Post- transcriptional gene silencing (PTGS) of tomato leaf curl virus (ToLCV) in tomato
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Date
2006
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UAS Dharwad
Abstract
Tomato, an economically important crop in many countries is plagued by many viral
diseases including leaf curl caused by Tomato leaf curl virus (ToLCV) belonging to the genus
begomovirus. Begomoviruses are small circular, single stranded DNA plant viruses. Yield
losses usually vary from 28-92% making tomato cultivation unprofitable. Genetically
engineering resistance is a viable alternative is to genetically engineer tomato for protection
against ToLCV. PTGS/RNAi is a novel gene regulatory mechanism that limits the transcript
level by either suppressing transcription or by activating a sequence-specific RNA
degradation process.
We have cloned and characterized ToLCV-coat protein (TCP), replicase (TRP) and
suppressor of PTGS (TRS) genes from a Dharwad local isolate. Constructs have been
developed using all the three genes for the available gene silencing strategies viz., sense (s),
antisense (as), ihp (sas) and HUTR (heterologous 3 -untranslated region). Coat protein (TCP)
gene has been cloned and expressed in a prokaryotic system. As a comparative study plant
expression vectors carrying TCP, TRP and TRS gene following different strategies were used
for transgenic development through Agrobacterium. Analysis of putative To-transgenics
showed positive for PCR, GUS, Dot blot and Southern blot analysis. Semi-quantitative PCR
analysis of plants from TRP constructs showed drastic reduction in the virus inoculum
compared to non-transgenic plants. The T1-generataion transgenic plants obtained from TRP
constructs were positive for PCR and Dot blot analysis. Among different strategies tested for
resistance to ToLCV in transgenics, those with sas/ihp construct showed significant
resistance against ToLCV followed by HUTR, antisense (as) and sense (s). Similarly, among
the three different genes tested, silencing was more in TRS constructs followed by TRP and
TCP.