Construction of a Mild Vaccine of Zucchini Yellow Mosaic Virus and Its Cross-protective Efficacy

doi:10.13560/j.cnki.biotech.bull.1985.2025-0944

Abstract

Abstract:

Objective By investigating the effects of the mutation at the 364th conserved lysine (K³⁶⁴) in helper component-proteinase (HC-Pro) on the virulence of zucchini yellow mosaic virus (ZYMV), an attenuated ZYMV mutant were developed, and its genetic stability and cross-protection efficacy were assessed. Our study provided the theoretical basis for developing sustainable control strategies against ZYMV using attenuated strains. Method Using site-directed mutagenesis, the K³⁶⁴ residue in HC-Pro was substituted with aspartic acid (D) to investigate the effect of the K364D mutation on the virulence of ZYMV and the RNA silencing suppression activity of HC-Pro, thereby constructing the attenuated ZYMV-HC-Pro_K364D mutant. Furthermore, the genetic stability of ZYMV-HC-Pro_K364D mutant was assessed through serial passage of progeny viruses. The cross-protection efficacy of ZYMV-HC-Pro_K364D mutant was evaluated through cross-protection assays. Result The K364D mutation in HC-Pro attenuated ZYMV virulence, alleviated typical yellowing symptoms and reduced ZYMV accumulation in Cucumis melo plants. Simultaneously, K364D mutation suppressed the RNA silencing suppression activity of ZYMV HC-Pro. The attenuated mutant ZYMV-HC-Pro_K364D-GFP maintained genetic stability without reversion mutation after three successive passages in C. melo plants. In cross-protection assays, ZYMV-HC-Pro_K364D mutant induced significant resistance in C. melo plants against the wild-type ZYMV infection when the protection interval was 11 d. Conclusion The conserved K³⁶⁴residue in HC-Pro was crucial in determining the RNA silencing suppression activity and the virulence of ZYMV. The ZYMV-HC-Pro_K436D mutant present excellent cross-protection efficacy and is a potential mild vaccine against the wild-type ZYMV infection in C. melo plants.

Key words: Zucchini yellow mosaic virus, helper component-proteinase, mild vaccine, genetic stability, cross-protection

ZHANG Yue, REN Qian, LUAN Ya-meng, LI Yi-chen, SUN Wu-xia, REN Shu-ying, XU Xiao-jie, SUN Xiao-hui. Construction of a Mild Vaccine of Zucchini Yellow Mosaic Virus and Its Cross-protective Efficacy[J]. Biotechnology Bulletin, 2026, 42(1): 271-278.

Figures/Tables 5

Table 1 Primer names and sequences used in this study

引物名称 Primer name	引物序列 Primer sequence （5'-3'）
ZYMV-HC-Pro_K364D-F	ATTTCACCGATATGATTCGTGATGTTTTGATCCCTATGCTTG
ZYMV-HC-Pro_K364D-R	CGAATCATATCGGTGAAATCCTTCGCCTCGTTCTCATTAAC
ZYMV-HC-Pro-F	GCGCTCTAGCAAGGCTATG
ZYMV-HC-Pro-R	CACCTAGTATGTATGCTGCAGT
GFP-F	ATGAGTAAAGGAGAAGAAC
GFP-R	TTTGTAGAGCTCATCCATG
GFP-qRT-F	GTGGAGAGGGTGAAGGTGAT
GFP-qRT-R	CGGATAACGGGAAAAGCATTGA
actin-qRT-F	CTGATGAAGATACTCACAGAAAGAG
actin-qRT-R	CAGGATACGGGGAGCTAATG
EF1α-qRT-F	CCACGAGTCTCTCCCAGAAG
EF1α-qRT-R	CACGCTTGAGATCCTTGACA

Fig. 1 Mutation of K364D reduced ZYMV virulence in C. melo plantsA: Alignment of the partial amino acid sequences of 6 potyvirus HC-Pros. The K364 residue in ZYMV HC-Pro were pointed in the red box. B: The symptoms observed on C. melo plants at day 11 after infection with the wild-type ZYMV and ZYMV-HC-ProK364D-GFP mutant. C: Western blotting was used to detect the accumulation of ZYMV CP in the upper leaves of C. melo plants. The loading controls were the Ponceau S staining (PSS) bands, and the band gray values were measured using ImageJ software. The accumulation of ZYMV CP is represented by the data normalized to PSS

Fig. 2 Mutation of K364D affected the activity of RNA silencing of ZYMV HC-ProA: Suppression activity of RNA silencing of wild-type ZYMV HC-Pro and HC-ProK364D mutant. B: The accumulation of GFP mRNA in different regions of 16c leaves. Tukey multiple range test was used for statistical analysis between groups. Significant differences are indicated by different letters (P<0.05). C: Accumulation of GFP protein in different regions of 16c leaves

Fig. 3 Genetic stability of ZYMV-HC-ProK364D-GFP mutantA: During three consecutive passages, the symptoms caused by the ZYMV-HC-ProK364D-GFP mutant on C. melo plants. B: The HC-Pro RNA accumulations of the ZYMV-HC-ProK364D-GFP mutant F3 generation in C. melo plants. C: Analysis of HC-Pro sequence in the F3 generation of ZYMV-HC-ProK364D-GFP mutant in C. melo plants. The codon encoding the 364th amino acid residues in ZYMV HC-Pro was underlined

Fig. 4 Cross protection efficacy mediated by ZYMV-HC-ProK364D mutantA: Symptoms of C. melo plants with an interval period of 7 or 11 d at the 11th day post ZYMV-GFP challenge. The numbers in parentheses indicate the number of C. melo plants with GFP fluorescence/the total number of inoculated C. melo plants. B: The accumulation of GFP mRNA in C. melo plants on the 11th day after ZYMV-GFP challenge. C: The accumulation of GFP protein in C. melo plants on the 11th day after ZYMV-GFP challenge. D: Analysis of HC-Pro sequence in C. melo plants on the 11th day after ZYMV-GFP challenge. The codon encoding the 364th amino acid residue in ZYMV HC-Pro was underlined. The HC-Pro coding sequence from the ZYMV progeny in each C. melo plant was individually sequenced three times

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