Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (13): 2552-2562.doi: 10.3864/j.issn.0578-1752.2014.13.007

• PLANT PROTECTION • Previous Articles     Next Articles

Molecular Cloning of T-DNA Targeted Genes of Ustilaginoidea virens Pathogenicity-Defective Mutant Strain B-1015

 HUANG  Lei-1, 2 , HU  Jian-Kun-2, YU  Mi-Na-2, YU  Jun-Jie-2, WANG  Ya-Hui-2, ZHENG  Meng-Ting-2, ZHENG  Rui-2, LIU  Yong-Feng-1, 2   

  1. 1、College of Life Sciences, Nanjing Agricultural University, Nanjing 210095;
    2、Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2013-12-15 Online:2014-07-01 Published:2014-02-10

RichHTML

1

PDF

729

Abstract: 【Objective】 The objective of this study is to analyze the biological phenotypes of the pathogenicity-defective mutant strain B-1015, to clone its T-DNA integration flanking sequences, and finally to reveal the functions of targeted genes in pathogenesis of Ustilaginoidea virens. The study will contribute to better understanding of the molecular pathogenesis of U. virens. 【Method】 Biological phenotypes of B-1015 were analyzed by testing growth rate, sporulation ability, spore germination rate and pathogenicity. The copy numbers of T-DNA inserted into B-1015 were identified by Southern blot. The flanking sequences of T-DNA were cloned by hiTail-PCR and the whole gene sequences of the T-DNA targeted genes were cloned by RACE PCR. The genes expression were detected by RT-PCR. 【Result】By comparing with wild type strain P1, the sporulation ability and growth rate of the B-1015 were significantly declined, but the spore germination rate had no significant difference. The inserted mutant lost its pathogenicity in rice. Molecular detection analysis confirmed that the B-1015 was a stable single T-DNA insertional mutant. The T-DNA flanking sequences were obtained by hiTail-PCR. Two genes, Uvt-1015R and Uvt-1015L, were cloned by RACE PCR. Gene sequence analysis showed that Uvt-1015R contained a 948 bp open reading frame, which could encode a 295 amino acid protein, a 341 bp 5′-UTR and a 271 bp 3′-UTR. Another gene, Uvt-1015L, comprised a 351 bp open reading frame coding a 116 amino acid protein, a 31 bp 5′-UTR and a 174 bp 3′-UTR. By searching in the known protein, the Uvt-1015R and Uvt-1015L genes showed no homology of any protein. Sequence alignment showed that the flanking sequences of T-DNA were non-adjacent in the wild type genome and the T-DNA disrupted the two genes sequence. RT-PCR analysis confirmed that compared to P1 strain, the transcripts of Uvt-1015R was significantly decreased, and the transcripts of Uvt-1015L was not detected any more. 【Conclusion】 In B-1015, both the T-DNA insertion and chromosomal rearrangement caused important biological phenotype mutant, thus leading to the loss of pathogenicity.

Key words: Ustilaginoidea virens , pathogenicity , T-DNA , gene clone

[1]胡东维, 王疏. 稻曲病菌侵染机制研究现状与展望. 中国农业科学, 2012, 45(22): 4604-4611.

Hu D W, Wang S. Progress and perspectives in infection mechanism of Ustilaginoidea virens. Scientia Agricultura Sinica, 2012, 45(22): 4604-4611. (in Chinese)

[2]田鸿, 陶家凤. 水稻稻曲病菌厚垣孢子萌发特性及稻曲病菌毒素对水稻、玉米、小麦种子萌发的影响. 西南农业学报, 2000, 13(3): 113-116.

Tian H, Tao J F. The germinating characters of falsesmut chalmydospores and the influence of toxin produced by Ustilaginoidea virens on the growth of radicle and plumule of rice, tritici and madyis. Southwest China Journal of Agricultural Science, 2000, 13(3): 113-116. (in Chinese)

[3]尹小乐, 陈志谊, 刘永锋, 于俊杰, 李燕, 俞咪娜. 稻曲毒素A的相对含量分析及其与致病力的相关性. 中国农业科学, 2012, 45(22): 4720-4727.

Yin X L, Chen Z Y, Liu Y F, Yu J J, Li Y, Yu M N. Detection of the relative content of ustiloxin A in rice false smut balls and correlation analysis between pathogenicity and ustiloxin A production of Ustilaginoidea virens. Scientia Agricultura Sinica, 2012, 45(22): 4720-4727. (in Chinese)

[4]De Groot M A, Bundock P, Hooykaas P J, Beijersbergen A G. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology, 1998, 16: 839-842.

[5]Jiang D, Zhu W, Wang Y, Sun C, Zhang K Q, Yang J K.    Molecular tools for functional genomics in filamentous fungi: Recent advances and new strategies. Biotechnology Advances, 2013, 31(8): 1562-1574.

[6]Combier J P, Melayah D, Raffier C, Gilles G, Marmeisse R. Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum. FEMS Microbiology Letters, 2003, 220(1): 141-148.

[7]Zwiers L H, De Waard M A. Efficient Agrobacterium tumefaciens- mediated gene disruption in the phytopathogen Mycosphaerella graminicola. Current Genetics, 2001, 39(5/6): 388-393.

[8]Sugui J A, Chang Y C, Kwon-Chung K J. Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption. Applied and Environmental Microbiology, 2005, 71(4): 1798-1802.

[9]Maruthachalam K, Klosterman S J, Kang S, Hays R J, Subbarao K V. Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis. Molecular Biotechnology, 2011, 49(3): 209-221.

[10]Nakamura M, Kuwahara H, Onoyama K, Iwai H. Agrobacterium tumefaciens-mediated transformation for investigating pathogenicity genes of the phytopathogenic fungus Colletorichum sansevieriae. Current Microbiology, 2012, 65: 176-182.

[11]张君成, 张炳欣, 陈志谊, 刘永锋, 陆凡. 稻曲病菌分生孢子的生物学研究. 植物病理学报, 2003, 33(1): 44-47.

Zhang J C, Zhang B X, Chen Z Y, Liu Y F, Lu F. Study on biology of conidia of Ustilaginoidea virens. Acta Phytopathologica Sinica, 2003, 33(1): 44-47. (in Chinese)

[12]张君成, 陈志谊, 张炳欣, 刘永锋, 陆凡. 稻曲病的接种技术研究. 植物病理学报, 2004, 34(5): 463-467.

Zhang J C, Chen Z Y, Zhang B X, Liu Y F, Lu F. Inoculation techniques used for inducing rice false smut efficiently. Acta Phytopathologica Sinica, 2004, 34(5): 463-467. (in Chinese)

[13]周永力, 樊金娟, 曾超珍, 刘小舟, 王疏, 赵开军. 稻曲病菌遗传多样性与群体结构的初步分析. 植物病理学报, 2004, 34(5): 442-448.

Zhou Y L, Fan J J, Zeng C Z, Liu X Z, Wang S, Zhao K J. Preliminary analysis of genetic diversity and population structure of Ustilaginoidea virens. Acta Phytopathologica Sinica, 2004, 35(5): 442-448. (in Chinese)

[14]陈莉, 许娟, 丁克坚, 李勋卓, 叶正和. 药剂对稻曲病菌的毒力测定及复配研究. 植物保护, 2012, 38(3): 159-161.

Chen L, Xu J, Ding K J, Li X Z, Ye Z H. Toxicity test of fungicides to Ustilaginoidea virens and mixed formulation. Plant Protection, 2012, 38(3): 159-161. (in Chinses)

[15]刘连盟, 王玲, 黄雯雯, 刘恩勇, 黄世文. 水稻稻曲病菌G蛋白β亚基基因的克隆, 表达与序列分析. 中国水稻科学, 2010, 24(4): 353-359.

Liu L M, Wang L, Huang W W, Liu E Y, Huang S W. Cloning, expression and sequence analysis of G protein β subunit gene of rice false smut pathogen Ustilaginoidea virens. Chinese Journal of Rice Science, 2010, 24(4): 353-359. (in Chinese)

[16]张震, 杜新法, 柴荣耀, 邱海萍, 王教瑜, 毛学琴, 孙国昌. 稻曲病菌PMK1类同源基因克隆及在稻瘟病菌遗传互补中的功能验证. 微生物学报, 2008, 48(11): 1473-1478.

Zhang Z, Du X F, Chai R Y, Qiu H P, Wang J Y, Mao X Q, Sun G C. Cloning of a homologous gene of Magnaporthe grisea PMK1 type MAPK from Ustilaginoidea virens and functional identification by complement in Magnaporthe grisea corresponding mutant. Acta Microbiologica Sinica, 2008, 48(11): 1473-1478. (in Chinese)

[17]俞咪娜, 胡建坤, 黄磊, 于俊杰, 尹小乐, 聂亚锋, 陈志谊, 刘永锋. 稻曲病菌T-DNA插入突变体5062的插入位点分析. 中国农业科学, 2013, 46(9): 1790-1798.

Yu M N, Hu J K, Huang L, Yu J J, Yin X L, Nie Y F, Chen Z Y, Liu Y F. Molecular characterization of T-DNA integration of the Ustilaginoidea virens mutant 5062. Scientia Agricultura Sinica, 2013, 46(9): 1790-1798. (in Chinese)

[18]黄磊, 俞咪娜, 胡建坤, 于俊杰, 尹小乐, 聂亚峰, 陈志谊, 刘永锋. 稻曲病菌突变体B-726生物学性状分析及其T-DNA插入位点侧翼序列的克隆. 中国农业科学, 2013, 46(16): 3344-3353.

Huang L, Yu M N, Hu J K, Yu J J, Yin X Y, Nie Y F, Chen Z Y, Liu Y F. Analysis of biological phenotypes and molecular cloning of T-DNA integration flanking sequences of Ustilaginoidea virens mutant strain B-726. Scientia Agricultura Sinica, 2013, 46(16): 3344-3353. (in Chinese)

[19]罗婵, 汤刚彬, 谢体三, 石德顺. 感受态细胞制备与保存方法的比较研究. 生物技术, 2005, 15(1): 52-54.

Luo C, Tang G B, Xie T S, Shi D S. Comparative study on preparation and storage of competent Escherichia coli cells. Biotechnology, 2005, 15(1): 52-54. (in Chinese)

[20]Liu Y G, Chen Y L. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. BioTechniques, 2007, 43(5): 649-656.

[21]顾志敏, 丁正中, 陈析丰, 郭龙彪, 曾大力, 钱前, 马伯军. 实时荧光定量PCR筛选稻曲病菌内参基因. 中国水稻科学, 2012, 26(5): 615-618.

Gu Z M, Ding Z Z, Chen X F, Guo L B, Zeng D L, Qian Q, Ma B J. Reference genes selection of Ustilaginoidea virens by real-time PCR. Chinese Journal of Rice Science, 2012, 26(5): 615-618. (in Chinese)

[22]Gupta A, Chattoo B B. A novel gene MGA1 is required for appressorium formation in Magnaporthe grisea. Fungal Genetics and Biology, 2007, 44(11): 1157-1169.

[23]Zhou Z, Li G, Lin C, He C. Conidiophore stalk-less1 encodes a putative zinc-finger protein involved in the early stage of conidiation and mycelial infection in Magnaporthe oryzae. Molecular Plant-Microbe Interactions, 2009, 22(4): 402-410.

[24]张震, 杜新法, 柴荣耀, 毛学琴, 邱海萍, 王艳丽, 王教瑜, 孙国 昌. 根癌农杆菌介导遗传转化稻曲病菌. 中国水稻科学, 2006, 20(4): 440-442.

Zhang Z, Du X F, Chai R Y, Mao X Q, Qiu H P, Wang Y L, Wang J Y, Sun G C. Agrobacterium tumefaciens-mediated transformation of the pathogen of Ustilaginoidea virens. Chinese Journal of Rice Science, 2006, 20(4): 440-442. (in Chinese)

[25]Zhu Q H,  Ramm K, Eamens A L, Dennis E S, Upadhyaya N M. Transgene structures suggest that multiple mechanisms are involved in T-DNA integration in plants. Plant Science, 2006, 171: 308-322.

[26]Müller A E, Atkinson R G, Sandoval R B, Jorgensen R A. Microhomologies between T-DNA ends and target sites often occur in inverted orientation and may be responsible for the high frequency of T-DNA-associated inversions. Plant Cell Reports, 2007, 26: 617-630.
[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] WANG ZhuangZhuang, DONG ShaoYun, ZHOU Qi, MIAO Han, LIU XiaoPing, XU KuiPeng, GU XingFang, ZHANG ShengPing. Cloning and Analysis of Key Genes for Vitamin C Synthesis in Cucumber Fruit [J]. Scientia Agricultura Sinica, 2023, 56(3): 508-518.
[11] 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.
[12] 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.
[13] 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.
[14] 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.
[15] 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.
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