Construction of the Soybean Membrane System cDNA Library and Interaction Proteins Screening for Effector PsAvr3a

doi:10.13560/j.cnki.biotech.bull.1985.2022-0972

Abstract

Abstract:

The plant plasma membrane is the first barrier in response to pathogen infection, and the receptor proteins on it are the radar for sensing pathogen invasion, which is an important part of the plant immune system. Phytophthora sojae disrupts the host immune activities of plants by secreting effectors, promoting Phytophthora colonization and spread. The construction of soybean split-ubiquitin based membrane yeast two-hybridization cDNA library is significant for exploring the interaction mechanisms between effectors and host cell membrane protein targets. The avirulent effector PsAvr3a was used as a bait protein to screen the library, and 198 candidate host targets were preliminarily obtained. The yeast two-hybrid technique between the interested target proteins and PsAvr3a revealed that five of them interacted with the bait vector. The enzyme that regulates tyrosine breakdown, the vesicle transport protein, the ethylene biosynthesis regulatory enzyme, the cell metabolic binding protein, the stress-resistant osmotic protein, and others were among the prospective host targets. The interactions amony GmACO, GmSec61 and PsAvr3a were verified by Bimolecular Fluorescence Complementation（BIFC）and Luciferase Complementation Assay（LCA）. Therefore, two soybean membrane proteins, ethylene precursor ACC oxidase GmACO and trimeric transposon GmSec61, were identified as the host target of Phytophthora sojae avirulent effector PsAvr3a.

Key words: soybean, effector, Phytophthora sojae, split-ubiquitin based membrane yeast two-hybridization, membrane protein, avirulence gene

HOU Xiao-yuan, CHE Zheng-zheng, LI Heng-jing, DU Chong-yu, XU Qian, WANG Qun-qing. Construction of the Soybean Membrane System cDNA Library and Interaction Proteins Screening for Effector PsAvr3a[J]. Biotechnology Bulletin, 2023, 39(4): 268-276.

Figures/Tables 9

Fig. 1 Subcelluar localization of pBIN-GFP2-PsAvr3a The pBIN-GFP2-PsAvr3a fusion protein was coexpressed with pCAMBIA1300-mCherry-PsAvh241 in N. benthamiana through agroinfiltration. Scale bar: 20 μm

Fig. 2 Results of self-activation detection and functional verification A：pNubG-Fe65 + pTSU2-APP；B：pBT3-N-APP + pPR3-N；C：pBT3-N-PsAvr3a + pOst1-NubI；D：pBT3-N-PsAvr3a + pPR3-N。100、10-1、10-2为酵母菌液稀释倍数。下同100, 10-1, 10-2 are the dilution ratio of yeast solution. The same below

Table 1 Retrieved partial interacting proteins

编号No.	基因名称Gene name	功能预测Function forecasting
1	Glycine max protein transport protein Sec61 subunit beta-like	介导信号肽依赖性蛋白质转运到内质网 Mediating signal peptide-dependent protein transport to endoplasmic reticulum
2	Glycine soja ER membrane protein complex subunit 4-like	参与eIF2B 介导的翻译Participating in eIF2B-mediated translation
3	Glycine max bax inhibitor 1-like	凋亡抑制基因Apoptotic inhibitor gene
4	Glycine max peroxisomal membrane protein 11	过氧化物酶体膜蛋白Peroxisomal membrane protein
5	Glycine max 1-aminocyclopropane-1-carboxylate oxidase	参与乙烯生物合成Participating in ethylene biosynthesis
6	Glycine max zinc finger CCCH domain-containing protein 23	DNA结合蛋白DNA binding protein
7	Glycine soja RING-H2 finger protein ATL8-like	泛素蛋白连接酶Ubiquitin-protein ligase enzyme
8	Glycine max SNF1-related protein kinase regulatory subunit beta-1	参与种子内部碳水化合物代谢和储藏物质积累 Involving in carbohydrate metabolism and storage material accumulation in seeds
9	Glycine max 4-hydroxyphenylpyruvate dioxygenase	α-酮酸依赖性加氧酶，催化酪氨酸分解代谢 α-ketoacid-dependent oxygenase, catalyzing tyrosine catabolism
10	Glycine max wound-induced protein	创伤诱导蛋白Wound-induced protein
11	Glycine max formin-like protein 20	钙依赖性脂质结合域蛋白Calcium-dependent lipid-binding domain protein
12	Glycine max osmotin-like protein, acidic（OLPa）	具有抗逆功能的渗透蛋白The stress-resistant osmotic protein
13	Glycine max probable enoyl-CoA hydratase 1	烯酰辅酶A水合酶Enoyl-CoA hydratase

Fig. 3 Results of yeast two-hybrid verification A: pPR3-N-GmOsmotin + pBT3-N-PsAvr3a; B: pPR3-N-GmSNF1 + pBT3-N-PsAvr3a; C: pPR3-N-GmHPPD + pBT3-N-PsAvr3a; D: pPR3-N-GmSec61 + pBT3-N-PsAvr3a; E: pPR3-N-GmACO + pBT3-N-PsAvr3a; F: pNubG-Fe65 + pTSU2-APP; G: pBT3-N-APP + pPR3-N

Fig. 4 Verifying protein interacting results in tobacco cells using the BiFC method BiFC of PsAvr3a and GmACO, GmSec61 in transiently transformed N. benthamiana leaves. The YFP fluorescent signal was observed using confocal microscopy. Scale bar: 20 μm

Fig. 5 Verifying protein interacting results in tobacco using LCA A: p1300-35S-cLUC-PsAvr3a/p1300-35S-Nluc; B: p1300-35S-cLUC/p1300-35S-nLUC-GmACO; C: p1300-35S-cLUC /p1300-35S-nLUC-GmSec61; D: p1300-35S-cLUC-PsAvr3a/p1300-35S-nLUC-GmACO; E: p1300-35S-cLUC-PsAvr3a/p1300-35S-nLUC-GmSec61

Fig. 6 Transmembrane domain prediction of GmACO using TMHMM

Fig. 7 Transmembrane domain prediction of GmSec61 using TMHMM

Fig. 8 Subcellular localization of GmACO and GmSec61 Subcellular localization of GmACO, GmSec61 or empty vector after agroinfiltration. The GFP signal was observed using confocal microscopy. Scale bar: 20 μm

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