Agroinoculation and Agroinfiltration: Simple Tools for Complex Gene Function Analyses

Abstract

Agroinoculation, first developed as a simple tool to study plant-virus interactions, is a popular method of choice for functional gene analysis of viral genomes. With the explosive growth of genomic information and the development of advanced vectors to dissect plant gene function, this reliable method of viral gene delivery in plants, has been recruited and morphed into a technique popularly known as agroinfiltration. This technique was developed to examine the effects of transient gene expression, with applications ranging from studies of plant-pathogen interactions, abiotic stresses, a variety of transient expression assays to study protein localization, and protein-protein interactions. We present a brief overview of literature which document both these applications, and then provide simple agroinoculation and agroinfiltration methods being used in our laboratory for functional gene analysis, as well as for fast-forward and reverse genetic screens using virus-induced gene silencing (VIGS).

AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings

“…Because Arabidopsis plants are less amenable for transient expression analysis, both fluorescent protein localization and bimolecular fluorescence complementation (BiFC) studies are often conducted in protoplasts via transfection or in N. benthamiana leaves via agroinfiltration because of the high transient expression efficiency [16,17]. Here, we co-infected two A. tumefaciens strains carrying a binary vector for 35S::Venus-intron or 35S::NLS-RFP in efr-1 seedlings and detected both cytoplasmic and nuclear fluorescence signals for Venus and nuclear localization of NLS-RFP in separate or the same cells (Figure 7K).…”

“…This fast, sensitive, and quantitative assay was routinely used with culture plates, which are easily scaled up for quick and systematic screens. Importantly, this method nicely compliments the commonly used Arabidopsis mesophyll-protoplast transfection [15,16] and Agrobacterium- mediated leaf infiltration in N. benthamiana [17] for gene functional studies and provides advantages for its high reproducibility without advanced skills. Furthermore, AGROBEST may be an alternative method for evaluating Agrobacterium virulence and discovering and dissecting gene functions involved in various steps of Agrobacterium -mediated DNA transfer.…”

“…In plant biology research, Arabidopsis mesophyll-protoplast transfection [15,16] and Agrobacterium- mediated leaf infiltration in Nicotiana benthamiana [17] are the well-established and commonly used platforms for transient gene expression analysis. The Arabidopsis mesophyll-protoplast transient expression system allows for versatile and high-throughput analyses of diverse gene functions and signal transduction pathways; advanced skills with training and practice are essential for successful use of this powerful tool for gene function studies [16,18,19].…”

Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing

“…Cloned ALSV cDNAs first need to be inoculated into Chenopodium quinoa plants by mechanical inoculation, and then the virus propagated in C. quinoa plants is used as an inoculum for target plants. Agroinoculation methods have been established to introduce many plant viruses into plant tissues because Rhizobium radiobacter (previously Agrobacterium tumefaciens ) can easily introduce viral genomes and foreign genes into plants by means of a simple inoculation method (Vaghchhipawala et al, 2011). …”

Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins

“…Moreover, it has the flexibility of either infiltrating the whole leaf with one target DNA construct or introducing multiple constructs into different areas of one leaf, permitting multiple assays to be performed on a single leaf [16]. These advantages have allowed syringe infiltration to become the method of choice for transient gene expression, in a broad range of applications, including studies of plantpathogen interactions, abiotic stresses, plant gene functional analysis with transient silencing assay, protein localization and function, and proteinprotein interactions [16]. Entire leaf syringe agroinfiltration can be applied to obtain laboratory scale of recombinant proteins, for their biochemical characterization, purification and preclinical functional studies [1,20,21].…”