Developing a Replication System for Maize Chlorotic Mottle Virus in Yeast

Annika Pratt Author

07/28/2021 Added

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Maize Lethal Necrosis Disease is a corn disease caused by the synergistic relationship between Maize chlorotic mottle virus (MCMV) and a Potyvirus. To combat the disease, we are developing a system to replicate MCMV in yeast, which will allow us to study MCMV in a time-efficient and effective way.

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[00:00:01.240]Hello, my name is Annika
[00:00:02.837]and this summer I have been developing a replication system for Maize chlorotic mottle virus in yeast.
[00:00:10.730]Understanding Maize chlorotic mottle virus, or MCMV, is essential in combating Maize Lethal Necrosis Disease
[00:00:18.467]which is caused by the coinfection of MCMV and a virus of the family Potyviridae
[00:00:23.490]such as Sugarcane mosaic virus.
[00:00:24.870]MCMV is found around the world and is currently devastating crops in Sub-Saharan Africa
[00:00:31.000]which is leading to a rise in corn prices
[00:00:34.170]as well as an increase in malnutrition in those countries.
[00:00:38.130]In figure A, we can see healthy corn plants here in Nebraska as
[00:00:43.100]compared to figure B
[00:00:44.970]which shows corn plants with Maize Lethal Necrosis Disease in Kenya
[00:00:49.710]Figure C shows, from left to right
[00:00:53.310]a mock infected corn plant, so a corn plant that is not infected with a virus
[00:00:59.070]a plant that's infected with Sugarcane mosaic virus
[00:01:02.730]a plant that's infected with Maize chlorotic mottle virus
[00:01:06.150]and then a plant that's infected with both Sugarcane mosaic virus and Maize chlorotic mottle virus.
[00:01:13.500]Here we can see that one virus does not have a huge impact on plant health
[00:01:20.760]but it's two viruses together that is causing Maize Lethal Necrosis Disease
[00:01:25.920]So, Maize chlorotic mottle virus
[00:01:29.070]plus any virus in Sugarcane mosaic virus' family
[00:01:32.730]is going to cause this disease and kill corn.
[00:01:37.860]Scientists have been unable to identify any natural
[00:01:42.330]resistance to MCMV,
[00:01:44.970]so our goal is to develop resistant plants. To accomplish this goal,
[00:01:50.430]we need to understand how Maize chlorotic mottle virus replicates, and to do that,
[00:01:55.600]we are using yeast because it has proven itself as an effective and time
[00:02:00.060]efficient model organism for plant viruses.
[00:02:03.240]To be able to study MCMV in yeast, we first need to ask
[00:02:08.070]Can Maize chlorotic mottle virus replicate in yeast?
[00:02:13.010]To answer this question
[00:02:14.980]we'll focus on proteins p50 and p111 of MCMV which are required for replication
[00:02:21.000]as well as sgRNA1 whose proteins are used for viral movement.
[00:02:28.360]We created clones of p50, p111, and sgRNA1.
[00:02:34.550]To p50 and p111
[00:02:37.570]we attached tags so that we will be able to detect these proteins.
[00:02:42.220]To all of the clones, we added a galactose inducible promoter
[00:02:46.730]so when these clones are put in yeast cells
[00:02:51.458]and those cells are put in a galactose media, transcription will start and proteins will form
[00:02:58.750]We inserted these three plasmids into yeast cells and called them family E.
[00:03:04.140]We also created a family N, which contains no viral genes and acts
[00:03:08.750]as a negative control.
[00:03:12.106]I grew these cells in galactose media to induce
[00:03:16.738]transcription, harvested the cells,
[00:03:19.350]and then did a protein and RNA extraction from those yeast cells.
[00:03:25.940]With the protein that was extracted from those cells
[00:03:29.890]I did a Western blot analysis for p50 and p111.
[00:03:35.080]So in figure B,
[00:03:38.190]we can see all the way on the right is our positive control
[00:03:42.370]which contains p50 from a plant.
[00:03:46.860]And here we can see p50 clearly
[00:03:52.780]in family E, which contains our p50.
[00:03:56.793]And then in our negative controls, we can't see any p50.
[00:04:00.894]So this was exactly what we were expecting to see. On the other hand
[00:04:06.280]figure A, we were trying to detect p111. So again,
[00:04:10.900]on the right hand side, we have our positive control from plants.
[00:04:15.880]And then in family E, we did not detect any p111.
[00:04:20.486]So this could be for two reasons.
[00:04:23.382]One of them is that p111 accumulates at much smaller amounts
[00:04:28.049]than p50. And we know that.
[00:04:31.026]So it could be that p111 is in our samples,
[00:04:35.095]but we're just unable to detect it because it's there in such small amounts,
[00:04:39.574]or it could be that p111 is not in our sample at all.
[00:04:46.118]In conclusion, Maize Lethal Necrosis Disease
[00:04:50.271]is a devastating disease caused by Maize chlorotic mottle virus.
[00:04:54.403]We're learning more about MCMV to develop resistance to this virus.
[00:04:59.823]We know from our study that MCMV can produce protein p50 in yeast
[00:05:05.873]and we are able to detect it. We will continue experiments
[00:05:10.850]to detect p111 as well.
[00:05:13.290]We will also do RNA analysis to determine whether MCMV is
[00:05:17.760]replicating in yeast
[00:05:19.650]and we'll determine whether p50, p111, or both proteins are necessary for replication.
[00:05:27.000]To end, I would like to thank everyone in the Garcia-Ruiz lab here at UNL
[00:05:31.989]as well as our partners at La Sierra University
[00:05:34.590]This has really been a team effort.
[00:05:37.000]I would also like to recognize the NIH and Nebraska Center for Virology for funding this work.
[00:05:44.280]Thank you so much for watching and have a wonderful day.

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Developing a Replication System for Maize Chlorotic Mottle Virus in Yeast

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