RNA-Dependent RNA Polymerase Gene's Effect of Viral Silencing

Benjamin Downing & Erica Schufeldt Author

04/05/2021 Added

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Plant Virus Interactions and Viral Silencing

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[00:00:01.950]Okay. Hello. My name is Erica and I,
[00:00:05.250]this is my partner Benjamin,
[00:00:07.380]and we'll be talking about our RNA dependent RNA polymerase genes
[00:00:12.120]effect on viral silencing. So plant, I mean,
[00:00:15.780]systems are relatively robust consisting of proteins and enzymes that are
[00:00:20.310]capable of protecting the plant. Specifically.
[00:00:23.070]The antiviral immune system in plants is mediated by RNA silencing and RNA
[00:00:27.840]decay mechanisms.
[00:00:29.580]Systemic silencing is mediated by enzymes called RNA dependent,
[00:00:33.480]RNA polymerases or RD RPS.
[00:00:36.300]That's synthesized double-stranded RNA from single-stranded RNA,
[00:00:40.140]allowing the cells to silence the viral genome,
[00:00:42.660]to protect the plant from infection. However,
[00:00:45.810]some viruses are able to utilize RD RPS for their own benefit.
[00:00:50.010]For our study, we use the turnip mosaic virus or tump,
[00:00:55.230]which is a well-known positive sense.
[00:00:57.180]Single-stranded RNA virus with a flexible filamentous structure
[00:01:02.640]upon infection in a plant to UMB is able to utilize host gene transcripts to
[00:01:07.530]produce S I RNs wellbeing, D C L R four,
[00:01:12.120]and already are dependent and ultimately results in the
[00:01:16.560]silencing of host genes.
[00:01:18.870]RDRP has been shown in previous research to be necessary for genome
[00:01:23.280]replication of Podio viruses, such as two MB.
[00:01:26.700]So we wanted to investigate how an amino acid substitution mutation of the
[00:01:31.260]RDRP gene would impact the proteins function in host genome
[00:01:35.880]silencing.
[00:01:40.610]Hi, I'm Ben, uh,
[00:01:42.140]built our half of this team and I'll kind of walk you through one of the
[00:01:46.730]projects that are one of the experiments that we did for this project.
[00:01:50.510]As you can see here,
[00:01:51.590]we have a wild type transient essay where we developed
[00:01:56.390]a plasmid that, um,
[00:01:58.610]contained our mutation in the tum V as long as along
[00:02:03.470]with a green fluorescent protein tag,
[00:02:07.130]which under UV light will show us the movement and location of the plasmid
[00:02:12.050]in our viral particles. So as you can see here through the,
[00:02:16.870]the figure we have our time course, um, uh,
[00:02:20.930]from one day past infiltration all the way to seven days past
[00:02:25.250]infiltration.
[00:02:26.480]And I think the key thing to take out of this assay and the results show
[00:02:31.010]that while the one 97 M
[00:02:35.570]um, RDRP mutant, um,
[00:02:38.780]does show infection it's at a much lower rate, much lower rate,
[00:02:43.580]or at a slower, uh,
[00:02:47.060]rate of infection compared to the ASM nine,
[00:02:50.720]which our lab is previously studied and characterized its, um,
[00:02:55.430]effects on gene silencing and viral.
[00:03:01.000]And so through these results,
[00:03:02.350]we concluded that there must be some mechanism of
[00:03:08.290]in the genetic change or through other
[00:03:12.460]processes that caused the,
[00:03:16.090]the virus particles in the RDRP mutant to, uh,
[00:03:21.100]have this lower response.
[00:03:25.860]And then,
[00:03:26.490]so with those results where we discovered the lower results and the
[00:03:31.320]one 97, uh, mutate,
[00:03:33.960]or the one 97 RDRP mutated, um,
[00:03:38.550]virus,
[00:03:39.240]we decided to get the leaves
[00:03:43.860]imaged through transmission electron microscopy,
[00:03:48.570]which we'll be able to show us, um, at a microscopic level,
[00:03:52.980]whether or not the virus particles, what their, um,
[00:03:56.100]what their confirmation was and in what level of concentration they were at.
[00:04:01.320]And so with help from, um, uh, for the professor,
[00:04:05.790]Dr. Joe in the fetal center,
[00:04:09.030]we are able to take these really good, um, transmission,
[00:04:12.450]electron microscope pictures, um,
[00:04:15.570]and this figure kind of just illustrates what we found with those results.
[00:04:20.610]Um,
[00:04:21.270]as you can see on the left side of the figure with the ASMR nine,
[00:04:27.240]there are clearly a large amount of viral particles
[00:04:32.370]and, um,
[00:04:35.520]they're in high concentration and they look very straight and kind of
[00:04:39.720]characteristic of what we would think the fibrous would look like normally. Um,
[00:04:44.460]and if that might be hard to see,
[00:04:45.810]I can kind of outline some of these right here.
[00:04:48.960]So you can see that the viruses particles are kind of these long,
[00:04:53.430]straight filamentous strands. And as we increase in,
[00:04:58.140]uh, magnification,
[00:04:59.400]obviously we can see a better picture of it here on the lower end.
[00:05:03.060]And then if we shift our focus more to the right side,
[00:05:05.820]we can see while the RDRP is
[00:05:10.620]creating virus particles, as I'm outlining here,
[00:05:14.820]they are in much lower concentration and much lower amounts
[00:05:20.160]within the same magnification and therefore same space.
[00:05:24.390]So we know that, um,
[00:05:28.410]the RDRP mutant is creating our viral viral particles,
[00:05:33.360]but there's something where it, the amount and concentration is a lot lower.
[00:05:46.830]And then.
[00:05:48.000]Following this, we decided to investigate what the systemic,
[00:05:52.860]um, silencing characteristics were of these mutants. Um,
[00:05:56.880]as you can see here in this figure, we used plants that, um,
[00:06:01.970]natively through a mutation, uh, expressed GFP.
[00:06:07.640]And so with the viral,
[00:06:12.290]uh, particles that we collected from wild type infected plants,
[00:06:16.640]we harvest those, uh, four days,
[00:06:19.280]four to five days after initial infiltration into the wild type plants
[00:06:24.050]and through a process of enrichment and another, uh,
[00:06:28.160]inoculation,
[00:06:29.420]we're able to introduce the viral particles from the wild type plants into the
[00:06:34.040]16 seed plants that express the GFP.
[00:06:37.550]And this is April to give us an illustration, um,
[00:06:41.030]very visually on whether or not the plant or the virus is able
[00:06:45.710]to, uh, have that, uh,
[00:06:47.660]systemic silencing effect on the plant and at what
[00:06:52.520]rate it does. And as you can see in the graph, um, through time,
[00:06:57.320]the [inaudible] plant, as we, as our lab has previously,
[00:07:01.550]already discovered is still able to create the systemic, uh,
[00:07:06.080]silencing that you see on the lower right hand portion of the
[00:07:10.700]figure. However, in the case of the RDRP gene,
[00:07:14.270]we found that while there are,
[00:07:16.910]might be some evidence for a minimal amount of silencing, there,
[00:07:20.690]isn't a strong amount that's noticeable in specific places,
[00:07:25.610]especially in a systemic, um, silencing fashion.
[00:07:34.570]All right. So while we did find to TuMV,
[00:07:36.760]RDRP mutant is making particles in the plants. Um,
[00:07:40.650]these viral particles have been unable to actually show the silencing of the GFP
[00:07:44.500]reported gene in the ne 16 C Nicotiana,
[00:07:47.440]but the Miata plants as seen in the last figure.
[00:07:50.410]So this leads us to conclude that there may be other genes such as the HC
[00:07:53.950]protein that are vital to viral silencing in plants.
[00:07:57.670]So future research into other proteins and genes of plant immune systems is
[00:08:01.480]needed to better understand the mechanism of viral silencing.
[00:08:06.310]Thank you. Um,v

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RNA-Dependent RNA Polymerase Gene's Effect of Viral Silencing

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