Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (13): 2243-2255.doi: 10.3864/j.issn.0578-1752.2019.13.005

• PLANT PROTECTION • Previous Articles     Next Articles

Screening of Interacting Proteins of Anti-Canker Transcription Factor CsBZIP40 in Citrus by GST Pull-Down Combined with LC-MS/MS

DOU WanFu,QI JingJing,HU AnHua,CHEN ShanChun,PENG AiHong,XU LanZhen,LEI TianGang,YAO LiXiao,HE YongRui(),LI Qiang()   

  1. Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712
  • Received:2019-03-12 Accepted:2019-04-03 Online:2019-07-01 Published:2019-07-11
  • Contact: YongRui HE,Qiang LI E-mail:heyongrui@cric.cn;liqiang@cric.cn

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Abstract:

【Objective】 CsBZIP40 is a citrus canker-related transcription factor. The objective of this study is to screen the interacting proteins of CsBZIP40 in response to Xanthomonas citri subsp. citri (Xcc) infection, and to analyze the interaction network of CsBZIP40, so as to provide a theoretical basis for molecular breeding of citrus canker resistance. 【Method】 The GST pull-down strategy was used to screen the interacting proteins of CsBZIP40 in the infection process of Xcc. Firstly, the vector of GST-CsBZIP40 was constructed and induced by IPTG (isopropyl β-D-thiogalactoside). The fusion protein of GST-CsBZIP40 was purified for the next GST pull-down. Then GST-CsBZIP40 was fixed on the GST-beads and incubated with total proteins extracted from CsBZIP40 over-expression plants infected by Xcc or LB medium as control. The protein complex bound to GST-CsBZIP40 bait protein was eluted and collected, and then verified by SDS-PAGE gel electrophoresis. The interacting proteins of CsBZIP40 were detected by LC-MS/MS and then annotated based on the genomic database of Citrus sinensis. The GO, KEGG pathways and the interaction network of the interacting proteins were also analyzed. 【Result】 The phenotype of transgenic plants over-expression CsBZIP40 was normal, and there was no significant difference between the transgenic plants and the control plants. The lesion area on the over-expression plant was significantly smaller compared to that on the wild-type (WT) (45%) and the disease index of CsBZIP40 over-expression plant was significantly lower than that of WT (54%). The total proteins were successfully extracted from CsBZIP40 over-expression plant infected by Xcc or LB medium, and the GST-CsBZIP40 was expressed and purified for the GST pull-down. GST-CsBZIP40 bait protein was used to catch the protein from the total proteins of Xcc-infected and uninfected citrus, then the protein was detected by LC-MS/MS. After comparison, annotation and screening, there are 53 interacting proteins specifically binding to GST-BZIP40 in the process of Xcc infection. These proteins are involved in many molecular functions and pathways. Among the 53 proteins, 6 proteins (Cs1g02310, Cs3g05280, Cs3g23950, Cs6g13880, Cs7g12130, orange1.1t04973) may be closely related to the plant disease resistance, 44 of the 53 proteins have been proven to interact directly or indirectly with CsBZIP40 in the database. 【Conclusion】 In the infection of Xcc, 53 proteins interacted with CsBZIP40 were detected. According to the annotation, 6 proteins are closely related to plant disease resistance. These proteins may play an important role in improving the stress resistance of citrus under biological stress.

Key words: Citrus sinensis, citrus bacteria canker, Xanthomonas citri subsp. citri (Xcc), BZIP, GST pull-down, interacting protein

Fig. 1

Verification and canker resistance evaluation of overexpressed CsBZIP40 lines"

Fig. 2

Construction of fusion expression vector"

Fig. 3

Induced expression and purification detection of fusion protein"

Fig. 4

The detection of GST pull-down results"

Table 1

Disease resistance-related interacting proteins of CsBZIP40"

基因 ID
Gene ID		 功能预测
Predicted protein function		 等电点
PI		 分子量
Molecular weight (kD)		 染色体位置
Chromosomal loci
Cs1g02310 TGA1 resistance (SAR) via its interaction with NPR1 7.07 40.83 chr1:1,637,432..1,643,794
Cs3g05280 Disease resistance protein 5.83 130.03 chr3:6,932,350..6,936,297
Cs3g23950 Transcription factor MYB 8.75 28.06 chr3:26,088,190..26,089,510
Cs6g13880 Peroxiredoxin-2 8.49 29.50 chr6:15,280,950..15,284,180
Cs7g12130 Heat shock protein 83 5.34 90.12 chr7:8,048,239..8,054,656
orange1.1t04973 Death-associated protein kinase 1 5.85 35.14 chrUn:40,320,115..40,324,317

Fig. 5

GO analysis of CsBZIP40 interacting proteins"

Fig. 6

KEGG analysis of CsBZIP40 interacting proteins"

Fig. 7

Interaction analysis of CsBZIP40 binding proteins"

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