The Mechanism Research Of Rice Blast Resistance Regulated By Rice Transcription Factor ZFP3

Rice blast is one of the most destructive diseases, which belongs to fungal disease. It occurs in rice producing areas of the world. Rice blast in the major producing area of Asia and Africa was most serious among the areas. Rice blast in mountain was more serious than that in plain, whereas rice blast in japonica was more serious than that in indica. Rice blast could significantly grain yield and end-use quality when large-scale outbreak occurs. Therefore, prevention of rice blast is always the focus of researches by breeders and pathologists. Currently, there are lots of methods to prevent rice blast, but breeding resistant varieties is a reasonable, effective and long-term approach of preventing rice blast because breeding resistant varieties is stable, safe and pollution-free by comparing to other approaches. Developing the genes that associated with rice blast resistance is the basis for breeding resistant varieties. In recent years, the development of sequencing technology and microarray technology provides a solid foundation of developing effective candidate genes that associated with rice blast resistanceIn this study, the leaf of Lijangxintuanheigu (LTH) (susceptible) and Digu (resistant) were use for transcriptome study at 20 hour post inoculation of Magnaporthe oryzae. Among the genes that were significantly regulated by M. oryzae, we found a gene that was up-regulated in LTH, not in Digu. The protein encoded by this gene had two zinc finger domain belonging to C2H2 type. This gene was named as ZFP3. The ZFP 3 might negatively regulate rice blast resistance because it was induced in LTH by M. oryzae. To test this hypothesis, we constructed the vector of ZFP3RNAi, and then transformed this vector to rice TP309. Subsequently, reveal the role of ZFP3 in the interaction of rice-M. oryzae by the techniques of pathology, cytology and molecular biology. The results are as follows:1. On the basis of microarray data, we screened the ZFP3 gene that was significant up-regulated in LTH, not in Digu. The expression pattern of the ZFP3 is reliable by qRT-PCR.2. ZFP3 was located in nuclear of rice cell. Meanwhile, ZFP3 could bind EP1S that is the DNA sequence combined by C2H2-type zinc finger protein. The results suggest that ZFP3 is a transcription factor.3. The specific fragment of ZFP3 was cloned after blasting the RGAP database and constructed to ZFP3RNAi vector. Subsequently, the ZFP3RNAi vector was transformed to TP309. We got the transgenic positive plants of ZFP3RNAi successfully.4. The homozygous transgenic plants were got from T2 plants of transgenic positive lines. Among the homozygous transgenic lines, two (ZFP3Ri2, ZFP3R14) with significant ZFP3RNAi were obtained by qRT-PCR.5. The lesions on leaves of ZFP3Ri2 and ZFP3Ri4 were smaller than that of TP309. The number of M. oryzae fungus on leaves of ZFP3Ri2 and ZFP3Ri4 were more that of TP309. The results suggested that ZFP3 is a negative factor of defending against M. oryzae.6. The development of appressorium of M. oryzae was delayed on the leaf sheath of the ZFP3RNAi transgenic plants by comparing to the control TP309. Meanwhile, when hypha of M. oryzae were growth, HR rapidly occurred in the cell of ZFP3RNAi transgenic plants by comparing to that of TP309, and cell that was infected dead rapidly, which prevented the hypha from spreading to neighboring cells.7. On the site of appressorium, the H2O2 on leave of the ZFP3RNAi transgenic plants was more than that of TP309 by DAB staining. Meanwhile, several genes that associated with degrading H2O2 were positively regulated by ZFP3 by qRT-PCR. The results suggest that the accumulation of H2O2 resulted into HR. however, the accumulation of H2O2 resulted from that the genes that associated with degrading H2O2 were depressed during to ZFP3RNAi.8. Salicylic Acid (SA) was associated with disease resistance. The expression of the genes that associated with SA synthesis and signal transduction were induced in ZFP3RNAi transgenic plants by qRT-PCR. The result suggests that ZFP3 negatively regulated the genes that associated with SA synthesis and signal transduction to defending against rice blast.In summary, the gene ZFP3 might negatively regulate the genes that associated with SA synthesis and signal transduction and positively regulate the genes that associated with H2O2 degradation, which changed the accumulation of H2O2 at the infected site of M. oryzae, and then affected development of appressorium and invasion of hypha of M. oryzae that result into the difference of resistant and susceptible to rice blast.