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.

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