Examining the Role of OCTOPUS in Arabidopsis thaliana Immunity

An Honors Thesis

Presented by

Kaitlyn Greenwood – Drury University

Completion Date:

December 2020

Approved By:

_____________________________________________

Richard Schur, Honors Director

_____________________________________________

Carina Collins, Professor of Biology

Kevin Jansen, Biology Department Chair

Drury University, 2019 © 2019, Kaitlyn Greenwood






ABSTRACT

Title: Examining the Role of OCTOPUS in Arabidopsis thaliana Immunity

Author: Kaitlyn Greenwood
Thesis/Project Type: (Choose either Thesis or Project)

The Food and Agriculture Association of the United Nations estimate that harm from
plant diseases on crops, globally effects 40% of crops and costs the economy $220 billion
annually. Past research has attempted to find ways of heightening a plants immune response
without sacrificing fruit production. It has been well documented that brassinosteriod contribute
to plant growth, but the balance between energy put towards growth or defense is a complex
process. One component in this process is the protein OCTOPUS (OPS) which is a presumed
positive regulator of brassinosteroid and shown to be a key element in root growth. Studying the
role of OPS in defense processes leads down the path towards an encouraging possible solution
to the afore mentioned issue. Through studying Arabidopsis thaliana OCTOPUS knockout
mutants treated with flg22, I show that OPS is a negative regulator of immune processes by
increased amounts of mRNA linked to immune signaling; without substantial hinderance of
growth.





Examining the Role of OCTOPUS in Arabidopsis thaliana Immunity

Introduction

A functioning immune system is critical for an organism’s survival. As humans, we rely

on our immune systems for protection and a breech of our defenses leaves us ill while our bodies

fight off the pathogen. Just like humans, plants have immune systems they rely on to keep them

healthy by fighting off pathogens. A number of viruses and bacterias can infect plants, and with a

poor immune system, the plant could die or become too ill to bear fruit. Determining immune

responses in plants would allow us a better understanding of how to work along side their

immune system to protect them.

The immune response in Arabidopsis begins with a Pattern Recognition Receptor

(PRR)1,2. These receptors are on the cell surface and recognize Pattern Associated Molecular

Patterns (PAMPs), which are typically peptides derived from bacteria. In my project, the PRR

utilized was Flagellin Sensitive 2 (FLS2), and the PAMP was flg22, a peptide chain made of 22

amino acids that resides in the flagellin of bacteria2.

Arabidopsis thaliana has been widely studied and is considered the ‘rat’ of plant

research. One gene in Arabidopsis that is relatively unknown is OCTOPUS (OPS). A majority of

the information on OPS is about the gene’s role in phloem differentiation and vasculature

patterns in cotyledons3. More recent studies have found that OPS is phosphorylated during an

immune response and that this phosphorylation may have an effect on plant growth and immune

response4,5. To further discover the function of OPS during immune response processes, I ran

experiments with wildtype (Col0) and ops Arabidopsis plants that had been artificially infected.





Our plants were treated with isolated flg22 so that they would elicit an immune response

without being harmed by the full pathogen. After recognizing infection from flg22, a cascade of

events occurs. Approximately 30 minutes after receptor binding, due to a calcium influx into the

cell, the PHI1 gene becomes activated and expressed within the cell. The WRKY33 gene is also

activated at this time but as a result of mitogen activated protein kinase (MAPK) becoming

activated by flg22 binding. Approximately three hours after receptor binding, the FRK1gene is

activated also due to activation of MAPK. The three genes mentioned do not have any known

effects in the cell and are specifically used as markers for the immune response. Higher

expression of the genes within the cell signal a stronger immune response. The gene expression

levels of PHI1, WRKY33, and FRK1 were used to determine immune response in our plants.

Figure 1





Through use of quantitative polymerase chain reaction (QPCR), we were able to measure

gene expression levels. In the results from QPCR, we expected to see increased expression levels

in plants treated with flg22, with even more expression in ops plants as it is believed that OPS

inhibits the immune response.

Materials and Methods

Plant Growth

All seeds were sterilized in a 10% bleach and 1% Titron solution for thirty minutes to one

hour in Eppendorf tubes. Under a sterilization hood, seeds were thoroughly rinsed with sterile,

deionized water before being transferred via pipette from Eppendorf tubes to plates. All plates

were then placed in fridge at 4oC for two days, to even germination, before being moved to a

growth chamber.

Treatment with flg22

When seedlings have grown for two weeks, they are ready for flg22 treatment. The day

before treatment, the seedlings are placed into wells, 4-6 seedlings per well, in 2 mL of deionized

water. For treatment, the water is taken out of each well and replaced with the flg22 solution.

After each specified time point, the seedlings are removed from the wells and transferred to

Eppendorf tubes before being placed in liquid nitrogen. The tubes are then placed in the freezer

at -20oC until ready for RNA isolation.



RNA Isolation

Seedlings are ground with pestles under liquid nitrogen before TRI reagent is added. All

tubes are kept on ice during this process until finished. The tubes then incubate at room

temperature for five minutes before the addition of chloroform to each tube. All tubes are

centrifuged at room temperature for 15 minutes. The aqueous layers are removed and transferred

to new Eppendorf tubes and isopropanol is added to precipitate RNA, and tubes are placed at -

20oC for one to two hours. Tubes are removed from the freezer and centrifuged at room

temperature for 15 minutes. The supernatant is removed, and the RNA pellet is washed with 70%

ethanol and briefly spun down again. The ethanol wash is removed, and the pellet is allowed to

dry for 10-15 minutes.

cDNA Synthesis

After allowing the RNA pellet to dry, the cDNA sysntesis process can begin. The RNA

pellet is resuspended in a mixture of DNAse I reaction buffer, RNAse OUT, DNAse I, and water.

This reaction is then incubated at 37oC for one hour. After incubation, EDTA solution is added

and mixed before the tubes incubate for five minutes at 65oC to heat-kill the DNAse enzyme.

The solution is then spun down to remove any debris and the supernatant is transferred to a new

Eppendorf tube. At this stage, RNA concentration is quantified using a nanodrop. Next, a master

mix of dNTP and oligo DT is divided equally into the tubes containing the RNA and water. This

reaction is incubated at 65oC for five minutes and then immediately transferred to ice in order to

denature the RNA. Then, a master mix of reaction buffer, DTT, and reverse transcriptase is made

and divided into each Eppendorf tube. This reaction is incubated at 42oC for one hour. To stop

the reactions, the tubes are incubated at 85oC for five minutes to heat-kill the reverse


transcriptase enzyme. The new cDNA is then diluted with DEPC-treated water to increase

volume amount without altering detection levels.

QPCR

To prepare for QPCR, 1:10 primer dilutions were made. The primer dilutions for actin

and the other desired gene, were added to Eppendorf tubes and mixed with Evagreen master mix,

cDNA, and water. The tubes were in sets of four connected, the first tube in each set contained

actin primers and the last three contained primers for our desired gene; all tubes in one set

contained the same cDNA. QPCR was then run for approximately one hour before data was

collected.

Results

In order to obtain results from QPCR, a reference gene (actin) was used. Actin was

chosen because it is present in every cell and thus levels of actin become the reference for

measurement of the other genes tested. To get an idea of expression levels over a period of time

for one of the marker genes, a time trial was done with PHI1I. The time points used (0 min, 30

min, 1 hr, and 3 hrs), were chosen based off previous papers stating these times as peak

expression points.

In the time trial performed, PHI1 expression in Col0 was initially low, as it would be

expected in a plant not experiencing an immune challenge. After 30 minutes of treatment with

flg22, PHI1 expression had increased a relatively small amount. The expression peaks at the one

hour mark in the Col0 plants. The expression levels shown after three hours of flg22 treatment,

resemble a non-treated plant, as there is considerably low expression of PHI1.






In the ops plants not exposed to any flg22, there were unexpected high levels of PHI1

expression. The peak expression levels in the ops plants was after 30 min of treatment with

flg22. Similar to the Col0 plants, in the time point after peak expression, the PHI1 levels

plummet to almost nothing. After three hours of treatment, PHI1 expression shows relatively

small increase from the plants treated for one hour.

Figure 2

Figure 2: A time trial was done with Col0 and ops plants to determine expression levels of PHI1. The dots show
expression levels relative to Actin at the given time point of treatment with flg22.

PHI1 expression was low at the no treatment (0 min) point and had increased expression at the

30 min treatment point across all plants tested.. Results for WRKY33 treatment echoed the PHI1

results. From these two gene expression results, we see that in the plants lacking the ops gene,

there is a higher expression after flg22 treatment than in Col0 plants.






Figure 3a

Figure 3b

Figure 3a: Col0 and ops plants treated with flg22 for 0 and 30 min, underwent QPCR. The graphs show
expression levels PHI1 in each plant genome at the given time point.

Figure 3b: Col0 and ops plants were treated with flg22 for 0 and 30 min before undergoing QPCR. The
graph shows expression levels of WRKY33 in each genome at each given time point.


Analysis of FRK1 expression in Col0 and ops were also done. The Col0 plants resembled

the same pattern as in the previous results. Our ops plants, on the other hand, were completely

opposite. We saw increases levels of expression in FRK1 at the no treatment point and then

declines in expression at the three-hour treatment point. There is a small difference in the initial

expression levels of FRK1 in the ops plants. This could be due to the differences in genomes and

the degree to which a functional OPS protein is available still.

Figure


Figure 4: Col0 and ops plants were treated with flg22 for 0 min and 3 hrs before undergoing QPCR. The
graph above shows expression levels of FRK1 in each genome of plant at the given time points.

Growth assays have been conducted with Col0 and ops plants in order to determine if

there is a detrimental effect on growth caused by the absences of the OPS gene. As shown below,

there is no hinderence on plant growth, only a visible change in root pattern.






Figure 5

Figure 5: Above is a growth assay conducted with Col0 and ops plants by a student prior to my
work in the lab.

Discussion

Our results show that plants lacking OPS exhibit increased expression of FLS2- induced

marker genes than Col0 plants, especially when there is no threat present. This demonstrates that

OPS is a negative regulator of the immune response in Arabidopsis thaliana. Combined with

past growth assays showing ops plants do not differ in size from wild types, we can speculate

that the increased immune response does not hinder the growth ability of the plants due to the

growth assays performed.

This novel finding that OPS is a negative regulator of the immune response could have

significant effects worldwide. There are numerous next steps that could be taken. Trials where

Arabidopsis plants (Col0 and ops) are exposed to full pathogens need to be done to determine the





survival rate. These would allow us to determine if the ops plants are actually able to survive

infection of a pathogen better compared to the wildtype plants. Next, the experiments should be

tested on a crop plant such as tomatoes. Since tomatoes have three different OPS genes, it would

need to be determined which one, if any, are active in the plant. After this determination,

knockout experiments can be run to eliminate the function of OPS. Growth assays of these

tomato plants would be critical to know if the ops plants can still produce fruit that is viable to

harvest. From here, the same experiments from this paper could be run on the tomato plants.

The ability to genetically alter crops so that they have enhanced immunity from

pathogens would save millions of dollars globally, and allow farmers to stop using harmful

pesticides. There would be a tremendous reduction in harm to the surrounding environment,

including the wildlife living there, if we eliminated pesticide use. If our preliminary thoughts

prove to be correct and ops plants have enhanced immune response without any detrimental

effects to growth, then we have began to find a solution to the issue of crops lost due to

pathogens and the harmful environmental effects of pesticides.






References

1. Asai, Tsuneaki, et al. “MAP Kinase Signalling Cascade in Arabidopsis Innate

Immunity.” Nature, vol. 415, no. 6875, 2002, pp. 977–983.,

doi:10.1038/415977a.

2. Belkhadir, Y., et al. “Brassinosteroids Modulate the Efficiency of Plant Immune

Responses to Microbe-Associated Molecular Patterns.” Proceedings of the

National Academy of Sciences, vol. 109, no. 1, 2011, pp. 297–302.,

doi:10.1073/pnas.1112840108.

3. Truernit, E., et al. “OCTOPUS, a Polarly Localised Membrane-Associated Protein,

Regulates Phloem Differentiation Entry in Arabidopsis Thaliana.” Development,

vol. 139, no. 7, 2012, pp. 1306–1315., doi:10.1242/dev.072629.

4. Anne, Pauline, et al. “OCTOPUS Negatively Regulates BIN2 to Control Phloem

Differentiation in Arabidopsis Thaliana.” Current Biology, vol. 25, no. 19, 2015,

pp. 2584–2590., doi:10.1016/j.cub.2015.08.033.

5. Benschop, Joris J, et al. Molecular and Cellular Proteomics, vol. 6, no. 7, 2007.