Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing

Abstract

Agroinfiltration--the infiltration of Agrobacterium tumefaciens into intact plant levels--provides a rapid and simple way of screening large numbers of transgene constructs for silencing in response to a resident transgene. Transgenic Nicotiana sylvestris plants homozygous for the tobacco class I chitinase A gene CHN48 under the control of the cauliflower mosaic virus 35S RNA promoter (P35S) show a high incidence of postranscriptional gene silencing. We forced suspensions of A. tumefaciens, carrying P35S-CHN48 in a binary Tiplasmid vector, into wild-type and transgenic N, sylvestris leaves with a blunt-tipped plastic syringe. The infiltrated CHN48 transgene was expressed in leaves transformed with the vector alone, but not in CHN48-transformed leaves showing the silent phenotype. In contrast, expression of a chimeric P35S-E. coli beta-glucuronidase gene (uidA) infiltrated into leaves was not affected by the presence of the CHN48 transgene stably integrated in the host genome. These results show that extra copies of CHN48 are silenced by resident, silent copies of the same gene and confirm that CHN48 silencing is not the result of promoter interactions. The results also suggest that silencing of the additional CHN48 copies does not require their integration into chromosomes.

A simple and efficient transient transformation for hybrid aspen (Populus tremula × P. tremuloides)

“…We first examined whether the syringe injection technique, used in tobacco species, was useful for hybrid aspen leaves [14]. An infiltration medium [10 mM MgCl 2 and 5 mM MES-KOH (pH 5.6)] in a blunt-tipped plastic syringe was forced into an abaxial epidermis of full-expanded aspen leaves that grew in soil for one month.…”

“…To avoid these drawbacks, another transient assay such as Agrobacterium -mediated infiltration would be useful for Populus trees. This assay allows for a simple transformation process, easy operation, and high transformation efficiency in several plant species including Arabidopsis , grapevine, potato, switchgrass, tobacco, and tomato [6,7,14-17]. …”

Passive immunoprotection targeting a secreted CAMP factor of Propionibacterium acnes as a novel immunotherapeutic for acne vulgaris

“…For syringe infiltration, the central lower epidermises (i.e., the centermost 25 mm 2 area) of potted seedlings leaves were wounded with a sterile scalpel (number 15, Feather Safety Razor Co., Osaka, Japan) and 0.1 ml of Agrobacterium bacterial suspension (5×10 7 CFU) was injected into the wound site, which was positioned between a finger and a 1 ml syringe (BD, Bioscience, San Diego, CA). Infiltration was confirmed by visually monitoring the diffusion of bacterial suspension toward the leaf margin [48]. Agroinfiltrated leaves were grown for five days before GUS assays and immunization was performed.…”

The tomato xylem sap protein XSP10 is required for full susceptibility to Fusarium wilt disease

“…Silencing in T 1 progeny and T 0 parents was assayed by screening for reduced GUS expression upon leaf infiltration with A. tumefaciens strain C58C1 harbouring plasmid pTFS40 (Jones et al , 1992), which allows GUS expression in planta (Wroblewski et al , 2005). Leaf infiltrations were performed as described previously (Schob et al , 1997). Histochemical GUS staining was done essentially as described before (Jefferson et al , 1987).…”