Demonstrate

1) Concept

Pines, which are essential for our culture and daily lives, are withering on a global scale due to the epidemic called pine wilt disease. Pine wilt disease is caused by the nematode Bursaphelenchus xylophilus. We planned to apply feeding RNAi, which is often used for nematodes, to B. xylophilus, to eradicate it from the pine forest, and save the pine trees.

We chose Saccharomyces cerevisiae as a carrier because some researchers believe that B. xylophilus might prey on S. cerevisiae, whose resources are rich. However, when we asked researchers for more details, there was no definitive information on whether or not B. xylophilus could feed on S. cerevisiae.

Therefore, we set out to prove definitively that B. xylophilus preyed on S. cerevisiae for the initial step. We examined the conditions of B. xylophilus feeding on yeast, established a system for recording live imaging, and succeeded in collecting a number of videos of B. xylophilus puncturing S. cerevisiae with their stylet and sucking out the yeast’s contents. In addition, we introduced a system of labeling B. xylophilus that had preyed on yeast, and established a method for measuring the phenotype of B. xylophilus incorporating dsRNA.

2) Proof of Concept

2-1) To establish a system for recording live imaging

We put medium containing yeast between a microscope slide and a cover glass, and injected nematodes from the side of the cover glass. In this way, we enabled yeasts to be fixed firmly in the medium and prevented them from moving when B. xylophilus tried to pierce them with their stylet.

Shown in the figure below is the moment when B. xylophilus preyed upon the yeast. We observed the nematode probing the yeast with its stylet, and inserting it into the yeast. The yeast pierced by the nematode’s stylet started to shrink rapidly and was finally crushed flat.

The moment when a nematode feeds a yeast

2-2) To establish a method for measuring the phenotype of B. xylophilus

The figure shows the results of observing nematodes cultured on eGFP-expressing yeast using a fluorescence microscope. The intestines of B. xylophilus were easily distinguished by green fluorescence penetrating through the center throughout the body. There discontinuous fluorescence near the center is consistent with the location where B. xylophilus’ gonads crossed over the intestines.

From the above results, it was found that by expressing eGFP in yeast, it was possible to clearly distinguish nematodes that preyed on budding yeast from non-predatory nematodes. By expressing dsRNA simultaneously with GFP, it should be possible to identify feeding worms and measure the effect of dsRNA efficiently.

2-3) To prepare the yeast expressing dsRNA and quantify hairpin RNA

Plasmids containing Gal1 promoter-AK1, GPD promoter-AK1, and GPF promoter-GFP were transformed into two yeast strains (MKY13 WT and MKY117 ski2Δ). The two strains containing Gal1 promoter plasmids were each cultured in glucose medium and galactose medium.

The loop region of hairpin RNA, AK1 mRNA, and 5 'end of 25S rRNA were each amplified by qPCR. The quantitative value of the loop and that of AK1 were divided by the quantitative value of 25S rRNA, to normalize variation in RNA recovery. The results are shown in the figure below.

The conditional Gal1promoter showed a higher expression in galactose medium and suppression in glucose medium. When the ski2Δ strain was used, the amount of hairpin RNA expressed under the Gal1 promoter on galactose medium markedly increased. The GPD promoter was not active in either ski2Δ or wild type strains.

As shown by the above results, we succeeded in quantifying hairpin RNA by qRT-PCR targeting the loop part and comparing yeast expression of AK1-target dsRNA.

2-4) Localization of dsRNA and nuclear export of RNA by Rev-RRE

The dsGFP and dsGFP with RRE prepared by combining GFPfwd and GFPrev were microinjected into Xenopus oocytes, and nuclei and cytoplasm were separated after a certain period of time, RNA was recovered from each and analyzed. The results are shown in the figure below.

The figure on the right of fig6-f shows that many of dsRNA stays in the nucleus. When U6 - RRE is injected together with buffer without Rev, U6-RRE remains in the nucleus. In contrast, when U6 - RRE is injected together with Rev - containing buffer, U6-RRE was located outside the nucleus. U6-RRE was remarkably transported. From these results, it was demonstrated that U6 RNA is dependent on both RRE sequence and Rev protein, promoting nuclear export of RNA. This result shows that parts BBa_K2403000 and BBa_K2403002 are promising as devices for efficiently transporting highly structured RNA to the cytoplasm.