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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (12): 3080-3090.doi: 10.3724/SP.J.1006.2022.14244

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

Interaction of sugarcane main facilitator superfamily member ScZIFL1 with 6K2 in response to Sugarcane mosaic virus infection

LIU Shu-Xian(), YANG Zong-Tao, CHENG Guang-Yuan, ZHANG Hai, ZHOU Ying-Shuan, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng()   

  1. Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs / National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University / Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fuzhou 350002, Fujian, China
  • Received:2021-12-22 Accepted:2022-03-25 Online:2022-12-12 Published:2022-04-19
  • Contact: XU Jing-Sheng E-mail:Liushuxian1010@163.com;xujingsheng@126.com
  • Supported by:
    National Natural Science Foundation of China(31971991);Science and Technology Innovation Project of Fujian Agriculture and Forestry University(CXZX2018026)

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Abstract:

The major facilitator superfamily (MFS) members are extensively distributed in the organisms. The zinc induced facilitator like proteins (ZIFL), members of the MFS, are involved in the transport of small organic molecules. In our previous study, the ZIFL homologue was screened from sugarcane (Saccharum spp. hybrid) and identified to interact with Sugarcane mosaic virus (SCMV) coding protein 6K2 by yeast two-hybrid (Y2H), then named as ScZIFL1. In the present study, the interaction of ScZIFL1 with SCMV-6K2 was further confirmed by bimolecular fluorescence complementation (BiFC) assays. Bioinformatics analysis showed that ScZIFL1 coded 484 aa, and was an unstable hydrophobic protein without signal peptide but 12 transmembrane domains. Sequences alignment revealed the conserved cysteine-containing motif, canonical MFS signature, and anti-porter signatures in ScZIFLl. Phylogenetic tree indicated that ZIFLl was divergent between monocotyledons and dicotyledons, as well as C3 and C4 plants. Subcellular localization referred that ScZIFL1 was localized in tonoplast but partially co-localized with SCMV-6K2. ScZIFL1 gene had obvious tissue specificity in sugarcane by RT-qPCR. The relative expression levels of ScZIFL1 genes were the highest in stems, followed by leaves, and the lowest in roots. Specifically, the relative expression levels of ScZIFL1 genes in the established morphogenesis and fully functional tissues such as the leaf +1 and the 8th internode were significantly higher than those in the immature tissues, i.e., leaf roll and the 3rd internode, or the fading tissues, i.e., leaf +7. Upon the infection of SCMV, ScZIFL1 was significantly up-regulated at the early stage and remained higher expression with time going on.

Key words: Sugarcane mosaic virus, 6K2, ZIFL1, 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
221-ScZIFL1-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGGCGGTGGCGGCGA 双分子荧光互补载体构建
221-ScZIFL1-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTGAATGCCTCATTGAATTTG Vector generation for BiFC
ScZIFL1-qF TGGACGAGGCGAACGAGACC 定量PCR
ScZIFL1-qR AGCGGCGGTGAGGCAGAC Real-time qPCR
GAPDH-F CACGGCCACTGGAAGCA 内参基因
GAPDH-R TCCTCAG GGTTCCTGATGCC Reference gene
Actin-F CCTGAAGATCACCCTGTGCT 内参基因
Actin-R GCAGTCTCCAGCTCCTGTTC Reference gene
SCMV-CP-F TACAGAGAGACACACAGCTG SCMV检测
SCMV-CP-R ACGCTACACCAGAAGACACT Detection of SCMV

Fig. 1

Transmembrane domain prediction of ScZIFL1"

Fig. 2

Amino acid sequence alignment of ScZIFL1 and ZIFL1 from other plant species The cysteine-containing motif CPGC, canonical signature WG(V/M/I)(F/V/A/I)AD(K/R)(Y/I//H/L)GRKP and anti-porter signature S(x8)G(x3)GP(A/T/G)(L/I)GG (x stands for any amino acid) were in red color, and were highlighted by yellow color. Sequences in I are from monocotyledon, while sequences in II are from dicotyledon. ZmZIFL1 (NP_001141824.1): Zea mays; SbZIFL1 (XP_002457667.1): Sorghum bicolor; PhZIFL1 (XP_025817905.1): Panicum hallii; SvZIFL1 (XP_034593088.1): Setaria viridis; BdZIFL1 (XP_003567304.1): Brachypodium distachyon; HvZIFL1 (KAE8779043.1): Hordeum vulgare; TaZIFL1 (KAF7029361.1): Triticum aestivum; OsZIFL1 (XP_025880517.1): Oryza sativa; PtZIFL1 (XP_024443823.1) Populus trichocarpa; PpZIFL1 (XP_007217932.1): Prunus persica; VvZIFL1 (XP_002277598.2): Vitis vinifera; NaZIFL1 (XP_019247413.1): Nicotiana attenuate; AtZIFL1 (OAP02507.1): Arabidopsis thaliana; StZIFL1 (XP_006347685.1): Solanum tuberosum; GmZIFL1 (XP_006594342.1): Glycine max; CaZIFL1 (XP_016582203.1): Capsicum annuum."

Fig. 3

Phylogenetic tree of ScZIFL1 and ZIFL1 proteins from other plant species The red box, the green box, and the orange box indicate the subgroup I-1, the subgroup I-2, and the group II, respectively."

Fig. 4

Subcellular localization of ScZIFL1 fused with YFP in the epidermal cells of N. benthamiana A: subcellular localization of ScZIFL1-YFP; B: subcellular colocalization of ScZIFL1-YFP with 6K2-CFP. Bar: 25 μm."

Fig. 5

Protein-protein interaction between ScZIFL1 and SCMV-6K2 by BiFC assays A: the N-terminal half of YFP was fused to the N-terminal of ScZIFL1to generate ScZIFL1-YN, while the C-terminal half of YFP was fused to the C-terminal of SCMV-6K2 to generate SCMV-6K2-YC; B: the N-terminal half of YFP was fused to the N-terminal of SCMV-6K2 to generate SCMV-6K2-YN, while the C-terminal half of YFP was fused to the C-terminal of ScZIFL1 to generate ScZIFL1-YC. Plasmid’s combination of 6K2-YC plus ScZIFL1-YN (A), ScZIFL1-YC plus 6K2-YN (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."

Fig. 6

Tissue specific expression analysis of ScZIFL1 genes LR: leaf roll; +1 L: leaf +1; leaf +7; +3 I: the 3rd internode; +8 I: the 8th internode; R: root. The error bar represents the standard error of each treating group (n = 3). Bars superscripted by different lowercase letters are significantly different at P < 0.05."

Fig. 7

Relative expression profile of ScZIFL1 genes under the infection of SCMV The error bar represents the standard error of each treatment group (n = 3). Bars superscripted by different lowercase letters are significantly different at P < 0.05."

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