Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (18): 3543-3555.doi: 10.3864/j.issn.0578-1752.2022.18.006

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning, Expression and Anti-TMV Function Analysis of Nicotiana benthamiana NbMBF1c

YuXia WEN(),Jian ZHANG,Qin WANG,Jing WANG,YueHong PEI,ShaoRui TIAN,GuangJin FAN,XiaoZhou MA,XianChao SUN()   

  1. Chongqing Key Laboratory of Plant Disease Biology, College of Plant Protection, Southwest University, Chongqing 400715
  • Received:2022-04-06 Accepted:2022-05-09 Online:2022-09-16 Published:2022-09-22
  • Contact: SUN XianChao E-mail:wenyuxia123456@163.com;sunxianchao@163.com

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Abstract:

【Objective】Tobacco mosaic virus (TMV) is an important virus that harms crops such as Solanaceae, Cruciferae and Cucurbitaceae, causing great losses to agricultural production. The objective of this study is to obtain Nicotiana benthamiana multiprotein bridging factor 1c (MBF1c) by molecular cloning, clarify the antiviral function and mechanism of NbMBF1c by bioinformatics, cell biology and molecular biology methods, and to provide a theoretical basis for the antiviral breeding of crops.【Method】Based on the full-length NbMBF1c sequence reported in Sol Genomics Network, primers were designed to clone the full-length sequence of NbMBF1c. GeneDoc and MEGA X were employed to align the homologous protein sequences of NbMBF1c and other species. The gene characteristics and protein structure of NbMBF1c were analyzed by bioinformatics. The tissue expression and its expression after TMV infection were detected by real-time fluorescence quantitative PCR (qRT-PCR). Tobacco rattle virus (TRV)-induced gene silencing technology was used to silence NbMBF1c and determine its effect on TMV-GFP infection. The transient overexpression vectors of pART27-GFP-NbMBF1c and pART27-Myc-NbMBF1c were constructed and the fusion protein GFP-NbMBF1c was expressed in N. benthamiana leaf to observe its subcellular localization under confocal microscope. Myc-NbMBF1c was expressed and the changes of TMV-GFP infection after NbMBF1c expressed were observed. qRT-PCR was employed to detect the expression of hormone related genes after NbMBF1c silencing, and the mechanism of NbMBF1c affecting virus infection was analyzed.【Result】NbMBF1c, which is 441 bp in full length, encodes 146 amino acids and contains a conserved domain HTH (helix-turn-helix). Phylogenetic analysis showed that NbMBF1c had the most closely relationship with Nicotiana tomentosiformis MBF1c (XP_009614458.1). NbMBF1c was localized in the cytoplasm and nucleus and had specific tissue expression, with the highest expression in roots, followed by stems, leaves and flowers. Silencing of NbMBF1c in N. benthamiana significantly reduced the tobacco plant height and promoted TMV-GFP infection, on the 5th day after TMV-GFP inoculation, the virus content of new leaf in the silencing treatment was higher than that in the control group. Transient overexpression of Myc-NbMBF1c suppressed TMV-GFP infection, after inoculation with TMV-GFP, virus infection was reduced. However, NbMBF1c did not interact with TMV components, but silencing NbMBF1c up-regulated the expression of abscisic acid (ABA) related gene NCED3, PYL1, ABAI, and SnRK2E and jasmonic acid (JA) synthesis pathway AOS1, while down-regulated the expression of JA signaling pathway receptor COI1.【Conclusion】NbMBF1c is mainly located in cytoplasm and nucleus, and acts as a positive regulator to inhibit TMV infection in N. benthamiana. NbMBF1c does not inhibit viral infection by directly interacting with TMV components, but by regulating phytohormone production and signal transduction.

Key words: Nicotiana benthamiana, NbMBF1c, tobacco mosaic virus (TMV), gene expression, abscisic acid (ABA), jasmonic acid (JA)

Table 1

Primer sequences used in this study"

用途
Use		 引物名称
Primer name		 引物序列F
Primer sequence F (5′-3′)		 引物序列R
Primer sequence R (5′-3′)
实时荧光定量PCR
qRT-PCR		 qNbMBF1c GACGCCGGTTTGAATAAGAA GGCCGTTCATTGATCTGTTT
qTMV-GFP GACCTGACAAAAATGGAGAAGATCT GAAAGCGGACAGAAACCCGCTG
qNbNCED3 ACCCGACTCGATTTTCAATG AACTTTTGGCCATGGTTCTG
qNbAOS1 CATGAAAGATTTGCCGGTTT ACTTGCAAGCATCAGCAATG
qNbPYL1 CAGGGTTGACACCAGAA TGTGCGAGTAGGGAAGA
qNbSnRK2E TTGCCACCGAGACTTGA CTGCGAATGTAACACTGAGGA
qNbABAI1 GCCGTTGTTTGTTCATCTC ACTGTATCACCTTGCCTCC
qNbCOl 1 CAGCAGCCCATTGTTTCTTAC TACTGGCCAAGTACTTCCAATC
qNbPR1 ATGGTCAATACGGCGAAAAC CCTAGCACATCCAACACGAA
qNbActin CTTGAAACAGCAAAGACCAGC CATCCTATCAGCAATGCCCG
沉默载体构建
Construction of silent vector		 TRV-NbMBF1c GCTCTAGAGCCGGTCCGACCAACT CCGCTCGAGTTCTAACATTAACGACAGCG
瞬时表达载体构建Construction of transient overexpression vectors GFP-NbMBF1c CGGAATTCATGCCGGTCCGACCAACT GCTCTAGATCACTTGTGAATTTTACCTC
Myc-NbMBF1c CGGAATTCATGCCGGTCCGACCAACT GCTCTAGATCACTTGTGAATTTTACCTC
酵母载体构建Construction of yeast-expression vectors
AD-NbMBF1c CCGGAATTCATGCCGGTCCGACCAACT CGCGTCGACTCACTTGTGAATTTTACCTC
BK-NbMBF1c CCGGAATTCATGCCGGTCCGACCAACT CGCGTCGACTCACTTGTGAATTTTACCTC
BK-TMV CP GCGAATTCATGTCTTACAGTATCACTAC CCGCTCGAGTCAAGTTGCAGGACCAGAGG
AD-TMV P50 CATGGAGGCCGAATTGAATTCATGGAGATAGAGTCTTTAGAGCAGTT GCCGCTGCAGGTCGAGTCGACCTATTGTGTTCCTGCA
BK-TMV MP GGAATTCCATATGCAATGACTCATTCTCATGAACCATTATTTTATCAACT CGGGATCCAAACGAATCC

Fig. 1

Amino acid sequence alignment and phylogenetic analysis of NbMBF1c and MBF1c from other plants NbMBF1c and its homologous phylogenetic tree, the numbers on the branches represented the values of Bootstrap test, the short line below indicated the distance scale 0.050"

Fig. 2

Expression analysis of NbMBF1cNbMBF1c expression after TMV infection. The statistical analyses were performed using Student’s t-test (***P<0.001). The experiments were repeated three times with three plants each time. Values represent means±SE from three biological replications"

Fig. 3

Subcellular localization of NbMBF1c and localization change of NbMBF1c after TMV infection eGFP:NbMBF1c with mCherry:00 and mCherry:H2B were transiently co-expressed in the epidermal cells of N. benthamiana leaves by agroinfiltration. mCherry and GFP fluorescence signals were visualized using laser confocal microscope at 48 h after infiltration and were depicted in red and green, respectively. Pearson’s coefficient of localization represents fluorescence coincidence degree, the intensity of GFP fluorescence (green line) and mCherry fluorescence (red line) were presented in the right panels. The white scale bars represented 20 μm; 313Location of eGFP:NbMBF1c after TMV infection"

Fig. 4

Phenotype and silencing efficiency detection in NbMBF1c silenced plant Silencing efficiency detection in TRV:NbMBF1c. The statistical analyses were performed using Student’s t-test (*0.01<P<0.05, ***P<0.001). The experiments were repeated three times with ten plants each time. Values represent means±SE from three biological replications"

Fig. 5

Silencing of NbMBF1c promotes TMV-GFP infection Detection of TMV-GFP content in TRV:NbMBF1c and TRV:00. The statistical analyses were performed using Student’s t-test (**0.001<P<0.01, ***P<0.001). The experiments were repeated three times with ten plants each time. Values represent means±SE from three biological replications"

Fig. 6

Overexpression of NbMBF1c inhibits TMV-GFP infection"

Fig. 7

The interaction of NbMBF1c and TMV virus components was analyzed by Y2H"

Fig. 8

Effect of NbMBF1c silencing on expression of ABA and JA related genes Actin is employed as an internal reference. The statistical analyses were performed using Student’s t-test (*0.01<P<0.05, **0.001<P<0.01, ns means no significant difference). The experiments were repeated three times with ten plants each time. Values represent means±SE from three biological replications"

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