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Virus-induced gene silencing (VIGS) is a useful tool for functional characterization of genes in plants. Unfortunately, the efficiency of infection by Tobacco rattle virus (TRV) is relatively low for some non-Solanaceae plants, which are economically important, such as rose (Rosa sp.). Here, to generate an easy traceable TRV vector, a green fluorescent protein (GFP) gene was tagged to the 3' terminus of the coat protein gene in the original TRV2 vector, and the silencing efficiency of the modified TRV-GFP vector was tested in several plants, including Nicotiana benthamiana, Arabidopsis thaliana, rose, strawberry (Fragaria ananassa), and chrysanthemum (Dendranthema grandiflorum). The results showed that the efficiency of infection by TRV-GFP was equal to that of the original TRV vector in each tested plant. Spread of the modified TRV virus was easy to monitor by using fluorescent microscopy and a hand-held UV lamp. When TRV-GFP was used to silence the endogenous phytoene desaturase (PDS) gene in rose cuttings and seedlings, the typical photobleached phenotype was observed in 75-80% plants which were identified as GFP positive by UV lamp. In addition, the abundance of GFP protein, which represented the concentration of TRV virus, was proved to correlate negatively with the level of the PDS gene, suggesting that GFP could be used as an indicator of the degree of silencing of a target gene. Taken together, this work provides a visualizable and efficient tool to predict positive gene silencing plants, which is valuable for research into gene function in plants, especially for non-Solanaceae plants.

Ethylene plays an important role in organ growth. In Arabidopsis, ethylene can inhibit root elongation by stabilizing DELLA proteins. In previous work, it was found that ethylene suppressed cell expansion in rose petals, and five unisequences of DELLA genes are induced by ethylene. However, the mechanism of transcriptional regulation of DELLA genes by ethylene is still not clear. The results showed that the expression of RhGAI1 was induced in both ethylene-treated and ETR gene-silenced rose petals, and the promoter activity of RhGAI1 was strongly induced by RhEIN3-3 in Arabidopsis protoplasts. What is more, RhEIN3-3 could bind to the promoter of RhGAI1 directly in an electrophoretic mobility shift assay (EMSA). Cell expansion was suppressed in RhGAI1-Δ17-overexpressed Arabidopsis petals and promoted in RhGAI1-silenced rose petals. Moreover, in RhGAI1-silenced petals, the expression of nine cell expansion-related genes was clearly changed, and RhGAI1 can bind to the promoter of RhCesA2 in an EMSA. These results suggested that RhGAI1 was regulated by ethylene at the transcriptional level, and RhGAI1 was a direct target of RhEIN3-3. Also, RhGAI1 was shown to be involved in cell expansion partially through regulating the expression of cell expansion-related genes. Furthermore, RhCesA2 was a direct target of RhGAI1. This work uncovers the transcriptional regulation of RhGAI1 by ethylene and provides a better understanding of how ethylene regulates petal expansion in roses.