Agrobacterium-mediated transformation of Verticillium dahliae with GFP gene to study cotton-pathogen interaction using a novel inoculation method. | [Pakistan Journal of Botany • 2016]

Author(s):

1. Feng Li: Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, People’s Republic of China

2. Noreen Bibi: Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, People’s Republic of China; Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan

3. Kai Fan: Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, People’s Republic of China

4. M. I. Ni: Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, People’s Republic of China

5. Shuna Yuan: Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, People’s Republic of China

6. Ming Wang: Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, People’s Republic of China

7. Xuede Wang: Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, People’s Republic of China

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 cottonpathogen interaction and identification of resistant plant cultivars.

Page(s): 1219-1227

DOI: DOI not available

Published: Journal: Pakistan Journal of Botany, Volume: 48, Issue: 3, Year: 2016

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