Unmarked gene editing in Clavibacter michiganensis using CRISPR/Cas9 and 5-fluorocytosine counterselection

Plant pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis (Cm), which couples the expression of cas9 and sgRNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the Cm rpsL gene to generate a streptomycin resistant mutant, and complete gene knockout, in which the deletion of the Cm celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 100 μg/ml 5-fluorocytosine (5-FC), in which an unmarked katA mutant lacking the transformation vector was recovered. Compared to conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient as it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into Cm and other Clavibacter species.

No Evidence of Unexpected Transgenic Insertions in T1190 – A Transgenic Apple Used in Rapid Cycle Breeding – Following Whole Genome Sequencing

Rapid cycle breeding uses transgenic early flowering plants as crossbreed parents to facilitate the shortening of breeding programs for perennial crops with long-lasting juvenility. Rapid cycle breeding in apple was established using the transgenic genotype T1190 expressing the BpMADS4 gene of silver birch. In this study, the genomes of T1190 and its non-transgenic wild-type PinS (F1-offspring of ‘Pinova’ and ‘Idared’) were sequenced by Illumina short-read sequencing in two separate experiments resulting in a mean sequencing depth of 182× for T1190 and 167× for PinS. The sequencing revealed 8,450 reads, which contain sequences of ≥20 bp identical to the plant transformation vector. These reads were assembled into 125 contigs, which were examined to see whether they contained transgenic insertions or if they are not using a five-step procedure. The sequence of one contig represents the known T-DNA insertion on chromosome 4 of T1190. The sequences of the remaining contigs were either equally present in T1190 and PinS, their part with sequence identity to the vector was equally present in apple reference genomes, or they seem to result from endophytic contaminations rather than from additional transgenic insertions. Therefore, we conclude that the transgenic apple plant T1190 contains only one transgenic insertion, located on chromosome 4, and shows no further partial insertions of the transformation vector.Accession Numbers: JQ974028.1.

Standardisation of an Agroinfiltration Protocol for Eggplant Fruits and Proving its Usefulness by Over-expressing the SmHQT Gene

Eggplant is a fruit vegetable of family Solanaceae, and eggplant fruits are of different shape and sizes that render them as an ideal system for metabolic engineering. Here, we have developed an agroinfiltration protocol for the transient expression of a gene in the eggplant fruit using GUS bearing; pCAMBIA1304 vector. Thereafter, to prove the effectiveness of the developed protocol, we have used the eggplant hydroxycinnamoyl CoA-quinate transferase (SmHQT), which is the central enzyme studied to increase the chlorogenic acid content, in a gene construct with the specific promoter in a plant transformation vector (pBIN19). Also, in our cassette, we also co-expressed the P19 protein of Tomato bushy stunt virus (native promoter) to overexpress the protein. Overall, using the protocol, the chlorogenic content was increased by more than two folds in the transgenic tissues.

Enhanced Tolerance against a Fungal Pathogen and Insect Resistance in Transgenic Tobacco Plants Overexpressing an Endochitinase Gene from Serratia marcescens

In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the corpse of a Diatraea magnifactella lepidopteran, which is an important sugarcane pest. The chitinase gene SmchiC amplified from the S. marcescens genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco (Nicotiana tabacum L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus Botrytis cinerea and to the pest Spodoptera frugiperda was evaluated: both the activity of chitinase as well as the resistance against B. cinerea and S. frugiperda was significantly higher in transgenic plants compared to the wild-type.