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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (11): 1722-1733.doi: 10.3724/SP.J.1006.2020.04030

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

Sugarcane PsbS subunit response to Sugarcane mosaic virus infection and its interaction with 6K2 protein

ZHANG Hai(), LIU Shu-Xian, YANG Zong-Tao, WANG Tong, CHENG Guang-Yuan, SHANG He-Yang, XU Jing-Sheng*()   

  1. Sugarcane Research & Development Center, China Agricultural Technology System, Fujian Agriculture and Forestry University / Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs / Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fuzhou 350002, Fujian, China
  • Received:2020-02-10 Accepted:2020-04-15 Online:2020-11-12 Published:2020-04-27
  • Contact: Jing-Sheng XU E-mail:zhanghai940410@163.com;xujingsheng@126.com
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31971991);the Science and Technology Innovation Project of Fujian Agriculture and Forestry University(CXZX2018026)

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Abstract:

Non-photochemical quenching (NPQ) is the main mechanism of photoprotective regulation in higher plants. The PsbS subunit of Photosystem II (PSII) plays a key role in NPQ. The involvement of PSII PsbS subunit in Sugarcane mosaic virus (SCMV) infection of sugarcane (Saccharum spp. hybrid) has not been reported. In the previous research, we cloned the coding sequence of the PsbS subunit from sugarcane and designated it as ScPsbS. ScPsbS had an open reading frame (ORF) length of 798 bp and encoded a protein of 265 aa. Bioinformatics analysis showed that ScPsbS was a stable hydrophobic protein with chloroplast localization signals and four transmembrane domains. The ScPsbS protein possesses a typical domain of PsbS protein. Phylogenetic tree analysis showed that ScPsbS was divergent between monocotyledons and dicotyledons, or C3 plants and C4 plants. Subcellular localization analysis showed that ScPsbS was located in chloroplasts and partially colocalized with SCMV-6k2 in chloroplasts. The interaction of ScPsbS with the SCMV-6K2 was further confirmed by bimolecular fluorescence complementation assays (BiFC). ScPsbS gene showed obvious tissue specificity in sugarcane tested by real-time quantitative PCR analysis. ScPsbS gene had highest expression in mature leaves, followed by immature leaves and leaves beginning to senesce, and hardly expressed in stems and roots. The expression of ScPsbS gene was significantly affected by SCMV infection, with significant upregulation in the early stage of SCMV infection, and no significant affection in the later stage of SCMV infection.

Key words: sugarcane, PsbS subunit, Sugarcane mosaic virus, 6K2, light protection

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 策略
Strategy
221-ScPsbS-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGCAGTCCATGCT 双分子荧光互补载体构建
221-ScPsbS-R GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACTCTTCGTCCTCGC Vector generation for BiFC
ScPsbS-qF CTCCATCATCGGCGAGATCATCAC 定量PCR
ScPsbS-qR GGCGGCGACGAAGAAGAAGAG Real-time-qPCR
GAPDH-F CACGGCCACTGGAAGCA 内参基因
GAPDH-R
eEF-1α-F
eEF-1α-R		 TCCTCAG GGTTCCTGATGCC
TTTCACACTTGGAGTGAAGCAGAT
GACTTCCTTCACAATCTCATCATAA		 Reference gene
内参基因
Reference gene
SCMV-CP-F TACAGAGAGACACACAGCTG SCMV检测
SCMV-CP-R ACGCTACACCAGAAGACACT Detection of SCMV

Fig. 1

Three-dimensional structure of ScPsbS, ZmPsbS, OsPsbS, and AtPsbS protein based on SWISS-MODEL ZmPsbS (NP_001105228.2) is from Zea mays; OsPsbS (XP_ 015621169.1) is from Oryza sativa; AtPsbS (NP_973971.1) is from Arabidopsis thaliana."

Fig. 2

Nucleotide sequence of the ScPsbS gene and its deduced amino acid sequence *: stop codon. The black underlined sequence indicates the transit peptide. The red underlined part indicates the PsbS domains. The red font marked amino acids indicate the conserved motif between transit peptide and mature protein in the precursor protein targeting to thylakoid lumen. ↓ indicates the digestion site."

Fig. 3

Amino acid sequence alignment of ScPsbS and PsbSs of other monocotyledon species Sorghum bicolor: SbPsbS (XP_002456704.1); Zea mays: ZmPsbS (NP_001105228.2); Panicum hallii: PhPsbS (XP_025818476.1); Setaria italic: SiPsbS (XP_004970722.1); Oryza sativa: OsPsbS (XP_015621169.1); Brachypodium distachyon: BdPsbS (XP_ 003564708.1); Triticum aestivum: TaPsbS (CDM85166.1)."

Fig. 4

Phylogenetic tree analysis of ScPsbS protein and PsbS proteins from other plant species"

Fig. 5

Subcellular localization of ScPsbS fused with YFP in the epidermal cells of N. benthamiana A: subcellular localization of ScPsbS-YFP; B: subcellular colocalization of ScPsbS-YFP with 6K2-CFP. White arrows indicate the chloroplasts; bar = 25 μm."

Fig. 6

BiFC assays for protein-protein interaction between ScPsbS and SCMV-6K2 A: the C-terminal half of YFP was fused to the C-terminal of ScPsbS to generate ScPsbS-YC, while the N-terminal half of YFP was fused to the N-terminla of SCMV-6K2 to generate YN-6K2; B: the N-terminal half of YFP was fused to the N-terminal of ScPsbS to generate YN- ScPsbS, while the C-terminal half of YFP was fused to the C-terminal of SCMV-6K2 to generate 6K2-YC. Plasmids combination of YN-6K2 plus ScPsbS-YC (A), YN-ScPsbS plus SCMV-6K2-YC (B) were individually co-injected into N. benthamiana leaves for transient expression. The fluorescent signal was monitored by confocal microscopy at 48 h after infiltration; bar = 25 μm."

Fig. 7

Expression profile of ScPsbS in different sugarcane tissues The error bars represent the standard error of each treatment group (n = 3). LR: leaf roll; +1 L: +1 leaf; +7 L: +7 leaf; +3 I: +3 internode; +8 I: +8 Internode; R: root."

Fig. 8

Expression profile of ScPsbS under the infection of SCMV The error bars represent the standard error of each treatment group (n = 3)."

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