Cloning and Transformation with Plasmid Vectors

Plasmids occupy a place of honor in molecular cloning: They were used in the first recombinant DNA experiments and, 40 or more years later, they remain as the carriage horses of molecular cloning. After almost half a century of sequential improvement in design, today's plasmid vectors are available in huge variety, are often optimized for specific purposes, and bear only passing resemblance to their forebears. Here, various features of plasmid vectors and methods for transforming E. coli cells are introduced.

Design and validation of plasmid vectors for characterizing protein-protein interactions in Spodoptera frugiperda insect cells

There are limited molecular biology resources for interrogating protein-protein interactions (PPI) in insect cells. To address this deficiency, we developed plasmid vectors for localization, bi-molecular fluorescence complementation (BiFC), and co-immunoprecipitation (co-IP) assays in Sf9 insect cells. Plasmids were designed to express a protein of interest as a fusion with epitope tags and autofluorescent proteins using the Gateway cloning system. Two robust interactors were utilized to validate this system, the nucleoprotein (N) and the phosphoprotein (P) of maize mosaic virus. The viral N was fused with the carboxy-terminal portion of eYFP and a FLAG epitope tag, and P was fused with the amino-terminal portion of eYFP and a c-myc epitope tag. The two expression plasmids were co-transfected into Sf9 cells, and fluorescence microscopy was used to visualize BiFC and co-IP was performed to confirm that this system was sensitive enough to detect PPI between the two proteins. BiFC was seen in cells co-transfected with N and P and co-IP validated the interaction. This plasmid-based system can be used to investigate a variety of PPI that occur in insects. We validated viral protein interactions that occur in the insect vector which provides further insights into the biology of rhabdoviruses that are transmitted by insects. The ability to express viral and insect proteins in insect cells for studying PPI with this streamlined system represents an advancement for protein research in insects. Future work will focus on identifying interacting viral and host proteins and discovery of targets for control of viruses and insect vectors.

IRI-CCE: A Platform for Evaluating CRISPR-based Genome Editing Tools and Its Components

Rapid assessment of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based tools and validation of cloned CRISPR-plasmid vectors are critical aspects for the successful application of genome editing (GE) in different organisms, including plants. Also, the selection of active guide RNAs (gRNAs) is a determining factor to achieve efficient GE at the targeted locus. Incidentally, plasmid vectors capable of ectopic expression of Cas enzyme and sgRNAs in bacteria will facilitate the quick screening and reliability of cloned plasmids and the functionality of designed gRNAs. We report a platform for in vivo rapid investigation of CRISPR components in Escherichia coli (IRI-CCE), which is compatible with plant-transforming binary vectors comprising plant promoters/terminators. Besides, IRI-CCE analyses reveal distinct features of cytidine (PmCDA1, evoCDA1, APOBEC3A) and adenine (ABE8e) base editors. Finally, we show the IRI-CCE platform as a reliable and rapid method for screening functional gRNAs to achieve successful GE outcomes. Base editor-based CRISPR components expressed by promoters of different strengths led to the establishment of IRI-CCE platform for three major applications: optimization of novel CRISPR tools, validation of cloned CRISPR plasmids, and to know gRNA functionality.

Acidic pH Decreases the Endonuclease Activity of Initiator RepB and Increases the Stability of the Covalent RepB-DNA Intermediate while Has Only a Limited Effect on the Replication of Plasmid pMV158 in Lactococcus lactis

Plasmid vectors constitute a valuable tool for homologous and heterologous gene expression, for characterization of promoter and regulatory regions, and for genetic manipulation and labeling of bacteria. During the last years, a series of vectors based on promiscuous replicons of the pMV158 family have been developed for their employment in a variety of Gram-positive bacteria and proved to be useful for all above applications in lactic acid bacteria. A proper use of the plasmid vectors requires detailed knowledge of their main replicative features under the changing growth conditions of the studied bacteria, such as the acidification of the culture medium by lactic acid production. Initiation of pMV158 rolling-circle replication is catalyzed by the plasmid-encoded RepB protein, which performs a sequence-specific cleavage on one of the parental DNA strands and, as demonstrated in this work, establishes a covalent bond with the 5′-P end generated in the DNA. This covalent adduct must last until the leading-strand termination stage, where a new cleavage on the regenerated nick site and a subsequent strand-transfer reaction result in rejoining of the ends of the cleaved parental strand, whereas hydrolysis of the newly-generated adduct would release the protein from a nicked double-stranded DNA plasmid form. We have analyzed here the effect of pH on the different in vitro reactions catalyzed by RepB and on the in vivo replication ability of plasmid pMV158. We show that acidic pH greatly impairs the catalytic activity of the protein and reduces hydrolysis of the covalent RepB-DNA adduct, as expected for the nucleophilic nature of these reactions. Conversely, the ability of pMV158 to replicate in vivo, as monitored by the copy number and segregational stability of the plasmid in Lactococcus lactis, remains almost intact at extracellular pHs ranging from 7.0 to 5.0, and a significant reduction (by ∼50%) in the plasmid copy number per chromosome equivalent is only observed at pH 4.5. Moreover, the RepB to pMV158 molar ratio is increased at pH 4.5, suggesting the existence of compensatory mechanisms that operate in vivo to allow pMV158 replication at pH values that severely disturb the catalytic activity of the initiator protein.

A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants

SUMMARYInvestigation of plant-bacteria interactions requires quantification of in planta bacterial titers by means of colony counting assays. However, colony counting assays are cumbersome and time-consuming, and are unable to detect spatial patterns of bacterial colonization in plants. Here, to overcome these shortcomings, we devised a broadly applicable genetic engineering tool for bioluminescence-based quantitative and spatial detection of bacteria in plants. We developed plasmid vectors that have broad host ranges and enable Tn7 transposon-mediated integration of the luxCDABE luciferase operon into a specific genomic location ubiquitously found across bacterial phyla. These vectors allowed for generation of bioluminescent transformants of various plant pathogenic bacteria belonging to the genera Pseudomonas, Rhizobium (Agrobacterium), and Ralstonia. The bioluminescent transformant of Pseudomonas syringae pv. tomato DC3000 (Pto-lux) was as virulent in Arabidopsis thaliana as its parental strain. Direct luminescence measurements of Pto-lux-inoculated plant tissues reported bacterial titers in A. thaliana, Solanum lycopersicum, Nicotiana benthamiana, and Marchantia polymorpha as accurately as conventional colony counting assays. We further showed the utility of our vectors for converting the previously generated Pto derivatives to isogenic bioluminescent strains. Importantly, quantitative bioluminescence assays using these Pto-lux strains accurately reported the effects of plant immunity and bacterial effectors on bacterial growth with a dynamic range of 4 orders of magnitude. Moreover, macroscopic bioluminescence imaging illuminated spatial colonization patterns of the Pto-lux in/on inoculated plant tissues. Taken together, our vectors offer untapped opportunities for developing bioluminescence-based quantitative and spatial analysis of bacterial growth in a variety of plant-bacteria interactions.SIGNIFICANCE STATEMENTWe developed broad-host-range plasmid vectors that integrate the luciferase operon, luxCDABE, into a specific genomic location ubiquitously found across bacterial phyla. Using these vectors, we established a high-throughput method for bioluminescence-based quantitative assays of in planta bacterial growth with a dynamic range of 4 orders of magnitude and visualized spatiotemporal patterns of bacterial colonization in/on inoculated plant tissues.