Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (15): 2919-2928.doi: 10.3864/j.issn.0578-1752.2023.15.007

• PLANT PROTECTION • Previous Articles     Next Articles

Function of Transcription Factor NbERF RAP2-1 in Cucumber Green Mottle Mosaic Virus Infection

YU LianWei1(), JIANG XingLin1, YANG LingLing1, WANG He1, ZHANG YuYang1, XIE LiNa1, XIA ZiHao5, LI HongLian1,3,4, YANG Xue1,2(), SHI Yan1()   

  1. 1 College of Plant Protection, Henan Agricultural University, Zhengzhou 450002
    2 Crop Science Postdoctoral Programme of Henan Agricultural University, Zhengzhou 450002
    3 Collaborative Innovation Centre of Henan Grain Crops, Zhengzhou 450002
    4 State Key Laboratory of Wheat and Maize Crop Science, Zhengzhou 450002
    5 College of Plant Protection, Shenyang Agricultural University, Shenyang 110866
  • Received:2023-05-11 Accepted:2023-06-10 Online:2023-08-01 Published:2023-08-05

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Abstract:

【Background】 Cucumber green mottle mosaic virus (CGMMV) is an important quarantine plant virus in China, which has caused serious economic losses to vegetable and melon industry. The ERF transcription factor is involved in biotic and abiotic stress and the defense response against a variety of plant pathogens. In previous study, it was demonstrated that the host transcription factor NbERF RAP2-1 was significantly down-regulated after CGMMV infection.【Objective】 The objective of this study is to clarify the function of the ERF transcription factor family members in CGMMV infection, and to provide a theoretical basis for disease control caused by CGMMV.【Method】 MEGA7.0 was used to construct a phylogenetic tree to analyze the amino acid sequence of NbERF RAP2-1. The expression vector NbERF RAP2-1-GFP was constructed to observe the subcellular localization of NbERF RAP2-1. The transcript level of NbERF RAP2-1 at different stages of CGMMV infection was analyzed by qRT-PCR. VIGS and transient overexpression of NbERF RAP2-1 were conducted to analyze the function of NbERF RAP2-1 during CGMMV infection.【Result】 Phylogenetic tree analysis showed that NbERF RAP2-1 was highly homologous to ERF transcription factors in tobacco, and was far from ERF transcription factors in Arabidopsis thaliana. The results of subcellular localization showed that NbERF RAP2-1 was localized in the nucleus and acted as a transcription factor. The effect of CGMMV infection on the transcript level of the NbERF RAP2-1 showed that the expression level of NbERF RAP2-1 did not significantly change at the 6, 9, and 12 d after inoculation, but at the 15 and 18 d after inoculation, the expression level of NbERF RAP2-1 was significantly down-regulated. Tobacco rattle virus (TRV) mediated VIGS was used to silence the NbERF RAP2-1, and CGMMV was inoculated in TRV: NbERF RAP2-1 and TRV: 00 plants. At 8 d after inoculation, the leaves of TRV: 00 plants showed mottling and curling, while the TRV: NbERF RAP2-1 plants showed no symptoms. At the same time, the detection results of CGMMV RNA and protein levels showed that TRV: NbERF RAP2-1 could effectively inhibit the accumulation of CGMMV. Similarly, transient overexpression of NbERF RAP2-1 inhibited the accumulation of CGMMV at 24, 48, and 72 h, respectively.【Conclusion】 NbERF RAP2-1 effectively inhibit the initial CGMMV replication, i.e. the viral RNA replication stage. With the invasion of CGMMV, i.e. the intercellular and systemic movements of CGMMV, CGMMV recognizes and inhibits the transcript level of NbERF RAP2-1. When NbERF RAP2-1 is knocked down, that may inhibit the transcription of downstream proteins, thereby inhibiting the accumulation of CGMMV at a later stage. NbERF RAP2-1 plays an important role during the CGMMV infection.

Key words: cucumber green mottle mosaic virus (CGMMV), NbERF RAP2-1, pathogenic mechanism

Table 1

Nucleotide sequences of primers for vector construction"

引物名称Primer name 核苷酸序列Nucleotide sequence (5′-3′)
NbERF RAP2-1 GFP-F GGGGACAAGTTTGTACAGCAGGCTTCATGGAAGGAGGGTCATGTTGTT
NbERF RAP2-1 GFP-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTATAGCTAATTCCTCCCAGGTTTCTGG
NbERF RAP2-1 VIGS-F GGGGACAAGTTTGTACAGCAGGCTTCGTGGGTGGCAGAAATAAGAG
NbERF RAP2-1 VIGS-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTCTTGCGAATATCAGAAGCAGAG

Table 2

Nucleotide sequences of qRT-PCR-related primers"

引物名称
Primer name		 核苷酸序列
Nucleotide sequence (5′-3′)
Nb UBC-RT-F GAGGAAGAGACTGGTGAGGGAT
Nb UBC-RT-R CACAGAGCAAAGACTGGATTGA
NbERF RAP2-1-RT-F ATGGAAGGAGGGTCATGTTG
NbERF RAP2-1-RT-R TTTAGCAGAAGGGCCTCTCA
CGMMV CP-RT-F CCAGACTCAAGCGGGAAGA
CGMMV CP-RT-R TCTACAACCTCAATGACCC

Fig. 1

Phylogenetic analysis of NbERF RAP2-1 based on its amino acid sequence"

Fig. 2

Subcellular localization of NbERF RAP2-1 and GFP"

Fig. 3

Transcription level of NbERF RAP2-1 at different stages of CGMMV infection Different letters on the bars represent significantly different at 0.05 level"

Fig. 4

Effect of NbERF RAP2-1 silencing on CGMMV infection Compared with control group, * means significant difference (P<0.05), ** means extremely significant difference (P<0.01). The same as below"

Fig. 5

Effect of transient overexpression of NbERF RAP2-1 on CGMMV infection"

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