If its are effective transmitters of plant viruses, if it micro injection of virus, the procedure we will show you today is a technique allowing researchers to inject virus directly into the hemos cell of the AFib, bypassing the gut, one of the two major barriers for virus transmission in a circulative manner. Hi, I am Cecilia Tamrin Dehi post doc in the plant pathology department in Cornell University. Today I'm going to show you how to inject virus into the AFI hemo cell virus that can then be transmitted to different plant species.
For example, this FE plant is infected with PLRV potato leaf virus. You can see the difference between a healthy and an infected plant. The most notable symptom is the taven sclerosis.
PLRV is a luthier virus that is carried by APHIS in their hemolymph, the insect equivalent of mammalian blood. When an uninfected AFI feeds on an infected plant, it contracts the virus through the plant flu, which you can see here. Once ingested, the virus must pass from the insect gut to the hemolymph and then must pass through the salivary gland in order to be transmitted back to the ESE plant.
And if it may take many viruses when feeding on a ESE plant, however, only as more fraction will pass through the gut and salivary gland. The two main barriers for transmission to infect more plants to test. If an AFI disabled to vector a virus, we feed the afis on infected tissue for two days, then we allow it to feed in a healthy plant for five days.
The plant is then fumigated and after three weeks we start searching for symptoms. When we find an AFib that cannot vector a virus, we can assess which of the two barriers is stopping the virus using the microinjection procedure, which I'm going to show you today. In this procedure, the virus is injected directly into the hemo cell there, right bypassing the gut transmission barrier.
So let's get started and I'm going to show you how to micro inject some apex. So this is the glass, the glass needles that we use. We pull down glass needles from this glass shields using APU apu.
So this is the micro injector. To do the micro injection of a virus. I'm going to start putting the needle into the needle holder.
So I'm just put the needle and and put it in place. So now I'm going to load the virus into the needle to the top of this helm. Now we need to position AFI for microinjection.
So I'm going to take an AFI with the pain pressure and I'm going to place it in the AFI holder. Ideally, I'm going to try to place the back of the aphid against the holder so I can have access to the abdominal part of the AFib. Okay, great.
Now we can go ahead and micro inject. Okay, now I'm going to position the needle and I'm going to bring it closer to the AFib. So now I'm going to bring the needle into contact with the AFib and to pierce the exoskeleton, the best thing is to try to pierce in between the segment at the base of the legs.
So I'm going to inject. Now I'm taking the AFI from with the brush and I putting into the recover paper where I'm going to allow it to record for a moment. So we need also to micro inject control afi.
This control micro injection is done only with the buffer without the virus. The buffer that we use is 0.1 more phosphate buffer pH seven. So I'm going to place the A fit in a healthy plant where I'm going to allow him to fit for five days after I'm going to fate the plant and wait for symptoms development after micro injection of the lower stranded NA during a week each day, the level of expression of the target gene is measured in the afi.
So we have just micro injected an afit that was unable to transmit the virus. If after the microinjection, the AFI is able to transmit the virus, it will mean that this AFI has a gut barrier that was blocking the virus. If in the other hand there's no symptoms development, it'll mean that this afit has a strong salivary gland barrier.
So we have just shown how to micro inject virus into the aphids. This same procedure is used to micro inject those stranded RNA into the aphids to perform RNI eye interference, which is a interesting technique to help studying gene function in aphid. So that's it for the micro injection.
I hope you enjoy it and it'll be helpful for your experiments. Thank you.
