RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

The overall goal of this work is to provide a reliable, accurate, inexpensive, and fast method for testing the efficacy of RNAi mediated silencing of aflatoxin synthesis genes of aspergillus in transgenic peanut using a minimum number of seeds, usually hundreds of grams of seeds are required to effectively quantify aflatoxins in samples. The main advantage of this technique is that it uses less than five seeds to test a particular treatment 10 days in advance of preparing the seeds set up a culture of aflatoxin positive aspergillus flavus NR RL 3 3 5 7 in ZAP Pex auger medium. Grow this culture at 25 degrees Celsius.

It will be used for seed inoculation. After preparing the transgenic plants expressing the RNAI, which is detailed in the text protocol, proceed with harvesting their seeds. First, remove the peric carp using abrasion from a pressure washer set to low, or scrape the peric carp off by hand.

Once the peri carp is removed, identify the color of the meso carp by placing the pods on a maturity board into the proper groups. Now remove the holes and process the yellow and brown seeds separately. Calculate the number of seeds needed for the experiment at least three seed pieces, meaning three co leadin halves are needed per peanut line being sampled.

Cover the bottom of a sterile beaker with a layer of seeds, and then just cover the seeds with a measured volume of 75%ethanol. Then double that volume. Let the seeds incubate in solution for 30 seconds, and then rinse them off in sterilized distilled water.

Next, treat the seeds with a volume of 2%hypochlorite as done with the F ethanol. Let this incubation proceed for five minutes. Then use three rinses in five-fold volumes of sterilized distilled water.

To remove the hypochlorite, it is critical that all the hypochlorite solution is washed off in this step. The seeds are now surface sterilized. Next, hydrate the seeds by submerging them in sterilized distilled water for two hours prepared to put the hydrated seeds on a sterile Petri dish.

All materials should be sterilized at this point. Remove the seed coats using forceps, then separate the cohain. Lastly, remove the embryos with a scalpel.

The embryos can be discarded or used to regenerate new plants. Next, cut the olein in half with a scalpel and submerge the halves and sterilized distilled water until they are ready for inoculation. Next blot excess water from half co leadin on sterile paper towels and place seed pieces in separate 1.5%auger plates with seed size divots and tuck the seeds into them with the cut sides up to inoculate the co leadin halves.

Add two microliters of a 10, 000 aspergillus spores per microliter suspension to the cut surfaces. Do not let the suspension run down the sides of the seed halve. Lastly, place the dishes in a dark incubator at 30 degrees Celsius for one to four days until they are tested.

After one day, after two days, and after three or four days sample the seeds gently remove randomly selected inoculated halves as needed. Use a tissue to wipe excess spores and auger and place the sample into a glass screw cap vial. Transfer non inoculated samples to two milliliter tubes for R-T-P-C-R analysis.

Free samples in liquid nitrogen and store frozen samples in a freezer until they are processed. For the aflatoxin analysis, get the inoculated half cot leadin in a four milliliter glass screw cap vial if needed. The sample will be stable for many months at negative 80 degrees Celsius.

When the seeds are at room temperature, extract the sample by adding four volumes of methanol and letting the tubes incubate overnight in the dark at room temperature without agitation. The next day, perform the UPLC first, prepare a 1.5 milliliter propylene mini column with a matching frit. Then add 200 milligrams of aluminum oxide.

And next, insert another frit. Next position, A-U-P-L-C autos sampler vial under and in contact with the mini column to minimize evaporation. Now add 0.5 milliliters of methanol extract from the sample to a disposable glass two not plastic.

Then add 0.5 milliliters of aceto nitrile to the tube and mix the solutions with a pipette. Now apply 0.5 milliliters of the mixture to the prepared mini column and collect the EIT using gravity only. Once the EIT is collected quickly attach a septic cap to the collection vial for use on the UPLC.

Place the vials in A-U-P-L-C already prepared with a mobile phase of water methanol and acedonite trial. Then quantify the aflatoxin content in the EITs. See the text protocol for more details along with UPLC analysis.

Set up the seed pieces used for extraction in individual glass vials. Then lyophilize the vials overnight and measure the dry weight of the tissue sample in the morning so aflatoxin concentrations can be expressed in nanograms per gram of sample. Then remove the samples from the freezer and add triol and grind the frozen tissues with a bead mill homogenizer at 3, 100 RPM for 40 seconds and proceed to RNA extraction an RNAI vector carrying small fragments of five aflatoxin synthesis genes from a flavis was used to transform peanut plants.

The identification numbers correspond to the broad institute's genome annotation numbers from 99 regenerated TRANSFORMANTS seven PCR positive lines were cloned and tested as described. All seven showed 60 to 100%less aflatoxin accumulation compared to control lines. Results from two of the seven lines are presented.

These two RN AI lines showed the presence of the NPT two selectable marker. According to the S-T-N-P-C-R results, the negative control used was a PCR negative line that went through the transformation process to test the RNAi lines for their ability to resist aflatoxins freshly harvested kedia of an aflatoxin positive. A flavis line NRRL 3 3 5 7 were applied to half cot leadin lacking embryo and testa after incubation for up to 96 hours.

The inoculated tissues were processed using UPLC to measure their aflatoxin levels as described. These results were further confirmed by liquid chromatography mass spectrometry. Overall, RNAi 2 88 72 showed a 94 to 100%reduction in aflatoxin B two and a 90 to 100%reduction in aflatoxin B one compared to the control.

Whereas RNAi 2 88 74 showed a 60 to 100%reduction in both aflatoxins. Following this procedure, the same seed extracts can be used to analyze phyto Xin in order to better understand the seed response to fungal invasion. This method will also provide a unique tool to help develop RNAI technology for the control of mycotoxins.Oxon.