Multiplicity of the Agrobacterium Infection of Nicotiana benthamiana for Transient DNA Delivery.
ACS Synth Biol
; 12(8): 2329-2338, 2023 08 18.
Article
em En
| MEDLINE
| ID: mdl-37558215
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 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 versus multiple transfection events per cell. Despite the value of Agrobacterium-mediated transient transformation of plants, MOI has not been quantified. Here, we analyze the Poisson probability distribution of the 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 OD600, plant cell perimeter, and leaf age, as well as plant cell coinfection rates. Our analysis 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 at infiltration OD600 above â¼0.2 for individual strains. In contrast, single-strain T-DNA delivery can be achieved at low strain infiltration OD600 at OD600 0.02, we observe that â¼40% of plant cells are infected, with 80% of those infected cells containing T-DNA product from just a single strain. We anticipate that these data will enable users to develop new approaches to in-leaf library development using Agrobacterium transient expression and reliable combinatorial assaying of multiple heterologous proteins in a single plant cell.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Nicotiana
/
Agrobacterium
Tipo de estudo:
Prognostic_studies
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article