Discussion

At the beginning of the project we considered using our dsRNA-expressing yeast (B. x. Busters/B. x. B.) directly as an agricultural pesticide, but we recognized some potential problems with this idea as a result of our HP activities.

• 1. Because pines have been almost annihilated in Japan, it is necessary to breed pines anew.
• 2. If B. x. Busters do not propagate on pines, they will need to be applied/injected every year.
• 3. It is difficult to control genetically modified yeast once released into the wild.

Thanks to being able to recognize these problems through our HP activities, we were able to consider more realistic uses of our B. x. B. technology related to pines resistance to B. xylophilus.

In Japan, attempts are now being made to make resistant pine trees by hybridizing pines which are surviving in areas where pine-wilt disease was pandemic without using genetic modification. In establishing a new strain of pines resistant to B. xylophilus, attempts are being made to select strains of pine that show natural resistance to B. xylophilus and mate them.
However, this method has several problems and has not been put to practical use.

• 1. The pines are not 100% resistant simply because they show improved resistance to B. xylophilus.
• 2. In the case of gardens and sightseeing spots, characteristics of the strains such as branching should be emphasized, but such considerations are limited using this method.
• 3. Under stress, B. xylophilus itself may evolve to become pathogenic to currently resistant pines.

These problems could be solved by making recombinant RNAi pine trees expressing dsRNA.

Figure RNAi pines will solve the problem of pine-wilt disease

However, applying the same method to make B. x. Busters may be difficult in pine trees, especially in a timely manner.

• 1. It takes at least several years for pines to grow until it becomes possible to evaluate resistance to B. xylophilus by injecting them into the trunk.
• 2. It is not yet known whether feeding RNAi is effective for B. xylophilus.
• 3. There is currently insufficient data on which essential gene(s) to target.

Establishing RNAi conditions by trial and error in pine trees would be too in efficient. Using our knowledge from experimentation with B.x.B to establish a delivery system (point 2 above), it is expected that RNAi may be used as a screening system to determine the effects on various essential genes both efficiently and quickly (point 3 above).
Methods to protect plants from plant parasitic nematodes by RNAi expression has attracted attention in recent years.
There is a report that a host plant of M. incognita, belonging to the same order Tylenchida as B. xylophilus, escaped from the parasitism of M. incognita by making the plant express dsRNA.[1] That is to say, the application of B. x. B may not be limited to feeding RNAi to B. xylophilus.
15% of all nematodes are plant parasitic nematodes which have stylet similar to B. xylophilus. These nematodes have caused enormous damage to important crop plants including potatoes, eggplants, rice, tomatoes, cucumbers, radishes, and carrots, resulting in an estimated annual loss of $8 billion in the United States and &#03678 billion worldwide. Therefore, urgent measures are required. RNAi expression in plants is a powerful weapon against these pathogenic nematodes,[2] so development of such technology has a strong potential for application.
The B. x. Busters system we developed can be used as a preliminary RNAi evaluation system to target plant-specific pathogens, before initiating breeding of recombinant crops. If efficient breeding is carried out and countermeasures progress, many plant diseases will be abolished, which will have a strong impact on resolving current threats to global food resources.

Reference

• [1]
• [2]