The study of NbEILP from Nicotiana benthamiana involved in the cell-to-cell movement of Bamboo mosaic virus

• Main Authors: Chung-Han Tsai, 蔡崇瀚
• Other Authors: 蔡慶修
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/65337367438434178777

碩士 === 國立中興大學 === 生物科技學研究所 === 104 === To study the interaction between the viral pathogens and their hosts, by cDNA-AFLP technique, we screened the differentially expressed genes from the Nicotiana benthamiana plants after inoculated with Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. Among those differentially expressed genes, one of the upregulated genes ACCT8-1 is an ortholog of N. tabacum elicitor inducible leucine-rich repeats (LRR) protein gene (EILP) and Cladosporium fulvum resistance genes cf-2 and cf-5. In the previous study, by Tobacco rattle virus (TRV)-based silencing system, we knocked down the expression of ACCT8-1 in plants and resulting in reduced BaMV coat protein accumulation and lesion size on the inoculated leaves. Furthermore, the accumulation of BaMV coat protein in the ACCT8-1-knockdown protoplasts showed no significant difference to that in the control protoplasts. The results suggest that ACCT8-1 might be involved in the movement step of the BaMV infection cycle. Since the similarity of this gene is very similar to the EILP of N. tabacum, thus we designated this gene as NbEILP. To further reveal how the NbEILP is involved in the movement of BaMV, we fused the green fluorescent protein (GFP) at the C-terminus of the NbEILP. Unfortunately, because of the large gene size about 3.2 kb in length, the expression of this gene in plants was shown very difficult to detect. We then construct a deletion mutant removing the leucine-rich repeats domain (ΔLRR). The accumulation of BaMV coat protein is enhanced in the NbEILP/ΔLRR-GFP transient expressed leaves. The results indicate that the leucine-rich repeats domain is not essential in assisting BaMV movement. We then based on this construct NbEILP/ΔLRR-GFP to create a few deletion mutants such as NbEILP/ΔLRRΔTMD (without transmembrane domain), NbEILP/ΔLRRΔCD (without cytoplasmic domain), and NbEILP/ΔLRRΔSP (without signal peptide) to examine if these domains are involved in BaMV movement. The results of transient expression indicated that the two mutants NbEILP/ΔLRRΔTMD and NbEILP/ΔLRRΔSP could not enhance the accumulation of BaMV as that of NbEILP/ΔLRR. These results also suggest that NbEILP requires the signal peptide to target the right location and the transmembrane domain to associate the membrane in assisting the cell-to-cell movement of BaMV.