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Involvement of OsNPR1/NH1 in rice basal resistance to blast fungus Magnaporthe oryzae

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Abstract

Rice blast disease, caused by the fungus Magnaporthe oryzae, is a major threat to worldwide rice production. Plant basal resistance is activated by virulent pathogens in susceptible host plants. OsNPR1/NH1, a rice homolog of NPR1 that is the key regulator of systemic acquired resistance in Arabidopsis thaliana, was shown to be involved in the resistance of rice to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae and benzothiadiazole (BTH)-induced blast resistance. However, the role of OsNPR1/NH1 in rice basal resistance to blast fungus M. oryzae remains uncertain. In this study, the OsNPR1 gene was isolated and identified from rice cultivar Gui99. Transgenic Gui99 rice plants harbouring OsNPR1-RNAi were generated, and the OsNPR1-RNAi plants were significantly more susceptible to M. oryzae infection. Northern hybridization analysis showed that the expression of pathogenesis-related (PR) genes, such as PR-1a, PBZ1, CHI, GLU, and PAL, was significantly suppressed in the OsNPR1-RNAi plants. Consistently, overexpression of OsNPR1 in rice cultivars Gui99 and TP309 conferred significantly enhanced resistance to M. oryzae and increased expression of the above-mentioned PR genes. These results revealed that OsNPR1 is involved in rice basal resistance to the blast pathogen M. oryzae, thus providing new insights into the role of OsNPR1 in rice disease resistance.

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Abbreviations

TMV:

Tobacco mosaic virus

SAR:

Systemic acquired resistance

HR:

Hypersensitive response

PR:

Pathogenesis-related

BTH:

Benzothiadiazole

NPR1:

Nonexpressor of PR genes 1

SA:

Salicylic acid

INA:

2,6-Dichloroisonicotinic acid

Xoo :

Xanthomonas oryzae pv. oryzae

TF:

Transcription factors

RT-PCR:

Reverse-transcriptase polymerase chain reaction

RACE:

Rapid Amplification of cDNA Ends

ORF:

Open reading frame

dsRNA:

Double-stranded RNA

References

  • Bai, W., Chern, M., Ruan, D., Canlas, P. E., Sze-to, W. H., & Ronald, P. C. (2011). Enhanced disease resistance and hypersensitivity to BTH by introduction of an NH1/OsNPR1 paralog. Plant Biotechnology Journal, 9, 205–215.

    Article  PubMed  CAS  Google Scholar 

  • Cao, H., Bowling, S. A., Gordon, A. S., & Dong, X. (1994). Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. The Plant Cell, 6, 1583–1592.

    Article  PubMed  CAS  Google Scholar 

  • Cao, H., Li, X., & Dong, X. (1998). Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proceedings of the National Academy of Sciences of the United States of America, 95, 6531–6536.

    Article  PubMed  CAS  Google Scholar 

  • Chern, M. S., Fitzgerald, H. A., Yadav, R. C., Canlas, P. E., Dong, X., & Ronald, P. C. (2001). Evidence for a disease-resistance pathway in rice similar to the NPR1-mediated signaling pathway in Arabidopsis. The Plant Journal, 27, 101–113.

    Article  PubMed  CAS  Google Scholar 

  • Chern, M., Fitzgerald, H. A., Canlas, P. E., Navarre, D. A., & Ronald, P. C. (2005). Over-expression of a rice NPR1 homolog leads to constitutive activation of defense response and hypersensitivity to light. Molecular Plant-Microbe Interactions, 18, 511–520.

    Article  PubMed  CAS  Google Scholar 

  • Durrant, W. E., & Dong, X. (2004). Systemic acquired resistance. Annual Review of Phytopathology, 42, 185–209.

    Article  PubMed  CAS  Google Scholar 

  • Endah, R., Beyene, G., Kiggundu, A., van den Berg, N., Schlüter, U., Kunert, K., et al. (2008). Elicitor and Fusarium-induced expression of NPR1-like genes in banana. Plant Physiology and Biochemistry, 46, 1007–1014.

    Article  PubMed  CAS  Google Scholar 

  • Fitzgerald, H. A., Chern, M. S., Navarre, R., & Ronald, P. C. (2004). Overexpression of (At)NPR1 in rice leads to a BTH and environment induced lesion mimic/cell death phenotype. Molecular Plant-Microbe Interactions, 17, 140–151.

    Article  PubMed  CAS  Google Scholar 

  • Friedrich, L., Lawton, K., Dietrich, R., Willitis, M., Cade, R., & Ryals, J. (2001). NIM1 overexpression in Arabidopsis potentiates plant disease resistance and results in enhanced effectiveness of fungicides. Molecular Plant-Microbe Interactions, 9, 1114–1124.

    Article  Google Scholar 

  • Glazebrook, J., Rogers, E. E., & Ausubel, F. M. (1996). Isolation of Arabidopsis mutants with enhanced disease susceptibility by direct screening. Genetics, 143, 973–982.

    PubMed  CAS  Google Scholar 

  • Grant, M., & Lamb, C. (2006). Systemic immunity. Current Opinion in Plant Biology, 9, 414–420.

    Article  PubMed  CAS  Google Scholar 

  • Helliwell, C., & Warterhouse, P. (2003). Constructs and methods for high-throughput gene silencing in plants. Methods, 30, 289–295.

    Article  PubMed  CAS  Google Scholar 

  • IRRI. (2002). Standard evaluation system for rice. Los Banos: International Rice Research Institute.

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Kim, S. T., Kim, S. G., Hwang, D. H., Kang, S. Y., Kim, H. J., Lee, B. H., et al. (2004). Proteomic analysis of pathogen-responsive proteins from rice leaves induced by rice blast fungus, Magnaporthe grisea. Proteomics, 4, 3569–3578.

    Article  PubMed  CAS  Google Scholar 

  • Kini, K. R., Vasanthi, N. S., & Shetty, H. S. (2000). Induction of β-1,3-glucanase in seedlings of pearl millet in response to infection by Sclerospora graminicola. European Journal of Plant Pathology, 106, 267–274.

    Article  CAS  Google Scholar 

  • Kusaba, M. (2004). RNA interference in crop plants. Current Opinion in Biotechnology, 15, 139–143.

    Article  PubMed  CAS  Google Scholar 

  • Le Henanff, G., Heitz, T., Mestre, P., Mutterer, J., Walter, B., & Chong, J. (2009). Characterization of Vitis vinifera NPR1 homologs involved in the regulation of pathogenesis-related gene expression. BMC Plant Biology, 9, 54.

    Article  PubMed  Google Scholar 

  • Lin, W. C., Lu, C. F., Wu, J. W., Cheng, M. L., Lin, Y. M., Yang, N. S., et al. (2004). Transgenic tomato plants expressing the Arabidopsis NPR1 gene display enhanced resistance to a spectrum of fungal and bacterial diseases. Transgenic Research, 13, 567–581.

    Article  PubMed  CAS  Google Scholar 

  • Makandar, R., Essig, J. S., Schapaugh, M. A., Trick, H. N., & Shah, J. (2006). Genetically engineered resistance to Fusarium head blight in wheat by expression of Arabidopsis NPR1. Molecular Plant-Microbe Interactions, 19, 123–129.

    Article  PubMed  CAS  Google Scholar 

  • Malnoy, M., Jin, Q., Borejsza-Wysocka, E. E., He, S. Y., & Aldwinckle, H. S. (2007). Overexpression of the apple MpNPR1 gene confers increased disease resistance in Malus × domestica. Molecular Plant-Microbe Interactions, 20, 1568–1580.

    Article  PubMed  CAS  Google Scholar 

  • Marĭa, C., Gisela, P., Jorge, G., Sonia, C., Cristina, B., Joaquima, M., et al. (2006). Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice. Planta, 223, 392–406.

    Article  Google Scholar 

  • Nishizawa, Y., Nishio, Z., Nakazono, K., Soma, M., Nakajima, E., Ugaki, M., et al. (1999). Enhanced resistance to blast (Magnaporthe grisea) in transgenic japonica rice by constitutive expression of rice chitinase. Theoretical and Applied Genetics, 99, 383–390.

    Article  CAS  Google Scholar 

  • Parkhi, V., Kumar, V., Campbell, L. M., Bell, A. A., Shah, J., & Rathore, K. S. (2010). Resistance against various fungal pathogens and reniform nematode in transgenic cotton plants expressing Arabidopsis NPR1. Transgenic Research, 19, 959–975.

    Article  PubMed  CAS  Google Scholar 

  • Potlakayala, S. D., DeLong, C., Sharpe, A., & Fobert, P. R. (2007). Conservation of NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 function between Arabidopsis thaliana and Brassica napus. Physiological and Molecular Plant Pathology, 71, 174–183.

    Article  CAS  Google Scholar 

  • Quilis, J., Peñas, G., Messeguer, J., Brugidou, C., & San Segundo, B. (2008). The Arabidopsis AtNPR1 inversely modulates defense responses against fungal, bacterial, or viral pathogens while conferring hypersensitivity to abiotic stresses in transgenic rice. Molecular Plant-Microbe Interactions, 21, 1215–1231.

    Article  PubMed  CAS  Google Scholar 

  • Rohilla, R., Singh, U. S., & Singh, R. L. (2002). Mode of action of acibenzolar-S-methyl against sheath blight of rice, caused by Rhizoctonia solani Kühn. Pest Management Science, 58, 63–69.

    Article  PubMed  CAS  Google Scholar 

  • Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual (3rd ed.). New York: Cold Spring Harbor Laboratory.

    Google Scholar 

  • Sandhu, D., Tasma, I. M., Frasch, R., & Bhattacharyya, M. K. (2009). Systemic acquired resistance in soybean is regulated by two proteins, orthologous to Arabidopsis NPR1. BMC Plant Biology, 9, 105.

    Article  PubMed  Google Scholar 

  • Schweizer, P., Schlagenhauf, E., Schaffrath, U., & Dudler, R. (1999). Different patterns of host genes are induced in rice by Pseudomonas syringae, a biological inducer of resistance, and the chemical inducer benzothiadiazole (BTH). European Journal of Plant Pathology, 105, 659–665.

    Article  CAS  Google Scholar 

  • Shi, Z., Maximova, S. N., Liu, Y., Verica, J., & Guiltinan, M. J. (2010). Functional analysis of the Theobroma cacao NPR1 gene in Arabidopsis. BMC Plant Biology, 10, 248.

    Article  PubMed  Google Scholar 

  • Shimono, M., Sugano, S., Nakayama, A., Jiang, C. J., Ono, K., Toki, S., et al. (2007). Rice WRKY45 plays a crucial role in benzothiadiazole-inducible blast resistance. The Plant Cell, 19, 2064–2076.

    Article  PubMed  CAS  Google Scholar 

  • Skamnioti, P., & Gurr, S. J. (2009). Against the grain: Safeguarding rice from rice blast disease. Trends in Biotechnology, 27, 141–150.

    Article  PubMed  CAS  Google Scholar 

  • Smith, J. A., & Metraux, J. P. (1991). Pseudomonas syringae pv. syringae induces systemic resistance to Pyricularia oryzae in rice. Physiological and Molecular Plant Pathology, 39, 451–461.

    Article  Google Scholar 

  • Spoel, S. H., Mou, Z., Tada, Y., Spivey, N. W., Genschik, P., & Dong, X. (2009). Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity. Cell, 137, 860–872.

    Article  PubMed  CAS  Google Scholar 

  • Sugano, S., Jiang, C. J., Miyazawa, S., Masumoto, C., Yazawa, K., Hayashi, N., et al. (2010). Role of OsNPR1 in rice defense program as revealed by genome-wide expression analysis. Plant Molecular Biology, 74, 549–562.

    Article  PubMed  CAS  Google Scholar 

  • Tada, Y., Spoel, S. H., Pajerowska-Mukhtar, K., Mou, Z., Song, J., Wang, C., et al. (2008). Plant immunity requires conformational changes of NPR1 via S-nitrosylation and thioredoxins. Science, 321, 952–956.

    Article  PubMed  CAS  Google Scholar 

  • Tang, J. L., Feng, J. X., Li, Q. Q., Wen, H. X., Zhou, D. L., Wilson, T. J., et al. (1996). Cloning and characterization of the rpfC gene of Xanthomonas oryzae pv. oryzae: involvement in exopolysaccharide production and virulence to rice. Molecular Plant-Microbe Interactions, 9, 664–666.

    Article  PubMed  CAS  Google Scholar 

  • Van Loon, L. C., & Van Strien, E. A. (1999). The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology, 55, 85–97.

    Article  Google Scholar 

  • Wally, O., Jayaraj, J., & Punja, Z. K. (2009). Broad-spectrum disease resistance to necrotrophic and biotrophic pathogens in transgenic carrots (Daucus carota L.) expressing an Arabidopsis NPR1 gene. Planta, 231, 131–141.

    Article  PubMed  CAS  Google Scholar 

  • Wang, D., Weaver, N. D., Kesarwani, M., & Dong, X. (2005). Induction of protein secretory pathway is required for systemic acquired resistance. Science, 308, 1036–1040.

    Article  PubMed  CAS  Google Scholar 

  • Yuan, Y., Zhong, S., Li, Q., Zhu, Z., Lou, Y., Wang, L., et al. (2007). Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility. Plant Biotechnology Journal, 5, 313–324.

    Article  PubMed  CAS  Google Scholar 

  • Zhang, Y., Wang, X., Cheng, C., Gao, Q., Liu, J., & Guo, X. (2008). Molecular cloning and characterization of GhNPR1, a gene implicated in pathogen responses from cotton (Gossypium hirsutum L.). Bioscience Reports, 28, 7–14.

    Article  PubMed  Google Scholar 

  • Zhang, X., Francis, M. I., Dawson, W. O., Graham, J. H., Orbović, V., Triplett, E. W., et al. (2010). Over-expression of the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker. European Journal of Plant Pathology, 128, 91–100.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the 973 Program of the Ministry of Science and Technology of China (2006CB101902), the special project of National Transgenic Plant Research and Commercialization (J99-A-029, JY04-A-01), the Foundation for University Key Teachers by the Ministry of Education of China (2000–65), and the Guangxi Natural Science Foundation (0007006).

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Correspondence to Jia-Xun Feng.

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Figure S1

The cloning of OsNPR1 gene from Indica rice cultivar Gui99. a The hybridization of Gui99 genomic DNA with the 228 bp internal sequence of OsNPR1 cDNA. 1-2, the λDNA digested with EcoRI and HindIII, respectively; 3, 1 kb DNA ladder; 4–6, Gui99 genomic DNA digested with EcoRI, BamHI, and EcoRI/BamHI, respectively. 7–9, the Southern blot autoradiogram of the lanes 4–6. b Colony hybridization of the partial library containing ca. 7.4 kb EcoRI/BamHI fragment of Gui99 DNA. The 228 bp internal sequence of amplified OsNPR1 cDNA was used as a probe. (PPT 345 kb)

Figure S2

Comparison of 422 bp RNAi fragment with the five NPR1-like homologous genes. Nucleotide sequences were aligned by AlignX multiple alignment software. The sequence in the box represents the longest ungapped homology (17 bp) between the 422 bp RNAi fragment and OsNPR5. (PPT 1765 kb)

Figure S3

Southern hybridization analysis of hygromycin resistant T1 plant lines. a Southern hybridization analysis of hygromycin resistant Gui99 OsNPR1-RNAi T1 plant lines. The 422 bp BamHI/SpeI DNA fragment released from digestion of plasmid pCAMBIA1301- OsNPR1i was used as a probe. Gui99 is the untransformed control plant. 1–6, Gui99 transgenic lines I1, I2, I3, I5, I6, and I7, respectively. b Southern hybridization analysis of hygromycin resistant TP309 OsNPR1-overexpressing T1 plant lines. The 3.1 kb EcoRV/XhoI DNA fragment (including partial OsNPR1 DNA) recovered from the digestion of plasmid pCAMBIA1301-UbiN-OsNPR1 was used as a probe. TP309 is the untransformed control plant; 1-8, TP309 transgenic lines 743, 758, 817, 878, 749, 862, 492, and 888, respectively. (PPT 1012 kb)

Figure S4

OsNPR1 is involved in BTH-induced blast resistance in rice. WT is the wild-type untransformed rice Gui99. The numbers 782 and 784 represent two independent Gui99 transgenic OsNPR1-overexpressing lines. The I1 and I3 are two independent Gui99 OsNPR1-RNAi lines. a Disease symptom of rice plants after infection with M. oryzae strain CHL0742. The tree-week-old rice seedlings were sprayed with 10 mM BTH (the commercial product Bion, Ciba-Geigy GmbH, Germany). The mock treatment was foliar spray with distilled water. The plants were inoculated with M. oryzae 48 h after treatment. The photograph was taken 7 days post inoculation. b Average disease index of the rice plants. Data are the means ± SD from three repeats, each using 10 leaves. Statistically significant differences are indicated by * (P < 0.05) and ** (P < 0.01). BTHtreated WT was compared with mock-treated WT. Each of BTH-treated OsNPR1-overexpressing lines was compared with BTH-treated WT. Each of BTH-treated OsNPR1-RNAi lines was compared with BTH-treated WT. c Northern blot analysis of the expression of OsNPR1 in rice plants. Total RNAs were extracted from rice leaves 0 h and 48 h after BTH treatment and also from leaves 48 h post inoculation (hpi). (PPT 1181 kb)

Figure S5

Resistance assay of the OsNPR1-RNAi plants to rice bacterial blight pathogen Xoo. Gui99 is the untransformed control plant, and I1-I7 are seven independent transgenic rice OsNPR1-RNAi plants. a Symptoms of OsNPR1-RNAi transgenic plant lines caused by Xoo. The photograph was taken 14 days post inoculation. b Leaf lesion length of OsNPR1-RNAi plant lines and wild-type plant 14 days post inoculation with Xoo. **indicate a significant difference (P<0.01) of the lesion length in OsNPR1-RNAi transgenic plants in comparison with the wild-type plant Gui99. The standard deviations are indicated on the figure. The experiments were repeated at least three times and similar results were obtained. (PPT 266 kb)

Figure S6

Resistance assay of the OsNPR1-overexpressing plants to rice bacterial blight pathogen Xoo. Gui99 and TP309 are untransformed control plants. The numbers 782, 783 and 784 represent three independent Gui99 transgenic lines. The numbers 357, 890, 485, 923, and 927 represent five independent TP309 transgenic OsNPR1-overexpressing lines. a Symptoms of OsNPR1-overexpressing plants caused by Xoo in Gui99 derived lines. The photograph was taken 14 days post inoculation. b Leaf lesion length of OsNPR1-overexpressing rice after infection with Xoo in Gui99 derived lines. **indicate a significant reduction (P<0.01) of the lesion length in the OsNPR1-overexpressing plant lines in comparison with their wild-type rice. The standard deviations are indicated on the figure. The experiments were repeated at least three times and similar results were obtained. c Symptoms of OsNPR1-overexpressing rice lines caused by Xoo in TP309 derived lines. The photograph was taken 14 days post inoculation. d Leaf lesion length of OsNPR1-overexpressing rice after infection with Xoo in TP309 derived lines. Statistically significant differences between transgenic plants and wild type plants are indicated by *(P < 0.05) and **(P < 0.01). The standard deviations are indicated on the figure. The experiments were repeated at least three times and similar results were obtained. (PPT 746 kb)

Figure S7

Transcription levels of defenserelated genes and OsNPR1 in transgenic plants after inoculation with Xoo 13751. Total RNAs were extracted from leaves at 0 h, 6 h, 12 h, 18 h, 24 h, and 48 h after pathogen inoculation. RNA gel blots were hybridized with probes for the PR-1a, PAL, POD, Actin, and OsNPR1 genes. a Increased expressions of defense genes PR-1a, PAL, and POD in OsNPR1- overexpressing lines 782 and 784 compared with the wild-type plant Gui99. b Increased expressions of defense genes PR- 1a, PAL, and POD in OsNPR1- overexpressing lines 890 and 927 compared with the wild-type plant TP309. c Decreased expressions of defense genes PR- 1a, PAL, and POD in OsNPR1-RNAi lines I1, I3 compared with the wild-type plant. (PPT 1093 kb)

Table S1

Primer pairs and primers used in this work (DOC 49 kb)

Table S2

Media used for rice tissue culture and transformation (DOC 38 kb)

Table S3

Gene probes used for Northern hybridization (DOC 40 kb)

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Feng, JX., Cao, L., Li, J. et al. Involvement of OsNPR1/NH1 in rice basal resistance to blast fungus Magnaporthe oryzae . Eur J Plant Pathol 131, 221–235 (2011). https://doi.org/10.1007/s10658-011-9801-7

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