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 leaves – 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 Ti-plasmid 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. coliβ-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.