Model pds 1000 he

For agroinfiltration, Agrobacterium EHA105 strain harboring the tested expression construct(s) was grown in LB medium supplemented with spectinomycin (100 μg/ml) overnight at 28°C. Cells were harvested by centrifugation and resuspended to optical density of A₆₀₀ = 0.1 in infiltration buffer [10 mM MgCl₂, 10 mM MES (pH 5.5), 100 μM acetosyringone]. Bacterial suspension was incubated for 2 h at room temperature and infiltrated into the abaxial sides of 3- to 4-week-old intact N. benthamiana leaves with a 1-ml needleless syringe. Plants were grown for 48–72 h under standard growth conditions before being harvested.
For biolistic delivery, DNA preparations of the tested constructs were mixed at a 1:1 w/w ratio, and 100 μg DNA was adsorbed onto 10 mg of 1-μm gold particles (Bio-Rad, Hercules, CA). These microprojectiles were bombarded into the leaf epidermis of N. benthamiana using a portable Helios gene gun system (Model PDS-1000/He, Bio-Rad) at a pressure of 90–150 psi, and tissues were analyzed 48 h after microbombardment.

Single-cell transient gene expression assay using biolistic delivery of plasmid DNA into barley epidermal cells was performed as previously described [77] (link). For MIRNAs overexpression, the β-glucuronidase (GUS) reporter gene was mixed with respective plasmids (molar ratio 1∶1) before coating of gold particles. Barley leaf epidermal cells were transformed with the biolistic particle delivery system (Bio-Rad, Model PDS-1000/He), and incubated 4 hrs before inoculation of Bgh spores. To identify transformed cells, bombarded leaves were fixed in solution at 48 hrs after Bgh infection and further stained for GUS activity. For MIRNAs and Mla co-expression, the GUS reporter and MIR9863b plasmids were co-coated with Mla1-mYFP or Mla10-mYFP plasmids (molar ratio 1∶1∶1).