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Expression-based genotyping of the rice blast resistance genes in the elite maintainer line Yixiang1B

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Abstract

Rice blast caused by Magnaporthe oryzae poses a major threat to rice production worldwide. Utilization of resistance is the most effective and economic means to control the disease. An elite maintainer line Yixiang1B (Y1B) has been widely exploited in three-line hybrid rice breeding. However, it remains unclear which blast resistance gene or genes contribute to its resistant phenotype. In this work, we found that a novel functional Pid2 allele is critical for the Y1B’s resistance against rice blast pathogen. First, we confirmed that Y1B is resistant to a number of M. oryzae isolates, with a resistance frequency of 65%. Then, we exploited expression-based approach to genotype the cloned rice blast resistance genes. Transcripts from Pid2, Pid3, Pib, Pi36, Pi5 and Pia were readily detected in Y1B upon M. oryzae infection. Sequencing analyses detected many SNPs in these genes, except that Pib had no substitution and Pid2 contained two tandem base-pair mutations at the nucleotide positions 1997 and 1998 leading to the H666R substitution. Knocking-down Pid2_Y1B via RNAi in Y1B resulted in susceptibility. In contrast, over-expression of Pid2_Y1B in a blast-susceptible accession led to enhanced resistance to M. oryzae. Therefore, Pid2_Y1B is the main contributor to the rice blast resistance in Y1B. Taken together, we identified a functional resistance allele of Pid2 that can be utilized as a resource in rice blast resistant breeding programs.

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References

  • Ashikawa, I., Hayashi, N., Yamane, H., Kanamori, H., Wu, J., Matsumoto, T., et al. (2008). Two adjacent nucleotide-binding site-leucine-rich repeat class genes are required to confer Pikm-specific rice blast resistance. Genetics, 180(4), 2267–2276.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ashikawa, I., Hayashi, N., Abe, F., Wu, J., & Matsumoto, T. (2011). Characterization of the rice blast resistance gene Pik cloned from Kanto51. Molecular Breeding, 30(1), 485–494.

    Article  Google Scholar 

  • Boller, T., & Felix, G. (2009). A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annual Review of Plant Biology, 60(1), 379–406.

    Article  CAS  PubMed  Google Scholar 

  • Bryan, G. T., Wu, K. S., Farrall, L., Jia, Y., Hershey, H. P., Mcadams, S. A., et al. (2000). A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene pi-ta. Plant Cell, 12(11), 2033–2046.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cesari, S., Thilliez, G., Ribot, C., Chalvon, V., Michel, C., Jauneau, A., et al. (2013). The Rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding. Plant Cell, 25(4), 1463–1481.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chauhan, R. S., Farman, M. L., Zhang, H. B., & Leong, S. A. (2002). Genetic and physical mapping of a rice blast resistance locus, pi-CO39(t), that corresponds to the avirulence gene AVR1-CO39 of Magnaporthe grisea. Molecular Genetics and Genomics, 267(5), 603–612.

    Article  CAS  PubMed  Google Scholar 

  • Chen, X. W., Shang, J. J., Chen, D. X., Lei, C. L., & Zou, Y. (2006). A B-lectin receptor kinase gene conferring rice blast resistance. Plant Journal, 46(5), 794–804.

    Article  CAS  PubMed  Google Scholar 

  • Chen, J., Liu, W. Z., Zhuang, J. Y., Shi, Y. F., Chai, R., Fu, Y. P., et al. (2011). A Pid3 allele from rice cultivar Gumei2 confers resistance to Magnaporthe oryzae. Journal of Genetics and Genomics, 38(5), 209–216.

    Article  CAS  PubMed  Google Scholar 

  • Costanzo, S., & Jia, Y. (2010). Sequence variation at the rice blast resistance gene. Pi-km locus: Implications for the development of allele specific markers Plant Science, 178(6), 523–530.

    CAS  Google Scholar 

  • Fukuoka, S., Saka, N., Koga, H., Ono, K., Shimizu, T., Ebana, K., et al. (2009). Loss of function of a proline-containing protein confers durable disease resistance in rice. Science, 325(5943), 998–1001.

    Article  CAS  PubMed  Google Scholar 

  • Hayashi, N., Inoue, H., Kato, T., Funao, T., Shirota, M., Shimizu, T., et al. (2010). Durable panicle blast-resistance gene Pb1 encodes an atypical CC-NBS-LRR protein and was generated by acquiring a promoter through local genome duplication. Plant Journal for Cell & Molecular Biology, 64(3), 498–510.

    Article  CAS  Google Scholar 

  • Hua, L., Wu, J., Chen, C., Wu, W., He, X., Lin, F., et al. (2012). The isolation of Pi1, an allele at the Pik locus which confers broad spectrum resistance to rice blast. Theoretical & Applied Genetics, 125(5), 1047–1055.

    Article  CAS  Google Scholar 

  • Huang, H., Huang, L., Feng, G., Wang, S., Wang, Y., Liu, J., et al. (2011). Molecular mapping of the new blast resistance genes Pi47 and Pi48 in the durably resistant local rice cultivar Xiangzi 3150. Phytopathology, 101(5), 620–626.

    Article  PubMed  Google Scholar 

  • Jones, J. D. G., & Dangl, J. L. (2006). The plant immune system. Nature. Nature, 444(7117), 323–329.

    Article  CAS  PubMed  Google Scholar 

  • Khush, G. S. (2005). What it will take to feed 5.0 billion rice consumers in 2030. Plant Molecular Biology, 59(1), 1–6.

    Article  CAS  PubMed  Google Scholar 

  • Lee, S. K., Song, M. Y., Seo, Y. S., Kim, H. K., Ko, S., Cao, P. J., et al. (2009). Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two coiled-coil–nucleotide-binding–leucine-rich repeat genes. Genetics, 181(4), 1627–1638.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li, W., Wang, B., Wu, J., Lu, G., Hu, Y., Zhang, X., et al. (2009). The Magnaporthe oryzae avirulence Gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in Rice mediated by the blast resistance Gene Piz-t. Molecular plant-microbe interactions: MPMI, 22(4), 411–420.

    Article  CAS  PubMed  Google Scholar 

  • Li, J., Sun, Y., Hui, L., Wang, Y., Jia, Y., & Xu, M. (2014a). Genetic variation and evolution of the pit blast resistance locus in rice. Genetic Resources and Crop Evolution, 61(2), 473–489.

    Article  CAS  Google Scholar 

  • Li, Y., Lu, Y. G., Shi, Y., & Wu, L. (2014b). Multiple rice microRNAs are involved in immunity against the blast fungus Magnaporthe oryzae. Plant Physiology, 164(2), 1077–1092.

    Article  CAS  PubMed  Google Scholar 

  • Li, J. B., Sun, Y. D., Liu, H., Wang, Y. Y., Jia, Y. L., & Xu, M. H. (2015). Natural variation of rice blast resistance gene pi-d2. Genetics & Molecular Research Gmr, 14(1), 1235–1249.

    Article  CAS  PubMed  Google Scholar 

  • Lin, F., Chen, S., Que, Z., Wang, L., Liu, X., & Pan, Q. (2007). The blast resistance gene Pi37 encodes a nucleotide binding site leucine-rich repeat protein and is a member of a resistance gene cluster on rice chromosome 1. Genetics, 177(3), 1871–1880.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu, W., & Wang, G. L. (2016). Plant innate immunity in rice: a defense against pathogen infection. National Science Review. doi:10.1093/nsr/nww015.

    PubMed  Google Scholar 

  • Liu, X. Q., Lin, F., Wang, L., & Pan, Q. H. (2007). The in silico map-based cloning of Pi36, a Rice coiled-coil nucleotide-binding site-leucine-rich repeat Gene that confers race-specific resistance to the blast fungus. Genetics, 176(4), 2541–2549.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu, Y., Liu, B., Zhu, X., Yang, J., Bordeos, A., Wang, G., et al. (2013). Fine-mapping and molecular marker development for Pi56(t), a NBS-LRR gene conferring broad-spectrum resistance to Magnaporthe oryzae in rice. Theoretical and Applied Genetics, 126(4), 985–998.

    Article  CAS  PubMed  Google Scholar 

  • Lv, Q., Xu, X., Shang, J., Jiang, G., Pang, Z., Zhou, Z., et al. (2013). Functional analysis of Pid3-A4, an ortholog of rice blast resistance gene Pid3 revealed by allele mining in common wild rice. Phytopathology, 103(6), 594–599.

    Article  CAS  PubMed  Google Scholar 

  • Ma, J., Lei, C., Xu, X., Hao, K., Wang, J., Cheng, Z., et al. (2015). Pi64, encoding a novel CC-NBS-LRR protein, confers resistance to leaf and neck blast in rice. Molecular Plant-Microbe Interactions, 28(5), 558–568.

    Article  CAS  PubMed  Google Scholar 

  • Okuyama, Y., Kanzaki, H., Abe, A., Yoshida, K., Tamiru, M., Saitoh, H., et al. (2011). A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes. Plant Journal for Cell & Molecular Biology, 66(3), 467–479.

    Article  CAS  Google Scholar 

  • Qu, S., Liu, G. B., Bellizzi, M., Zeng, L., Dai, L., Han, B., et al. (2006). The broad-spectrum blast resistance gene Pi9 encodes a nucleotide-binding site-leucine-rich repeat protein and is a member of a multigene family in rice. Genetics, 172(3), 1901–1914.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rafiqi, M., Ellis, J. G., Ludowici, V. A., Hardham, A. R., & Dodds, P. N. (2012). Challenges and progress towards understanding the role of effectors in plant-fungal interactions. Current Opinion in Plant Biology, 15(4), 477–482.

    Article  CAS  PubMed  Google Scholar 

  • Schwessinger, B., Bahar, O., Thomas, N., Thomas, N., Holton, N., Nekrasov, V., et al. (2015). Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand-dependent activation of defense responses. Plos Pathogens, 11(3), e1004809. doi:10.1001371/journal.ppat. 1004809.

    Article  PubMed  PubMed Central  Google Scholar 

  • Shang, J. J., Tao, Y., Chen, X. W., Zou, Y., Lei, C. L., Wang, J., et al. (2009). Identification of a new rice blast resistance gene, Pid3, by genomewide comparison of paired nucleotide-binding site--leucine-rich repeat genes and their pseudogene alleles between the two sequenced rice genomes. Genetics, 182(4), 1303–1311.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shi, J., Li, D., Li, Y., Li, X., Guo, X., Luo, Y., et al. (2015). Identification of rice blast resistance genes in the elite hybrid rice restorer line Yahui2115. Genome, 58(3), 91–97.

    Article  CAS  PubMed  Google Scholar 

  • Su, J., Wang, W., Han, J., Chen, S., Wang, C., Zeng, L., et al. (2015). Functional divergence of duplicated genes results in a novel blast resistance gene Pi50 at the Pi2/9 locus. Theoretical and Applied Genetics, 128(11), 2213–2225.

    Article  CAS  PubMed  Google Scholar 

  • Takahashi, A., Hayashi, N., Miyao, A., & Hirochika, H. (2010). Unique features of the rice blast resistance Pish locus revealed by large scale retrotransposon-tagging. BMC Plant Biology, 10, 175. doi:10.1186/1471-2229-10-175.

    Article  PubMed  PubMed Central  Google Scholar 

  • Tao, Z., Liu, H., & Wang, S. P. (2009). A pair of allelic WRKY genes play opposite roles in rice-bacteria interactions. Plant Physiology, 151(2), 936–948.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Toki, S., Naho, H., Kazuko, O., Haruko, O., Akemi, T., Oka, S., et al. (2006). Early infection of scutellum tissue with agrobacterium allows high-speed transformation of rice. Plant Journal for Cell & Molecular Biology, 47(6), 969–976.

    Article  CAS  Google Scholar 

  • Vasudevan, K., Gruissem, W., & Bhullar, N. K. (2015). Identification of novel alleles of the rice blast resistance gene Pi54. Scientific Reports, 5, 15678. doi:10.1038/srep15678.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang, Z. X., Yano, M., Yamanouchi, U., Iwamoto, M., Monna, L., Hayasaka, H., et al. (1999). The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes. Plant Journal, 19(1), 55–64.

    Article  PubMed  Google Scholar 

  • Wu, Y., Yu, L., Pan, C., Dai, Z., Li, Y., Xiao, N., et al. (2016). Development of near-isogenic lines with different alleles of Piz locus and analysis of their breeding effect under Yangdao 6 background. Molecular Breeding, 36, 12. doi:10.1007/s11032-016-0433-7.

    Article  CAS  Google Scholar 

  • Xu, H. T., Jun, Z. Y., Yang, F. Y., Ru, Y. Q., Peng, Z., Dong, Y., et al. (2013). DNA-marker based analysis of the allelic constitution and subspecies attribution of super hybrid Rice Yiyou 673. Scientia Agricultura Sinica, 46(10), 1965–1973 (In Chinese).

    Google Scholar 

  • Xu, X., Hayashi, N., Wang, C. T., Fukuoka, S., Kawasaki, S., Takatsuji, H., et al. (2014). Rice blast resistance gene Pikahei −1 (t), a member of a resistance gene cluster on chromosome 4, encodes a nucleotide- binding site and leucine-rich repeat protein. Molecular Breeding, 34(2), 691–700.

    Article  CAS  Google Scholar 

  • Yuan, B., Zhai, C., Wang, W. J., Zeng, X. S., Xu, X. K., Hu, H. Q., et al. (2011). The Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely linked CC-NBS-LRR genes. Theoretical & Applied Genetics, 122(5), 1017–1028.

    Article  Google Scholar 

  • Zhai, C., Lin, F., Dong, Z., He, X., Yuan, B., Zeng, X., et al. (2011). The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication. New Phytologist, 189(1), 321–334.

    Article  CAS  PubMed  Google Scholar 

  • Zhai, C., Zhang, Y., Yao, N., Lin, F., Liu, Z., Dong, Z., et al. (2014). Function and interaction of the coupled genes responsible for Pik-h encoded rice blast resistance. PloS One, 9(6), e98067. doi:10.1371/journal.pone.0098067.

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhou, B., Qu, S., Liu, G., Dolan, M., Sakai, H., Lu, G., et al. (2006). The eight amino-acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea. Molecular Plant-Microbe Interactions, 19(11), 1216–1228.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was partially supported by National Natural Science Foundation of China (Grant # 31430072 to W. W), grants from the China Agriculture Rice Research System Projects (CARS-075 to D. Z) and Public Relation Project of Rice Breeding in Sichuan Province (2016NYZ0028 to F. H). We thank Jian Wang (Sichuan Academy of Agricultural Sciences) for kindly providing M. oryzae isolates.

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Authors and Affiliations

  1. Rice Research Institute & Key Laboratory for Major Crop Diseases, and College of Agronomy, Sichuan Agricultural University, Chengdu, 611130, China

    Li Wang, Xiao-Hong Hu, Zhi-Xue Zhao, Rui Zeng, Heng-Jing Li, De-Qiang Li, Jing Fan, Yan Li, Fu Huang & Wen-Ming Wang

  2. Yibin Academy of Agricultural Sciences, Yibin, Sichuan, 644000, China

    Li Wang, Gang Lin & De-Ming Zhao

  3. Mianyang Academy of Agricultural Sciences, Mianyang, Sichuan, 621000, China

    Jun Shi

  4. Collaborative Innovation Center for Hybrid Rice in Yangtze River Basin, Sichuan Agricultural University, Chengdu, Sichuan, 611130, China

    Jing Fan, Yan Li & Wen-Ming Wang

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  1. Li Wang
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  2. Xiao-Hong Hu
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  3. Gang Lin
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  11. Yan Li
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  12. Fu Huang
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Correspondence to Fu Huang or Wen-Ming Wang.

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Table S1

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Fig. S1

R gene alleles of amino acid sequence alignment. (a) Amino acid sequence alignment of Pid3. Pid3_Y1B has 10 amino acid substitutions compared with Pid3_Digu (FJ745364.1), and four of them were found in the functional allele Pid3_A4 (FJ745368.1), three were found in LTH. Susceptible allele of Pid3_LTH (FJ745367.1) has a premature stop codon at residue 731. (b) Amino acid sequence alignment of Pi36. Pi36_Y1B has 22 amino acid substitutions compared with Pi36_Kasalath (DQ900896.1). Susceptible allele of Pi36_LTH (GU169402.1) mutated at residue S590 N. Amino acid polymorphism was marked by shaded letters. (GIF 895 kb)

High resolution image (TIFF 12812 kb)

Fig. S2

Vector construction and identified by restriction enzyme digestion. (a) Construction of the binary vectors used for rice transformation. Pid2_Y1B cDNA sequences designed with KpnI inserted into the binary vector pCAMBIA1300 under the control of the CaMV 35S promoter designated as pBWA (V) HS-1Bpid2 (above). Two β-lectin domain of Pid2_Y1B containing small hairpin structure were designed, named as pBWA (V) HU-1Bpid2 (below). (b) Enzyme digestion of pBWA (V) HS-1B pid2 with KpnI, digestion of pBWA (V) HU-1Bpid2 with MfeI and HindIII. (GIF 29 kb)

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Wang, L., Hu, XH., Lin, G. et al. Expression-based genotyping of the rice blast resistance genes in the elite maintainer line Yixiang1B. Eur J Plant Pathol 148, 955–965 (2017). https://doi.org/10.1007/s10658-017-1149-1

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