Team:Kyoto/Results

Results

1) Observe that B. xylophilus feeds on yeast

Dr. Y Takeuchi, a researcher of B. xylophilus, gave us a strain that was bred in the laboratory. These nematodes were cultivated with gray mold. It was also possible to grow nematodes on the plate growing gray mold.

After we washed nematodes cultured in mold with lactic acid to remove mold, we placed them with budding yeast on a new plate. At first we observed nematodes by microscope, but we could not observe them eating yeast. Although they tried to pierce yeast with their stylet, it seemed to be difficult for them to penetrate it well because the yeast was not fixed on the plate then moved, and also originally the yeast was too small.

So we thought it would be easier to pierce with a stylet if the yeast was bigger, and we modified the conditions and used diploid yeast instead of haploid yeast, which had been used thus far. Visually, diploid yeast is different from haploid with respect to shape and size, where diploid is bigger than haploid. Therefore, we decided to continue observation with diploid yeast.

We also improved the filming conditions. We stopped observing the nematodes on the medium directly, and changed to a method of sandwiching the medium and yeast between the slide glass and the cover glass and injecting nematodes from the side. By doing so, the yeast was fixed firmly on the medium and we could solve the problem that the yeast moved when nematodes were trying to pierce it with their stylet.

Shown in the figure is the moment B. xylophilus preyed on budding yeast. This was filmed for the first time in the world. The needle-shaped structure found near the nematode's mouth is a stylet. The nematode pushed the stylet against the yeast and inserted it in the yeast. The yeast started to shrink rapidly sometime after the nematode pierced it, and in the end its shape almost disappeared. Yeast has a robust cell wall, but it seems to be crushed with a strong force.

The results of experiments on whether or not B. xylophilus that ate S. cerevisiae would grow was shown in the figure.(fig1-b)

Fig1-b Diploid vs haploid
Spread haploid yeast or diploid yeast on an agar medium and dry a little, then put B. xylophilus in the medium. "no food" is B. xylophilus alone in an agar medium. We showed transition of survival rate of nematodes from Day 0 to Day 4 with the number of nematodes on the first day as 100%. (n = 3) The nematode counts twice per plate and the mean value is taken as the survival number.


In the case of haploid or no food, the survival rate decreases at Day 4, but when using diploids the survival rate increases. The large cell diploid yeast seems easier to ingest for B. xylophilus.

2) Identify B. xylophilus which ate yeast by fluorescence

In order to confirm the effect of RNAi, we needed a marker to distinguish nematodes that preyed on yeast from the others within the experimental period. Cultured nematodes included many growth stages, so it was anticipated that they included some nematodes at the stages where they could not eat yeast. In order to distinguish only the individuals which ate yeast and identify the effect, we decided to repeat the feeding experiment using yeast which fluoresces with eGFP.

The results of observation of nematodes which ate yeast with a fluorescence microscope are shown in the figure.

The intestines of B. xylophilus were highlighted by green fluorescence penetrating through the center throughout the entire body. The mouth is on the left side and the anus is on the right side. eGFP seemed to be very stable in the intestine, and the entire intestine fluoresced uniformly. There was some fluorescence discontinued in the center, and this was correspondent with the part where B. xylophilus gonads crossed over the intestine.

fig2-a eGFP expression cassette
We used a yeast that introduced a plasmid with a cassette as shown in the figure
fig2-b eGFP expression yeasts
A photograph of fluorescence microscope of yeast introduced with the plasmid of fig2-a. The left is the one irradiated with the excitation light, the right is the normal image. The fluorescence of eGFP is observed in many yeasts.
fig2-c Fluorescence micrograph of eGFP labbeled nematodes
Left: Fluorescence micrograph of nematode fed eGFP + yeast.
Right: Fluorescence micrograph of nematode fed yeast without eGFP expression plasmid.
The nematode esophagus is marked by GFP.
fig2-d Confocal micrograph of eGFP labbled nematodes
Left: Confocal micrograph of nematode fed eGFP + yeast.
Right: Confocal micrograph of nematode fed yeast without eGFP expression plasmid.
Since the cross section is also marked, it can be seen that the nematode body surface is not labeled


From the above results, it was found that by expressing eGFP, it became possible to clearly distinguish nematodes that preyed on yeast from the others. By expressing dsRNA simultaneously with eGFP, it should be possible to measure the effect of dsRNA efficiently. As can be seen from this photograph, feeding nematodes on yeast expressing GFP also made it possible to clearly observe the structure of B. xylophilus intestine. This method seemed to be effective for closely observing abnormalities occurring in an intestine of B. xylophilus, like gastroscopy using barium in humans.

Among the nematodes being cultured, how many nematodes were thought to surely eat yeast? We collected nematodes on the 3rd and 7th day from the start of the culture by eGFP yeast and observed what proportion of them were fluorescent using a fluorescence microscope. The results are shown in the figure (fig2-e).

Fig2-e Rate of eGFP+ nematodes
After spreading the eGFP expression yeast to the elementary agar medium, it was dried slightly and then B. xylophilus was placed in the medium. At the time of day 0, day 3, and day 7 from the start of culture, the proportion of those labeled by fluorescence of GFP among the total number of nematodes (including dead worms) was shown. A piece of agar medium cut into quarter corners was mounted on a preparation and the number was counted with a fluorescence microscope. (n = 18)


3) Choose dsRNA

In order to kill B. xylophilus, it is necessary to efficiently knock-down genes essential for growth. Examining the literature, we found a paper that succeeded in knocking down essential genes and reducing the survival rate by submerging B. xylophilus in high concentrations of dsRNA for a certain period of time (soaking RNAi). In this paper, the target was mRNA of arginine kinase AK1 which was an essential gene expressed in the intestines and RNAi of AK1 showed a fatal effect also in C. elegans. AK1 is an invertebrate-specific key enzyme of energy metabolism so it is often used as a target for development of invertebrate-specific inhibitors. Ip was a promising candidate for dsRNA expressed in yeast. In order to select the most ideal target, we prepared dsRNA of several target mRNAs including AK1 in vitro and tried soaking RNAi. We obtained target sequence from a public database, designed oligos, and cloned genes by PCR. At this time, we put the T7 promoter on both ends of the DNA (Fig.) so that dsRNA was synthesized by in vitro transcription. After transcription, association of dsRNA was induced by an annealing operation, and the template was removed with DNase. We confirmed the dsRNA finally obtained by electrophoresis. This is the results.

We prepared these RNAs, adjusted to a concentration of 2 μg /μL, and tried soaking RNAi The results is shown in the figure. As is clearly shown, we could not see the phenotype due to the introduction of dsRNA which was inconsistent with the previously reported example. As a result of contacting several B.xylophilus researchers and gathering information, it turned out that even several Japanese researchers have attempted to reproduce B. xylophilus soaking RNAi, but no group was able to observe a clear effect. The reason may be that soaking RNAi of B. xylophilus contains technically unstable steps. Alternatively, since B. xylophilus used this time is derived from wild nematodes collected from the field, there may be a difference between the strain we used and nematodes in the publication where soaking RNAi was effective.

Although the effect of soaking RNAi was not observed, we decided to target the AK1 gene because there is already the report, and constructed the expression system of dsRNA in yeast.

4) Conduct feeding RNAi in yeast

In order to express AK1-dsRNA, we placed inverted repeat derived from AK1 ORF downstream of the Gal1 promoter of the part (BBa_K517000), and inserted a small loop sequence of 80-nt between repeats. There was a report that this loop sequence was effective when S. cerevisiae expressed long dsRNA. Since S. cerevisiae has no Dicer homolog, dsRNA is not processed into siRNA. However, overexpression of dsRNA may be toxic to S. cerevisiae, so we adopted the Gal1 conditional promoter. When S.cerevisiae is cultured in the presence of glucose, this promoter is inactive, and many mRNAs are expressed when the carbon source of the medium is replaced with galactose. At the same time, we also used the GPD promoter which is a constitutive expression type promoter. dsGFP with a sequence specific to GFP and was designed as a negative control. Outline of construction is shown below.

We cultured plasmid-containing yeasts in several media, collected RNA, and quantified by qRT-PCR with the "loop" part as a target.

It is known that various viruses of dsRNA type exist in S. cerevisiae. As a factor closely related to the life cycle of such a virus, Ski gene group is known. Many of these are now revealing detailed functions. The Ski complex binds to the 3 'end of RNA and serves as a cofactor for RNA exosome, which is an exonuclease complex that degrades RNA in the 3-5 direction. By binding to the 3 'end to disband the higher-order structure of RNA, it makes the recognition of substrate by exosome efficient. Since the dsRNA virus is known to proliferate in the ski2Δ strain, it was hoped that the use of this strain would greatly increase the yield of the target dsRNA. As a result quantified by actual experiment, as shown in the figure,???

5) Observe that B. xylophilus feeds on yeast expressing dsRNA

We let B. xylophilus prey on the yeast prepared as described above and recorded the survival rate and behavior of nematodes as follows.

We counted the number of surviving nematodes which fed on dsRNA / eGFP expressing yeast every other day. We also confirmed the survival rate among nematodes that showed fluorescence of eGFP.

From the above results, no effect of dsAK1 was observed even when the Gal1promoter or GPD promoter was used. We thought that dsRNA was not well incorporated into the nematode, so we sought out the cause.

As shown in the figure, the diameter of the stylet is very small, about a fraction of a single cell of yeast. For this reason, B. xylophilus seemed to draw out the cytoplasmic fraction, but large cellular componentns such as the nucleus may not be efficiently consumed by B. xylophilus.

A number of studies have been done on the nuclear export of mRNA, and the basic mechanism has been elucidated. It is known that various RNAs such as mRNA, rRNA, tRNA, etc. are recognized by transporters specific to each type and pass through the nuclear pore complex. However, since the dsRNA as prepared this time does not exist in nature, it is not known whether there is a transport factor that recognizes this RNA or whether it is efficiently transported out of nucleus.

In order to prepare remedies for this problem, we tried experiments utilizing the REV factor of HIV-1 RNA, which is known to have the function of improving the efficiency of nuclear export of RNA.

6) Improve transport of mRNA to cytosol

As shown in the figure, REV plays the role of carrying an unspliced RNA genome to the cytoplasm in the life cycle of HIV-1. (fig 6 - a) In the case of ordinary mRNA, there is a retention mechanism that prevent molecules retaining introns from transferring out of the nucleus, thus preventing the transport of immature mRNA. REV binds to a specific part (RRE: Rev responsive element) of the intron on the HIV-1 RNA genome and binds itself to the nuclear export factor CRM1, and overcomes such a retention mechanism and transports RNA to the cytoplasm. Even if the dsRNA is not recognized as a nuclear export factor or even if it is retained in the nuclear retention factor, we thought that it is possible that the efficiency of nuclear export can be improved by inserting REV-RRE system, and provide these new parts to the iGEM community ( http://parts.igem.org/Part:BBa_K2403000 http://parts.igem.org/Part:BBa_K2403002).

We show the figure of the result of actually connecting RRE to dsRNA and microinjecting it into the nucleus of Xenopus oocytes. We separated nuclei and cytoplasm after a certain period of time following injection, and we collected RNA from each and analyzed it. As shown in the figure,

Reference