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稻曲病侵染过程以及水稻响应稻曲菌侵染的转录组变化
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摘要
稻曲病是近年流行的一种发生于水稻穗部的真菌性病害。自上世纪七十年代以来,随着优质稻的逐渐推广和化肥的大量施用,稻曲病在世界主要稻谷产区迅速蔓延,甚至有些地区已经取代稻瘟病和白叶枯病成为首要病害。稻曲病不仅严重影响水稻产量,其散发的孢子还污染环境,影响稻米品质,威胁人类身体健康。目前有关稻曲病的研究主要集中在病原菌生物学性状观察、群体结构、遗传转化、毒素鉴定以及水稻抗性资源的筛选上,而对于稻曲菌侵染过程中水稻-病原菌互作的分子生物学研究还没有报道。本研究中,我们从细胞学水平对稻曲菌侵染水稻过程进行了观察,并且用数字表达谱对病原菌侵染水稻时水稻转录组变化情况进行了监测,发现了一些水稻-病原菌互作的代谢途径和受稻曲病特异调控的基因。主要结果如下:
     1、胚芽鞘是水稻营养生长期侵染的一个重要部位。将催芽后的水稻和稻曲菌分生孢子悬浮液共培养,十天后通过显微镜观察可在胚芽鞘内层细胞发现明显的侵染痕迹:部分宿主细胞被稻曲菌膨大孢子填充,形成一条条孢子带,有的孢子萌发成菌丝沿着细胞间隙蔓延,在适当部位刺穿细胞壁进而在水稻细胞中生长,说明根部可能是稻曲病初期侵染途径之
     2、稻曲菌对水稻小穗的侵染是一个多器官连续侵染的过程,根据侵染部位的不同我们将其分成三个时期。扫描电子显微镜观察发现稻曲菌首先在雌蕊的花柱上形成侵染钉和侵染垫,大量菌丝缠绕在柱头上,我们称其为时期1(S1);随之菌丝开始缠绕雄蕊基部,并形成特殊的类似于“脚”的结构向上攀爬,此时为时期2(S2);最后菌丝包裹整个花器官,形成白色的花器官-菌丝复合体,进一步解剖发现稻曲菌从药隔部位进入药室,在药室内部产生大量的分生孢子,并导致部分花粉粒空瘪化,为时期3(S3)。
     3、连续两年(2010-2011)的转录组测序表明,S1,S2,S3时期分别检测到423、879和1620个差异表达基因。排除褐飞虱、钻心虫、稻瘟病、条纹叶枯病等生物胁迫和早、盐、热、冷等非生物胁迫共同诱导的表达基因,在三个时期分别得到126、304和580个稻曲菌特异调控基因,其中,有53个上调基因和60个下调基因在三个时期均检测到,我们称为核心调控基因。
     4、对核心调控基因启动子区域序列分析发现,有9个保守性调控元件明显富集,其中上调部分有六个,包括两个转录调节元件(TTATTT、YTCANTYY),两个WRKY结合元件(TTGAC、TGACT),一个MYB结合元件(TGTCA),一个Dof结合元件(TAAAG),其中,WRKY、MYB和Dof是典型的胁迫相关转录因子。下调部分有三个,包括两个ABA调节元件(CANNTG、CATGCA),一个钙调素结合元件(VCGCGB),ABA一般认为对非生物胁迫是正调控因子,对生物胁迫是负调控因子;钙调素是植物体内第二信使重要组成成员,对于维护植物体正常的生理活动代谢有重要作用。
     5、对稻曲菌特异调控基因GO enrich分析结果表明,有大量的GO类别被显著富集。其中“Cell Part"部分有4个,‘"Molecular Functions"部分有55个,"Biological Process"部分有52个。进一步分析发现在稻曲菌侵染初期上调部分主要是一些蛋白修饰方面的类别富集,下调部分主要是一些碳水化合物代谢类别富集,这说明稻曲菌侵染初期就激活了水稻的防御系统,并且操纵宿主碳水化合物代谢;随着病情的发展,上调部分一些与细胞死亡相关的代谢被富集,下调部分中一些离子转运相关的类别被富集;进入S3期,与花粉识别特异相关的类别在上调部分富集,而下调部分主要是一些细胞壁合成代谢类富集,这也与我们观察到的在这个时期病原菌通过破坏药隔结构进入药室,并且产生大量分生孢子吸附与花粉粒表面这一现象相吻合。
Rice false smut is a typical fungal disease occurred in rice spikelets caused by Ustilaginoidea virens. In the last decades, with popularization of good rice cultivars and intensive application of nitrogen fertilizer, rice false smut is rapidly spreading around rice-growing regions worldwidely. In some areas, it has replaced rice blast and bacterial blight and becames a primary disease. Aparts causing severe yield losses, the toxins produced by false smut can greatly inhibit cell division which threatens to people life. However, the infection only occurs on booting spikelet strongly hinders the reaserch go on, thus the results mainly focon on the pathogen diversity, infection process, heredity, toxity and damage to rice production up to known. The infection process and molecular interaction between rice and pathogen are still poorly understood. In this study, we observed the infection process after inoculation and analyzed the expression profile of a spikelet infected with U. virens using digital gene expression tag profiling (DGE). Some interesting results were obtained as follow.
     1. Coleoptiles can be infected by U. virens. After inoculation rice bud with coindia, we obesved coleoptiles can be infected by pathogen at10day post inoculation (dpi). Several host cell were filled with swell spores and formed "spore stripes". Some spore germinated hyphe, grew alone with intercellular cell and entered host cell by directly pierced into cell wall. These results showed that false smut might be a systemic disease, and fungus carried by seed might be the source of primary infection.
     2. Cytological observation showed the early development of false smut took place in a stepwise invasion process and could be divided into three distinctive stages based on morphology of the infected floral organs and tissues. Initially, the two pistils in the infected spikelet were separate and only latter intertwined together by the hyphae, this phase here was named stage S1. As the fungi reproduced, mass of hyphae surrounded pistils and formed a penetration peg on column. Then, visible mycelial mass reached at the bottom of anthers, hyphae climbed up the anther using some special organs like "feet" and "feelers", this stage was here named stage S2. As the disease continued to develop, the whole floral organ became surrounded by mycelium, forming a floral-hyphae complex. Finally, the hyphae entered the anther chambers by piercing through anther wall at the weak connectivum, this stage was here named stage S3. Cytological observation showed mass of conidia wrapped on the surfaces of pollen grains and innate anther wall, and some of pollen became collapsed. Carbohydrate analysis showed the content of soluble sugar increased significantly whereas starch content significantly reduced. This was further confirmed by the decrease of expression level of starch synthase and increase of the starch-degrading enzyme glycosyl hydrolase. In addition, two sugar transporter members were also highly induced in the present study, hinting stamen is an important tissue for U. virens infection and reproduction.
     3. By integrative two years (2010-2011) DGE data, totally423,879and1620DE genes across both years were monitored in S1, S2and S3, respectively. To obtain the genes specially regulated by false smut, we compared our data to that of the openly available biotic and abiotic databases to exclude genes generally regulated by other abiotic and biotic factors by bioinformatics.126(58up,68down),304(136up,168down), and580(243up,373down) expressional genes which be considered specially regulated by the invasion of U. virens were detected in S1, S2, and S3respectively. Of which,53and68core genes were detected among three infection stages in up-and down-regulation part.
     4. Analysis of the highly conserved c/s-acting elements between core up-and down-regulation genes, we obtained9elements (6up and3down). In up parts, YTCANTYY and TTATTT are basic transcription initiation elements for genes expression, TTGAC, TGACT for W-box, TGTCA for Myb and TAAAG for Dof were typically stress related motifs. In down parts, two (CANNTG and CATGCA) out of three motifs were detected for abscisic acid (ABA)-response. Previous studies showed ABA is usually a positive regulator in response to abiotic stress, whereas a negative one in response to biotic stress. CGCG box is regulated by calmodulin. As an important second message, calmodulin play a key role in plant development.
     5. GO analysis showed mass of caterious differently enriched. Of which,4categories in "Cell part",55categories in "Molecular functions",52categories in "Biological process". Further analysis showed in the beginning infection,"Phosphorylation" and "Protein modification" were greatly activated whereas "Carbohydrate metabolic process" was repressed. With disease development, categories as "Cell death" was widely up-regulated whereas "Ion transport" ere widely repressed at stage S2. During final infection (S3),"Cell recognition" was enriched in up-regulation and "Cell wall metabolism" was enriched in down-regulation part, which was in accordance with the cytological observation that the pathogen hyphae intertwined with microspores and great amount of conidia produced in the anther chambers.
引文
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