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Single amino acid substitutions in the coat protein and RNA-dependent RNA polymerase alleviated the virulence of Cucumber green mottle mosaic virus and conferred cross protection against severe infection

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Abstract

Cross protection is a promising alternate to control Cucumber green mottle mosaic virus (CGMMV) which is of increasing economic importance to cucurbit production worldwide. One major factor confronting the application of cross protection to control CGMMV is the scarcity of available mild mutants. The objective of this paper was to screen attenuated mutants of CGMMV and evaluate their potential in cross protection. An infectious cDNA clone of CGMMV, pCGMMV, was obtained by cloning intron-containing CGMMV genome to modified pCambia0390 vector with the Cauliflower mosaic virus 35S promoter. Five pCGMMV-derived mutants were obtained via site-directed mutagenesis and inoculated to Nicotiana benthamiana plants for symptom observation. The attenuated CGMMV mutants were evaluated for their efficiency in cross protection. The intron-containing clone pCGMMV induced similar disease symptoms and accumulated similar titres of virus in N. benthamiana plants as wild-type CGMMV. Mutations of aspartic acid at position 89 in the coat protein to alanine (D89A) or glutamic acid at position 1069 in the ORF1/2 read-through protein, in the RNA-dependent RNA polymerase domain to alanine (E1069A) alleviated the symptoms of pCGMMV in N. benthamiana plants significantly. In cross protection assay, the two mutants pCGMMV-CP-D89A and pCGMMV-RdRp-E1069A could prevent the superinfection of CGMMV, with protection efficiency of 91.7% and 100%, respectively. The intron-containing clone pCGMMV was stable and highly infectious. The D89 in the coat protein and E1069 in the RNA-dependent RNA polymerase played an important role in regulating the virulence of CGMMV. Mutants pCGMMV-CP-D89A and pCGMMV-RdRp-E1069A were of great potential in the control of CGMMV via cross protection.

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Abbreviations

bp:

Base pairs

CGMMV:

Cucumber green mottle mosaic virus

CP:

Coat protein

MP:

Movement protein

ORF:

Opening reading frames

PMMoV:

Pepper mild mottle mosaic virus

PTA-ELISA:

Plate-trapped antigen enzyme-linked immunosorbent assay

qRT-PCR:

Quantitative real-time polymerase chain reaction

RdRP:

RNA-dependent RNA polymerase

ToMV:

Tomato mosaic virus

UTR:

Untranslated region

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Acknowledgements

This study was supported by National Natural Science Foundation of China (NSFC, 31720103912; 31571984) and Funds of Shandong ‘Double Top’ Program (SYL2017XTTD11). We thank Xue-ying Wang from Jiyang Protection Station of Shandong Province for providing the CGMMV isolate.

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Authors and Affiliations

  1. College of Landscape Science and Engineering, Shandong Agriculture and Engineering University, Jinan, 250100, Shandong, China

    Jin Liu

  2. Shandong Provincial Key Laboratory for Agricultural University, Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Tai’an, 271018, Shandong, China

    Xiang-Dong Li & Shuai Xu

Authors
  1. Jin Liu
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  2. Xiang-Dong Li
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  3. Shuai Xu
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Contributions

JL and SX designed and performed the experiments, analyzed the data, and wrote the manuscript. X-DL conceived the study and participated in design and coordination and writing. Reagents/materials/analysis tools were contributed by JL, SX, and X-DL. All authors read and approved the final manuscript.

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Correspondence to Jin Liu.

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Liu, J., Li, XD. & Xu, S. Single amino acid substitutions in the coat protein and RNA-dependent RNA polymerase alleviated the virulence of Cucumber green mottle mosaic virus and conferred cross protection against severe infection. Virus Genes 56, 228–235 (2020). https://doi.org/10.1007/s11262-019-01726-3

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  • DOI: https://doi.org/10.1007/s11262-019-01726-3

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