Team:Kyoto/Project

Description

1) Why is pine tree important?

Throughout Japanese history we have valued beautiful sightseeing spots such as Matsushima ("Matsu no Shima"), Miyajima, Amanohashidate as "Japan's three scenic spots". If you look at the photos of these places, you will notice that certain plants form an important background in every area. It is "pine".

Pine has been widely used as a building material, fuel, and as a component of windbreak forest to prevent the sea breeze from the sea. Pine trees are strong against salt and can grow even in soil dried with poor nutrition, so even now it is no longer used as building materials, planting activities on the coast are continuing as windbreak forests. It is also important as a raw material for industrially important rosin.

Thus, pine which is an important plant in many ways has been lost on a global scale in recent years due to the epidemic of pathogenic microorganisms.

2) Pines are being lost due to pine wilt disease

In 1905 the first report on pine was issued in Japan. From that time, the disease gradually spreaded among Japan, and now this disease has been confirmed in all prefectures except Hokkaido. In regions other than Japan, pine wilt disease is also reported in East Asia such as Korea, Taiwan, China, Europe such as Poland, Spain, Portugal and even North America including Canada and the USA.

Because of death of pine by this disease, beautiful landscapes around the world have been greatly damaged. In addition to that, it is estimated that huge economic damage of tens of millions of dollars has been caused.

In the forest where the pines die, it is known that the forestry phase changes, and the evergreen broad-leaved trees such as the vertebrae will take precedence. An increase in evergreen broad-leaved trees reduces the light reaching the forest floor, causing the harmful effects of herbs and the seedlings responsible for the next generation, which has a serious impact on the ecosystem.

3) The cause of pine wilt disease is nematode

In 1971, it was clarified that the cause of pine was nematode called Bursaphelenchus xylophilus. B. xylophilus adheres to spiracles of Japanese pine sawyer and is delivered to pine branches by it. When an insect bites a pine branch, B. xylophilus invades the tree trunk through the resin path and reaches the trunk, ingesting pine cells. Through this process, the pine releases defense substances such as volatile terpenes excessively, and bubbles are generated in the tracheid, so that the pine can not absorb water and this induces death of pines. This is the mechanism of pine wilting.

B. xylophilus is a nematode of dioecy and pierces a stylet like straw into a pine cell, blue stain, yeast etc feed and sucks their contents. It is known that species with such stylets cover 15% of the whole nematodes. Unlike bacterial feeding nematodes like C. elegans, this group of nematodes does not prey on bacteria. It is known that many phytopathogenic nematodes are of the type using stylet.

4) It is difficult to prevent B. xylophilus

There are basically three methods taken to stop pine wilting. However, all of them have several problems, and it does not mean that we are getting the expected results.
· Spraying of medicine by helicopter targeting Camember leaves
This is a method targeting beetle, which is a carrier that spreads nematodes. Of course, this has a harmful effect to humans and forest creatures. It is inevitable to have a huge impact on the ecosystem.
· Removal of dead wood to prevent infection spread
The longhoon beetle produces eggs on pine trees killed by B. x. It is known that when longhoon beetle emerges in the following year, nematodes adhere to beetle. For this reason, attempts have been done to prevent the spread of infection by disposing of dead pine through incision or incineration. Even in this case, there are places where human beings can not go, so there are many restrictions, such as not being able to do for all dead woods completely This fact means a limit to the effect of preventing pine withering.
· Prevention by drug stem injection
In order to combat the nematodes in the pine, in the winter, a method of injecting medicine into all the pine in the target mountain is also done. Although this method is effective, it is necessaly to do it every year, and if neglecting it, the damage will increase (there are actual cases), etc. Therefore, it causes economic and physical costs.

5) RNAi is a powerful weapon to fight against nematodes

In order to develop weapons fighting this nematode, we applied naive feeding RNAi which is often used for nematodes to B. xylophilus and planned to eradicate B. xylophilus from the pine forest. If we create various genetically modified organisms, and are able to create a genetically modified machine that kills nematodes using budding yeast whose resources are rich , we may be able to produce biological pesticides that we had never before. Once ignoring how to prevent our recombinant yeast from leaking into the environment and deliver yeast to nematodes (after that, a powerful solution for these problems emerges through activities of Human Practices. Please click here and you can see our human pracitices), we first decided to answer the question of whether yeast which kills nematodes can be made.

RNAi is a gene expressing suppression mechanism discovered in 1998 by Fire et al. In the body of C. elegans incorporating double-stranded RNA, rapid degradation of mRNA having a complementary sequence to that dsRNA is induced. If the target gene is essential for growth, C. elegans dies. C. elegans belongs to bacterial feeding nematode and feeds E. coli. Therefore, by preparing E. coli expressing dsRNA as bait, gene expression of C. elegans can be controlled. However, B. xylophilus belongs to the Stylet type nematode, so it can not be expected that we can use E. coli for bait. In order to deliver dsRNA to B. xylophilus it was essential to develop a new dsRNA carrier.

6) Deliver dsRNA to nematode with stylet

We chose budding yeast as a career.

Originally B. xylophilus was known to prey on blue stain fungi. In addition, although there is no report, some researchers gave information that they might prey on budding yeast. But when we asked the details, there is only the impression that the number of live B. xylophilus reduces slowly when he/she cultured B. xylophilus with yeast, or the impression seems to be increasing with yeast.There was no definitive information on whether or not B. xylophilus could feed Saccharomyce celevisiae.

For the initial step, we started to prove definitively that B. xylophilus preyed on budding yeast. For this purpose, we examined the conditions of B. xylophilus' feeding yeast and established a system of recording live imaging, and first succeeded in photographing a number of videos of B. xylophilus sucking content by puncturing Stylet in budding yeast. In addition, we introduced a system for labeling B. x that preyed yeast, and established a method for measuring the phenotype of B. xylophilus incorporating dsRNA.

Something moving is B. xylophilus and you can see it eating a small dot (S. cereviseae).