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.

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.

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.

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.

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.

Cucumber green mottle mosaic virus (CGMMV) is an important quarantine plant virus in China, which has seriously reduced the production of vegetables and melons worldwide. The MYB protein family is large, multifunctional and present in all eukaryotes. Most MYB proteins act as transcription factors that control development, and metabolism in plants, and regulate biotic and abiotic stress responses. Previous studies have shown that during CGMMV infection, the expression of transcription factor gene NbMYB1R1 was significantly up-regulated.

The objective of this study is to clarify the mechanism of NbMYB1R1 involved in CGMMV infection, and to provide a theoretical basis for controlling CGMMV infection.

MEGA 7.0 was used to construct a phylogenetic tree to analyze the amino acid sequence of NbMYB1R1 protein. The expression vector NbMYB1R1-GFP was constructed, transformed into Agrobacterium GV3101 and infiltrated the tobacco leaves to observe the subcellular localization of NbMYB1R1 by confocal microscopy. The transcriptional levels of NbMYB1R1 at different stages of CGMMV infection and ROS related genes in silenced NbMYB1R1 plants were analyzed using qRT-PCR technology. TRV-mediated gene silencing (VIGS) was utilized to analyze the function of NbMYB1R1, NbAOX1a, and NbAOX1b during CGMMV infection. Transient overexpression of NbMYB1R1 and NbMYB1R1 mutant was conducted to analyze the effect of NbMYB1R1 on CGMMV infection. Trypan blue staining and DAB staining were used to observe whether the cell death caused by the transient overexpression of NbMYB1R1 was related to the programmed cell death (PCD) and the accumulation of ROS. Yeast two-hybrid system was used to verify whether NbMYB1R1 interacted with CGMMV related proteins.

Phylogenetic tree analysis showed that NbMYB1R1 belonged to the 1R MYB category and had high homology with MYB transcription factors in a variety of tobaccos. The results of subcellular localization showed that NbMYB1R1 was localized in the nucleus. In CGMMV-infected tobacco plants, the transcript level of NbMYB1R1 was significantly changed compared with healthy plants, which was significantly up-regulated at 8 and 12 d of CGMMV infection. CGMMV was inoculated into TRV:NbMYB1R1 and TRV:00 plants. The NbMYB1R1-silenced plants showed the mottled and curled symptoms at 3 dpi, while the control plant appeared the symptoms at 3.5 dpi. Silencing NbMYB1R1 could effectively promote the accumulation of CGMMV at mRNA and protein levels. The CGMMV accumulation in NbMYB1R1 and NbMYB1R1 mutant transiently overexpression leaves was detected, and the results showed that NbMYB1R1 overexpression could effectively inhibit CGMMV infection and DNA binding domain deletion mutant reduced the inhibition of CGMMV by NbMYB1R1. The results of trypan blue and DAB staining showed that NbMYB1R1 overexpression could induce the ROS accumulation and cell death. The transcription level of ROS-related genes in TRV:NbMYB1R1 and TRV:00 plant was also detected, and the results showed that silencing NbMYB1R1 could specifically up-regulate the transcription of AOX1a and AOX1b. After CGMMV inoculation on silencing endogenous genes NbAOX1a and NbAOX1b, the systemic leaves of silenced NbAOX1a and NbAOX1b plants showed mottled and curled symptoms at 4 dpi, while the control plants appeared symptoms at 3.5 d. Meanwhile, the results of CGMMV CP mRNA and protein levels also indicated that silencing NbAOX1a and NbAOX1b could effectively inhibit the accumulation of CGMMV. The yeast two-hybrid results showed that NbMYB1R1 did not directly interact with the CGMMV proteins.

During the CGMMV infection, the defense-related gene NbMYB1R1 is up-regulated. It speculates that the upregulation of NbMYB1R1 inhibits the transcription of the downstream genes AOX1a and AOX1b and activates the production of ROS in cells, thereby inhibiting viral infection. However, NbMYB1R1 does not produce this effect through a direct interaction with the CGMMV viral protein. NbMYB1R1 plays an important role in CGMMV infection.