Genetic manipulation in the genome of Soybean yellow mottle mosaic virus (SYMMV) and infectivity analysis for exploring its potentiality as a transient gene delivery system
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Date
2016
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Division of Plant Pathology Indian Agricultural Research Institute New Delhi –1
Abstract
Genetic engineering with the genome of plant viruses resulted in the development of
vectors that could be utilized for expression of foreign proteins, silencing of host genes and
delivery of other genetic constructs into plants. Virus-derived transient vector system also
provided an alternative to transgenic approach. Though many viruses have been utilized
for such purposes in many crops, relatively less attention was given to pulse crops
particularly those grown in tropical or sub-tropical climates. In the present investigation,
an infectious clone of a pulse infecting carmovirus, Soybean yellow mottle mosaic virus
was manipulated using inverse PCR and overlapping extension PCR methods and two
different mutant amplicons were generated. Among these mutants, the coat protein deleted
mutant was not stable after cloning. However, the other mutant, where a multiple cloning
site (MCS) with four restriction sites (Kpn2I, Bst1107I, KspAI, and RruI) was inserted
successfully at the beginning of the coat protein ORF of SYMMV genome, was found to be
stable in E. coli. This MCS-added full-length mutant successfully reproduced the disease
symptoms upon agroinoculation. However, compared to the wild type, the mutant
produced milder symptoms and took longer time for expression of symptoms in the
different legume species (black gram, mung bean, guar bean, French bean, and soybean).
The mutant could produce virion after inoculation and could move systemically as
evidenced by electron microscopy, ELISA, and RT-PCR. The real-time estimation of
transcript abundance showed that the replication of this mutant is markedly reduced in the
legume host species. The approach for inserting an MCS into the infectious construct has
been standardized and the same strategy will be helpful for introducing more manipulation
in the SYMMV genome in future to enhance the efficacy of the construct so that it could be
used as a vector for gene delivery in the plant
Description
T-9529
Keywords
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