Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (22): 4473-4482.doi: 10.3864/j.issn.0578-1752.2024.22.007

• PLANT PROTECTION • Previous Articles     Next Articles

Research on Prevention and Control Technology of Citrus Yellow Vein Clearing Virus Based on VIGS

LI ChuXin(), SONG ChenHu(), ZHOU JinHuan, LI JiaXin, WANG XinLiang, TIAN XuBin, SONG Zhen()   

  1. Citrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712
  • Received:2024-07-09 Accepted:2024-07-31 Online:2024-11-16 Published:2024-11-22
  • Contact: SONG Zhen

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Abstract:

【Objective】Citrus yellow vein clearing virus (CYVCV) is a novel virus that poses a significant threat to the citrus industry. Currently, there is no effective therapeutic agent. The primary strategies for prevention and management involve utilizing virus-free seedlings and implementing stringent control measures against insect vectors. The objective of this study is to investigate the technology of virus-induced gene silencing (VIGS) as a means of developing antiviral “vaccines” for CYVCV, and to offer innovative approaches for the prevention and management of citrus viral diseases.【Method】Based on the previously constructed VIGS vector pCLBV201 in the laboratory, a series of recombinant vectors were designed and developed to target the conserved regions of the CYVCV genome, specifically open reading frame 1 (ORF1), open reading frame 6 (ORF6), coat protein (CP), and triple gene block (TGB). Agrobacterium-mediated vacuum infiltration was inoculated on Eureka lemon, followed by RT-PCR detection. After acquiring several positive plants including pCLBV201-ORF1, pCLBV201-ORF6, pCLBV201-CP, and pCLBV201-TGB, CYVCV infectious clones were inoculated via Agrobacterium-mediated injection, with plants infiltrated with the pCLBV201 empty vector serving as controls. Subsequent RT-qPCR, Western blot analysis, symptom observation, and disease index statistics were conducted to elucidate the preventive and control effects of the various VIGS recombinant vectors on CYVCV. 【Result】A series of recombinant vectors of pCLBV201 were constructed, and the results of RT-PCR following inoculation indicated that multiple positive plants for pCLBV201-ORF1, pCLBV201-ORF6, pCLBV201-CP, and pCLBV201-TGB were obtained, respectively. CYVCV infectious clones were inoculated by injection. The results of RT-qPCR at 7, 14, 28, 70, and 150 dpi (days post infection) showed that the relative expression of CYVCV in the pCLBV201-CP and pCLBV201-TGB treatment groups was significantly lower than that in the control group. Western blot analyses conducted at 70 and 150 dpi showed a significant decrease in the expression of CP protein in the pCLBV201-CP and pCLBV201-TGB treatment groups. Observational symptoms indicated that the control group exhibited typical manifestations of CYVCV infection, such as severe vein clearing and leaf distortion, whereas the pCLBV201-CP treatment group displayed mild symptoms, and the pCLBV201-TGB treatment group showed no apparent symptoms. The disease index statistics at 70 and 150 dpi demonstrated that the disease indices of pCLBV201-CP and pCLBV201-TGB were the lowest, recorded at 17.6, 41.2 and 15.6, 29.1, respectively, while the control group had indices of 52.1 and 80.0, showing obvious differences.【Conclusion】The VIGS-based technology for the prevention and control of CYVCV was developed, which clarified that pCLBV201-CP and pCLBV201-TGB could significantly reduce the virus titers and alleviate the symptoms caused by the virus. They have the potential to be used as “vaccines” for CYVCV prevention and control.

Key words: citrus yellow vein clearing virus (CYVCV), pCLBV201, virus-induced gene silencing (VIGS), RNA interference (RNAi)

Table 1

Information of the primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 扩增产物长度
Product length (bp)		 用途
Usage
CitActin-F CATCCCTCAGCACCTTCC 195 柑橘内参基因
Citrus reference gene
CitActin-R CCAACCTTAGCACTTCTCC
qCYVCV F GGTGCCATCGAACGCATTAC 361 CYVCV qPCR检测
CYVCV qPCR detection
qCYVCV R TAGAAACGTCGCGTGGTTCA
pCLBV201-orf1 F *ttagaaatgtagccc AAAGAGATTCGTATGACAAT 327 ORF1序列扩增
ORF1 sequence amplification
pCLBV201-orf1 R gggtcccgaattctg GGTGTATGTTTCAAGCTCCT
pCLBV201-orf6 F ttagaaatgtagccc AGCTATCATTTCGTGTGTGT 340 ORF6序列扩增
ORF6 sequence amplification
pCLBV201-orf6 R gggtcccgaattctg ATTTATGTTTCATCTGGGGT
pCLBV201-CP F ttagaaatgtagccc GCTCAACCTCAACCTAAGAT 341 CP序列扩增
CP sequence amplification
pCLBV201-CP R gggtcccgaattctg CCTTGATCATGAGGTTCCAT
pCLBV201-TGB F ttagaaatgtagccc GATACACGAGAACGTCGAGT 369 TGB序列扩增
TGB sequence amplification
pCLBV201-TGB R gggtcccgaattctg GCAGTAAGTTCGTGAGAGAT
CYVCV 614 F TACCGCAGCTATCCATTTCC 612 CYVCV检测
CYVCV detection
CYVCV 614 R GCAGAAATCCCTAACCACTA
H2206 F TGAGGCTAACTCTGCTATG 249 CLBV检测
CLBV detection
H2206 R TGAGGCTCCGCACAATCT

Fig. 1

PCR detection of pCLBV201 recombinant vector"

Fig. 2

Results of RT-PCR detection of lemon seedlings inoculated with pCLBV201-based vectors"

Fig. 3

RT-PCR detection of CYVCV (7 dpi)"

Fig. 4

RT-qPCR detection of CYVCV"

Fig. 5

Titer monitoring of CYVCV"

Fig. 6

Western blot detection of CYVCV"

Fig. 7

Symptoms for each treatment group at 70 dpi"

Fig. 8

Symptoms for each treatment group at 150 dpi"

Table 2

Disease index statistics of different treatments"

处理
Treatment		 病情指数Disease index
70 dpi 150 dpi
pCLBV201 52.1 80.0
pCLBV201-ORF1 29.6 62.9
pCLBV201-ORF6 33.3 66.1
pCLBV201-CP 17.6 41.2
pCLBV201-TGB 15.6 29.1
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