Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (19): 3810-3822.doi: 10.3864/j.issn.0578-1752.2024.19.008

• PLANT PROTECTION • Previous Articles     Next Articles

Complete Genome Sequence Analysis and Infectious Clone Construction of Mume Virus A Peach Isolate pp in Xinjiang

REN CaiXia1(), LIU Lin1, LIU ShengXue2, BU Fang DI1, XIANG BenChun3, ZHENG YinYing1(), CUI BaiMing1()   

  1. 1 College of Life Sciences, Shihezi University, Shihezi 832003, Xinjiang
    2 Analysis and Test Center, Shihezi University, Shihezi 832003, Xinjiang
    3 Agricultural College, Shihezi University, Shihezi 832003, Xinjiang
  • Received:2024-06-16 Accepted:2024-07-14 Online:2024-10-01 Published:2024-10-09
  • Contact: ZHENG YinYing, CUI BaiMing

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Abstract:

【Objective】Mume virus A peach isolate (MuVA pp) is a newly discovered virus infecting peach (Prunus persica) trees, and its complete genome sequence study has not been reported in China. Therefore, the purpose of this study is to analyze the genome, phylogenetic evolution and pathogenicity of MuVA pp isolate, and to investigate its prevalence in peach in Xinjiang, China, so as to provide scientific basis for the prevention and control of MuVA.【Method】RT-PCR was used to detect MuVA in field samples of peach, and 5′/3′ rapid amplification of cDNA ends (RACE) technology was used to determine the complete genome sequence of MuVA pp isolate. The genome organization and phylogenetic relationships were analyzed by bioinformatics methods. A full-length infectious cDNA clone was constructed using Gibson assembly, and its infectivity was tested by inoculation with Agrobacterium tumefacieas.【Result】The results of RT-PCR showed that 10 of the 30 suspected viral disease samples were infected with MuVA, and the infection rate of different peach varieties was from high to low, namely ‘July flat peach’ (4/9), ‘flat peach on August 1’ (5/13) and ‘medium mature August 1’ (1/8), respectively. The genome of MuVA pp is 7 647 nt in length and consists of 5′ UTR, 3′ UTR and two overlapping open reading frames (ORFs), encoding methyltransferase (Met), RNA helicase (Hel), RNA-dependent RNA polymerase (RdRp), coat protein (CP) and movement protein (MP). The sequence analysis showed that MuVA pp isolate has 80.8% and 82.7% identities with previously reported MuVA isolate pm14 at the nucleotide sequence and amino acid sequence levels, respectively. The polyproteins encoded by these two isolates differ by 406 amino acid residues, which are distributed in Met (21), Hel (29), RdRp (19), CP (18), and other (319). In addition, the 5′ UTR of the genome was more different, and the identity of the nucleotide sequence was only 74.6%. Of these encoded proteins, MP has the greatest variability (80.9% identity), while RdRp is the most conserved (94.0% identity). Phylogenetic analysis showed that MuVA pp isolate was closely related to MuVA pm14 isolate, and MuVA showed a tendency of host and geographic specificity. Plum isolates and mume isolates clustered into clusters, while peach isolates formed clays alone. The Chenopodium amaranticolor plants could be systemically infected by MuVA infectious clone, but symptomless. Tobacco (Nicotiana tabacum var. Samsun NN) agroinfiltrated showed allergic necrosis in inoculated leaves, but no systemic infection occurred. Other indicator plants, including C. quinoa, N. glutinosa, N. occidentalis, N. benthamiana, or Solanum lycopersicum, Cucumis sativus and Cucurbita moschata, could not be infected by MuVA infectious clones.【Conclusion】The complete genome sequence of the peach isolate pp of MuVA was sequenced successfully. The genome is a single-stranded positive-sense RNA with a length of 7 647 nt, lacking a 3′ terminal poly(A) tail, and encoding two ORFs. Mechanical inoculation proved that MuVA could not infect herbaceous plants. pCB301-MuVA, a MuVA pp full-length cDNA infectious cloning vector, was constructed. It could infect C. amaranticolor systematically and N. tabacum var. Samsun NN locally. The results provide a reference for further study of the pathogenic molecular mechanism of MuVA.

Key words: mume virus A (MuVA), RT-PCR, genome structure, evolutionary analysis, identification of infectivity

Table 1

Primers used in this study"

引物名称
Primer name		 序列
Sequence (5′-3′)		 引物位置
Primer location		 退火温度
Tm (℃)
DT_MVA_F CTTCACATCGGAGCTGTCCT 5728-5747 57
DT_MVA_R TGCTGATTGCTTCAGACCCT 6270-6289 57
MuVA1_F GTTCATTTCATTTGGAGAGGGGAAAACCATCATCAACTTAAC 1-22 56
MuVA1_R TCTCCTTCTTGAACTCTCTGGATTCTCTAG 3741-3770 56
MuVA2_F CAGAGAGTTCAAGAAGGAGAGGGTTGGATG 3751-3780 58
MuVA2_R TGGAGATGCCATGCCGACCCCTTAAGGAAAAAGGAAATAAAGTCC 7623-7647 58
MuVA5′-RACE TTCCGACACAGAGACGTCGGGAGAGGT 606-632 68
MuVA3′-RACE GCTTGCATGGGGTGCTGTAGGTGCAGGG 6823-6850 68

Fig. 1

Construction of pCB301-MuVA expression vector"

Fig. 2

RT-PCR detection of MuVA in P. persica samples"

Fig. 3

The genome structure of MuVA pp"

Table 2

Genomic sequence comparison between MuVA pp and MuVA pm14"

基因组区域
Genomic region		 核苷酸位置 Nucleotide position (nt) 长度Length (nt/aa) 一致性 Identity (%)
MuVA pp MuVA pm14 MuVA pp MuVA pm14 nt aa
5′ UTR 1-145 1-146 145 146 74.6 -
Met 272-1198 273-1199 926/308 926/308 83.0 93.2
Hel 2579-3412 2580-3413 833/277 833/277 76.9 89.6
RdRp 3971-4924 3972-4925 953/317 953/317 80.2 94.0
CP 6650-7123 6651-7124 473/157 473/157 77.6 88.6
MP 5464-6861 5465-6856 1397/465 1391/463 83.0 80.9
3′ UTR 7147-7647 7146-7644 500 498 86.6 -
Replicase 146-7147 147-7148 7001/2333 7001/2333 78.2 82.7
全基因组Complete genome 1-7647 1-7644 7647 7644 80.8 -

Fig. 4

Phylogenetic analysis of MuVA and representative species of Capillovirus"

Fig. 5

Complete genome PCR amplified fragment of MuVA pp isolate"

Fig. 6

RT-PCR detection results of pCB301-MuVA infectious clone inoculated C. amaranticolor system leaves (35 dpi)"

Fig. 7

Symptoms and RT-PCR detection results of pCB301-MuVA infectious clone inoculated N. tabacum var. Samsun NN inoculated leaves"

Fig. 8

DAB dyeing (A) and trypan blue dyeing (B)"

[1]
MARAIS A, FAURE C, THEIL S, CANDRESSE T. Molecular characterization of a novel species of Capillovirus from Japanese apricot (Prunus mume). Viruses, 2018, 10(4): 144.
[2]
LEE J, LEE D S, RYU H, LIM S, LEE S J. First report of mume virus A infecting Prunus salicina worldwide and Prunus mume in Korea. Plant Disease, 2023, 107(3): 972.
[3]
ZHANG Y, ZHOU J, ZHAN B, LI S, ZHANG Z. First report of peach leaf pitting-associated virus (PLPaV), plum bark necrosis stem pitting-associated virus (PBNSPaV), and mume virus A (MuVA) from Mei (Prunus mume) in China. Plant Disease, 2021, 105(8): 2259.
[4]
ZHENG Y Y, BU F D, WU C J, CHEN J G, LIU Z, XIANG B C, CUI B M. First report of mume virus A infection of Prunus persica in China. Plant Disease, 2020, 104(10): 2741.
[5]
TUO D, SHEN W, YAN P, LI X, ZHOU P. Rapid construction of stable infectious full-length cDNA clone of papaya leaf distortion mosaic virus using in-fusion cloning. Viruses, 2015, 7(12): 6241-6250.

doi: 10.3390/v7122935 pmid: 26633465
[6]
DAI Z, HE R, BERNARDS M A, WANG A. The cis-expression of the coat protein of turnip mosaic virus is essential for viral intercellular movement in plants. Molecular Plant Pathology, 2020, 21(9): 1194-1211.

doi: 10.1111/mpp.12973 pmid: 32686275
[7]
SUN K, ZHAO D, LIU Y, HUANG C, ZHANG W, LI Z. Rapid construction of complex plant RNA virus infectious cDNA clones for agroinfection using a yeast-E. coli-Agrobacterium shuttle vector. Viruses, 2017, 9(11): 332.
[8]
RIECHMANN J L, LAÍN S, GARCÍA J A. Infectious in vitro transcripts from a plum pox potyvirus cDNA clone. Virology, 1990, 177(2): 710-716.
[9]
LI C, YAEGASHI H, KISHIGAMI R, KAWAKUBO A, YAMAGISHI N, ITO T, YOSHIKAWA N. Apple russet ring and apple green crinkle diseases: Fulfillment of Koch’s postulates by virome analysis, amplification of full-length cDNA of viral genomes, in vitro transcription of infectious viral RNAs, and reproduction of symptoms on fruits of apple trees inoculated with viral RNAs. Frontiers in Microbiology, 2020, 11: 1627.
[10]
SIMKOVICH A J, LI Y, KOHALMI S E, GRIFFITHS J S, WANG A. Molecular identification of prune dwarf virus (PDV) infecting sweet cherry in Canada and development of a PDV full-length infectious cDNA clone. Viruses, 2021, 13(10): 2025.
[11]
CUI H, HONG N, WANG G, WANG A. Genomic segments RNA1 and RNA2 of prunus necrotic ringspot virus codetermine viral pathogenicity to adapt to alternating natural Prunus hosts. Molecular Plant-Microbe Interactions, 2013, 26(5): 515-527.
[12]
LI Z N, JELKMANN W, SUN P P, ZHANG L. Construction of full-length infectious cDNA clones of apple stem grooving virus using Gibson assembly method. Virus Research, 2020, 276(15): 197790.
[13]
OHIRA K, NAMBA S, ROZANOV M, KUSUMI T, TSUCHIZAKI T. Complete sequence of an infectious full-length cDNA clone of citrus tatter leaf capillovirus: Comparative sequence analysis of capillovirus genomes. The Journal of General Virology, 1995, 76(9): 2305-2309.
[14]
NOORANI M S, AWASTHI P, SINGH R M, RAM R, SHARMA M P, SINGH S R, AHMED N, HALLAN V, ZAIDI A A. Complete nucleotide sequence of cherry virus A (CVA) infecting sweet cherry in India. Archives of Virology, 2010, 155(12): 2079-2082.

doi: 10.1007/s00705-010-0826-6 pmid: 20938696
[15]
BHARDWAJ P, HALLAN V. Molecular evidence of apple stem grooving virus infecting Ficus palmata. Trees, 2019, 33(1): 1-9.
[16]
张丽, 王德富, 裴燕妮, 咸珅, 牛颜冰. 大豆花叶病毒半夏分离物侵染性克隆构建及鉴定. 生物工程学报, 2020, 36(5): 949-958.
ZHANG L, WANG D F, PEI Y N, XIAN S, NIU Y B. Construction and characterization of an infectious clone of soybean mosaic virus isolate from Pinellia ternata. Chinese Journal of Biotechnology, 2020, 36(5): 949-958. (in Chinese)
[17]
卜方迪, 陈俊光, 刘贞, 向本春, 申冕, 崔百明, 郑银英. 新疆蟠桃中发现油桃茎痘相关病毒和亚洲李属病毒. 园艺学报, 2021, 48(1): 49-59.

doi: 10.16420/j.issn.0513-353x.2020-0215
BU F D, CHEN J G, LIU Z, XIANG B C, SHEN M, CUI B M, ZHENG Y Y. Nectarine stem pitting-associated virus and Asian Prunus virus found in Prunus persica of Xinjiang. Acta Horticulturae Sinica, 2021, 48(1): 49-59. (in Chinese)
[18]
ADAMS M J, ANTONIW J F, BAR-JOSEPH M, BRUNT A A, CANDRESSE T, FOSTER G D, MARTELLI G P, MILNE R G, ZAVRIEV S K, FAUQUET C M. The new plant virus family Flexiviridae and assessment of molecular criteria for species demarcation. Archives of Virology, 2004, 149(5): 1045-1060.
[19]
LÜTCKE H A, CHOW K C, MICKEL F S, MOSS K A, KERN H F, SCHEELE G A. Selection of AUG initiation codons differs in plants and animals. The EMBO Journal, 1987, 6(1): 43-48.
[20]
PETRZIK K, PŘIBYLOVÁ J, KOLONIUK I, ŠPAK J. Molecular characterization of a novel capillovirus from red currant. Archives of Virology, 2016, 161(4): 1083-1086.

doi: 10.1007/s00705-016-2752-8 pmid: 26754736
[21]
COMMANDEUR U, JARAUSCH W, LI Y, KOENIG R, BURGERMEISTER W. cDNAs of beet necrotic yellow vein virus RNAs 3 and 4 are rendered biologically active in a plasmid containing the cauliflower mosaic virus 35S promoter. Virology, 1991, 185(1): 493-495.

pmid: 1926790
[22]
DOMIER L L, FRANKLIN K M, HUNT A G, RHOADS R E, SHAW J G. Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus. Proceedings of the National Academy of Sciences of the United States of America, 1989, 86(10): 3509-3513.
[23]
LIU Q, YANG L, XUAN Z, WU J, QIU Y, ZHANG S, WU D, ZHOU C, CAO M. Complete nucleotide sequence of loquat virus A, a member of the family Betaflexiviridae with a novel genome organization. Archives of Virology, 2020, 165(1): 223-226.
[24]
HIRATA H, YAMAJI Y, KOMATSU K, KAGIWADA S, OSHIMA K, OKANO Y, TAKAHASHI S, UGAKI M, NAMBA S. Pseudo- polyprotein translated from the full-length ORF1 of Capillovirus is important for pathogenicity, but a truncated ORF1 protein without variable and CP regions is sufficient for replication. Virus Research, 2010, 152(1/2): 1-9.
[25]
WYLIE S, JONES M. Hardenbergia virus A, a novel member of the family Betaflexiviridae from a wild legume in Southwest Australia. Archives of Virology, 2011, 156(7): 1245-1250.
[26]
KOMATSU K, HIRATA H, FUKAGAWA T, YAMAJI Y, OKANO Y, ISHIKAWA K, ADACHI T, MAEJIMA K, HASHIMOTO M, NAMBA S. Infection of capilloviruses requires subgenomic RNAs whose transcription is controlled by promoter-like sequences conserved among flexiviruses. Virus Research, 2012, 167(1): 8-15.

doi: 10.1016/j.virusres.2012.02.019 pmid: 22401846
[27]
GARCIA-RUIZ H. Susceptibility genes to plant viruses. Viruses, 2018, 10(9): 484.
[28]
MITSUHARA I, UGAKI M, HIROCHIKA H, OHSHIMA M, MURAKAMI T, GOTOH Y, KATAYOSE Y, NAKAMURA S, HONKURA R, NISHIMIYA S, et al. Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant & Cell Physiology, 1996, 37(1): 49-59.
[29]
CHENG J H, PENG C W, HSU Y H, TSAI C H. The synthesis of minus-strand RNA of bamboo mosaic potexvirus initiates from multiple sites within the poly(A) tail. Journal of Virology, 2002, 76(12): 6114-6120.
[30]
KÜHN U, GÜNDEL M, KNOTH A, KERWITZ Y, RÜDEL S, WAHLE E. Poly(A) tail length is controlled by the nuclear poly(A)-binding protein regulating the interaction between poly(A) polymerase and the cleavage and polyadenylation specificity factor. The Journal of Biological Chemistry, 2009, 284(34): 22803-22814.
[31]
CHEN I H, CHENG J H, HUANG Y W, LIN N S, HSU Y H, TSAI C H. Characterization of the polyadenylation activity in a replicase complex from bamboo mosaic virus-infected Nicotiana benthamiana plants. Virology, 2013, 444(1/2): 64-70.
[32]
SHIEN J H, SU Y D, WU H Y. Regulation of coronaviral poly(A) tail length during infection is not coronavirus species- or host cell-specific. Virus Genes, 2014, 49(3): 383-392.
[33]
WU H Y, KE T Y, LIAO W Y, CHANG N Y. Regulation of coronaviral poly(A) tail length during infection. PLoS ONE, 2013, 8(7): e70548.
[34]
NIU S, CAO S, HUANG L J, TAN K C, WONG S M. The length of an internal poly(A) tract of hibiscus latent Singapore virus is crucial for its replication. Virology, 2015, 474 (1): 52-64.
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