Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (10): 1885-1891.doi: 10.3864/j.issn.0578-1752.2016.10.005

• PLANT PROTECTION • Previous Articles     Next Articles

Development of the Agrobacterium tumefaciens Mediated Transformation System of Alternaria alternata Apple Pathotype

GU Xue-ying1, SHI Wen-xiao2, WANG Hong-kai 2, GUO Qing-yuan1   

  • Received:2015-12-29 Online:2016-05-16 Published:2016-05-16

RichHTML

20

PDF

642

Abstract: 【Objective】 Alternaria alternata apple pathotype is one of the important pathogens on apple which can infect leaves and fruits seriously. The objective of this study is to establish the Agrobacterium tumefaciens mediated transformation system of Al. alternata apple pathotype, provide a technical basis for study molecular mechanisms of pathogenesis. 【Method】 Vector pKO1-HPH is a shuttle plasmid which is constructed on the backbone of pCAMBIA1300 containing hygromycin phosphotransferase gene (hph) and GFP gene. Fungal transformation was carried out by co-incubation of Ag. tumefaciens strain AGL1 containing plasmid pKO1-HPH with conidia of Al. alternata apple pathotype. Transformants were screened at PDA plates containing hygromycin B. Hph gene expression was detected by incubation of transformants on PDA plates containing 50 μg·mL-1 hygromycin B, GFP was detected using Green fluorescence microscope. Influences of fungal cultural time and pretreatment method of conidia on transformation were evaluated. The stability of transformants was determined by growing on PDA plates containing hygromycin B after five-generation successive growth. PCR assay was used to confirm the existence of the hph gene and GFP gene in transformants. The copy number of inserted T-DNA in transformants was determined by Southern blot. Pathogenicity test of some transformants was performed on apple fruits and leaves. 【Result】 Spores of Al. alternata apple pathotype were formed enough when 100 μL of spore suspension (about 105 spores) was spread on PCA plate and incubated for 36-48 h. The maximum of transformation efficiency was up to 200 transformants/107 conidia when pretreatment of conidia at 4℃ for 6 h before transformation. The resistance to hygromycin B in transformants was stable after five successive generations. Results of specific PCR showed the exogenous genes (GFP and hygromycin B) were integrated into genome of Al. alternata apple pathotype strain and single copy insertion was detected in genome of majority of the transformants according to southern blot analysis. Green fluorescence was detected in the mycelia and spores under the fluorescent microscopy, indicating GFP was expressed in spores and hyphae of transformants. Results of pathogenicity test on fruits and leaves showed that the pathogenicity of several transformants was reduced.【Conclusion】Ag. tumefaciens mediated transformation system of Al. alternata apple pathotype is developed in this research and useful for further studies on molecular interaction of apple and Al. alternata apple pathotype.

Key words: Alternaria alternate, apple pathotype, ATMT, pathogenicity

[1]    Li Y, Aldwinckle H S, Sutton T, Tsuge T, Kang G D, Cong P H, Cheng Z M. Interactions of apple and the Alternaria alternata apple pathotype. Critical Reviews in Plant Sciences, 2013, 32(3): 141-150.
[2]    Jung K H. Growth inhibition effect of pyroligneous acid on pathogenic fungus, Alternaria mali, the agent of Alternaria blotch of apple. Biotechnology and Bioprocess Engineering, 2007, 12: 318-322.
[3]   Saha D, Fetzner R, Burkhardt B, Podlech J, Metzler M, Dang H, Lawrence C, Fischer R. Identification of a polyketide synthase required for alternariol (AOH) and alternariol-9- methyl ether (AME) formation in Alternaria alternata. PLoS One, 2012, 7(7): e40564.
[4]    Pavón Moreno M Á, González Alonso I, Martín de Santos R, García Lacarra T. The importance of genus Alternaria in mycotoxins production and human diseases. Nutricion Hospitalaria, 2012, 27(6): 1772-1781.
[5]    孙霞. 链格孢属真菌现代分类方法研究[D]. 泰安: 山东农业大学, 2006.
Sun X. The methodological study on taxonomy of the genus Alternaria Nees[D]. Taian: Shandong Agricultural University, 2006. (in Chinese)
[6]    Tanaka A, Shiotani H, Yamamoto M, Tsuge T. Insertional mutagenesis and cloning of the genes required for biosynthesis of the host-specific AK-toxin in the Japanese pear pathotype of Alternaria alternata. Molecular Plant-Microbe Interactions, 1999, 12(8): 691-702.
[7]    Markham J E, Hille J. Host-selective toxins as agents of cell death in plant-fungus interactions. Molecular Plant Pathology, 2001, 2(4): 229-239.
[8]    Tsuge T, Harimoto Y, Akimitsu K, Ohtani K, Kodama M, Akagi Y, Egusa M, Yamamoto M, Otani H. Host-selective toxins produced by the plant pathogenic fungus Alternaria alternata. FEMS Microbiology Review, 2013, 37(1): 44-66.
[9]    Shtienberg D. Effects of host physiology on the development of core rot, caused by Alternaria alternata, in red delicious apples. Phytopathology, 2012, 102(8): 769-778.
[10]   王金友, 朱虹, 李美娜, 方永玮, 吴明勤, 林晃, 张互助, 陆敏. 苹果斑点落叶病侵染及发生规律研究. 中国果树, 1992(2): 11-14, 28.
Wang J Y, Zhu H, Li M N, Fang Y W, Wu M Q, Lin H, Zhang H Z, Lu M. Research on the occurrence and infection of Alternaria mali on apple[J]. China Fruits, 1992(2): 11-14, 28. (in Chinese)
[11]   胡同乐, 王树桐, 宋萍, 张凤巧, 甄文超, 曹克强. 苹果斑点落叶病菌大量侵染的决定性天气条件初探. 河北农业大学学报, 2006, 29(1): 63-66.
Hu T L, Wang S T, Song P, Zhang F Q, Zhen W C, Cao K Q. Primary study on the crucial weather conditions of mass infection of Alternaria mali on apple. Journal of Agricultural University of Hebei, 2006, 29(1): 63-66. (in Chinese)
[12]   Thomma B P. Alternaria spp.: from general saprophyte to specific parasite. Molecular Plant Pathology, 2003, 4(4): 225-236.
[13]   Johnson R D, Johnson L, Kohmoto K, Otani H, Lane C R, Kodama M. A polymerase chain reaction-based method to specifically detect Alternaria alternata apple pathotype (A. mali), the causal agent of Alternaria blotch of apple. Phytopathology, 2000, 90(9): 973-976.
[14]   Miyashita M, Nakamori T, Miyagawa H, Akamatsu M, Ueno T. Inhibitory activity of analogs of AM-toxin, a host-specific phytotoxin from the Alternaria alternata apple pathotype, on photosynthetic O2 evolution in apple leaves. Bioscience, Biotechnology, Biochemistry, 2003, 67(3): 635-638.
[15]   Harimoto Y, Tanaka T, Kodama M, Yamamoto M, Otani H, Tsuge T. Multiple copies of AMT2 are prerequisite for the apple pathotype of Alternaria alternata to produce enough AM-toxin for expressing pathogenicity. Journal of General Plant Pathology, 2008, 74: 222-229.
[16]   Harimoto Y, Hatta R, Kodama M, Yamamoto M, Otani H, Tsuge T. Expression profiles of genes encoded by the supernumerary chromosome controlling AM-toxin biosynthesis and pathogenicity in the apple pathotype of Alternaria alternata. Molecular Plant-Microbe Interactions, 2007, 20(12): 1463-1476.
[17]   Ito K, Tanaka T, Hatta R, Yamamoto M, Akimitsu K, Tsuge T. Dissection of the host range of the fungal plant pathogen Alternaria alternata by modification of secondary metabolism. Molecular Microbiology, 2004, 52(2): 399-411.
[18]   李彩红, 李志芳, 冯自力, 赵丽红, 师勇强, 王玲飞, 刘义杰, 朱荷琴. 农杆菌介导的基因敲除技术在丝状真菌基因功能研究中的应用. 棉花学报, 2013, 25(3): 262-268.
Li C H, Li Z F, Feng Z L, Zhao L H, Shi Y Q, Wang L F, Liu Y J, Zhu H Q. Applications of gene knockout by ATMT in filamentous fungus functional genomics. Cotton Science, 2013, 25(3): 262-268. (in Chinese)
[19]   周芳芳, 李志芳, 冯自力, 师勇强, 赵丽红, 李彩红, 朱荷琴. 根癌农杆菌介导的真菌遗传转化研究进展. 棉花学报, 2012, 24(5): 461-467.
Zhou F F, Li Z F, Feng Z L, Shi Y Q, Zhao L H, Li C H, Zhu H Q. Progress on transformation in fungi mediated by Agrobacterium tumefaciens. Cotton Science, 2012, 24(5): 461-467. (in Chinese)
[20]   Chen L, Sun G W, Wu S J, Liu H X, Wang H K. Efficient transformation and expression of gfp gene in Valsa mali var. mali. World Journal of Microbiology and Biotechnology, 2015, 31: 227-235.
[21]   孔慧敏. 农杆菌介导的稻瘟病菌转化及致病突变体的筛选[D]. 杭州: 浙江大学, 2008.
Kong H M. Agrobacterium tumefaciens-mediated transformation of Magnaporthe grisea[D]. Hangzhou: Zhejiang University, 2008. (in Chinese)
[22]   冯晓晓. 稻瘟病菌致病相关基因MgS11、MgATG5和MgATG8的克隆与功能分析[D]. 杭州: 浙江大学, 2006.
Feng X X. Cloning and functional analysis of MgS11, MgATG5 and MgATG8 gene in Magnaporthe grisea[D]. Hangzhou: Zhejiang University, 2006. (in Chinese)
[23]   Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual. Beijing: Science Press, 2002: 26-27.
[24]   Bundock P, Den Dulk-Ras A, Beijersbergen A, Hooykaas P J. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. The EMBO Journal, 1995, 14(13): 3206-3214.
[25]   黄卫, 汪天虹, 吴志红, 吴静, 李滢冰. 丝状真菌遗传转化系统研究进展. 微生物学杂志, 2000, 20(3): 40-44.
Huang W, Wang T H, Wu Z H, Wu J, Li Y B. Advance in genetic transformation system of filamentous fungi. Journal of Microbiology, 2000, 20(3): 40-44. (in Chinese)
[26]   袁亮, 李玉蓉, 张希福, 郭威, 车亦舟, 陈功友. 热不对称PCR (TAIL-PCR)和Tn5转座子质粒拯救法快速分离水稻条斑病菌致病相关基因. 农业生物技术学报, 2009, 17(6): 1089-1095.
Yuan L, Li Y R, Zhang X F, Guo W, Che Y Z, Chen G Y. Quick identification of pathogenicity-related genes in Xanthomonas oryzae pv. oryzicola by thermal asymmetric interlaced PCR (TAIL-PCR) and Tn5 transposon rescue. Journal of Agricultural Biotechnology, 2009, 17(6): 1089-1095. (in Chinese)
[27]   Wang Z, Ye S, Li J, Zheng B, Bao M, Ning G. Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning. BMC Biotechnology, 2011, 11: 109.
[28]   徐后娟, 徐荣燕, 李多川. 链格孢ATMT转化体系的构建及弱毒突变株验证. 基因组学与应用生物学, 2009, 28(6): 1056-1062.
Xu H J, Xu R Y, Li D C. Agrobacterium tumefaciens-mediated transformation of Alternaria alternata and identification of hypovirulent mutant. Genomics and Applied Biology, 2009, 28(6): 1056-1062. (in Chinese)
[29]   张彩霞, 陈莹, 李壮, 康国栋, 张利义, 周宗山, 丛佩华. 苹果斑点落叶病致病菌的鉴定及生物学特性研究. 生物技术, 2011, 21(4): 58-61.
Zhang C X, Chen Y, Li Z, Kang G D, Zhang L Y, Zhou Z S, Cong P H. Identification and biological characteristics of a pathogenic strain causing Alternaria blotch of apples. Biotechnology, 2011, 21(4): 58-61. (in Chinese)
[1] DONG Yu, WU Qian, FENG Xuan, ZHENG YinYing, CUI BaiMing. A Novel Plasmid pEA60 of Erwinia amylovora Enhances the Pathogenicity of Strains by Regulating the Synthesis of Virulence Factors [J]. Scientia Agricultura Sinica, 2026, 59(5): 996-1007.
[2] CONG QiQi, ZHANG JingYi, MENG XiangLong, DAI PengBo, LI Bo, HU TongLe, WANG ShuTong, CAO KeQiang, WANG YaNan. Identification of Hypovirus in Apple Ring Rot Fungus Botryosphaeria dothidea and Detection of Virus-Carrying Status in China [J]. Scientia Agricultura Sinica, 2025, 58(3): 478-492.
[3] TONG ZhaoYang, LIU WenHua, ZHANG GuoXin, DONG ChunYan, ZHANG YanXia, XU XiaoWei, HE Dong, LIU HeChun, LI Yang, WANG FengTao, FENG Jing, YAO XiaoBo, LIU MeiJin, LIN RuiMing. The Relationship Between Occurrence of Hulless Barley Ear Rot and Population Migration of Grass Mite (Siteroptes spp.) [J]. Scientia Agricultura Sinica, 2025, 58(3): 493-506.
[4] YANG WenJuan, GAO JiaCheng, WANG YanTing, LI Yan, GUO Ming, WANG JunCheng, MENG YaXiong, WANG HuaJun, SI ErJing. Function of Effector Pg00778 Regulation on the Pathogenicity of Pyrenophora graminea to Barley [J]. Scientia Agricultura Sinica, 2025, 58(15): 3020-3035.
[5] DONG ZaiFang, DING TengTeng, SHAN YiXuan, LI HongLian, CHEN LinLin, XING XiaoPing. Autophagy-Related Gene FpAtg3 Involves in Growth and Pathogenicity of Fusarium pseudograminearum [J]. Scientia Agricultura Sinica, 2024, 57(6): 1080-1090.
[6] ZHANG AiHong, YANG Fei, ZHAO YuanYe, ZHAO YiHan, DI DianPing, MIAO HongQin. Pathogenicity and Epidemic Risk of Barley Yellow Striate Mosaic Virus [J]. Scientia Agricultura Sinica, 2024, 57(23): 4686-4697.
[7] WANG Yuan, DU MengDan, LI ZhengGang, SHE XiaoMan, YU Lin, LAN GuoBing, DING ShanWen, HE ZiFu, TANG YaFei. Identification of Pathogen Causing Tomato White Tip and Curl Leaf Disease and Its Pathogenicity in Guangdong Province [J]. Scientia Agricultura Sinica, 2024, 57(12): 2350-2363.
[8] ZHANG Jian, ZHAO BinSen, FENG Hao, HUANG LiLi. Function and Mechanism Analysis of Vm-milRN7 Regulating the Pathogenicity of Valsa mali [J]. Scientia Agricultura Sinica, 2024, 57(10): 1930-1942.
[9] GAO XiaoXiao, TU LiQin, YANG Liu, LIU YaNan, GAO DanNa, SUN Feng, LI Shuo, ZHANG SongBai, JI YingHua. Construction of an Infectious Clone of Tobacco Mild Green Mosaic Virus Isolate Infecting Pepper from Jiangsu Based on Genomic Clone [J]. Scientia Agricultura Sinica, 2023, 56(8): 1494-1502.
[10] GONG AnDong, LEI YinYu, WU NanNan, LIU JingRong, SONG MengGe, ZHANG YiMei, YANG Guang, YANG Peng. The Effect of 3-Oxyacyl ACP Reductase Gene FgOAR1 on the Growth, Development and Pathogenicity of Fusarium graminearum [J]. Scientia Agricultura Sinica, 2023, 56(24): 4854-4865.
[11] LI HuiXin, SONG WenPing, HAN ZongXi, LIU ShengWang. Isolation and Pathogenicity of Fowl Adenovirus Serotype 8a Strain [J]. Scientia Agricultura Sinica, 2023, 56(16): 3226-3236.
[12] HUANG JiaQuan,LI Li,WU FengNian,ZHENG Zheng,DENG XiaoLing. Proliferation of Two Types Prophage of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and their Pathogenicity [J]. Scientia Agricultura Sinica, 2022, 55(4): 719-728.
[13] YANG ShiMan, XU ChengZhi, XU BangFeng, WU YunPu, JIA YunHui, QIAO ChuanLing, CHEN HuaLan. Amino Acid of 225 in the HA Protein Affects the Pathogenicities of H1N1 Subtype Swine Influenza Viruses [J]. Scientia Agricultura Sinica, 2022, 55(4): 816-824.
[14] ZHANG JinLong,ZHAO ZhiBo,LIU Wei,HUANG LiLi. The Function of Key T3SS Effectors in Pseudomonas syringae pv. actinidiae [J]. Scientia Agricultura Sinica, 2022, 55(3): 503-513.
[15] LI ZhengGang,TANG YaFei,SHE XiaoMan,YU Lin,LAN GuoBing,HE ZiFu. Molecular Characteristics and Pathogenicity Analysis of Youcai Mosaic Virus Guangdong Isolate Infecting Radish [J]. Scientia Agricultura Sinica, 2022, 55(14): 2752-2761.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd