing a vehicle for tagging proteins in the periplasmic compartment. A higher copy number version of select plasmids has been produced by introduction of a previously described repA mutation, affording an increase in protein expression levels. The utility of these plasmids for fluorescence-based imaging is demonstrated by root colonization of Solanum lycopersicum seedlings by P. fluorescens SBW25 in a hydroponic growth system. The plasmids are stably maintained during root colonization in the absence of selective pressure for more than 2 weeks.
Highlights
Plant growth and crop productivity are functions of nutrient resource supply and acquisition
In order to simplify future cloning tasks, site-directed mutagenesis was used to remove several restriction sites from pME6031. These include NotI(844), NotI(2377), NotI(3667), XbaI(8291) and BamHI(1)
In addition to the utility of pME6031 based vectors in Pseudomonas spp., vectors containing the pVS1 origin of replication have been found to be stable in a wide range of Gram-negative hosts including Rhizobium spp. (Stuurman et al, 2000) and Agrobacterium spp
Summary
Plant growth and crop productivity are functions of nutrient resource supply and acquisition. Plants have developed a complex association with different soil microbes, with microbes forming an intimate association with the plant roots (Danhorn and Fuqua, 2007). In a laboratory model system, it was shown that capacities of different Pseudomonas plant growth promotion (PGP) strains to protect plants from nutrient stress are a function of the respective bacterial transportomes (Shinde et al, 2017). We describe development of an enhanced set of plasmid vectors which will be useful for future studies of the molecular mechanisms of nutrient exchange processes and in particular to observe and study the association of plants with different microbes in the rhizosphere
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