DEVELOPMENT OF INFECTIOUS CLONE AND DESIGNING OF GENE EXPRESSION VECTOR BASED ON LEGUME INFECTING CARMOVIRUS IN INDIA
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Date
2016
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DIVISION OF PLANT PATHOLOGY ICAR- INDIAN AGRICULTURAL RESEARCH INSTITUTE NEW DELHI
Abstract
Soybean yellow mottle mosaic virus (SYMMV, genus Carmovirus) was previously
known to occur in South Korea and USA causing bright yellow mosaic in soybean.
However, so far occurrence of SYMMV is not known in India. In this study, a
carmovirus species obtained from mungbean (Vigna radiata L.) exhibiting systemic
mild mottling and puckering symptoms in the experimental field at Indian
Agricultural Research Institute, was characterized as a distinct strain of SYMMV-Mb
based on complete genome sequence, host reactions and serology. Further, an
infectious clone of SYMMV-Mb was used for designing a vector for expression of
heterologous protein in plants. The complete genome sequence of Indian isolate of
SYMMV-Mb contained 3974 nt (KP259608) which was 35 nt shorter than the
isolates of SYMMV reported from Korea and USA. In overall genome sequence
SYMMV-Mb shared 75% identity with the other SYMMV isolates and 63% with
cowpea mottle virus followed by 36-43% with other carmovirus species. The
phylogenetic analysis based on complete genome showed that SYMMV-Mb
belonged to the cluster formed by legume infecting carmoviruses. The virus isolate,
SYMMV-Mb induced veinal mottling, mild mottling, chlorotic blotching, local and
systemic necrosis in soybean, mungbean, blackgram, French bean and guar bean,
respectively. The symptomatology of the present isolate was distinct from the
previously reported South Korean isolate, as the later did not induce symptoms in
any of the above legumes other than soybean. In order to know the serological
relationships, the CP gene of the present isolate was over expressed as a 39 kDa
protein in E. coli and an antiserum of 1:16,000 titer against the recombinant CP was
produced. Serological cross reactivity analysis revealed that SYMMV-Mb was
serologically related to blackgram mottle virus but not to cowpea mottle virus, the
other legume infecting carmoviruses. A full-length cDNA of SYMMV-Mb was
constructed under the control of T7 RNA polymerase promoter as well as under
CaMV 35S promoter. The direct inoculation of pT7SYMMV RNA transcript
generated from pUC18 clone by rub inoculation did not result into any infection but
in vitro transcript from direct PCR product resulted in chlorotic blotches and
systemic mottling symptoms. However, agro-inoculation of SYMMV-Mb with
CaMV 35S promoter and OCS terminator resulted in 100% infection in the different
legume host species. The Progeny virions recovered from agro-infected symptomatic
leaves had the same morphological properties as the parental virions and showed
similar symptoms upon mechanical sap inoculation to healthy plants. The SYMMVMb
genome was further utilized to develop vector for transient gene expression in
plants based on full virus vector strategy. To examine the ability of the vector
system, GFP gene was cloned in the MCS region of SYMMV-Mb. When in vitro
runoff transcript was mechanically inoculated to French bean leaves, good level of
GFP expression was detected through confocal microscopy. However, further
investigation is necessary to improve the efficacy of the SYMMV-Mb based vector
for gene expression in plants. The system developed here will be useful for further
studies of SYMMV-Mb gene functions and exploitation of SYMMV-Mb as gene
expression and silencing vector. The present study provided evidence of occurrence
of a distinct strain of SYMMV-Mb in India. This also constitutes the study on
biological and molecular characterization of a legume infecting carmovirus in India
and its utilization as a foreign gene expression vector in legume plant.
Description
t-9302
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