Hiroshi Takatsuji, Shingo Goto, Fuyuko Shimoda-Sasakura, Mai Suetsugu, Michael Gomez Selvaraj, Nagao Hayashi, Muneo Yamazaki, Manabu Ishitani, Masaki Shimono, Shoji Sugano, Akane Matsushita, Takanari Tanabata

Disease Resistant Crops Research Unit

［Abstract］

Driving the gene for the rice transcription factor WRKY45 with a strong promoter rendered the rice plants extremely resistant to multiple diseases at the cost of significant reduction in yield. We have succeeded in developing rice plants that are resistant to multiple diseases while retaining good yield.

［Keywords］

Keywords: rice, blast disease, chemical defense inducer, WRKY45, OsUbi7 promoter

［Background］

Yield of rice decreases due to various infectious diseases such as blast disease caused by fungal pathogen and leaf-blight disease caused by bacterial pathogen. WRKY45 is a rice transcription factor that plays a central role in the functioning of chemical defense inducers, such as probenazole and benzothiadiazole. Overexpression of its gene in rice rendered rice plants extremely resistant to multiple diseases including rice blast and leaf blight (multi-disease resistance), which should lead to drastically reduced necessity of pesticides. However, driving WRKY45 gene under the control of strong promoter (P_ZmUbi) severely reduced the yield. Here, we attempted to optimize WRKY45-expressing rice by driving WRKY45 cDNA with several rice-derived promoters of various strengths to develop practical multi-disease rice.

［Results and Discussion］

1. We fused 2-kb upstream sequences of 16 rice genes, which have different constitutive expression levels, upstream of WRKY45 cDNA and obtained a large number of transformant lines (T0). Of these, we selected homozygous lines that showed resistance to both rice blast and leaf blight diseases.
   
2. Evaluation of the growth and yield of the homozygous lines in confined greenhouse revealed that OsUbi7-promoter-driven WRKY45 expression rice lines (P_OsUbi7) showed the best balance in terms of multi-disease resistance and rice yield.
   
3. Field trials in confined fields in Japan and overseas (Korea and Columbia) showed that P_OsUbi7 lines showed growth and yield very similar to those of control untransformed Nipponbare rice (Fig. 1). A nursery upland experiment revealed that P_OsUbi7 plants have strong resistance to rice blast.
   
4. P_OsUbi7 lines were resistant to all 4 races of blast fungus tested and 6 races of leaf blight pathogens (Xoo) tested including 3 races isolated in foreign countries, indicating that the disease resistance of P_OsUbi7 lines is race-nonspecific (Fig. 2).
   
5. Exposure to low temperature (8℃, 7 d) followed by room temperature in nursery killed most of P_ZmUbi plants, while Nipponbare plants survived (Fig. 3). Treatment with 250 mM NaCl for 3 d also killed only the the P_ZmUbi plants. Defense genes such as PR genes were induced under both the stress conditions in the P_ZmUbi plants but not in Nipponbare suggesting a relationship between the gene expression and the stress hypersensitivity of the P_ZmUbi plants. Unlike the P_ZmUbi plants, P_OsUbi7 plants survived the stress conditions similar to Nipponbare (Fig. 3). These results indicate that the use of the OsUbi7 promoter for driving WRKY45 saved the stress hypersensitivity resulting from WRKY45 overexpression.

1. We are now selecting the best P_OsUbi7 lines with a genetic background of forage rice towards the development of practical multi-disease resistant forage rice.
   
2. We are also developing a WRKY45 expression line in which WRKY45 is driven by a pathogen-inducible promoter, and can be cultivated without using pesticides.
   
3. Foreign companies and organizations are interested in our technology.

Fig. 1. Field trial in a confined field in Colombia. PZmUbi plants (right) show poorer growth and less yield but POsUbi7 plants (left) showed comparable agronomical traits comparable with Nipponbare.

 

Fig. 2. Disease resistance of WRKY45 expression lines. PZmUbi, POsUbi7, and Nipponbare plants were tested for disease resistance. (A) Blast resistance against 2 races of fungal pathogen. (B) Leaf blight resistance against 3 races of bacterial pathogen isolated in Japan.

 

Fig. 3. Low-temperature sensitivity of WRKY45 expression lines. Most of PZmUbi plants exposed to low temperature (8℃) for 7d, and transferred to room temperature died,whereas the POsUbi7 and Nipponbare plants survived.

 

[Reference]

1. Goto S, Sasakura-Shimoda F, Suetsugu M, Selvaraj M.G, Hayashi N, Yamazaki M, Ishitani M, Shimono M, Sugano S, Matsushita A, Tanabata T, Takatsuji H (2014) Development of disease-resistant rice by optimized expression of WRKY45 Plant Biotechnology Journal DOI:10.1111/pbi.12303
   
2. International publication #WO2012/121093