Rice OsWRKY42 is a Novel Element in Xa21-mediated Resistance Pathway Against Bacterial Leaf Blight

doi:10.11983/CBB21025

Abstract

Abstract:

Xa21 was the earliest resistant gene cloned in rice, which has broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo). Our previous study demonstrated that, OsWRKY42 might play a role in Xa21-mediated resistance pathway. In this study, OsWRKY42-RNA interference (OsWRKY42-RNAi) transgenic rice were generated under the background of Xa21, Western blot (WB) verified transgenic plants were used to inoculate Xoo, it was found that the lesion length was longer than that resistance control 4021 (carrying Xa21 gene), indicating that the down-regulation of OsWRKY42 protein abundance impaired Xa21-mediated resistance. In OsWRKY42-RNAi transgenic rice, it was also found by WB analysis that the abundance of OsPR6, OsPR15 and OsPR16 protein were decreased, while the abundance of OsPR1A, OsPR1B, OsPR2 and OsPR10A protein were increased, implying that these pathogenesis-related proteins were regulated by OsWRKY42 and play roles in Xa21-medicated resistance to Xoo at the downstream of OsWRKY42. In conclusion, the results showed that OsWRKY42 is a novel element in Xa21-mediated resistance pathway against Xoo and enhanced our understanding for the mechanism of rice Xa21-mediated resistance.

Key words: rice, bacterial leaf blight, resistant gene Xa21, WRKY transcription factor, pathogenesis-related protein

Tianxingzi Wang, Zheng Zhu, Yue Chen, Yuqing Liu, Gaowei Yan, Shan Xu, Tong Zhang, Jinjiao Ma, Shijuan Dou, Liyun Li, Guozhen Liu. Rice OsWRKY42 is a Novel Element in Xa21-mediated Resistance Pathway Against Bacterial Leaf Blight[J]. Chinese Bulletin of Botany, 2021, 56(6): 687-698.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB21025

https://www.chinbullbotany.com/EN/Y2021/V56/I6/687

Figures/Tables 6

Figure 1 Verification of rice OsWRKY42-RNAi transgenic lines Equal amounts of total protein from 4021 and OsWRKY42-RNAi transgenic lines were separated by Tricine-SDS-PAGE. The protein signal was detected by Mini Chemiluminescent Imager and Sage Capture software. PCR: PCR verification; WB: Western blot verification; CK: 4021 control of transgenic donor; R01-R03: Independent OsWRKY42-RNAi transgenic lines; 1-8: Individual plants within same transgenic lines; W42: Anti-OsWRKY42 polyclonal antibody detected band; HSP: Anti-OsHSP82 monoclonal antibody detected band.

Figure 2 Chlorophyll contents in rice OsWRKY42-RNAi transgenic lines (A)-(D) Chlorophyll a (Chl a) content, chlorophyll b (Chl b) content, total chlorophyll (total Chl) content and the ratio of Chl a/b in OsWRKY42-RNAi transgenic lines. TP309: Wild type; 4021: Homozygous transgenic TP309 line expressing Xa21; R01-R03: OsWRKY42-RNAi transgenic lines. Error bars are standard deviation (SD) (n=3); * indicate significant differences at P<0.05; ** indicate significant differences at P<0.01 (Student's t-test).

Figure 3 Phenotype of rice OsWRKY42-RNAi transgenic lines inoculated with Xoo (A) The phenotype of OsWRKY42-RNAi transgenic lines at 14-day post inoculation (bars=1 cm); (B) Lesion development of inoculated plant; (C) Lesion length of inoculated plants at 14 day post inoculation. Error bars are standard deviation (SD) (n>9); ** significant differences at P<0.01 level. R01-R03: OsWRKY42-RNAi transgenic lines; TP309: Wild type; 4021: Transgenic TP309 expressing Xa21

Figure 4 Expression abundance of total Xoo protein in inoculated rice leaves Equal amounts of total protein from multiple samples were resolved by SDS-PAGE (appendix figure 1), antibodies are anti-XOO polyclonal antibody and anti-OsHSP82 monoclonal antibody. Western blot (WB) signals were detected by Mini Chemiluminescent Imager and Sage Capture software. Lane 1D software was used to extract signals of Western blot. Average and standard deviation was calculated for three repeats. Error bars are standard deviation (SD). * P<0.05; ** P<0.01; TP309: Wild type; 4021: Transgenic TP309 expressing Xa21; R01 and R02 were homozygous OsWRKY42- RNAi transgenic lines.

Figure 5 Expression profiling of the OsWRKY42 protein in rice-Xoo interaction (A) Expression profiling of OsWRKY42 protein at different time points after inoculation. Samples were collected at 0, 1, 2, 4, 6-day post inoculation, respectively; (B) Schematic diagram for sample collection of inoculated leaves. Based on the lesion line (0), samples were taken at ±1, 1-3, 3-5, and 5-7 cm of the leaves at 6-day post inoculation, respectively; (C) Expression profiling of OsWRKY42 protein at different position of leaves at 6-day post inoculation. Based on lesion line, different portion of leaf tissues were collected after inoculation. Equal amounts of total protein from multiple samples were resolved by Tricine-SDS-PAGE. Antibodies are anti-OsWRKY42 polyclonal antibody and anti-OsHSP82 monoclonal antibody. OsWRKY42 protein signal were detected by Sage Capture software. TP309: Wild type; 4021: Transgenic TP309 expressing Xa21; Xoo: Total protein isolated from Xoo cells; W42 and HSP are shown in Figure 1.

Figure 6 Expression profiling of pathogenesis-related proteins in rice OsWRKY42-RNAi transgenic lines Equal amounts of total protein, isolated from inoculated leaves (lesion line ±1 cm) of 3-day and 6-day, were resolved by SDS-PAGE or Tricine-SDS-PAGE. The antibodies were anti-OsPR1A, -OsPR1B and -OsPR10A polyclonal antibodies (Wu et al., 2011); anti-OsPR2, -OsPR6, -OsPR15 and -OsPR16 polyclonal antibodies (Hou et al., 2012) and anti-OsHSP82 monoclonal antibody (Li et al., 2011). TP309: Wild type; 4021: Transgenic TP309 individual expressing Xa21; R01-R03: OsWRKY42-RNAi transgenic lines; HSP is shown in Figure 1.

References 49

[1]	白辉, 王宪云, 曹英豪, 李晓明, 李莉云, 陈浩, 刘丽娟, 朱健辉, 刘国振 (2010). 水稻叶绿体蛋白质在生长发育过程中的表达研究. 生物化学与生物物理进展 37, 988-995.
[2]	窦世娟, 关明俐, 李莉云, 刘国振 (2014). 水稻的病程相关基因. 科学通报 59, 245-258.
[3]	李雪姣, 范伟, 牛东东, 关明俐, 缪刘杨, 史佳楠, 窦世娟, 魏健, 刘丽娟, 李莉云, 刘国振 (2014). 水稻病程相关PR1家族蛋白质在叶片生长及与白叶枯病菌互作反应中的表达. 植物学报 49, 127-138.
[4]	缪刘杨, 周亮, 杨烁, 李莉云, 李雪姣, 范伟, 兰金苹, 史佳楠, 刘丽娟, 刘国振 (2014). 水稻转录因子WRKY42的转录、表达及其与W-box的结合特征分析. 生物化学与生物物理进展 41, 682-692.
[5]	Chen Q, Huang XE, Chen XH, Shamsunnaher, Song WY (2019). Reversible activation of XA21-mediated resistance by temperature. Eur J Plant Pathol 153, 1177-1184. DOI URL
[6]	Chen XW, Chern M, Canlas PE, Ruan DL, Jiang CY, Ronald PC (2010). An ATPase promotes autophosphorylation of the pattern recognition receptor XA21 and inhibits XA21-mediated immunity. Proc Natl Acad Sci USA 107, 8029-8034. DOI URL
[7]	Chen XW, Zuo SM, Schwessinger B, Chern M, Canlas PE, Ruan DL, Zhou XG, Wang J, Daudi A, Petzold CJ, Heazlewood JL, Ronald PC (2014). An XA21-associated kinase (OsSERK2) regulates immunity mediated by the XA21 and XA3 immune receptors. Mol Plant 7, 874-892. DOI URL
[8]	Cheng HT, Liu HB, Deng Y, Xiao JH, Li XH, Wang SP (2015). The WRKY45-2 WRKY13 WRKY42 transcriptional regulatory cascade is required for rice resistance to fungal pathogen. Plant Physiol 167, 1087-1099. DOI URL
[9]	Choi NY, Lee E, Lee SG, Choi CH, Park SR, Ahn I, Bae SC, Hwang CH, Hwang DJ (2017). Genome-wide expression profiling of OsWRKY superfamily genes during infection with Xanthomonas oryzae pv. oryzae using real- time PCR. Front Plant Sci 8, 1628. DOI URL
[10]	Das A, Pramanik K, Sharma R, Gantait S, Banerjee J (2019). In-silico study of biotic and abiotic stress-related transcription factor binding sites in the promoter regions of rice germin-like protein genes. PLoS One 14, e0211887.
[11]	Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000). The WRKY superfamily of plant transcription factors. Trends Plant Sci 5, 199-206. PMID
[12]	Guo MC, Lan JP, Shi J, Guan ML, Wei J, Liu LJ, Li LY, Dou SJ, Liu GZ (2015). Western blot detection of Xanthomonas oryzae pv. oryzae in rice. J Plant Pathol Microbiol S4, 005.
[13]	Han M, Kim CY, Lee J, Lee SK, Jeon JS (2014). OsWRKY42 represses OsMT1d and induces reactive oxygen species and leaf senescence in rice. Mol Cells 37, 532-539. DOI URL
[14]	Hou MM, Xu WJ, Bai H, Liu YM, Li LY, Liu LJ, Liu B, Liu GZ (2012). Characteristic expression of rice pathogenesis-related proteins in rice leaves during interactions with Xanthomonas oryzae pv. oryzae. Plant Cell Rep 31, 895- 904.
[15]	Hwang SH, Yie SW, Hwang DJ (2011). Heterologous expression of OsWRKY6 gene in Arabidopsis activates the expression of defense related genes and enhances resistance to pathogens. Plant Sci 181, 316-323. DOI URL
[16]	Jiang N, Yan J, Liang Y, Shi YL, He ZZ, Wu YT, Zeng Q, Liu XL, Peng JH (2020). Resistance genes and their interactions with bacterial blight/leaf streak pathogens (Xanthomonas oryzae) in rice (Oryza sativa L.)-an updated review. Rice (NY) 13, 3.
[17]	Jiang YN, Chen XH, Ding XD, Wang YS, Chen Q, Song WY (2013). The XA21 binding protein XB25 is required for maintaining XA21-mediated disease resistance. Plant J 73, 814-823. DOI URL
[18]	Jones JDG, Dangl JL (2006). The plant immune system. Nature 444, 323-329. DOI URL
[19]	Kim SG, Kim ST, Wang YM, Yu S, Choi IS, Kim YC, Kim WT, Agrawal GK, Rakwal R, Kang KY (2011). The RNase activity of rice probenazole-induced protein 1 (PBZ1) plays a key role in cell death in plants. Mol Cells 31, 25-31. DOI URL
[20]	Lamb C, Dixon RA (1997). The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48, 251-275. DOI URL
[21]	Lee SW, Han SW, Sririyanum M, Park CJ, Seo YS, Ronald PC (2009). A type I-secreted, sulfated peptide triggers XA21-mediated innate immunity. Science 326, 850-853. DOI URL
[22]	Li XM, Bai H, Wang XY, Li LY, Cao YH, Wei J, Liu YM, Liu LJ, Gong XD, Wu L, Liu SQ, Liu GZ (2011). Identification and validation of rice reference proteins for Western blotting. J Exp Bot 62, 4763-4772. DOI URL
[23]	Lichtenthaler HK (1987). Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Method Enzymol 148, 350-382.
[24]	Liu XQ, Bai XQ, Wang XJ, Chu CC (2007). OsWRKY71, a rice transcription factor, is involved in rice defense response. J Plant Physiol 164, 969-979. DOI URL
[25]	McGee JD, Hamer JE, Hodges TK (2001). Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthe grisea. Mol Plant Microbe Interact 14, 877-886.
[26]	Pandey SP, Somssich IE (2009). The role of WRKY transcription factors in plant immunity. Plant Physiol 150, 1648- 1655. DOI PMID
[27]	Park CJ, Bart R, Chern M, Canlas PE, Bai W, Ronald PC (2010). Overexpression of the endoplasmic reticulum chaperone BiP3 regulates XA21-mediated innate immunity in rice. PLoS One 5, e9262. DOI URL
[28]	Park CJ, Peng Y, Chen XW, Dardick C, Ruan DL, Bart R, Canlas PE, Ronald PC (2008). Rice XB15, a protein phosphatase 2C, negatively regulates cell death and XA21- mediated innate immunity. PLoS Biol 6, e282. DOI URL
[29]	Park CJ, Sharma R, Lefebvre B, Canlas PE, Ronald PC (2013). The endoplasmic reticulum-quality control component SDF2 is essential for XA21-mediated immunity in rice. Plant Sci 210, 53-60. DOI URL
[30]	Park CJ, Wei T, Sharma R, Ronald PC (2017). Overexpression of rice auxilin-like protein, XB21, induces necrotic lesions, up-regulates endocytosis-related genes, and confers enhanced resistance to Xanthomonas oryzae pv. oryzae. Rice (NY) 10, 27.
[31]	Peng Y, Bartley LE, Canlas P, Ronald PC (2010). OsWRKY IIa transcription factors modulate rice innate immunity. Rice (NY) 3, 36-42. DOI URL
[32]	Peng Y, Bartley LE, Chen XW, Dardick C, Chern M, Ruan R, Canlas PE, Ronald PC (2008). OsWRKY62 is a negative regulator of basal and Xa21-mediated defense against Xanthomonas oryzae pv. oryzae in rice. Mol Plant 1, 446-458. DOI URL
[33]	Pillai SE, Kumar C, Patel HK, Sonti RV (2018). Overexpression of a cell wall damage induced transcription factor, OsWRKY42, leads to enhanced callose deposition and tolerance to salt stress but does not enhance tolerance to bacterial infection. BMC Plant Biol 18, 177. DOI URL
[34]	Qiu DY, Xiao J, Ding XH, Xiong M, Cai M, Cao YL, Li XH, Xu CG, Wang SP (2007). OsWRKY13mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling. Mol Plant Microbe Interact 20, 492-499. DOI URL
[35]	Rice WRKY Working Group (2012). Nomenclature report on rice WRKY's-conflict regarding gene names and its solution. Rice (NY) 5, 3.
[36]	Ryu HS, Han M, Lee SK, Cho JI, Ryoo N, Heu S, Lee YH, Bhoo SH, Wang GL, Hahn TR, Jeon JS (2006). A comprehensive expression analysis of the WRKY gene superfamily in rice plants during defense response. Plant Cell Rep 25, 836-847. DOI URL
[37]	Sahu A, Das A, Saikia K, Barah P (2020). Temperature differentially modulates the transcriptome response in Oryza sativa to Xanthomonas oryzae pv. oryzae infection. Genomics 112, 4842-4852. DOI URL
[38]	Schwessinger B, Zipfel C (2008). News from the frontline: recent insights into PAMP-triggered immunity in plants. Curr Opin Plant Biol 11, 389-395. DOI PMID
[39]	Seo YS, Chern M, Bartley LE, Han M, Jung KH, Lee I, Walia H, Richter T, Xu X, Cao PJ, Bai W, Ramanan R, Amonpant F, Arul L, Canlas PE, Ruan R, Park CJ, Chen XW, Hwang S, Jeon JS, Ronald PC (2011). Towards establishment of a rice stress response interactome. PLoS Genet 7, e1002020. DOI URL
[40]	Shimono M, Koga H, Akagi A, Hayashi N, Goto S, Sawada M, Kurihara T, Matsushita A, Sugano S, Jiang CJ, Kaku H, Inoue H, Takatsuji H (2012). Rice WRKY45plays important roles in fungal and bacterial disease resistance. Mol Plant Pathol 13, 83-94. DOI PMID
[41]	Son S, An HK, Seol YJ, Park SR, Im JH (2020). Rice transcription factor WRKY114 directly regulates the expression of OsPR1a and Chitinase to enhance resistance against Xanthomonas oryzae pv. oryzae. Biochem Biophys Res Commun 533, 1262-1268. DOI URL
[42]	Song WY, Wang GL, Chen LL, Kim HS, Pi LY, Holsten T, Gardner J, Wang B, Zhai WX, Zhu LH, Fauquet C, Ronald P (1995). A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science 270, 1804-1806. PMID
[43]	Tanaka A, Christensen MJ, Takemoto D, Park P, Scott B (2006). Reactive oxygen species play a role in regulating a fungus-perennial ryegrass mutualistic interaction. Plant Cell 18, 1052-1066. PMID
[44]	Vo KTX, Kim CY, Hoang TV, Lee SK, Shirsekar G, Seo YS, Lee SW, Wang GL, Jeon JS (2017). OsWRKY67 plays a positive role in basal and XA21-mediated resistance in rice. Front Plant Sci 8, 2220. DOI URL
[45]	Wang X, Li BB, Ma TT, Sun LY, Tai L, Hu CH, Liu WT, Li WQ, Chen KM (2020). The NAD kinase OsNADK1 affects the intracellular redox balance and enhances the tolerance of rice to drought. BMC Plant Biol 20, 11. DOI URL
[46]	Wang YS, Pi LY, Chen XH, Chakrabarty PK, Jiang JD, De Leon AL, Liu GZ, Li LC, Benny U, Oard J, Ronald PC, Song WY (2006). Rice XA21 binding protein 3 is a ubiquitin ligase required for full Xa21-mediated disease resistance. Plant Cell 18, 3635-3646. DOI URL
[47]	Wu Q, Hou MM, Li LY, Liu LJ, Hou YX, Liu GZ (2011). Induction of pathogenesis-related proteins in rice bacterial blight resistant gene XA21-mediated interactions with Xanthomonas oryzae pv. oryzae. J Plant Pathol 93, 455- 459.
[48]	Yang S, Zhou L, Miao LY, Shi JN, Sun CQ, Fan W, Lan JP, Chen H, Liu LJ, Dou SJ, Liu GZ, Li LY (2016). The expression and binding properties of the rice WRKY68 protein in the Xa21-mediated resistance response to Xanthomonas oryzae pv. oryzae. J Integr Agr 15, 2451- 2460. DOI URL
[49]	Zhang XC, Li DY, Zhang HJ, Wang XE, Zheng Z, Song FM (2010). Molecular characterization of rice OsBIANK1, encoding a plasma membrane-anchored ankyrin repeat protein, and its inducible expression in defense responses. Mol Biol Rep 37, 653-660. DOI URL