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AGROBACTERIUM-MEDIATED TRANSFORMATION OF VERTICILLIUM DAHLIAE WITH GFP
GENE TO STUDY COTTON-PATHOGEN INTERACTION USING
A NOVEL INOCULATION METHOD
FENG LI1, NOREEN BIBI1,2, KAI FAN1, MI NI1, SHUNA YUAN1, MING WANG1 AND
XUEDE WANG1*
Abstract:
Verticillium dahliae is a soil-born fungal pathogen which causes
Verticillium wilt in economically important crops including cotton. We
conducted a study to monitor the interaction between the fungus and
cotton. V. dahliae was transformed with the gene encoding green
fluorescent protein. The gene can be constitutively expressed and
fluorescence was clearly visible in both hyphae and spores. Due to
heterogeneous gene insertion, the growth rate, colony morphology and
pathogenicity of fungus transformants showed differences compared with
corresponding wild type. Similarly, quantitative real-time PCR analysis
also indicated significant differences in the gene expression among
different V. dahliae transformants. To study cotton-pathogen
interaction, we devised a novel inoculation method and developed a
successful infection by keeping GFP-expressed mycelial plug alongwith
aseptic cotton seedlings. After 6-day inoculation, the LSM microscopic
image showed that the fungus rapidly formed a mycelial network on the
surface of the stems and colonized into plant tissue, displayed an
intercellular infection pattern. The early events during cotton
colonization by V. dahliae can be successfully observed in 10 days
including the plant growth period. Besides, pathological changes of
seedlings like tissue discoloration, wilting, stem dehiscence and
necrosis can be clearly observed without the influences of soil and
other microbes. This inoculation method provides a rapid, effective and
environmental friendly technique for the study of cotton-pathogen
interaction and identification of resistant plant cultivars.
Key words: Agrobacterium, Transformation, Verticillium dahliae, GFP,
Cotton.
1Institute of Crop Science, College of Agriculture and Biotechnology,
Zhejiang University,
Hangzhou 310058, People’s Republic of China
2Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan
*Corresponding author. E-mail: xdwang@zju.edu.cn Tel: (86)-571-88982683
Fax: (86)-571-88982683
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