Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae

文献类型： 外文期刊

第一作者： Rehman, Latifur

作者： Rehman, Latifur;Su, Xiaofeng;Guo, Huiming;Qi, Xiliang;Cheng, Hongmei

作者机构：

关键词： Verticillium dahliae;Driselase;Transformation;siRNAs

期刊名称：BMC BIOTECHNOLOGY （ 影响因子：2.563； 五年影响因子：3.292 ）

ISSN： 1472-6750

年卷期： 2016 年 16 卷

页码：

收录情况： SCI

摘要： Background: Large efforts have focused on screening for genes involved in the virulence and pathogenicity of Verticillium dahliae, a destructive fungal pathogen of numerous plant species that is difficult to control once the plant is infected. Although Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used for gene screening, a quick and easy method has been needed to facilitate transformation. Results: High-quality protoplasts, with excellent regeneration efficiency (65 %) in TB3 broth (yeast extract 30 g, casamino acids 30 g and 200g sucrose in 1L H(2)0), were generated using driselase (Sigma D-9515) and transformed with the GFP plasmid or linear GFP cassette using PEG or electroporation. PEG-mediated transformation yielded 600 transformants per microgram DNA for the linear GFP cassette and 250 for the GFP plasmid; electroporation resulted in 29 transformants per microgram DNA for the linear GFP cassette and 24 for the GFP plasmid. To determine whether short interfering RNAs (siRNAs) can be delivered to the protoplasts and used for silencing genes, we targeted the GFP gene of Vd-GFP (V. dahliae GFP strain obtained in this study) by delivering one of four different siRNAs-19-nt duplex with 2-nt 3' overhangs (siRNA-gfp1, siRNA-gfp2, siRNA-gfp3 and siRNA-gfp4)-into the Vd-GFP protoplasts using PEG-mediated transformation. Up to 100 % silencing of GFP was obtained with siRNA-gfp4; the other siRNAs were less effective (up to 10 % silencing). Verticillium transcription activator of adhesion (Vta2) gene of V. dahliae was also silenced with four siRNAs (siRNA-vta1, siRNA-vta2, siRNA-vta3 and siRNA-vta4) independently and together using the same approach; siRNA-vta1 had the highest silencing efficiency as assessed by colony diameter and quantitative real time PCR (qRT-PCR) analysis. Conclusion: Our quick, easy transformation method can be used to investigate the function of genes involved in growth, virulence and pathogenicity of V. dahliae.

分类号：

  • 相关文献

[1]Expression of alfalfa antifungal peptide gene and enhance of resistance to Verticillium dahliae in upland cotton. Zhang, Haiping,Wang, Xuede,Shao, Mingyan,Yuan, Shuna,Ni, Mi,Zhang, Haiping.

[2]Improved Pathogenicity of a Beet Black Scorch Virus Variant by Low Temperature and Co-infection with its Satellite RNA. Xu, Jin,Liu, Deshui,Zhang, Yongliang,Wang, Ying,Han, Chenggui,Li, Dawei,Yu, Jia-Lin,Wang, Xian-Bing,Xu, Jin. 2016

[3]Histological and Ultrastructural Observation Reveals Significant Cellular Differences between Agrobacterium Transformed Embryogenic and Non-embryogenic Calli of Cotton. Shang, Hai-Hong,Liu, Chuan-Liang,Zhang, Chao-Jun,Li, Feng-Lian,Hong, Wei-Dong,Li, Fu-Guang.

[4]Differential expression of induced resistance by an aqueous extract of killed Penicillium chrysogenum against Verticillium wilt of cotton. Dong, HZ,Li, WJ,Zhang, DM,Tang, W. 2003

[5]Comparative analyses of secreted proteins from the phytopathogenic fungus Verticillium dahliae in response to nitrogen starvation. Jun Chu,Wei-Fang Li,Wang Cheng,Mo Lu,Ke-Hai Zhou,He-Qin Zhu,Fu-Guang Li,Cong-Zhao Zhou.

[6]The endochitinase VDECH from Verticillium dahliae inhibits spore germination and activates plant defense responses. Xiao-Xiao Cheng,Li, Zhi-Fang,Zhu, He-Qin,Li-Hong Zhao,Steven J. Klosterman,Hong-Jie Feng,Zi-Li Feng,Feng Wei,Yong-Qiang Shi,Zhi-Fang Li,He-Qin Zhu.

[7]Isolation and functional analysis of the pathogenicity-related gene VdPR3 from Verticillium dahliae on cotton. Zhang, Ya-Lin,Li, Zhi-Fang,Feng, Zi-Li,Feng, Hong-Jie,Zhao, Li-Hong,Shi, Yong-Qiang,Hu, Xiao-Ping,Zhu, He-Qin.

[8]Analysis of sea-island cotton and upland cotton in response to Verticillium dahliae infection by RNA sequencing. Quan Sun,Huaizhong Jiang,Xiaoyan Zhu,Weina Wang,Xiaohong He,Yuzhen Shi,Youlu Yuan,Xiongming Du,Yingfan Cai. 2013

[9]Two Lysin-Motif Receptor Kinases, Gh-LYK1 and Gh-LYK2, Contribute to Resistance against Verticillium wilt in Upland Cotton. Li, Fangfang,Qian, Shasha,Zhou, Xueping,Gu, Zhouhang,Wang, Qian,Ye, Fei,Liu, Tingli,Chen, Tianzi,Yang, Yuwen,Wang, Jinyan,Zhang, Baolong,Ding, Bo,Wang, Guoliang,Zhou, Xueping. 2017

[10]Effect of temperature on conidial germination, mycelial growth and aggressiveness of the defoliating and nondefoliating pathotypes of Verticillium dahliae from cotton in China. Xu, Fei,Yang, Long,Zhang, Jing,Li, Guoqing,Xu, Fei,Yang, Long,Zhang, Jing,Li, Guoqing,Xu, Fei,Guo, Xiaoping,Zhang, Xianlong. 2012

[11]Mutational analysis of the Verticillium dahliae protein elicitor PevD1 identifies distinctive regions responsible for hypersensitive response and systemic acquired resistance in tobacco. Liu, Zhipeng,Liu, Wenxian,Zeng, Hongmei,Yang, Xiufen,Guo, Lihua,Qiu, Dewen. 2014

[12]Characterization of the Verticillium dahliae Exoproteome Involves in Pathogenicity from Cotton-Containing Medium. Chen, Jie-Yin,Xiao, Hong-Li,Gui, Yue-Jing,Zhang, Dan-Dan,Li, Lei,Bao, Yu-Ming,Dai, Xiao-Feng. 2016

[13]Comparative expression analysis in susceptible and resistant Gossypium hirsutum responding to Verticillium dahliae infection by cDNA-AFLP. Wang, Sheng-Zheng,Liu, Kai,Si, Ning,Qi, Fang-Jun,Jian, Gui-Liang.

[14]Cotton gene expression profiles in resistant Gossypium hirsutum cv. Zhongzhimian KV1 responding to Verticillium dahliae strain V991 infection. Jian, Gui-Liang,Jiang, Teng-Fei,Wang, Sheng-Zheng,Qi, Fang-Jun,Xu, Shi-Chang.

[15]Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection. Yang, Liu,Mu, Xiaoying,Liu, Chao,Cai, Jinghui,Shi, Ke,Zhu, Wenjiao,Yang, Qing,Yang, Liu. 2015

[16]The genes involved in the protective effects of phytohormones in response to Verticillium dahliae infection in Gossypium hirsutum. Zhang, Wenwei,Jian, Guiliang,Qi, Fangjun,Si, Ning.

[17]Generation of transcriptome profiling and gene functional analysis in Gossypium hirsutum upon Verticillium dahliae infection. Zhang, Huachong,Qi, Fangjun,Jian, Guiliang.

[18]Production of methyl sulfide and dimethyl disulfide from soil-incorporated plant materials and implications for controlling soilborne pathogens. Tharayil, N.,Gerik, J.,Rosen, C.,Kinkel, L.,Cao, A..

[19]Differential expression analysis by cDNA-AFLP of Solanum torvum upon Verticillium dahliae infection. Wang, Z.,Zhang, F.,Yang, Q.,Wang, Z.,Guo, J. L.,Huang, Q. S.,Huang, L. P.. 2010

[20]Characterization of Two Fungal Isolates from Cotton and Evaluation of their Potential for Biocontrol of Verticillium Wilt of Cotton. He-Qin Zhu,Zi-Li Feng,Zhi-Fang Li,Yong-Qiang Shi,Li-Hong Zhao,Jia-Rong Yang. 2013

作者其他论文 更多>>

微信小程序