Multiplicity ofAgrobacteriuminfection ofNicotiana benthamianafor transient DNA delivery

Abstract Biological DNA transfer into plant cells mediated by Agrobacterium represents one of the most powerful tools for the engineering and study of plant systems. Transient expression of transfer DNA (T-DNA) in particular enables rapid testing of gene products and has recently been harnessed for facile combinatorial expression of multiple genes. In analogous mammalian cell-based gene expression systems, a clear sense of the multiplicity of infection (MOI) allows users to predict and control viral transfection frequencies for applications requiring single vs. multiple transfection events per cell. Despite the value of Agrobacterium -mediated transient transformation of plants, MOI has not been quantified. Establishing MOI for Agrobacterium T-DNA delivery at the level of single plant cells would allow users to design genomic library delivery conditions (at most 1 event/cell), or maximize co-delivery of T-DNA loads from separate Agrobacterium (>1 event/cell). Here, we analyze the Poisson probability distribution of T-DNA transfer in leaf pavement cells to determine the MOI for the widely used model system Agrobacterium GV3101/ Nicotiana benthamiana . These data delineate the relationship between an individual Agrobacterium strain infiltration OD 600 , plant cell perimeter and leaf age, as well as plant cell co-infection rates. Our analysis also establishes experimental regimes where the probability of near-simultaneous delivery of >20 unique T-DNAs to a given plant cell remains high throughout the leaf. We anticipate that these data will enable users to develop new approaches to in-leaf library development using Agrobacterium transient expression and the reliable combinatorial assaying of multiple heterologous proteins in a single plant cell.