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http://krishi.icar.gov.in/jspui/handle/123456789/67902
Title: | Transcient expression study to identify RNA silencing suppressor (RSS) in Sugarcane streak mosaic virus |
Other Titles: | Not Available |
Authors: | Bagyalakshmi, K., B. Parameswari, K. Lakshmi, V. G. Malathi and R. Viswanathan |
ICAR Data Use Licennce: | http://krishi.icar.gov.in/PDF/ICAR_Data_Use_Licence.pdf |
Author's Affiliated institute: | ICAR::Sugarcane Breeding Institute |
Published/ Complete Date: | 2015-02-12 |
Project Code: | Not Available |
Keywords: | Viral genes, RNA silencing suppressor (RSS), Sugarcane streak mosaic virus (SCSMV) , |
Publisher: | TNAU, Coimbatore., Indian Society of Mycology and Plant Pathology, Udaipur, Rajasthan. |
Citation: | Not Available |
Series/Report no.: | Not Available; |
Abstract/Description: | Viral genes encode proteins that suppress the RNA silencing defense mechanism of plants. It helps in unveiling a new and promising way to understand the regulatory pathway in the host at the time of viral infection. Potyviral HC-Pro gene was the first proven RNA silencing suppressor (RSS) belonging to Potyviridae family. Sugarcane streak mosaic virus (SCSMV) of the same family earlier referred as an unclassified virus was characterized as a new genus Susmovirus based on its more evolutionary divergence with other genera of Potyviridae. Later, Poacevirus was proposed in place of Susmovirus in which SCSMV along with Triticum mosaic virus were included. Recently we characterized complete genome of SCSMV from India as SCSMV-IND with a size of 9,786 nucleotides excluding the poly (A) tail and encoded a polyprotein of 3,131 amino acid residues. We continued further to identify and characterize RSS part of the viral genome viz. SCSMV P1 and HC-Pro in model plant tobacco through silencing assays. The complete coding regions of both P1 and HC-Pro were first cloned into pTZ57R/T vector and sequenced. The sequences were analysed for the absence of restriction sites within the coding regions suitable for cloning into gene silencing hairpin vector pHANNIBAL specific for dicot. Further new primers for P1 and HC-Pro genes flanking with the restriction sites KpnI and XbaI were designed, PCR amplified and cloned into pTZ57R/T. The recombinant plasmids were then restricted with KpnI and XbaI to release the target gene from the vector. The PDK (pyruvate dehydrogenase kinase) intron from the pHANNIBAL was removed and the vector was linearized followed by ligation with the restricted target gene and cloning into the E.coli DH5α strain. The confirmed gene expression cartridge expressing under the 35S CaMV promoter and OCS terminator was then mobilized into the binary vector pART27 as a NotI fragment. The positive colonies were selected by blue/white screening for β-galactosidase and mobilized into the Agrobacterium strain LBA4404. pING71-GFP binary vector anchored in the Agrobacterium strain C58C1 (DSMZ, Germany). Agro-infiltration of the culture was carried out in the five leaf stage of eight weeks old wild type Nicotiana tabacum. To one side of the leaf blade GFP, P1 and HC-Pro were infiltrated separately whereas on the other side GFP+P1 and GFP+HC-Pro were given. GFP fluorescence was recorded using a portable UV-Lamp by 2-4 dpi. The GFP expression level increased after 2dpi and at the 7dpi the GFP signal was meagre in the GFP, HC-Pro+GFP infiltrated leaves and not in case of GFP+P1 which confirmed that P1 has the potent suppressor activity. Further confirmation on GFP expression was carried out through PCR assays and this confirmed that P1 is the suppressor that allowed the GFP gene to express at high level by overcoming the PTGS of the host tobacco. This study revealed that SCSMV P1 is the potent viral RNA suppressor rather than HC-Pro. Thus, transient expression in the model plant shortens the timeline to further focus on P1 gene than HC-Pro to map the silencing pathways in the highly complicated polyploid sugarcane crop through RNAi approach. |
Description: | Not Available |
ISBN: | 9788192330693 |
Type(s) of content: | Proceedings |
Sponsors: | Not Available |
Language: | English |
Name of Journal: | Not Available |
Volume No.: | Not Available |
Page Number: | 41 |
Name of the Division/Regional Station: | Division of crop protection |
Source, DOI or any other URL: | Not Available |
URI: | http://krishi.icar.gov.in/jspui/handle/123456789/67902 |
Appears in Collections: | CS-SBI-Publication |
Files in This Item:
File | Description | Size | Format | |
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36 th conf bagya 2015.pdf | 4.04 MB | Adobe PDF | View/Open |
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