Interaction analysis between rice ubiquitin ligase D3 and the disease resistance associated protein VOZ2

doi:10.3969/j.issn.1004-1524.20221670

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (1): 9-17.DOI: 10.3969/j.issn.1004-1524.20221670

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Interaction analysis between rice ubiquitin ligase D3 and the disease resistance associated protein VOZ2

LUO Yingjie¹^,²(), CUI Weijun², WANG Zhonghua¹, WU Yueyan¹, LIN Hongyou², ZHOU Jie², YAN Chengqi¹^,³^,^*(), WANG Xuming²^,^*()

1. College of Biological & Enviromental Sciences, Zhejiang Wanli University, Ningbo 315100, Zhejiang, China
2. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
3. Institute of Biotechnology, Ningbo Academy of Agricultural Sciences, Ningbo 315100, Zhejiang, China

Received:2022-11-22 Online:2024-01-25 Published:2024-02-18

Abstract

Abstract:

The tiller dwarf gene dwarf-3 (D3) is an important node gene in strigolactone signal transduction in rice. It has been reported that the MAX2 gene in Arabidopsis thaliana is homologous to D3, and MAX2 is involved in the defense response of A. thaliana. In this study, the yeast two-hybrid library was screened with D3 protein as bait, and it was found that there was a potential interaction between rice disease resistance-related protein vascular plant one-zinc finger protein 2 (VOZ2) and D3. The interaction between D3 and VOZ2 was confirmed by yeast two-hybrid validation. Real-time quantitative PCR experiments showed that the expression of VOZ2 gene was significantly induced at the transcriptional level after inoculation with Xanthomonas oryzae pv. oryzae. Subcellular co-localization experiment using rice protoplasts showed that D3 and VOZ2 were co-localized in the nucleus. Bimolecular fluorescence complementation experiments showed that D3 and VOZ2 produced strong fluorescence in the nucleus and cytoplasm of tobacco mesophyll cells, further confirming the interaction between D3 and VOZ2. The study result laid a foundation for further exploring of the function and molecular mechanism of D3 and VOZ2 in rice disease resistance and defense response.

Key words: rice, ubiquitin ligase D3, vascular plant one-zinc finger protein VOZ2, protein interaction, yeast two-hybrid, subcellular co-localization

CLC Number:

S511

LUO Yingjie, CUI Weijun, WANG Zhonghua, WU Yueyan, LIN Hongyou, ZHOU Jie, YAN Chengqi, WANG Xuming. Interaction analysis between rice ubiquitin ligase D3 and the disease resistance associated protein VOZ2[J]. Acta Agriculturae Zhejiangensis, 2024, 36(1): 9-17.

Figures/Tables 7

Table 1 Primer information

名称Name	引物序列Primer sequence (5'→3')	用途Usage
D3-CDS-F	ATGGCGGAAGAGGAGGAGG	基因克隆
D3-CDS-R	CTAATCATCAATTTGCCGGCT	Gene cloning
VOZ2-CDS-F	AGTCCTCTTCTTCGCCCAC	基因克隆(含部分UTR)
VOZ2-CDS-R	AATATGCTATCTTCATGTCCCGT	Gene cloning (contains some UTR)
D3-pB2E-F	TCCGAATTCGCCCTTATGGCGGAAGAGGAGGAGG	D3-pB2E载体构建
D3-pB2E-R	GTCGAATTCGCCCTTCTAATCATCAATTTGCCGGCT	Construction of D3-pB2E vector
VOZ2-pB2E-F	TCCGAATTCGCCCTTATGGCCGGCGATCCGGCC	VOZ2-pB2E载体构建
VOZ2-pB2E-R	GTCGAATTCGCCCTTTCATGTCCCGTCACTAGGGTTCC	Construction of VOZ2-pB2E vector
D3-pGBKT7-F	AGGAGGACCTGCATATGGCGGAAGAGGAGGAGG	D3-pGBKT7载体构建
D3-pGBKT7-R	GCAGGTCGACGGATCCCTAATCATCAATTTGCCGGCTG	Construction of D3-pGBKT7 vector
VOZ2-pGBKT7-F	CAGATTACGCTCATATGGCCGGCGATCCGGCC	VOZ2-pGBKT7载体构建
VOZ2-pGBKT7-R	CGAGCTCGATGGATCCTCATGTCCCGTCACTAGGGTTCC	Construction of VOZ2-pGBKT7 vector
VOZ2-35S-Flag-F	CGGTACCCGGGGATCCATGGCCGGCGATCCGGCCG	VOZ2-3×flag载体构建
VOZ2-35S-Flag-R	GGGCGAATTGGTCGACTGTCCCGTCACTAGGGTTCCAGT	Construction of VOZ2-3×flag vector
VOZ2-RT-F	AGGGCGATTGCGGAGGAATG	实时荧光定量PCR
VOZ2-RT-R	ATTGGGGCCGTCTGGGTTTG	qRT-PCR

Fig.1 Self-activation verification of D3

Fig.2 The protein interaction between D3 and VOZ2 verified by yeast two-hybrid method

Fig.3 β-Galactosidase activity of yeast The average Michaelis constant values of β-galactosidase in pGBKT7-D3 and pGADT7-VOZ2 co-transformed yeast, positive control yeast (con-transformed with pGBKT7-53 and pGADT7-T) and negative control yeast (co-transformed with pGBKT7-Lam and pGADT7-T) were 0.396,0.629 and 0.214, respectively. Three biological replicates and two technical replicates were carried out, and t-test was used to compare the data differences between the groups. ** indicated significant difference (P≤0.01) compared with the control. The same as below.

Fig.4 The expression level of VOZ2 gene after Xoo infection in rice *** represented P≤0.001.

Fig.5 Subcellular localization of D3 and VOZ2 BF, Bright field; GFP, Green fluorescent; RFP, Red fluorescent; Merge, Merge channel.

Fig.6 The protein interaction between D3 and VOZ2 verified by bimolecular fluorescent complimentary method BF, Bright field; YFP, Yellow fluorescent; Chl, Chloroplast fluorescent; Merge, Merge channel.

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Interaction analysis between rice ubiquitin ligase D3 and the disease resistance associated protein VOZ2

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