The Role of NbJAZ3 in the Infection of Nicotiana benthamiana by Alfalfa Mosaic Virus

doi:10.13560/j.cnki.biotech.bull.1985.2025-0084

Abstract

Abstract:

Objective To explore the effect of NbJAZ3, a gene encoding jasmonate ZIM-domain protein 3 (JAZ3), on the infection of Nicotiana benthamiana by alfalfa mosaic virus (AMV), and to provide a theoretical basis for further analyzing the mechanism of AMV infection. Method Using N. benthamiana and AMV as materials, AMV was inoculated into N. benthamiana by friction inoculation method. The leaves of N. benthamiana at 1, 7, 15 and 21 d after inoculation were used for transcriptome sequencing analysis. The differential genes in jasmonic acid (JA) signal transduction pathway were screened, and the expression of NbJAZ3 in AMV infection was detected by real-time fluorescence quantitative PCR (RT-qPCR). NbJAZ3 was cloned, biological information was analyzed, phylogenetic tree was constructed, and multiple sequence alignment was performed.The transient overexpression vector of pCAMBIA1300-NbJAZ3 was constructed. The subcellular localization of NbJAZ3 protein was observed by laser confocal microscopy, and the protein expression was detected by Western blot. NbJAZ3 was transiently overexpressed in N. benthamiana leaves and inoculated with AMV, and the relative expression of AMV CP at 5 d after inoculation was detected by RT-qPCR. The pTRV2-NbJAZ3 silencing vector was constructed by gene silencing technology induced by tobacco brittle fracture virus. After silencing NbJAZ3, AMV was inoculated by friction. The relative expression of AMV CP was detected by RT-qPCR. Result Transcriptome sequencing and RT-qPCR further verified that NbJAZ3 was down-regulated on different days after AMV inoculation, and the relative expression of NbJAZ3 was the lowest on the day15, which was 84.43% lower than that of CK. The NbJAZ3 CDS sequence was 1 143 bp in length, encoding 380 amino acids, containing two conserved domains of ZIM and Jas-motif. NbJAZ3 had the highest homology with NsJAZ3 of Nicotiana sylvestris, and the similarity was 96.58%. Microscopic observation showed that NbJAZ3 was localized in the nucleus, cytoplasm and cell membrane of N. benthamiana leaves. Compared with the control group, the relative expression of AMV CP decreased by 83.82% and increased by 78.58% in N. benthamiana with transient overexpression and transient silencing of NbJAZ3 on the day 5 of AMV inoculation, respectively. Conclusion AMV infection can cause the down-regulation of NbJAZ3 in N. benthamiana. Transient overexpression of NbJAZ3 can inhibit the infection of AMV, while silencing this gene can promote the infection of AMV, further indicating that NbJAZ3 can be used as a positive regulator of N. benthamiana resistance to AMV infection and play an important role in resisting virus infection.

Key words: alfalfa mosaic virus (AMV), infection, Nicotiana benthamiana, NbJAZ3, subcellular localization, transient overexpression, virus-induced gene silencing (VIGS), bioinformatics analysis

LAI Shi-yu, LIANG Qiao-lan, WEI Lie-xin, NIU Er-bo, CHEN Ying-e, ZHOU Xin, YANG Si-zheng, WANG Bo. The Role of NbJAZ3 in the Infection of Nicotiana benthamiana by Alfalfa Mosaic Virus[J]. Biotechnology Bulletin, 2025, 41(8): 186-196.

Figures/Tables 8

Table 1 Primers and sequences were used in this experiment

引物名称	上游引物序列	下游引物序列
Primer name	Forward primer sequence （5′-3′）	Reverse primer sequence （5′-3′）
NbJAZ3	ATGGAGAGAGATTTTATGGG	CGTCTCCTTGACCAAATT
Kpn Ⅰ/Sac I-NbJAZ3	GGTACCATGGAGAGAGATTTTATGGG	GAGCTCCGTCTCCTTGACCAAATT
si-NbJAZ3	GGTACCCTATCTCCAGTTCAGGC	GAGCTCCCGAGCCAATGAT
T-vector-M13	CGCCAGGGTTTTCCCAGTCAC	AGCGGATAACAATTTCACACAGGA
pCAMBIA1300	CTATCCTTCGCAAGACCCTTC	TGACGAACGTTGTCGAAACC
pTRV2-PCR	ACATTGTTACTCAAGGAAGCACGA	AACTTCAGACACGGATCTACTT
25S	AAGGCCGAAGAGGAGAAAGGT	CGTCCCTTAGGATCGGCTTAC
AMV-CP	GCATCCCTAGGGGCATTCATGCA	ATCATTGATCGGTAATGGGCCGTT
qPCR-NbJAZ3	CTGGTGTCGGGCAGAAAA	TGGGTTGGAAACTGGGAG
Actin	CTTGAAACAGCAAAGACCAGC	CATCCTATCAGCAATGCCCG

Fig. 1 Heat map of differential genes (A-C) and the relative expressions of AMV CP (D) and NbJAZ3 (E) of AMV-infected N. benthamiana at different timesA: 1 d after AMV infection. B:15 d after AMV infection. C:21d after AMV infection. D: Relative expressions of AMV CP. E: Relative expressions of NbJAZ3. Student's t test was used for statistical analysis (**: 0.001<P<0.01, ***: P<0.001). Each treatment was repeated three times, and three plants were used for repeated each time. The value indicates the mean ± standard error of the three biological replicates, the same below

Fig. 2 Phylogenetic development analysis (A) and amino acid sequence alignment (B) of NbJAZ3 and homologous genes

Fig. 3 Analysis for physicochemical properties ofNbJAZ3 proteinA: Prediction of protein hydrophilic structure. B: Prediction of protein secondary structure. C: Prediction of protein tertiary structure

Fig. 4 Double enzyme digestion of T-Vetor-NbJAZ3 (A) and pCAMBIA1300-NbJAZ3 (B) transient overexpressionM1: DNA marker (2 000 bp). M2: DNA marker (15 000 bp). The lengths of T-vector, pCAMBIA1300 and NbJAZ3 are 2 693, 11 189 and 1 143 bp, respectively

Fig. 5 Subcellular localization of NbJAZ3 in N. benthamiana leavesA: Expression of pCAMBIA1300-NbJAZ3 in N. benthamiana. B: Expression of pCAMBIA1300-35S-eGFP in N. benthamiana

Fig. 6 Detection of transient overexpression of NbJAZ3 in N. benthamiana and its effect on AMV replicationA: The relative expression of NbJAZ3 on the day 5 after transient overexpression of pCAMBIA1300-NbJAZ3.B: The expression of pCAMBIA1300-NbJAZ3 was detected by Western blot. C: The phenotype of N. benthamiana on the day 5 after transient overexpression of NbJAZ3 and inoculation with AMV. D: Effect of transient overexpression of NbJAZ3 on the relative expression of AMV CP

Fig. 7 Detection of silencing efficiency of NbJAZ3 and its effect on AMV replicationA: Silencing efficiency of NbJAZ3 on the day 9. B: The leaf phenotype of NbJAZ3 silenced plants on the day 5 after inoculation with AMV; C: The relative expression of AMV CP in NbJAZ3 silenced plants was detected by RT-qPCR on the day 5 after inoculation with AMV

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