Campus Units
Plant Pathology and Microbiology
Document Type
Article
Publication Version
Published Version
Publication Date
2-2021
Journal or Book Title
Journal of Visualized Experiments
Volume
168
First Page
e62277
DOI
10.3791/62277
Abstract
Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22) or phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.
Copyright Owner
JoVE
Copyright Date
2021
Language
en
File Format
application/pdf
Recommended Citation
Beernink, Bliss M.; Holan, Katerina L.; Lappe, Ryan R.; and Whitham, Steven A., "Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones" (2021). Plant Pathology and Microbiology Publications. 305.
https://lib.dr.iastate.edu/plantpath_pubs/305
Included in
Agricultural Science Commons, Agriculture Commons, Plant Breeding and Genetics Commons, Plant Pathology Commons
Comments
This article is published as Beernink, B.M., Holan, K.L., Lappe, R.R., Whitham, S.A. Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones. J. Vis. Exp. (168), e62277, doi:10.3791/62277 (2021). doi:10.3791/62277.