This protocol allows the inoculation of viral vectors directly into maize plants that can be readily applied in most lab settings without the need for any expensive specialized equipment. Direct inoculation eliminates the use of an alternative host as an inoculum source, saving both time and resources. This method could be applied to additional viral vectors, maize lines, and potentially other monocot species.
Begin by planting 1 to 2 maize seeds in the peat-based growing medium in small inserts placed inside trays. Place the trays in a growth chamber under 16 hour days at 25 degrees Celsius and 8 hour nights at 22 to 25 degrees Celsius, or in a greenhouse at 22 to 25 degrees Celsius with 16 hour days and 8 hour nights. Regularly water plants and fertilize once a week with 15-5-15 liquid fertilizer at 330 parts per million concentration.
Prepare agrobacterium for injection by inoculating the agrobacterium strain containing the desired viral construct in LB media with antibiotics. Then, incubate the flask or tubes at 28 degrees Celsius while shaking at 225 RPM for 24 hours. On the next day, pellet the bacteria by centrification at 4, 000 times G for 10 minutes at room temperature and discard the supernatant.
Then, wash the pellet with one milliliter of deionized water. Re-suspend the pellet in one milliliter of 10 millimolar magnesium sulfate solution, and measure the optical density at 600 nanometers. Then adjust it to 1.0.
Use 4 to 7 day old maize seedlings for injecting agrobacterium. Assemble the syringe and needle. Gently inject the bacterial suspension 2-3 millimeters above the coleoptilular node until it fills up the coleoptile, or is visible in the whorl, pending on the growth stage of the plants.
Inject all seedlings and change the syringe and needles for injecting each construct. Confirm phenotypic infection by observing lesions from silencing the control genes, lesion mimic 22 or phytoene desaturase on the leaves. Use a fluorescence imaging device for quick screening of plants and fluorescence microscopy to detect the presence of GFP expression.
Perform gene expression analysis for molecular detection of infection. Extract total RNA from leaves of infected plants 14 to 21 days after infection, and synthesized first strand cDNA as described in the text manuscript. Perform reverse transcriptase PCR to confirm infection and determine the gene's integrity or gene fragment of interest using specific primers designed for different viral constructs.
Visualize the PCR product on a 1%agarose gel containing a nucleic acid stain to determine the presence or absence of virus and gene or gene fragment. This protocol was used to insert recombinant viruses engineered for gene into maize seedlings. Approximately 12 days after injection, silencing phenotypes were observed on leaves as necrosis and a photobleaching.
The presence of construct in leaves after infection was detected by observing green fluorescent protein expression under a fluorescence using a different GFP filter. Green fluorescent protein expression in foxtail mosaic virus infected leaves was visualized as small punctate areas of fluorescence distributed across the leaves and as large patches in sugarcane mosaic virus infected leaves. Using the gene expression analysis, systemic foxtail mosaic virus infection was confirmed and gene silencing or suppression of phytoene desaturase and lesion mimic 22 was observed.
The construct also used influenced infection efficiency. In the case of foxtail mosaic virus infection, foxtail mosaic virus empty vector, and foxtail mosaic virus lesion mimic 22 typically had the highest infection efficiencies at 53%and 54%respectively. Foxtail mosaic virus phytoene desaturase at a slightly lower efficiency at 39%and foxtail mosaic virus green fluorescent protein, at the lowest efficiency at 16%Meanwhile, the infection efficiency of sugarcane mosaic virus green fluorescent protein was 8%Timing and symptoms will be based on the viral vector and maize line used.
Lack of a visual phenotype may not mean a lack of silencing or expression. This method can also be applied to gene editing technologies by improving delivery methods for guide RNAs.
