Rice MPK17 Plays a Negative Role in the Xa21-Mediated Resistance Against Xanthomonas oryzae pv. oryzae

Abstract

Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases affecting rice production worldwide. Xa21 was the first disease resistance gene cloned in rice, which encodes a receptor kinase and confers broad resistance against Xoo stains. Dozens of components in the Xa21-mediated pathway have been identified in the past decades, however, the involvement of mitogen-activated protein kinase (MAPK) genes in the pathway has not been well described. To identify MAPK involved in Xa21-mediated resistance, the level of MAPK proteins was profiled using Western blot analysis. The abundance of OsMPK17 (MPK17) was found decreased during the rice-Xoo interaction in the background of Xa21. To investigate the function of MPK17, MPK17-RNAi and over-expression (OX) transgenic lines were generated. The RNAi lines showed an enhanced resistance, while OX lines had impaired resistance against Xoo, indicating that MPK17 plays negative role in Xa21-mediated resistance. Furthermore, the abundance of transcription factor WRKY62 and pathogenesis-related proteins PR1A were changed in the MPK17 transgenic lines when inoculated with Xoo. We also observed that the MPK17-RNAi and -OX rice plants showed altered agronomic traits, indicating that MPK17 also plays roles in the growth and development. On the basis of the current study and published results, we propose a "Xa21-MPK17-WRKY62-PR1A" signaling that functions in the Xa21-mediated disease resistance pathway. The identification of MPK17 advances our understanding of the mechanism underlying Xa21-mediated immunity, specifically in the mid- and late-stages.

Keywords: Bacterial blight; MAPK; Resistance gene Xa21; Rice; Transcription factor; WRKY.

Fig. 1

Expression of MPK17 in rice leaves at different time points following inoculation with Xoo. A Representative Western blot. B Expression level relative to HSP82. MPK17: anti-MPK17 antibody-detected band; HSP82: anti-HSP82 antibody detected-band used as loading control. 4021: transgenic TP309 lines harboring the Xa21 gene. Protein signals generated by WB were detected by Mini Chemiluminescent Imager and Sage Capture software. Lane 1D software was used to extract signals of WB. Relative intensity to HSP82 were calculated according to the intensity of MPK17 divided by the intensity of HSP82 signal. Mean was calculated for three repeats. Error bars are standard deviation (SD)

Fig. 2

Identification of MPK17-RNAi transgenic lines by PCR and WB. i118, i120: independent MPK17-RNAi transgenic lines; CK: 4021 (homozygous TP309 transgenic line harboring the Xa21 gene); 1–8: individual transgenic plants in T₃ generation; PCR: PCR genotyping results done with primers (5’-GAGTCGTAAGAGACTCTGTATG-3’ and 5’- GCGAGCTCGGTTTTCAGTTGAGCAAC-3’). MPK17: anti-MPK17 antibody-detected band; HSP82: anti-HSP82 antibody-detected band used as loading control. PCR: PCR product separated on agarose gel. WB: detection of MPK17 using anti-MPK17 antibody

Fig. 3

Agronomic traits of MPK17-RNAi transgenic plants. A and B 4021 control and MPK17-RNAi transgenic lines at mature stage (bar = 30 cm). C Panicles of 4021 and MPK17-RNAi transgenic lines at mature stage (Bar = 5 cm). D Internodes of 4021 and MPK17-RNAi transgenic lines at mature stage (Bar = 10 cm). I: the third internode from the top; II: the second internode from the top; III: the upmost internode. E Columns showing plant height. F Columns showing number of grains per spike. G Columns showing for panicle length. H Columns showing seed setting rate. I Columns showing 1000-grain weight. i118 and i120: independent MPK17-RNAi transgenic lines; 4021: TP309 transgenic line harboring the Xa21 gene. Mean was calculated for eight individual plants. Error bars are standard deviation (SD). “**” designate difference at p < 0.05 level

Fig. 4

Identification of MPK17-OX transgenic lines by PCR and WB. OX202 and OX208: independent MPK17-OX transgenic lines; CK: 4021 (homozygous TP309 transgenic line harboring the Xa21 gene); 1–8: individual transgenic plants in T₃ generation; PCR: PCR genotyping results done with primers (5’-GCGGTACCATGGGCGGCCGCGCCCGCTC-3’, 5’-GCGAGCTCGGTTTTCAGTTGAGCAAC-3’). MPK17-OX: the transgenic MPK17 protein; MPK17-Endogenous: the endogenous MPK17 protein in rice; HSP82: anti-HSP82 antibody-detected band used as loading control

Fig. 5

Agronomic characters of MPK17-OX transgenic plants. A, B 4021 and MPK17-OX transgenic lines at mature stage (Bar = 30 cm). C Panicles of 4021 and MPK17-OX transgenic lines at mature stage (Bar = 5 cm). D Columns showing plant height. E Columns showing numbers of grains per spike. F Columns showing for panicle length. G Columns showing for seed setting rate. H Columns showing 1000-grain weight. OX202 and OX208: independent MPK17-OX transgenic lines; CK: 4021 (TP309 transgenic line harboring the Xa21 gene). Mean was calculated for eight individual plants. Error bars are standard deviation (SD). “*” and “**” designate difference at p < 0.05 and p < 0.01 levels respectively

Fig. 6

MPK17 knockdown enhanced Xa21-mediated resistance. A Disease lesions in inoculated leaves of i118 and i120 and control plants at 12 dpi. B Lesion length at different dpi. C Lesion length at 12 dpi. Arrows indicate the frontline of lesions. **denotes difference at p < 0.01 level. Bar = 1 cm. i118 and i120: independent MPK17-RNAi transgenic lines; TP309: Japonica rice; 4021: homozygous TP309 transgenic line harboring the Xa21 gene

Fig. 7

Xa21-mediated resistance in rice following overexpression of MPK17 at 27 °C. A Lesions in inoculated leaves of OX202, OX208 and control plants at 12 dpi. B Lesion length at different dpi. C Lesion length at 12 dpi. Arrows indicate the frontline of lesions. **designate difference at p < 0.01 level. Bar = 1 cm. OX202 and OX208: independent MPK17-OX transgenic lines; TP309: Japonica rice variety; 4021: TP309 transgenic line harboring the Xa21 gene

Fig. 8

Abundance of Xoo proteins in rice expressing different levels of MPK17 following Xoo inoculation. A MPK17-RNAi transgenic plants. B MPK17-OX transgenic plants. Inoculated rice plants were cultivated at 31℃ (MPK17-RNAi transgenic rice) or 27℃ (MPK17-OX transgenic rice). The whole leaves were collected at 0, 6, 9 dpi, total proteins were isolated and separated by SDS-PAGE, then detected by anti-Xoo antibody (Additional file 1: Fig. S2). Protein signal were detected by Mini Chemiluminescent Imager and Sage Capture software. Lane 1D software was used to capture the signals of WB. Mean was calculated for three repeats. Error bars are standard deviation (SD). **denotes difference at p < 0.01 level. i118, i120: independent MPK17-RNAi transgenic lines; OX202, OX208: independent MPK17-OX transgenic lines; TP309: Japonica rice variety; 4021: homozygous transgenic TP309 line harboring the Xa21 gene

Fig. 9

Expression of WRKY in MPK17-regulated transgenic rice after inoculated with Xoo. A Expression of five WRKY proteins in MPK17-RNAi transgenic rice after inoculated with Xoo. B Expression of five WRKY proteins in MPK17-OX transgenic rice after inoculated with Xoo. C Quantitative comparison of WB intensities of WRKY62 protein in panel (A). D Quantitative comparison of WB intensities of WRKY62 protein in panel (B). WRKY42: anti-WRKY42 antibody-detected band; WRKY62: anti-WRKY62 antibody-detected band; WRKY67: anti-WRKY67 antibody-detected band; WRKY68: anti-WRKY68 antibody-detected band; WRKY76: anti-WRKY76 antibody-detected band; HSP82: anti-HSP82 antibody-detected band used as loading control. i118, i120: independent MPK17-RNAi transgenic lines; OX202, OX208: independent MPK17-OX transgenic lines; TP309: Japonica rice variety; 4021: transgenic TP309 line harboring the Xa21 gene. Signal of WRKY62 protein in panel A and B 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). **designate difference at p < 0.01 level

Fig. 10

Expression of PR1A in MPK17-regulated transgenic plants following Xoo inoculation. A Expression of PR1A protein in MPK17-RNAi transgenic rice after inoculated with Xoo. B Expression of PR1A protein in MPK17-OX transgenic rice after inoculated with Xoo. C Quantitative comparison of WB intensities of WRKY62 protein in panel (A). D Quantitative comparison of WB intensities of WRKY62 protein in panel (B). PR1A: anti-PR1A antibody detected band; HSP82: anti-HSP82 antibody detected band used as loading control. i118, i120: independent MPK17-RNAi transgenic lines; OX202, OX208: independent MPK17-OX transgenic lines; TP309: Japonica rice variety; 4021: transgenic TP309 line harboring the Xa21 gene. Signal of PR1A protein in panel (A) and (B) 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). * and **designate difference at p < 0.05 and p < 0.01 levels respectively

Fig. 11

A model for the mid- and late-stages of the Xa21-mediated immunity. G⁺ and G⁻ denote the genetic evidence that supports a positive or negative regulator, respectively; solid and dotted lines denote WB or RT-PCR evidence, respectively