Cloning of Sugarcane ShPR10 Gene and Study on the Interaction Between ShPR10 Protein and P1 Protein Encoded by Sugarcane Streak Mosaic Virus

doi:10.13560/j.cnki.biotech.bull.1985.2023-0403

Abstract

Abstract:

Pathogenesis related protein 10（PR10）plays an important role in plant resistance to viral infection. In the early stage, the RNA silencing suppressor P1 encoded by sugarcane streak mosaic virus（SCSMV）was used as bait to screen and obtain a sugarcane ShPR10 protein. In order to explore the function of ShPR10 in sugarcane response to SCSMV infection, the sugarcane ShPR10 gene was cloned by homologous cloning technology, and its coding protein was analyzed via bioinformatics. The subcellular localization of ShPR10 protein was analyzed by fusion expression with green fluorescent protein. The interaction between ShPR10 and SCSMV P1 was validated by yeast two hybrid and bimolecular fluorescence complementation techniques. The effect of ShPR10 on P1 silencing suppressor activity was analyzed using the Agrobacterium tumefaciens co-infiltration transient expression system and Western blot technology. The results showed that the open reading frame of sugarcane ShPR10 gene was 570 bp, which encoded an unstable hydrophilic protein with a molecular weight of 21.17 kD, an isoelectric point of 4.77, one P-loop motif, and no transmembrane domains and signal peptides. The secondary structure of ShPR10 contained 51.85% random coil, 35.98% α-helix, 7.41% extended-strand, and 4.76% β-turn. The amino acid sequence similarity between ShPR10 protein and ZmPR10 protein of Zea mays was as high as 91.53%, and the two proteins were clustered into one branch on the evolutionary tree. ShPR10 was located in the cytoplasm and nucleus, and interacted with SCSMV P1 in yeast and tobacco cells. ShPR10 itself did not have silencing suppressor activity, and its expression weakened the silencing suppressor activity of P1, but had no significant effect on the content of P1 protein. In summary, ShPR10 may weaken the silencing suppressor activity of P1 by binding to P1, thereby improving the resistance of sugarcane to SCSMV.

Key words: pathogenesis related protein, PR10, sugarcane streak mosaic virus, RNA silencing suppressor, P1 protein, sugarcane

HUANG Jia-yan, FENG Xiao-yan, SHEN Lin-bo, WANG Wen-zhi, HU Hai-yan, ZHANG Shu-zhen. Cloning of Sugarcane ShPR10 Gene and Study on the Interaction Between ShPR10 Protein and P1 Protein Encoded by Sugarcane Streak Mosaic Virus[J]. Biotechnology Bulletin, 2023, 39(10): 163-174.

Figures/Tables 11

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	引物用途 Purpose of primer
ShPR10-F	ATGGGTTGGCGTTGGCACGA	基因克隆Gene cloning
ShPR10-R	TTACACATCTGAAATCTTGC	基因克隆Gene cloning
GFP-p-F	AACACGGGGGACTTTGCAACATGGTGAGCAAGGGCGAGGAG	亚细胞定位载体构建 Vector construction for subcellular localization
GFP-p-R	GCCAACCCATGTACAGCTCGTCCATGCCGTG
ShPR10-p-F	CGAGCTGTACATGGGTTGGCGTTGGCACGAC
ShPR10-p-R	TGAAGACAGAGCTAGTTACATTACACATCTGAAATCTTGCTCCTGAATTCTGAG
ShPR10-JT-F	AGTGGTCTCTGTCCAGTCCTATGGGTTGGCGTTGGCACGA	Y2H、BiFC和植物表达载体构建 Vector construction for Y2H, BiFC and plant expression
ShPR10-JT-R	GGTCTCAGCAGACCACAAGTTTACACATCTGAAATCTTGC	Y2H和植物表达载体构建 Vector construction for Y2H and plant expression
ShPR10-JT-R1	GGTCTCAGCAGACCACAAGTCACATCTGAAATCTTGCTCCTG	BiFC载体构建Vector construction for BiFC
P1-JT-F	AGTGGTCTCTGTCCAGTCCTATGGCTACTATCACTAAGAAGC	BiFC和植物表达载体构建 Vector construction for BiFC and plant expression
P1-JT-R	GGTCTCAGCAGACCACAAGTGTAAAATACTAAATCTTCACAGC	BiFC载体构建Vector construction for BiFC
P1-JT-R1	GGTCTCAGCAGACCACAAGTTCAGTAAAATACTAAATCTTCAC	植物表达载体构建Vector construction for plant expression

Fig. 1 Cloning results of ShPR10 gene A: Amplification product of ShPR10 gene, M represents DL2000 DNA marker. B: Nucleotide acid sequence and deduced amino acid sequence of ShPR10 gene, P-loop motif is marked with black underline

Fig. 2 Secondary structure prediction of ShPR10 protein The blue is α-helix. Red is extended-strand. Green is β-turn. Purple is random coil

Fig. 3 Transmembrane structure prediction of ShPR10 protein

Fig. 4 Signal peptide prediction of ShPR10 protein

Fig. 5 Amino acid sequence alignment between ShPR10 protein from sugarcane and PR10 from other species LpPR10: XP_051226795.1; LrPR10: XP_039849257.1; ObPR10: XP_040382970.1; OgPR10: XP_052140876.1; PvPR10: XP_039810901.1; TaPR10: XP_044357998.1; TdPR10: XP_037414265.1; TuPR10: XP_048564659.1; ZmPR10: NP_001152669.1

Fig. 6 Phylogenetic tree analysis of ShPR10 protein from sugarcane and PR10 proteins from other species

Fig. 7 Localization of ShPR10 in N. benthamiana epidermal cells

Fig. 8 Verification of the interaction between ShPR10 and SCSMV P1 by Y2H assay

Fig. 9 Verification of the interaction between ShPR10 and SCSMV P1 by BiFC assay

Fig. 10 Effect of ShPR10 on the activity of SCSMV P1 silencing suppressor A: Fluorescence expression in N. benthamiana leaves after co-injection of three A. tumefaciens cultures carrying different plasmids. GFP represents the pCam3304-35S-GFP plasmid, HA-ShPR10 represents the pNC-Cam33HN-ShPR10 plasmid, Flag-P1 represents the pNC-Cam33FN-P1 plasmid, HA represents the pNC-Cam33HN empty plasmid, and Flag represents the pNC-Cam33FN empty plasmid. B: The protein levels in the injection areas of N. benthamiana leaves were detected by Western blot. The plasmid combinations shown at the top of the figure were the same as that in Figure A. Levels of ShPR10, P1, and GFP proteins were confirmed with HA, Flag, and GFP antibodies, respectively. The large subunit of Rubisco stained with Coomassie brilliant blue（CBB）was used as a loading control. The error indicates the standard deviation of three repeated experiments

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