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Bacteriocins are a diverse group of bacterial antimicrobial peptides (AMPs) that represent potential replacements for current antibiotics due to their novel modes of action. At present, production costs are a key constraint to the use of bacteriocins and other AMPs. Here, we report the production of bacteriocins in planta - a potentially scalable and cost-effective approach for AMP production. Nine bacteriocin genes with three different modes of action and minimal or no post-translational modifications were synthesized, cloned and used to transform Arabidopsis thaliana. To confirm bacteriocin functionality and the potential to use these plants as biofactories, Arabidopsis T3 crude leaf extracts were subjected to inhibition assays against the bacterial pathogens Clavibacter michiganensis subsp. michiganensis (Cmm) and Pseudomonas syringae pv. tomato DC3000 (Pst). Six and seven of nine extracts significantly inhibited Cmm and Pst, respectively. Three bacteriocin genes (plantaricin, enteriocin, and leucocin) were then selected for over-expression in tomato (Solanum lycopersicum). In vitro plant pathogen inhibition assays of T0, T1 and T2 transgenic tomato leaf extracts confirmed antimicrobial activity against both pathogens for all three generations of plants, indicating their potential use as stable biopesticide biofactories. Plantaricin and leucocin-expressing T2 tomato plants were resistant to Cmm, and leucocin-expressing T2 plants were resistant to Pst. This study highlights that plants can be used as biofactories for AMP production and that the expression of bacteriocins in planta may offer new opportunities for disease control in agriculture.

In the present study, we developed an efficient and reliable protocol for in vitro plant regeneration and transient gene expression in medicinal plant Artemisia annua L. For in vitro regeneration, leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.5 mg/l NAA plus 2 mg/l BA (86.6%) and MS medium supplemented with 1 mg/l IAA plus 2 mg/l BA (80%). Root induction was obtained on MS medium supplemented with 0.1 mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. annua L. in short period via adventitious shoot induction approach. Based on the sequence of a putative sesquiterpene cyclase from A. annua , 1.7 kb fragment was amplified by PCR from Iranian variety of this plant after cDNA synthesis. Sesquiterpene cyclase gene was used for sub-cloning into pBI121 between Xba I and Sac I sites to allow the expression of the gene driven by CaMV35S promoter. The recombinant plasmids were transferred to competent cells of Agrobacterium tumefaciens strain GV 3850 for vacuum agro-infiltration. HPLC results showed 30.09% increase in atremisinin content (0.15%) in the agroinfiltrated samples (0.67%) in comparison to the control (0.51%).