Molecular Characterization of T-DNA Integration into Ustilaginoidea virens Mutant B1464

doi:10．3969/j．issn．1001G7216．2015．03．01

Abstract

Abstract:

With an avirulent Ustilaginoidea virens strain B1464 as material,we analyzed the changes in biological morphology and pathogenic mechanism. Compared to the wild-type U. virens strain P1, the mutant strain B1464 showed no pathogenicity in the field. The rate of growth on MM medium was not different to P1 and B1464. On PSA and TB3 medium the rates of growth were nutritionally endowed. B1464 grew slower and produced few conidiophores in PS medium, and its conidiophores morphology and spore size became smaller. Genomic Southern bolt analysis confirmed that B1464 was single T-DNA insertional events. The flanking sequences of T-DNA obtained by TAIL-PCR were non-adjacent in the wild type. It was founds that the mutant strain lost about 20 kb sequences in the insertion site comparing with the wild type strain. The expression of the flanking genes of the T-DNA was down-regulated, detecting by semi-quantitative RT-PCR. The reason of the chromosome rearrangement and abnormal expression of some genes is the insertion of T-DNA, probably, leading to the mutant strain B1464 lost its pathogenicity.

Key words: Ustilaginoidea virens, T-DNA insertion mutant, pathogenicity, malate synthase, copper transporter

摘要：

以稻曲病菌致病力丧失的突变菌株B1464为材料,对其生物学形态和分子遗传变异进行分析。B1464田间接种表现为不致病,与野生型菌株相比, 在营养贫瘠的MM培养基上生长速率没有显著差异,而在PSA和TB3培养基中生长速率较慢,并且在PS液体培养基中产孢能力下降,同时突变菌株的分生孢子与野生型相比形态和大小均发生变化。Southern杂交结果显示T-DNA在突变菌株B1464中以单拷贝形式插入,利用TAIL-PCR技术扩增得到的紧邻T-DNA两侧的侧翼序列在野生型中不相邻,与野生型菌株基因组比对发现突变菌株插入位点处丢失约20 kb的DNA片段。RT-PCR结果表明突变菌株中T-DNA插入位点两侧基因表达量均下调。推断突变菌株B1464的致病能力丧失可能是由于T-DNA的插入导致了染色体重排及破坏了某些基因的正常表达。

关键词: 稻曲病菌, T-DNA插入突变, 致病力, 苹果酸合成酶, 铜转运蛋白

CLC Number:

Ya-hui WANG, Yong-feng LIU, Fan LU, Mi-na YU, Lei HUANG, Meng-ting ZHENG, Jun-jie YU, Xiao-le YIN. Molecular Characterization of T-DNA Integration into Ustilaginoidea virens Mutant B1464[J]. Chinese Journal OF Rice Science, 2015, 29(3): 311-318.

王亚会, 刘永锋, 陆凡, 俞咪娜, 黄磊, 郑梦婷, 于俊杰, 尹小乐. 稻曲病菌T-DNA插入突变体B1464插入位点分析[J]. 中国水稻科学, 2015, 29(3): 311-318.

Figures/Tables 6

Table 1 Primers applied in the research.

引物名称 Name	引物序列 Sequence(5' → 3')
hyg1.4F	ACAGAAGATGATATTGAAGGAGC
hyg1.4R	TACTCTATTCCTTTGCCCTCG
LAD10	ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT^*
GFP-1	CCATCCTGGTCGAGCTGGA
GFP-2	CGAACTCCAGCAGGACCATG
GLB1	AAGATGGTGCGCTCCTGGACGTAG
GLB2	ACGATGGACTCCAGAGGCACTGCACGCCGTAGGTCAGGGTGG
GLB3	TGGGCACCACCCCGGTGAACAGCTC
HLB1	GATTCCCAATACGAGGTCGCCAA
HLB2	TGGAGGCCGTGGTTGGCTTGTATGGAG
HLB3	AGGGTCGATGCGACGCAATCGTCCGA
AC-S2	GAGTTTAGGTCCAGCGTCCGTCGACGATGGACTCCAGAG
AC-S3	GAGTTTAGGTCCAGCGTCCGT
G-1	TGGGATTGAGCGGGAGCT
H-1	GGCTGCTGTACGACGAGATC
U-1	CCAGTAGACGCAAGCACAGG
G-2	CACCACGACCACCGTCTCCT
G-3	CTGTACGCAAGCTCGTGGAC
H-2	CGAGGCGGAACTTGTTGTGC
H-3	GCTGCGTGCCCATCAACTAC
β-actin F^[22]	CCGTGAGAAGATGACCCAGA
β-actin R	GGCGAAACCCTCGTAGATGG

Fig.1. Molecular detection of Ustilaginoidea virens mutant B1464 strain. (A, PCR analysis of the gfp gene and HPH gene. Lanes 1 and 2, PCR detection of the gfp genes of P1 and B1464, respectively. Lanes 3 and 4, PCR detection of the HPH of P1 and B1464. Lane M, DNA marker. B, Southern blot detection of B1464, showing that a single T-DNA insertion event occurred in the B1464 genome. Lane 1, Genomic DNA of P1 was digested with EcoR Ⅰ, Hind Ⅲ. Lanes 2 and 3, Genomic DNA of B1464 was digested with EcoRⅠ,Hind Ⅲ, respectively.)

Fig. 2. Analysis of the phenotypie of Ustilaginoidea virens mutant B1464. (A, Colony diameters on TB3, PSA and MM medium; **indicate significant difference between B1464 and P1 at 0.01 level; B, Colonies of B1464 and P1 on different media; C, Conidium morphology of B1464 and P1; D, Sizes of conidium.)

Fig. 3. Structure of T-DNA and flanking sequence. (A, The structure of T-DNA; B, Structure of T-DNA RB and flanking sequence, the underlined fragments were not found in the genome of U.virens and T-DNA; C, Structure of T-DNA LB and flanking sequence.)

Fig. 4. Verification of the molecular characterization of the chromosomal rearrangement in B1464. (A, Insertion site to B1464 and primer design;B, PCR detection. )

Fig.5. RT-PCR analysis of the expression level of the two genes in B1464. (A, Loss of sequence and the two flanking genes in B1464; B, RT-PCR analysis of the expression level of the two genes in B1464. )

References 34

[1]	邹克琴, 胡东维, 王为民, 等. 水稻稻曲病的研究进展. 浙江农业科学, 2012(5): 704-706.
[2]	Rush M C, Shahjahan A K M, Jones J P. Outbreak of false smut of rice in Louisiana.Plant Dis, 2000, 84(1): 100.
[3]	Atia M M M. Rice false smut (Ustilaginoidea virens) in Egypt.J Plant Dis & Prot, 2004,111(1): 71-82.
[4]	Ladhalakshmi D, Laha G S, Singh R, et al.Isolation and characterization of Ustilaginoidea virens and survey of false smut disease of rice in India.Phytoparasitica, 2012, 40: 171-176.
[5]	Guo X Y, Li Y, Fan J, et al.Progress in the study of false smut disease in rice.J Agric Sci & Technol, 2012 (A2): 1211-1217.
[6]	张震, 王教瑜, 杜新法, 等. 稻曲病菌cDNA文库的构建. 植物病理学报, 2008, 38(5): 462-467.
[7]	刘连盟, 王玲, 黄雯雯, 等. 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.
[8]	于俊杰, 聂亚锋, 俞咪娜, 等. 稻曲病菌分生孢子高产突变体A2588的T-DNA 插入位点侧翼基因分析. 中国农业科学, 2013, 46(24): 5132-5141.
[9]	de Groot M J A, Bundock P, Hooykaas P J J, et al. Agrobacterium tumefaciens-mediated transformation of filamentous fungi.Nat Biotechnol, 1998, 16(9): 839-842.
[10]	Michielse C B, Hooykaas P J, van den Hondel C A, et al. Agrobacterium-mediated transformation as a tool for functional genomics in fungi.Curr Genet, 2005, 48(1): 1-17.
[11]	吴小燕, 王教瑜, 张震, 等. 稻瘟病菌致病性增强突变体B11 的基因分析. 中国水稻科学, 2009, 23(6): 611-615.
[12]	Maruthachalam K, Klosterman S J, Kang S, et al.Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis.Mol Biotechnol, 2011, 49(3): 209-221.
[13]	张震, 杜新法, 柴荣耀, 等. 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.
[14]	Ashizawa T, Takahashi M, Arai M, et al.Rice false smut pathogen, Ustilaginoidea virens, invades through small gap at the apex of a rice spikelet before heading.J Gener Plant Pathol, 2012, 78(4): 255-259.
[15]	Tang Y X, Jin J, Hu D W, et al.Elucidation of the infection process of Ustilaginoidea virens (teleomorph: Villosiclava virens) in rice spikelets.Plant Pathol, 2013, 62(1): 1-8.
[16]	Chen Y, Zhang Y, Yao J, et al.Frequency distribution of sensitivity of Ustilaginoidea virens to four EBI fungicides, prochloraz, difenoconazole, propiconazole and tebuconazole, and their efficacy in controlling rice false smut in Anhui Province of China.Phytoparasitica, 2013, 41: 277-284.
[17]	尹小乐, 陈志谊, 刘永锋, 等. 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.
[18]	范允卿, 张舒, 喻大昭, 等. 稻曲病原菌毒素研究进展. 湖北农业科学, 2012, 51(21): 4701-4704.
[19]	张君成, 陈志谊, 张炳欣, 等. 稻曲病菌的形态学观察研究. 植物病理学报, 2003, 33(6): 517-523.
[20]	Kim K W, Park E W.Ultrastructure of spined conidia and hyphae of the rice false smut fungus Ustilaginoidea virens.Micron, 2007, 38(6): 626-631.
[21]	于俊杰, 尹小乐, 陈志谊, 等. 稻曲菌交配型初探. 植物病理学报, 2012, 42(6): 561-568.
[22]	顾志敏, 丁正中, 陈析丰, 等. 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.
[23]	张君成, 陈志谊, 张炳欣. 稻曲病的接种技术研究. 植物病理学报, 2004, 34(5): 463-467.
[24]	Mullins E D, Chen X, Romaine P, et al.Agrobacterium-mediated transformation of Fusarium oxysporum: An efficient tool for insertional mutagenesis and gene transfer.Phytopathology, 2001, 91(2): 173-180.
[25]	黄骥, 王建飞, 张红生. 植物C2H2型锌指蛋白的结构与功能. 遗传, 2004, 26(3): 414-418.
[26]	Ciftci-Yilmaz S, Mittler R.The zinc finger network of plants.Cell Mol Life Sci, 2008, 65, 1150-1160.
[27]	Wang Y, Gracheva E O, Richmond J, et al.The C2H2 zinc-finger protein SYD-9 is a putative posttranscriptional regulator for synaptic transmission.Pro Natl Agric Sci USA, 2006, 103(27): 10 450-10 455.
[28]	Ashizawa T, Kataoka Y.Detection of Ustilaginoidea virens in rice (Oryza sativa) panicles before and after heading in the field using nested-PCR technique with species specific primers.Ann Phytopathol Soc Jpn, 2005, 71(1): 16-19.
[29]	Balasubramanian S, Kim S, Podila G K.Differential expression of a malate synthase gene during the preinfection stage of symbiosis in the ectomycorrhizal fungusLaccaria bicolor. New Phytol, 2002, 154: 517-527.
[30]	Borghouts C, Scheckhuber C Q, Stephan O, et al.Copper homeostasis and aging in the fungal model system Podospora anserina: Differential expression of PaCtr3 encoding a copper transporter.Int J Biochem & Cell Biol, 2002, 34: 1355-1371.
[31]	Barhoom S, Kupiec M, Zhao X H, et al.Functional characterization of CgCTR2, a putative vacuole copper transporter that is involved in germination and pathogenicity in Colletotrichum gloeosporioides.Eukaryotic Cell, 2008, 7(7): 1098-1108.
[32]	Bellemare D R, Shaner L, Morano K A, et al.Ctr6, a vacuolar membrane copper transporter in Schizosaccharomyces pombe.J Biol Chem, 2002, 277(48): 46 676-46 686.
[33]	Zhu Q H, Ramm K, Eamens A L, et al.Transgene structures suggest that multiple mechanisms are involved in T-DNA integration in plants.Plant Sci, 2006, 171: 308-322.
[34]	Zhu C F, Wu J H, He C Z.Induction of chromosomal inversion by integration of T-DNA in the rice genome.J Genet Genom, 2010, 37: 189-196.