TITLE Production and evaluation of transgenic tobacco plants expressing the coat protein gene of passionfruit woodiness virus
AUTHOR Shyi-Dong Yeh
Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, Republic of China
Fang-Hua Chu
Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, Republic of China
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ABSTRACT Passionfruit woodiness virus (PWV), a member of the plant potyvirus group, is the major limiting factor for growing passionfruit in Taiwan. The coding sequence of the coat protein (CP) gene of PWV was modified to have an initiation codon and a leader sequence for translation. Analyses by in vitro transcription, in vitro translation, and immunoprecipitation verified that the modified PWV CP gene correctly encoded and expressed a protein of 36 kDa reacting to PWV CP antiserum. The CP gene was further inserted in a Ti binary vector and transferred to tobacco Nicotiana benthamiana by Agrobacterium-mediated transformation. Analyses of the putative transgenic plants by PCR amplification coupled with Southern blotting revealed an expected DNA fragment of 0.7 kb reacting with the probe specific to the PWV CP gene. Western blotting and Northern blotting were also used to further detect the expression of the PWV CP gene. The transgene-derived PWV CP was not detected but the degraded forms of the CP transcript were present. The putative transgenic tobacco lines of w-6, 9, 10, 12, 14, 15, and 16 were subjected to inoculation with different concentrations of PWV prepared from PWV-infected tobacco leaves. All plants of the four lines w-9, 10, 14, and 15 remained symptomless up to six weeks after inoculation. Some plants of two lines (w-6 and 9) showed a delay of 3 to 10 days in symptom development. All plants of line w-12 showed symptoms at the same time as the controls. Inoculated plants of lines w-9, 10, 14, and 15 were further tested by ELISA and bioassays on the local lesion host Chenopodium quinoa and the systemic host N. benthamiana. The negative results indicated that the complete resistance was provided by the apparent inhibition of PWV replication.
KEYWORD Passionfruit woodiness virus; Transgenic tobacco plants; Coat protein gene; Potyvirus;
ARTICLE INFO Botanical Bulletin of Academia Sinica, Volume 37 Number 3 July 1996, page 181-190, 10 pages
PUBLISHER Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China