Differing Responses to Rust Pathogen in two Varieties of Switchgrass (P. virgatum)

Samuel Polk Author

04/04/2021 Added

16 Plays

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Research was conducted doing protein analysis to determine change in gene response due to rust pathogen in two varieties of switchgrass, a crop generally grown for biofuel and forage.

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[00:00:01.280]Hi, my name is Sam Polk.
[00:00:02.880]I'm a senior here at UNL studying plant biology.
[00:00:06.110]And today I'll be presenting my UCARE project.
[00:00:08.710]Differing responses to rust pathogen in two varieties
[00:00:12.410]of switchgrass supervised by
[00:00:14.230]Dr. Donald Lee and Dr. Gautam Sarath.
[00:00:18.870]So switchgrass scientific name, Panicum virgatum.
[00:00:22.491]Is a C4 grass in the family Poaceae
[00:00:25.500]It's generally grown for both biofuel and forage.
[00:00:29.180]Biofuel, generally, meaning ethanol production
[00:00:31.840]and forage, meaning food for grazing animals
[00:00:35.300]in a field environment.
[00:00:37.280]It's native to North America
[00:00:39.110]and is actually a part of the tall grass Prairie ecosystem
[00:00:42.040]we have here in Eastern Nebraska.
[00:00:45.000]Sometimes this plant is also used
[00:00:47.070]for restoration of these lost habitats.
[00:00:51.260]There are two main varieties of switchgrass
[00:00:53.510]or ecotypes of switchgrass
[00:00:55.513]Upland varieties, which grow in higher altitude environments
[00:01:00.710]and lowland varieties which
[00:01:02.250]grow in lower altitude environments.
[00:01:05.470]These two different varieties can have differences
[00:01:08.430]in their responses to biotic and abiotic factors
[00:01:12.160]based on the environment they generally
[00:01:14.180]grow in and have evolved in
[00:01:19.250]One such thing biotic factor that can differ
[00:01:21.430]between the two varieties is response to pathogens.
[00:01:25.150]So rust pathogen, the genus Puccinia
[00:01:28.870]is a fungal plant pathogen that infects many
[00:01:31.210]different types of crops grown throughout the world.
[00:01:33.800]It's a foliar pathogen, meaning that it affects
[00:01:36.320]the leaves of the plant and a biotrophic pathogen
[00:01:40.120]meaning that it saps the nutrients from a plant
[00:01:42.460]in order to live and grow on the plant.
[00:01:46.350]One sign of this disease
[00:01:48.210]on a plant includes dark raised pustules
[00:01:51.780]and one symptom includes yellowing of the leaves
[00:01:54.710]which is also called fluorosis.
[00:01:58.250]This can affect the yield of switchgrass
[00:02:00.330]since the rust pathogen saps the nutrients from the leaves
[00:02:04.070]and the switchgrass plant must put in resources
[00:02:08.640]in order to defend itself from the rest pathogen.
[00:02:13.700]So for my research goals
[00:02:15.220]I wanted to characterize the response to rust pathogen
[00:02:18.410]that switchgrass plants have
[00:02:20.660]in order to do that.
[00:02:21.720]I wanted to look at gene expression
[00:02:24.170]in order to look at gene expression.
[00:02:25.980]I decided to analyze the proteins
[00:02:28.540]in all of my samples
[00:02:30.560]this way I could find what proteins were higher
[00:02:33.110]for which treatment group
[00:02:36.100]I wanted to also look
[00:02:37.140]at the differing responses in different varieties.
[00:02:40.100]So my upland variety was named Summer
[00:02:42.800]and my lowland variety was named Kanalow.
[00:02:46.640]Hopefully this would characterize the differences
[00:02:49.420]in response between the two types of varieties.
[00:02:53.620]For my methods, my plants were grown
[00:02:56.120]in a greenhouse environment until the third leaf stage
[00:03:00.050]then inoculated with rust pathogen
[00:03:02.550]in placed in a humidity chamber for a few days.
[00:03:06.210]This allowed the spores to germinate.
[00:03:08.840]And after a few days
[00:03:10.250]they were taken back into the greenhouse.
[00:03:12.640]At this point, there were
[00:03:14.350]four collection times done over 14 days.
[00:03:17.473]These were four days after infection,
[00:03:19.990]seven days, 10 days, and 14 days.
[00:03:24.670]Three replicates were taken
[00:03:25.840]for each treatment time, point, and variety.
[00:03:29.420]And once those samples were collected,
[00:03:31.300]they were ground using liquid nitrogen.
[00:03:34.670]Then they were analyzed using
[00:03:36.380]SDS polyacrylamide gel electrophoresis standard procedures
[00:03:40.960]and Western blot standard procedures.
[00:03:43.150]This would allow me to separate my proteins
[00:03:47.300]from my samples out on a gel,
[00:03:49.290]and then transfer them to a blot
[00:03:50.870]in order to analyze the signal
[00:03:52.630]or the amount of those proteins in the sample.
[00:03:58.460]For my quantification
[00:03:59.920]I used software that allowed me to compare the volume
[00:04:04.370]of a signal with the background of the blot.
[00:04:07.400]This allowed me to quantify my various spans
[00:04:10.100]into numbers that I could use to compare to one another
[00:04:13.790]actin was used as a control.
[00:04:15.630]It's a protein that should be the same across treatments
[00:04:19.100]and correction factors were gained
[00:04:21.694]using the data that I got from my actin quantification.
[00:04:24.550]This allowed me to normalize the data in order to
[00:04:27.580]analyze it even more accurately.
[00:04:30.990]For statistical methods,
[00:04:32.320]I used a single factor, ANOVA to compare the control groups
[00:04:36.010]to the infected groups, with significance being given
[00:04:39.940]to a P-value of less than 0.05.
[00:04:44.770]The blots shown here give the results
[00:04:47.010]from the Prx4 or peroxidase 4 protein.
[00:04:51.470]The general setup of the blocks were a ladder on the left.
[00:04:55.040]Then three Kanalow control samples, three Kanalow infested,
[00:04:59.870]a blank lane, three Summer control samples,
[00:05:04.160]three Summer infested samples, another blank lane,
[00:05:07.580]and then another ladder on the right.
[00:05:11.100]We can see from these blocks
[00:05:11.933]there's a clear difference in peroxidase 4 enzyme
[00:05:15.310]between the controls and the infested
[00:05:17.560]as well as between the,
[00:05:19.540]varieties Summer and Kanalow.
[00:05:24.670]Once the quantification process
[00:05:26.450]was performed on the samples
[00:05:27.870]we got an average adjusted volume for the three
[00:05:30.360]different samples for each treatment in time point
[00:05:33.130]a standard error and a P-value.
[00:05:35.710]We found significance between the control
[00:05:37.860]and infested samples for Summer at all time points and
[00:05:41.620]for Kanalow at seven, 10, and 14 days after infection.
[00:05:48.830]Once the table was completed,
[00:05:50.400]I was able to make a graphical representation of my data.
[00:05:54.190]The first thing we can notice is that between
[00:05:57.409]between the Kanalow and the Summer,
[00:05:59.000]both varieties had an increase in peroxidase 4 protein
[00:06:03.530]after infection compared to the control groups.
[00:06:08.090]What we can also find is that the infested Kanalow plants
[00:06:11.910]had the highest total adjusted volume
[00:06:14.840]out of any of the treatments
[00:06:17.230]followed by the infested Summer plants
[00:06:19.430]than the Kanalow control plants.
[00:06:22.199]And finally, the Summer control plants.
[00:06:28.240]We also find that for the Summer plants,
[00:06:31.440]the peak of peroxidase 4 signal was found
[00:06:35.090]at about four days after infection.
[00:06:37.680]And the peak for the infested Kanalow plants
[00:06:41.330]was found about seven days after infection.
[00:06:48.326]A few other genes were also analyzed.
[00:06:50.635]However, we did not find any statistical significance
[00:06:54.455]between the control and the infested plants
[00:06:57.287]for these proteins.
[00:06:59.209]This includes PPDK, which is a C4 photosynthesis protein
[00:07:04.833]and CCOMT, which is a lignin biosynthesis protein
[00:07:11.108]It's been found in other studies that peroxidase 4
[00:07:14.445]in switchgrass might have a role in lignin biosynthesis,
[00:07:18.189]lignin being a molecule that strengthens cell walls.
[00:07:23.208]However, this can come with a downside
[00:07:25.997]higher lignin content generally leads
[00:07:28.023]to lower digestibility by animals
[00:07:30.708]and lower ease of conversion into biofuel.
[00:07:35.144]This can be negative
[00:07:36.224]because those are the two main reasons
[00:07:38.306]for why switchgrass is grown.
[00:07:41.188]So looking at our results,
[00:07:42.610]we find that once rust pathogen infects,
[00:07:46.058]both upland and lowland switchgrass
[00:07:50.098]that the lowland switchgrass produces
[00:07:53.315]a lot more of this lignin biosynthesis protein.
[00:07:56.925]Whereas the upland switchgrass produces
[00:07:59.428]more than it would normally.
[00:08:01.477]However, not as much as the lowland variety.
[00:08:05.303]For growers, this could mean that in upland variety
[00:08:08.972]of switchgrass will produce less
[00:08:11.143]of this lignin biosynthesis protein
[00:08:13.926]compared to the lowland variety
[00:08:16.495]of switchgrass upon infection by rust pathogens.
[00:08:19.927]As for future directions of this study,
[00:08:22.394]quantitative PCR which measures mRNA
[00:08:25.793]could be done to give another measure of gene expression
[00:08:30.258]and protein assays could also be done
[00:08:32.553]to bolster the data we already have on proteins.
[00:08:37.862]And that is all I hope you enjoyed my presentation.

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Differing Responses to Rust Pathogen in two Varieties of Switchgrass (P. virgatum)

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