A Dual Coat Protein Construct Establishes Resistance to Passionfruit Woodiness and Cucumber Mosaic Viruses

Authors
N. Sokhandan- Bashir 1
M. Gillings 2
J. Bowyer 3
1 Department of Plant Protection, College of Agriculture, University of Tabriz, Tabriz, Islamic Republic of Iran.
2 Department of Biological Sciences, Macquarie University, Sydney, Australia.
3 Department of Crop Sciences, The University of Sydney, New South Wales 2006 Australia.
Abstract
There is a high degree (>95%) of intraspecies similarity in the coat protein (CP) amino acid sequences within Passionfruit woodiness virus (PWV) and Cucumber mosaic virus (CMV), both infecting passionfruit vine in New South Wales. On this basis, a dual transgene containing the translatable cDNAs coding for the CPs of PWV and CMV was constructed in the binary vector pBI121 and used for transformation of Nicotiana benthamiana, a susceptible host to both viruses. The transformation was achieved by co-cultivation of the agrobacteria with the leaf disks prepared from the surface- sterilized leaves. Five transgenic lines including 1-1, 1-5, 1-7, 1-12 and 1-24 were regenerated. Insertion and transcription of the dual construct were confirmed, however, only the CMV CP was feasibly detectable by DAS-ELISA in the lines. Low level accumulation of CMV and/or PWV was evident in the lines. In the initail challenge trial where 1:10 dilution of plant sap was used, a 5-day delay in symptom was generally shown. Inoculations with 1:100 plant sap also gave similar results as with 1:10 dilution. Lines 1-5 and 1-12, which were inoculated with 1:1000 dilution of sap, remained uninfected by CMV till 27 dpi, whereas with PWV, 1-12 became infected by 11 dpi. Four cuttings of line 1-12 reacted diffferently to the challenge inoculations i.e. three of them resisted PWV, whereas two of them were susceptible to CMV. Since PWV CP was not detectable in the transgenic lines but evidence of resistance to PWV was found in them, this was suggestive of an RNA silencing mechnaism involved in the resistance. Because the CMV CP was detectable in the transgenic lines, this suggested requirement for the CP expression in the resistance . The resistance, or apparent immunity, was manifested by an apparent delay in symptom expression and accumulation of relatively low levels of the viruses.

Keywords

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Journal of Agricultural Science and Technology
Volume 14, Issue 5
September and October 2012
Pages 1105-1120