Regulation of OsPR10a Promoter Activity by Phytohormone and Pathogen Stimulation in Rice

doi:10.1016/j.rsci.2021.07.005

Abstract

Abstract:

OsPR10a is one of the well known pathogenesis-related genes in rice, and is induced by multiple plant hormones and pathogens. However, the underlying transcriptional regulation mechanisms in response to different signals and their crosstalks are still largely unknown. In order to find new players participated in the activation of OsPR10a, we systematically analyzed the basal expression patterns as well as the expression responses of a 2.5 kb OsPR10a promoter in rice transgenic plants after phytohormone and pathogen stimulations. In agreement with the native gene expression, the OsPR10a promoter can drive glucuronidase (GUS) gene expressing in spots of leaf cells, leaf trichomes, lemmas and paleae, germinating embryos, calli and root tips. The leaf expression of OsPR10a::GUS was dramatically increased upon jasmonic acid (JA) and cytokinin (CK) treatments, or challenges of the pathogen Magnaporthe grisea and Xanthomonas oryzae pv. oryzae. Thus, the OsPR10a promoter reported here can faithfully reflect its native gene expression. The effects of several JA and CK responsive OsWRKY genes on the regulation of OsPR10a promoter were then inspected by luciferase transient expression assay, and the JA inducible OsWRKY10 transcription factor was found as a new positive regulator of OsPR10a. However, the key transcription factors of JA and CK signaling pathways, OsMYC2 and B-type response regulators, were not responsible for the activation of OsPR10a promoter. Our findings provided new insights into the regulation of OsPR10a expression during plant-hormone/pathogen interactions, and the OsPR10a reporter system can be useful to unravel novel regulators from both pathogen and host.

Key words: OsPR10a promoter, β-glucuronidase, phytohormone, pathogen, OsWRKY10, rice

Ersong Zheng, Xuming Wang, Rumeng Xu, Feibo Yu, Chao Zheng, Yong Yang, Yang Chen, Jianping Chen, Chengqi Yan, Jie Zhou. Regulation of OsPR10a Promoter Activity by Phytohormone and Pathogen Stimulation in Rice[J]. Rice Science, 2021, 28(5): 442-456.

Figures/Tables 16

Fig. S1. Map of the OsPR10a::GUS vector used for transformation. LB, Left border; RB, Right border; Pnos, Nos promoter; HPT, Hygromycin phosphotransferase gene; T35S:CaMV35S terminator; GUS, β-glucuronidase gene; Tnos, Nos terminator.

Fig. S2. Histochemical staining of OsPR10a::GUS expression in leves of T0 rice transgenic plants.A, B and C, GUS expression with weak or strong blue spots scattered randomly on the leaf surface of three independent transgenic lines. D, GUS expression only induced at the cutting edges. Scale bars, 0.5 mm.

Fig. 1. Characterization of tissue expression pattern of OsPR10a.A?H, Histochemical staining of the OsPR10a::GUS activity in the 5-day-old rice transgenic plants. Parts of the root and shoot, including mature region with lateral roots (A and B), elongation zone (C), root meristem (D), the first true leaf (E), semi true leaf (F and G) and coleoptile (H) with GUS staining were shown. Scale bars are 200 μm in A to D and 1 mm in E to H, respectively.I?P, GUS expression in the young leaf of 14-day-old seedling (I and J), flag leaf (K and L), floret before flowering (M and N), germinating seed (O) and resistant callus (P). The areas indicated by rectangles in I and K were enlarged to show the GUS activity in the trichomes on the surface of leaf (J and L) as indicated by white arrows. Scale bars, 1 mm.

Fig. S3. OsPR10a gene expression in tissues and organs of wild type plants.OsPR10a gene expression was analyzed in different tissues and organs of wild type plant by qRT-PCR. The expression level was represented as fold change (FC) relative to the level of root was shown. Data are Mean?± SD (n?= 3).

Fig. 2. Expression of OsPR10a mRNA transcript level in response to hormone treatments for 24 h compared to the control (Cont).A, Expression in leaf. B, Expression in root. Data are Mean ± SD (n = 3).SA, Salicylic acid; IAA, Indoleacetic acid; GA, Gibberellin; ET, Ethylene; JA, Jasmonic acid; CK, Cytokinin; ABA, Abscisic acid.

Fig. S4. Dynamic changes of OsPR10a gene transcript levels in response to JA and CK treatments under different concentrations and time periods.A, OsPR10a gene expression in response to MeJA treatment. B, OsPR10a gene expression in response to KN treatment. The OsPR10a expression level was represented as fold change (FC) relative to the value at 0 h.

Fig. 3. Expression of OsPR10a::GUS in response to jasmonate (JA) and cytokinin (CK) treatments.A and C, GUS activity in the leaves of OsPR10a::GUS transgenic plants treated with JA (100 μmol/L) and CK (100 μmol/L) for 24 h. Data are shown as Mean ± SD (n = 3). ** indicates significant difference at the 0.01 level by the Student’s t-test. B and D, GUS staining in the leaves of OsPR10::GUS transgenic plants treated with JA (100 μmol/L) and CK (100 μmol/L) for 24 h. Two entire leaves for each treatment were shown. Scale bars, 1 cm. Magnified images of the areas with rectangles were shown aside. Scale bars, 1 mm.

Fig. 4. Expression of OsPR10a::GUS in response to M. grisea infection.A, GUS staining in leaves of OsPR10a::GUS transgenic plants sprayed with H2O + 0.05% Tween 20 (Control) or M. grisea strain Guy 11 for 3 d. Two entire leaves for each treatment were shown. Scale bar, 1 cm.B, Magnified images of the area indicated by rectangles in A. Scale bars, 1 mm.C, GUS staining in detached leaves of OsPR10a::GUS transgenic plants spot inoculated with H2O + 0.05% Tween 20 (Control) or M. grisea strain Guy 11 for 2 d. Two entire leaves for each treatment were shown. Scale bar, 1 cm.D, Magnified images of the area indicated by rectangles in C. Scale bars, 1 mm.E and F, GUS activities in leaves of OsPR10a::GUS transgenic plants sprayed (E) or spot inoculated (H) with H2O + 0.05% Tween 20 (Control) or M. grisea strain Guy 11. Data are shown as Mean ± SD (n = 3). ** and *** indicate significant differences at the 0.01 and 0.001 levels by the Student’s t-test.

Fig. 5. Expression of OsPR10a::GUS in response to Xoo infection.A, GUS staining in leaves of OsPR10a::GUS transgenic plants with clip inoculation by H2O (Control) or Xoo strain PXO341 for 24 h. Two entire leaves for each treatment were shown. Scale bar, 1 cm. B, Magnified images of the inoculated area indicated by the rectangle in A. Scale bars, 1 mm. C, GUS activity in leaves of OsPR10a::GUS transgenic plants with clip inoculation by H2O (Control) or Xoo strain PXO341 for 24 h. Data are shown as Mean ± SD (n = 3). ** indicates a significant difference at the 0.01 level by the Student’s t-test.

Fig. 6. Transactivation assays of OsPR10a promoter on JA and CK responsive transcription factors.A, Schematic diagram of OsPR10a promoter (2 523 bp). The numbers indicate the distance from the start codon. The predicted cis-elements (GCC box, G-like box, G/A box, ARR1 element, W box and TGA element) are indicated on the forward DNA strand. B, Schematic diagram of double-reporter and effector plasmids used in the dual luciferase (LUC) assay. The OsPR10a promoter was fused to firefly LUC and the renilla luciferase (REN) driven by CaMV35S was served as an internal control. The effector plasmids contain the OsMYC2, OsRR21/22/ 23/24 and OsWRKY8/10/28/72/111 genes driven by Ubiquitin, respectively. C?E, Transient transcriptional activities of OsMYC2 (C), OsRR21/22/23/24 (D) and OsWRKY8/10/28/72/111 (E) on the expression of OsPR10a promoter. The relative LUC activity was shown as fold change to the empty vector whose LUC/REN ratio was set to 1. All data are shown as Mean ± SD (n = 6). ** and *** indicate significant differences at the 0.01 and 0.001 levels by the Student’s t-test.

Table S1 List of OsWRKY genes induced greater than 2-fold in leaves of wild type rice plants treated with 100 μmol/L MeJA.

Probe Set ID	RAP_Locus	Gene symbol	Fold Change (FC)
Os.29979.1.S1_at	Os01g0186000	OsWRKY10	9.7
Os.30512.1.S1_at	Os01g0626400	OsWRKY11	2.8
Os.30386.1.S1_at	Os01g0734000	OsWRKY23	2.7
Os.11773.1.S1_at	Os05g0343400	OsWRKY53	2.0
Os.14882.1.S1_at	Os12g0116600	OsWRKY56	4.0
Os.50015.1.S1_at	Os06g0649000	OsWRKY28	9.6
Os.27227.1.S1_at	Os09g0334500	OsWRKY74	2.3
Os.49656.1.S1_at	Os04g0287400	OsWRKY51	2.2
Os.33990.1.S1_at	Os11g0116900	OsWRKY46	3.6
Os.33131.1.A1_at	Os03g0321700	OsWRKY55	3.9
Os.55827.1.S1_at	Os11g0490900	OsWRKY72	13.2
Os.29987.2.S1_at	Os05g0583000	OsWRKY8	21.9
Os.2160.2.S1_x_at	Os01g0750100	OsWRKY13	2.2
Os.12032.1.S1_at	Os02g0181300	OsWRKY71	6.9
OsAffx.11050.2.S1_x_at	Os01g0246700	OsWRKY1	6.4

Fig. S5. The spatial temporal expression profile of OsPR10a (Os12g0555500) analysed on RiceXpro platform (http://ricexpro.dna.affrc.go.jp/)

Fig. S6. Expression of OsPR10a::GUS in response to SA treatment. A, GUS staining in leaves of OsPR10::GUS transgenic plants with no treatment (control) or treated with SA (100 μmol/L) for 24 h. Two entire leaves for each treatment were shown. Scale bars, 1 cm. B, Magnified images of the areas with rectanges were shown aside. Scale bars, 1 mm.

Fig. S7. Expression of OsPR10a::GUS in response to JA+SA treatment. A, GUS staining in leaves of OsPR10::GUS transgenic plants with MeJA (100 μmol/L) treatment alone or co-treatment with SA (100 μmol/L) for 24 h. Two entire leaves for each treatment were shown. Scale bars, 1 cm. B, Magnified images of the areas with rectanges were shown aside. Scale bars, 1 mm. C, GUS staining in leaves of OsPR10::GUS transgenic plants with KN (100 μmol/L) treatment alone or co-treatment with SA (100 μmol/L) for 24 h. Two entire leaves for each treatment were shown. Scale bars, 1 cm. D, Magnified images of the areas with rectanges were shown aside. Scale bars, 1 mm.

Fig. S8. The transcriptional effects of several Xoo responsive OsWRKY genes on the regulation of OsPR10a promoter.The transient transcriptional activity of OsWRKY6/45/51 on the expression of OsPR10a promoter. The relative LUC activity of each effector was shown as fold change to the empty vector whose LUC/REN ratio was set to 1. All data are shown as Mean ± SD (n = 6). * indicate the significant difference at P<0.01 by Student’s t-test analysis.

Table S2 Sequences of the primers used in this study.

Primer	Sequence (5'-3')
OsPR10a-Pr-F1	AGGATCCATGCGTTTTTGCTGTAGGA
OsPR10a-Pr-R1	AGGTACCCACTGAAGATATAATCTAACTAG
OsPR10a-Pr-F2	GCAGCCCGGGGGATCCATGCGTTTTTGCTGTAGGAC
OsPR10a-Pr-R2	ACCGCGGTGGCGGCCGCCACTGAAGATATAATCTAACTAGCT
OsMYC2-CDS-F1	ATGAACCTTTGGACGGACGACAACG
OsMYC2-CDS-R1	TTACCGGGCGGCGGTGCCA
OsMYC2-CDS-F2	TATCAAGCTAATTCGAGCTCATGAACCTTTGGACGGACGACAACG
OsMYC2-CDS-R2	CGACTCTAGAGGATCCTTACCGGGCGGCGGTGCC
OsRR21-CDS-F1	ATGGCGCCGGTGGAGGATG
OsRR21-CDS-R1	TCACATCTGTCCACTAAATCCGAAAATAT
OsRR21-CDS-F2	GCTAATTCGAGCTCGGTACCATGGCGCCGGTGGAGGAT
OsRR21-CDS-R2	CGACTCTAGAGGATCCTCACATCTGTCCACTAAATC
OsRR22-CDS-F1	ATGCTTCTGGGTGCTTTGAGGATG
OsRR22-CDS-R1	TCATATGCAGGCACCAAGTGGAAA
OsRR22-CDS-F2	GCTAATTCGAGCTCGGTACCATGCTTCTGGGTGCTTTGA
OsRR22-CDS-R2	CGACTCTAGAGGATCCTCATATGCAGGCACCAAGT
OsRR23-CDS-F1	ATGAGGGCGGCGGAGGAGAGGAAG
OsRR23-CDS-R1	TCATATGCAAGCTCCAAGGGAGTAGAAA
OsRR23-CDS-F2	GCTAATTCGAGCTCGATGAGGGCGGCGGAGGAG
OsRR23-CDS-R2	CGACTCTAGAGGATCTCATATGCAAGCTCCAAGG
OsRR24-CDS-F1	ATGACGGTGGAGGAGAGGCAGGG
OsRR24-CDS-R1	CTAGACCAGCTCCCAGTCCCCATC
OsRR24-CDS-F2	GCTAATTCGAGCTCGGTACCATGACGGTGGAGGAGAGG
OsRR24-CDS-R2	CGACTCTAGAGGATCCCTAGACCAGCTCCCAGTC
OsWRKY8-CDS2-F1	ATGTCTGGACCTGGAGGAGGA
OsWRKY8-CDS2-R1	TCACGGAAGGAAGAGGTCTTG
OsWRKY8-CDS2-F2	TATCAAGCTAATTCGAGCTCATGTCTGGACCTGGAGGAGGAG
OsWRKY8-CDS2-R2	CTAGAGGATCCCCGGGTACCTCACGGAAGGAAGAGGTCTTGC
OsWRKY10-CDS-F1	ATGGCGGCTTCGCTGGGAC
OsWRKY10-CDS-R1	TCAGAACGACGATTCCGACGAGT
OsWRKY10-CDS-F2	TATCAAGCTAATTCGAGCTCATGGCGGCTTCGCTGGGAC
OsWRKY10-CDS-R2	CTAGAGGATCCCCGGGTACCTCAGAACGACGATTCCGACGAG
OsWRKY28-CDS-F1	ATGACCGCCGCGCCGGGG
OsWRKY28-CDS-R1	TCAGTTCTTGGTCGGCGA
OsWRKY28-CDS-F2	GCTAATTCGAGCTCGGTACCATGACCGCCGCGCCGGGG
OsWRKY28-CDS-R2	CGACTCTAGAGGATCCTCAGTTCTTGGTCGGCGA
OsWRKY72-CDS-F1	ATGGAGAACTTCCCGATACTCTT
OsWRKY72-CDS-R1	CTACTGGAACATGTGGGAAGC
OsWRKY72-CDS-F2	TATCAAGCTAATTCGAGCTCATGGAGAACTTCCCGATACTC
OsWRKY72-CDS-R2	CTAGAGGATCCCCGGGTACCCTACTGGAACATGTGGGAAGC
OsWRKY111-CDS-F1	ATGACCATCACACACGCTAG
OsWRKY111-CDS-R1	CTAGAGCGACAGCCGGCGTCCA
OsWRKY111-CDS-F2	TATCAAGCTAATTCGAGCTCATGACCATCACACACGCTAGC
OsWRKY111-CDS-R2	CTAGAGGATCCCCGGGTACCCTAGAGCGACAGCCGGCG
OsWRKY6-CDS-F1	ATGGATGGGGTGGAGGAG
OsWRKY6-CDS-R1	TCAGGTCTGGGGATTAGCGG
OsWRKY6-CDS-F2	TATCAAGCTAATTCGAGCTCATGGATGGGGTGGAGGAG
OsWRKY6-CDS-R2	CTAGAGGATCCCCGGGTACCTCAGGTCTGGGGATTAGCGG
OsWRKY45-CDS-F1	ATGACGTCATCGATGTCGC
OsWRKY45-CDS-R1	TCAAAAGCTCAAACCCATAATGTCGT
OsWRKY45-CDS-F2	TATCAAGCTAATTCGAGCTCATGACGTCATCGATGTCGC
OsWRKY45-CDS-R2	CTAGAGGATCCCCGGGTACCTCAAAAGCTCAAACCCATAATGT
OsWRKY51-CDS-F1	ATGATTACCATGGATCTGATGGGTGGG
OsWRKY51-CDS-R1	TCAGGCGAGCGGCAGCGCCAG
OsWRKY51-CDS-F2	TATCAAGCTAATTCGAGCTCATGATTACCATGGATCTGATGGGTGGG
OsWRKY51-CDS-R2	CTAGAGGATCCCCGGGTACCTCAGGCGAGCGGCAGCGCCAG
OsPR10a-RT-F	GGGCACCATCTACACCATGAA
OsPR10a-RT-R	TCGTACTCCACCTTGAGCTT
OsAct1-RT-F	GAGTATGATGAGTCGGGTCCAG
OsAct1-RT-R	ACACCAACAATCCCAAACAGAG

Table S2 Sequences of the primers used in this study.

Primer	Sequence (5'-3')
OsPR10a-Pr-F1	AGGATCCATGCGTTTTTGCTGTAGGA
OsPR10a-Pr-R1	AGGTACCCACTGAAGATATAATCTAACTAG
OsPR10a-Pr-F2	GCAGCCCGGGGGATCCATGCGTTTTTGCTGTAGGAC
OsPR10a-Pr-R2	ACCGCGGTGGCGGCCGCCACTGAAGATATAATCTAACTAGCT
OsMYC2-CDS-F1	ATGAACCTTTGGACGGACGACAACG
OsMYC2-CDS-R1	TTACCGGGCGGCGGTGCCA
OsMYC2-CDS-F2	TATCAAGCTAATTCGAGCTCATGAACCTTTGGACGGACGACAACG
OsMYC2-CDS-R2	CGACTCTAGAGGATCCTTACCGGGCGGCGGTGCC
OsRR21-CDS-F1	ATGGCGCCGGTGGAGGATG
OsRR21-CDS-R1	TCACATCTGTCCACTAAATCCGAAAATAT
OsRR21-CDS-F2	GCTAATTCGAGCTCGGTACCATGGCGCCGGTGGAGGAT
OsRR21-CDS-R2	CGACTCTAGAGGATCCTCACATCTGTCCACTAAATC
OsRR22-CDS-F1	ATGCTTCTGGGTGCTTTGAGGATG
OsRR22-CDS-R1	TCATATGCAGGCACCAAGTGGAAA
OsRR22-CDS-F2	GCTAATTCGAGCTCGGTACCATGCTTCTGGGTGCTTTGA
OsRR22-CDS-R2	CGACTCTAGAGGATCCTCATATGCAGGCACCAAGT
OsRR23-CDS-F1	ATGAGGGCGGCGGAGGAGAGGAAG
OsRR23-CDS-R1	TCATATGCAAGCTCCAAGGGAGTAGAAA
OsRR23-CDS-F2	GCTAATTCGAGCTCGATGAGGGCGGCGGAGGAG
OsRR23-CDS-R2	CGACTCTAGAGGATCTCATATGCAAGCTCCAAGG
OsRR24-CDS-F1	ATGACGGTGGAGGAGAGGCAGGG
OsRR24-CDS-R1	CTAGACCAGCTCCCAGTCCCCATC
OsRR24-CDS-F2	GCTAATTCGAGCTCGGTACCATGACGGTGGAGGAGAGG
OsRR24-CDS-R2	CGACTCTAGAGGATCCCTAGACCAGCTCCCAGTC
OsWRKY8-CDS2-F1	ATGTCTGGACCTGGAGGAGGA
OsWRKY8-CDS2-R1	TCACGGAAGGAAGAGGTCTTG
OsWRKY8-CDS2-F2	TATCAAGCTAATTCGAGCTCATGTCTGGACCTGGAGGAGGAG
OsWRKY8-CDS2-R2	CTAGAGGATCCCCGGGTACCTCACGGAAGGAAGAGGTCTTGC
OsWRKY10-CDS-F1	ATGGCGGCTTCGCTGGGAC
OsWRKY10-CDS-R1	TCAGAACGACGATTCCGACGAGT
OsWRKY10-CDS-F2	TATCAAGCTAATTCGAGCTCATGGCGGCTTCGCTGGGAC
OsWRKY10-CDS-R2	CTAGAGGATCCCCGGGTACCTCAGAACGACGATTCCGACGAG
OsWRKY28-CDS-F1	ATGACCGCCGCGCCGGGG
OsWRKY28-CDS-R1	TCAGTTCTTGGTCGGCGA
OsWRKY28-CDS-F2	GCTAATTCGAGCTCGGTACCATGACCGCCGCGCCGGGG
OsWRKY28-CDS-R2	CGACTCTAGAGGATCCTCAGTTCTTGGTCGGCGA
OsWRKY72-CDS-F1	ATGGAGAACTTCCCGATACTCTT
OsWRKY72-CDS-R1	CTACTGGAACATGTGGGAAGC
OsWRKY72-CDS-F2	TATCAAGCTAATTCGAGCTCATGGAGAACTTCCCGATACTC
OsWRKY72-CDS-R2	CTAGAGGATCCCCGGGTACCCTACTGGAACATGTGGGAAGC
OsWRKY111-CDS-F1	ATGACCATCACACACGCTAG
OsWRKY111-CDS-R1	CTAGAGCGACAGCCGGCGTCCA
OsWRKY111-CDS-F2	TATCAAGCTAATTCGAGCTCATGACCATCACACACGCTAGC
OsWRKY111-CDS-R2	CTAGAGGATCCCCGGGTACCCTAGAGCGACAGCCGGCG
OsWRKY6-CDS-F1	ATGGATGGGGTGGAGGAG
OsWRKY6-CDS-R1	TCAGGTCTGGGGATTAGCGG
OsWRKY6-CDS-F2	TATCAAGCTAATTCGAGCTCATGGATGGGGTGGAGGAG
OsWRKY6-CDS-R2	CTAGAGGATCCCCGGGTACCTCAGGTCTGGGGATTAGCGG
OsWRKY45-CDS-F1	ATGACGTCATCGATGTCGC
OsWRKY45-CDS-R1	TCAAAAGCTCAAACCCATAATGTCGT
OsWRKY45-CDS-F2	TATCAAGCTAATTCGAGCTCATGACGTCATCGATGTCGC
OsWRKY45-CDS-R2	CTAGAGGATCCCCGGGTACCTCAAAAGCTCAAACCCATAATGT
OsWRKY51-CDS-F1	ATGATTACCATGGATCTGATGGGTGGG
OsWRKY51-CDS-R1	TCAGGCGAGCGGCAGCGCCAG
OsWRKY51-CDS-F2	TATCAAGCTAATTCGAGCTCATGATTACCATGGATCTGATGGGTGGG
OsWRKY51-CDS-R2	CTAGAGGATCCCCGGGTACCTCAGGCGAGCGGCAGCGCCAG
OsPR10a-RT-F	GGGCACCATCTACACCATGAA
OsPR10a-RT-R	TCGTACTCCACCTTGAGCTT
OsAct1-RT-F	GAGTATGATGAGTCGGGTCCAG
OsAct1-RT-R	ACACCAACAATCCCAAACAGAG

References 80

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