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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (9): 2472-2484.doi: 10.3724/SP.J.1006.2023.24244

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Interaction of sugarcane VAMP associated protein ScPVA12 with SCMV P3N-PIPO

YU Quan-Xin1(), YANG Zong-Tao1, ZHANG Hai1, CHENG Guang-Yuan1, ZHOU Ying-Shuan1, JIAO Wen-Di1, ZENG Kang1, LUO Ting-Xu1, HUANG Guo-Qiang1, ZHANG Mu-Qing2,*(), XU Jing-Sheng1,*()   

  1. 1Fujian Agriculture and Forestry University / Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs / National Engineering Research Center for Sugarcane / Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fuzhou 350002, Fujian, China
    2Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning 530004, Guangxi, China
  • Received:2022-10-28 Accepted:2023-02-10 Online:2023-09-12 Published:2023-02-28
  • Supported by:
    National Natural Science Foundation of China(31971991);Science and Technology Innovation Project of Fujian Agriculture and Forestry University(CXZX2019132G);Open Project Program of Guangxi Key Laboratory of Sugarcane Biology(GXKLSCB-202003)

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Abstract:

PVA12 (plant vesicle-associated membrane protein (VAMP)-associated proteins homolog 12) is a member of the VAP27 family of proteins that mediate endoplasmic reticulum (ER) vesicle transport and membrane fusion in cells. Sugarcane (Saccharum spp. hybrid) PVA12 responding to Sugarcane mosaic virus (SCMV) infection has not been reported. In the present study, the coding gene of PVA12 was cloned from sugarcane cultivar ROC22 and designated as ScPVA12. The open reading frame (ORF) of ScPVA12 was 735 bp in length, which encoded a protein with 244 amino acids. Bioinformatics analysis showed that ScPVA12 was an unstable hydrophilic liposoluble protein with a transmembrane at the C-terminal domain. The ratio of the random coil ranked the highest in the secondary structure. Phylogenetic tree analysis revealed that the ScPVA12 was differentiated in monocotyledons and dicotyledons. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that ScPVA12 interacted with SCMV-P3N-PIPO. Subcellular localization experiments indicated that ScPVA12 was localized in the ER. Co-localization experiments demonstrated that ScPVA12 and SCMV-P3N-PIPO co-localized in the ER. Real-time quantitative PCR analysis showed that ScPVA12 gene was expressed in all sugarcane tissues, with the lowest expression level in the eighth internode and the highest expression level in the 7th leaves. The relative expression level of ScPVA12 gene was significantly affected under the stress of SCMV. ScPVA12 was down-regulated upon SCMV infection and then recovered to the regular level compared with the control.

Key words: PVA12, Sugarcane mosaic virus, P3N-PIPO, protein interaction

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 策略
Strategy
ScPVA12-F ATGGCCACCCCCGCCCCTGCCA 克隆
ScPVA12-R TGACTTCATCAGATAGCCCAGGATG Gene cloning
pPPR3-ScPVA12-F ATTAACAAGGCCATTACGGCCATGATGGCCACCCCCGCCCCTGCCA 酵母双杂交载体构建
Construction of yeast two-hybrid vector
pPPR3-ScPVA12-R TTGACTAAGGCCGAGGCGGCCGTTATGACTTCATCAGATAGCCCAG
221-ScPVA12-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGATGGCCACCCCCGCCCCTGCCA 亚细胞定位及双分子荧光互补载体构建
Construction of yeast subcellular localization and bimolecular fluorescence complementation vector
221-ScPVA12-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTGACTTCATCAGATAGCCCAGGATG
ScPVA12-qF ATGGAAGAGGTCAAGTTGCGAGT 定量PCR
Real-time qPCR
ScPVA12-qR TGAATCCTCCAGCAGGTCCATCTAC
Actin-F CACGGCCACTGGAAGCA 内参基因
Reference gene
Actin-R TCCTCAG GGTTCCTGATGCC
eEF-1α-F TTTCACACTTGGAGTGAAGCAGAT 内参基因
Reference gene
eEF-1α-R GACTTCCTTCACAATCTCATCATAA
SCMV-CP-F TACAGAGAGACACACAGCTG SCMV检测
Detection of SCMV
SCMV-CP-R ACGCTACACCAGAAGACACT

Fig. 1

ScPVA12 protein domain A: the amino acid comparison between ScPVA12 and AtVAP27-3 (The red marks indicate MSD, the orange marks indicate CCD, and the green marks indicate TMD. B: the schematic diagram of ScPVA12 and AtVAP27-3 domain (Ruler: 250 amino acids)."

Fig. 2

Amino acid sequence alignment of ScPVA12 and PVA12s of other monocotyledon species Sorghum bicolor: SbPVA12 (XP_002445075.1); Miscanthus lutarioriparius: MlPVA12 (CAD6266147.1); Zea mays: ZmPVA12 (NP_001130606.1); Panicum miliaceum: PmPVA12 (RLN03983.1); Setaria italica: SiPVA12 (XP_004972806.1); Brachypodium distachyon: BdPVA12 (XP_003573476.1); Triticum dicoccoides: TdPVA12 (XP_037458000.1)."

Fig. 3

Phylogenetic tree of ScPVA12 protein and PVA12 proteins from other plant species Group I is monocot, in which I-1 is C4 plant subgroup and I-2 is C3 plant subgroup. Group II is a dicotyledonous group."

Fig. 4

Subcellular localization of ScPVA12 fused with YFP in the epidermal cells of N. benthamiana A: the subcellular localization of ScPVA12-YFP; B: the subcellular colocalization of ScPVA12-YFP with SCMV-P3N-PIPO-CFP. Pairwise combination of three fluorescent protein signals and observation of colocalization presented by using pseudo-color, and the combinations were ScPVA12-YFP (green pseudo-color)+HDEL-RFP (red pseudo-color), ScPVA12-YFP (red pseudo-color)+P3N-PIPO-CFP (green pseudo-color) and HDEL-RFP (red)+P3N-PIPO-CFP (green pseudo-color). Bar: 25 μm. The white arrow marks the location of colocalization."

Fig. 5

Y2H assays for the interaction between ScPVA12 and SCMV-P3N-PIPO The positive and negative controls are yeast cotransformants with pNubG-Fe65 plus pTSU2-APP and pNubG-Fe65 plus pPR3-N, respectively. DDO+X-Gal: synthetic defined yeast minimal medium lacking Leu and Trp with the treatment of 5-Bromo-4Chlor-3-Indoly1 β-D-Galactopyranoside; QDO+X-Gal: synthetic defined yeast minimal medium lacking Leu, Trp, His, and Ade but plus the X-Gal."

Fig. 6

BiFC assays for the interaction between ScPVA12 and SCMV-P3N-PIPO A: the N-terminal half of YFP was fused to the N-terminal of ScPVA12 to generate YN-ScPVA12, while the C-terminal half of YFP was fused to the C-terminal of SCMV-P3N-PIPO to generate P3N-PIPO-YC; B: the C-terminal half of YFP was fused to the C-terminal of ScPVA12 to generate ScPVA12-YC, while the N-terminal half of YFP was fused to the N-terminal of SCMV-P3N-PIPO to generate YN-P3N-PIPO. Plasmids combination of YN-ScPVA12 plus P3N-PIPO-YC (A), YN-P3N-PIPO plus ScPVA12-YC (B) were individually co-injected into N. benthamiana leaves for transient expression. The fluorescent signal was monitored by confocal microscopy at 48 hours post infiltration. Bar: 25 μm. The white arrow marks the fluorescent signal with a dotted structure."

Fig. 7

Relative expression profile of ScPVA12 gene in different sugarcane tissues The error bars represent the standard error of each treating group (n = 3). Bars super-scripted by different lowercase letters are significantly different at P < 0.05."

Fig. 8

Relative expression profile of ScPVA12 gene under the infection of SCMV The error bars represent the standard error of each treating group (n = 3). Bars super-scripted by different lowercase letters are significantly different at P < 0.05."

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