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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (5): 1129-1140.doi: 10.3724/SP.J.1006.2022.12010

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Rice transcription factor WRKY68 plays a positive role in Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae

ZHU Zheng1,2(), WANG Tian-Xing-Zi1,2, CHEN Yue1,2, LIU Yu-Qing1,2, YAN Gao-Wei1,2, XU Shan1,2, MA Jin-Jiao1,2, DOU Shi-Juan1,2, LI Li-Yun1,2,*(), LIU Guo-Zhen1,2,*()   

  1. 1College of Life Sciences, Hebei Agricultural University, Baoding 071001, Hebei, China
    2Hebei Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071001, Hebei, China
  • Received:2021-02-10 Accepted:2021-09-09 Online:2022-05-12 Published:2021-10-19
  • Contact: LI Li-Yun,LIU Guo-Zhen E-mail:zhuzheng_mbb@163.com;Liliyun@hebau.edu.cn;gzhliu@hebau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31171528);National Natural Science Foundation of China(31400700)

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Abstract:

Leaf blight is a bacterial disease of rice which leads to heavy yield losses. Xa21 which harbors broad spectrum resistance to leaf blight is the first resistant gene cloned in rice and is widely used in rice breeding program. The identification of transcription factor involved in Xa21-mediated resistance to bacterial leaf blight is of great significance. In this study, transgenic WRKY68-RNAi lines with reduced expression level of WRKY68 protein in the background of Xa21 were generated. The WRKY68-RNAi transgenic lines showed reduced resistance to Xanthomonas oryzae pv. oryzae (Xoo), indicating that WRKY68 played a positive role in Xa21-mediated resistance in rice. No detectable changes of WRKY68 protein abundance in leaves at different stages and positions after inoculation were identified by Western blot, indicating that the expression of WRKY68 protein were independent of Xa21 and Xoo, and thus WRKY68 gene may function as a transcription factor to regulate downstream genes. It was further found that the abundance of a number of pathogenesis-related proteins, including PR1A, PR5, PR10A, PR-pha, and PAL1 were dramatically changed in WRKY68-RNAi transgenic lines when inoculated with Xoo, suggested that these pathogenesis-related proteins were regulated by WRKY68 and the corresponding genes were involved in the downstream defenses responses.

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

Fig. 1

Identification of WRKY68-RNAi transgenic lines by PCR and WB R#1, R#2, and R#3: independent WRKY68-RNAi transgenic lines; CK: 4021 (transgenic lines of TP309 harbor Xa21 gene); 1-8: transgenic plants in T2 generation; WRKY68: anti-WRKY68 antibody detected band; HSP: anti-HSP82 antibody detected band used as loading control."

Fig. 2

The agronomic traits investigation of WRKY68-RNAi transgenic lines A: plant height; B: the number of tillers; C: 1000-seed weight. CK: 4021 (transgenic lines of TP309 harbor Xa21 gene); R#1, R#2, and R#3: independent WRKY68-RNAi transgenic plants in T2 generation; **: P < 0.01. Error bars indicate the standard derivations."

Fig. 3

Responses of WRKY68-RNAi transgenic lines when inoculated with Xoo A: photographs of leaves at 12 dpi (days post inoculation). B: the development of lesion length on leaves inoculated with Xoo. C: lesion length at 12 dpi. Arrows indicate the frontline of lesions. **: P < 0.01. Bar: 1 cm. R#1, R#2, and R#3: independent WRKY68-RNAi transgenic lines; TP309: wild type; 4021: transgenic lines of TP309 harbor Xa21 gene."

Fig. S1

Abundance Xoo protein in inoculated rice leaves Equal amounts of total protein from multiple samples were resolved by SDS-PAGE. Antibodies: anti-Xoo polyclonal antibody, anti-HSP82 monoclonal antibody. R#2, R#3: WRKY68-RNAi transgenic lines; TP309: wild type; 4021: transgenic TP309 expressing Xa21."

Fig. S2

Expression abundance of pathogenesis-related proteins in leaves inoculated with Xoo Equal amounts of total protein, isolated from inoculated leaves (±1 cm within lesion line) of 5 d or 6 d, were resolved by SDS-PAGE or Tricine-SDS-PAGE. The red frames indicate the portion demonstrated in figure 6. PR1A: anti-PR1a antibody detected band; PR2: anti-PR2 antibody detected band; PR5: anti-PR5 antibody detected band; PR10A: anti-PR10a antibody detected band; PR-pha: anti-PR-pha antibody detected band; PAL1: anti-PAL1 antibody detected band; HSP: anti-HSP82 antibody detected band. R#1, R#2, and R#3: WRKY68-RNAi transgenic lines; TP309: wild type; 4021: transgenic TP309 expressing Xa21."

Fig. 4

Abundance Xoo protein in inoculated rice leaves The whole inoculated leaves were collected, total proteins were isolated and resolved by SDS-PAGE, then detected by anti-Xoo antibody (Fig. S1). Protein signal were detected by Mini Chemiluminescent Imager and Sage Capture software. Lane 1D software was used to extract signals of WB. Average was calculated for three repeats. Error bars are standard deviation (SD). Xoo cells (OD = 0.5) were used as the common reference (CR) to normalized the signal intensity among different WB analysis. R#2, R#3: independent WRKY68-RNAi transgenic lines; TP309: wild type; 4021: transgenic lines of TP309 harbor Xa21 gene."

Fig. 5

Expression profiling of WRKY68 protein in Xoo- inoculated leaves A: the abundance of WRKY68 protein in rice leaves at 3, 5, 7, and 9 dpi, respectively; B: schematic diagram of tissue collection in leaves inoculated with Xoo; C: the abundance of WRKY68 protein at ±1 cm, 1-3 cm, 3-5 cm, and 5-7 cm positions in Xoo-inoculated rice leaves. WRKY68: anti-WRKY68 antibody detected bands; HSP: anti-HSP82 antibody detected bands. TP309: wild type; 4021: transgenic lines of TP309 harbor Xa21 gene."

Fig. 6

Expression abundance of pathogenesis-related proteins in leaves inoculated with Xoo PR1A: anti-PR1A antibody detected band; PR2: anti-PR2 antibody detected band; PR5: anti-PR5 antibody detected band; PR10A: anti-PR10A antibody detected band; PR-pha: anti-PR-pha antibody detected band; PAL1: anti-PAL1 antibody detected band; HSP: anti-HSP82 antibody detected band used as loading control. R#1, R#2, and R#3: independent WRKY68-RNAi transgenic lines; TP309: wild type; 4021: transgenic lines of TP309 harbor Xa21 gene."

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