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. 2019 Aug 8;20(16):3867.
doi: 10.3390/ijms20163867.

Identification of Sugarcane Host Factors Interacting with the 6K2 Protein of the Sugarcane Mosaic Virus

Hai Zhang  1 , Guangyuan Cheng  1 , Zongtao Yang  1 , Tong Wang  1 , Jingsheng Xu  2   3
Affiliations

Identification of Sugarcane Host Factors Interacting with the 6K2 Protein of the Sugarcane Mosaic Virus

Hai Zhang et al. Int J Mol Sci. .

Abstract

The 6K2 protein of potyviruses plays a key role in the viral infection in plants. In the present study, the coding sequence of 6K2 was cloned from Sugarcane mosaic virus (SCMV) strain FZ1 into pBT3-STE to generate the plasmid pBT3-STE-6K2, which was used as bait to screen a cDNA library prepared from sugarcane plants infected with SCMV based on the DUALmembrane system. One hundred and fifty-seven positive colonies were screened and sequenced, and the corresponding full-length genes were cloned from sugarcane cultivar ROC22. Then, 24 genes with annotations were obtained, and the deduced proteins were classified into three groups, in which eight proteins were involved in the stress response, 12 proteins were involved in transport, and four proteins were involved in photosynthesis based on their biological functions. Of the 24 proteins, 20 proteins were verified to interact with SCMV-6K2 by yeast two-hybrid assays. The possible roles of these proteins in SCMV infection on sugarcane are analyzed and discussed. This is the first report on the interaction of SCMV-6K2 with host factors from sugarcane, and will improve knowledge on the mechanism of SCMV infection in sugarcane.

Keywords: 6K2; interaction; sugarcane mosaic virus; yeast two-hybrid.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Subcellular localization of Sugarcane mosaic virus (SCMV)-6K2. (A) The schematic diagram of the amino acids of the SCMV-6K2 protein. GXXXG motif (‘X’ being any amino acid) was highlighted by the red color. The predicted transmembrane domain (TMD) was marked by an underline. (B) Prediction of SCMV-6K2 TMD by TMHMM. The horizontal axis indicates the amino acid position. (C) Subcellular localization of 6K2-CFP in the leaves epidermal cells of N. benthamiana by 48-h post agroinfiltration. White arrows point to endoplasmic reticulum (ER), yellow arrows point to chloroplast. Scale bars, 25 μm.
Figure 2
Figure 2
Evaluation of the pBT3-STE-6K2 bait vector. Plasmid combinations of pBT3-STE-6K2 and pOst1-NubI, pBT3-STE-6K2, and pPR3-N were co-transformed into yeast NMY51 cells in a 10× dilution series of 10-μL aliquots, which were then cultured on DDO+5-bromo-4-chloro-3-indolyl β-D-galactoside (X-Gal) or QDO+X-Gal agar plates to evaluate the toxicity or auto-activation of 6K2, respectively. Yeast cells co-transformed with pTSU2-APP and pNubG-Fe65 were used as positive controls, pTSU2-APP and pPR3-N were used as negative controls. DDO+X-Gal: SD/-Trp/-Leu, supplemented with X-Gal,; QDO+X-Gal: SD/-Trp/-Leu/-His/-Ade, supplemented with X-Gal.
Figure 3
Figure 3
Verification of protein interaction by yeast two hybrid assays. The coding sequences of 24 proteins were individually infused into the prey vector pPR3 and co-transformed with the bait vector pBT3-STE-6K2 into the yeast NMY51 cells in a 10× dilution series of 10-μL aliquots, which were then plated on non-selective medium (DDO+X-Gal) or a high-stringency selective medium (QDO+X-Gal). Yeast cells co-transformed with pTSU2-APP and pNubG-Fe65 were used as positive controls, pTSU2-APP and pPR3-N were used as negative controls.
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