Investigation of Bamboo Mosaic Virus Satellite RNA(satBaMV)-Encoded p20 in the Movement of satBaMV in p20 Transgenic Tobacco

The satellite RNA of Bamboo mosaic virus (BaMV) contains an open reading frame for a 20-kDa nonstructural protein, which facilitates the long distance movement of satBaMV in plants. To determine whether the frame shift mutant of satBaMV (F6) could move systemically or could be complemented or promoted by P20, sucrose-synthasepromoter-driven P20 (SSP20) transgenic Nicotiana benthamiana plants have been developed via Agrobacteriummediated transformation. Homozygous transgenic plants have been obtained through two generations of selection on MS medium supplemented with 20 mg/L of hygromycin. The integrated transgene in transgenic plants was confirmed by PCR, but not by Southern blot, which maybe due to too low level of integration of transgene in host chromosome. RT-PCR results have shown that the transgene could be successfully expressed at transcriptional level. Positive results have been obtained in tissue printing of stem section of F2 transgenic plants. No signal could be detected from RNA blot of tissue printing and phloem sap. There are more branches in F2 transgenic plants than in WT. Both dot blot and Northern blot showed that BSF4 and its frame shift mutant BSF6 could move systemically in both WT and SSP20 transgenic N. benthamiana line SP3-4 and SP4-4, which indicates that P20 is not essential for the of replication and movement BaMV and satBaMV. P20 is helpful for satBaMV replication and movement because the stronger signal of BSF4 was detected in systemic leaves than that in BSF6 and BSGFP both in WT and transgenic plants by dot blot and Northern blot assay. No much difference between WT and transgenic plants in the expression of BSF4 and BSF6 both in inoculated and systemic leaves. The expression level of BSGFP in transgenic plants was slightly higher than that in WT. RT-PCR and subsequent sequencing data revealed the correct sequence of inoculated BSF4, BSF6, and BSGFP, respectively.