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白粉菌(Powdery mildew)是一种专性活体寄生真菌,可以侵染多种植物,造成巨大的经济损失[1-2]。白粉菌与寄主相互作用的过程中,首先刺入寄主细胞壁形成吸器(haustorium),从寄主细胞内获取水分和营养物质。同时,白粉菌通过在吸器转录,翻译形成大量的效应蛋白(Effector proteins),分泌到寄主细胞内,从而实现在寄主体内的增殖和侵染[3-4]。目前,利用基因组、转录组测序及蛋白质组分析,通过对大麦白粉菌(Bgh,Blumeria graminisf. sp.Hordei)、小麦白粉菌(Bgt,Blumeria graminisf. sp.tritici)、拟南芥白粉菌(Gor,Golovinomyces orontii)、葡萄白粉菌(En,Erysiphe necator)和葫芦白粉菌(Pxa,Podosphaera xanthii)等进行预测,发现了一些潜在的效应蛋白[5]。大麦和小麦白粉菌编码的效应蛋白数量最多,通常是一些小的蛋白质,并且与已知蛋白同源性很低[6]。另外,大麦白粉菌形成的效应蛋白N端通常含有一个保守的结构域:第一个氨基酸是芳香族氨基酸(酪氨酸,苯丙氨酸或色氨酸),最后一个氨基酸是半胱氨酸(Y/F/WXC)[7]。然而,双子叶植物白粉菌编码的效应蛋白并不是都含有该保守结构域[8-9]。白粉菌是专性活体寄生,无法进行遗传操作,因此,目前对白粉菌效应蛋白功能的了解还非常少。最近,通过寄主诱导的基因沉默技术(HIGS,host-induced gene silencing),发现大麦白粉菌的一些效应蛋白在刺入寄主植物和形成吸器的过程中发挥毒性功能[10]。橡胶树白粉病是由橡胶树白粉菌(Oidium heveae)引发的一种真菌病害,对天然橡胶的生产造成了严重的经济损失。该病于1918年在印尼爪哇首次发现[11-13],迄今已遍布全球各橡胶种植区。由于橡胶树白粉菌与橡胶树很难进行遗传操作,因此严重阻碍了二者相互作用机理的研究。最近研究发现,橡胶树白粉菌在野生型拟南芥Col-0激发依赖于EDS1(enhanced disease susceptibility 1)和PAD4(Phytoalexin Deficient 4)的抗病反应,推测拟南芥TIR-NB-LRR(Toll-Interleukin1 Receptor-nucleotide binding-leucine-rich repeat)类抗性基因参与了对橡胶树白粉菌的识别过程[14]。
通过基因组和转录组测序分析,预测出橡胶树白粉菌含有133个潜在的效应蛋白[8],然而,这些效应蛋白的功能还是未知的。笔者克隆了橡胶树白粉菌潜在的效应蛋白基因OhEF 2(Oidium heveaeeffector protein 2),并对该效应蛋白结构进行分析,构建在Col-0背景下的OhEF 2基因过表达转基因植株。结果表明,过表达植株对橡胶树白粉菌敏感性增加,并且有效降低了橡胶树白粉菌在拟南芥上诱导的胼胝体沉积和致病相关基因的表达。
Effector protein OhEF 2 enhances the susceptibility ofArabidopsistoOidium heveae
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摘要:白粉菌通过在植物细胞内形成吸器,产生大量的效应蛋白,从而实现在寄主细胞内的侵染。前期有研究人员对 Oidium heveae进行基因组和转录组测序分析,预测出133个潜在的效应蛋白。笔者克隆了其中的一个基因 OhEP2( Oidium heveaeEffector Protein 2),并构建了 OhEF 2基因在拟南芥Col-0背景下的过表达转基因植株。通过接种发现,过量表达OhEF 2可以明显促进拟南芥对橡胶树白粉菌的感病性,但不能提高假单胞菌DC 3000的毒性功能,表明OhEF 2可能只在白粉菌侵染的过程中发挥作用。进一步研究发现,OhEF 2显著降低了橡胶树白粉菌在拟南芥上激发的胼胝体沉积和 PR1(Pathogen-Related Gene1)基因表达,这为进一步研究效应蛋白OhEF 2在植物体内的毒性作用机理奠定了基础。Abstract:Powdery mildew fungi complete its infection process on living host plants through the establishment of haustoria which secretes a plethora of effectors into plant cells. A previous study showed that 133 potential effectors of Oidium heveaehave been predicted by genome and transcriptome sequencing. However, the biological function of these effectors are still unknown. In this study, an effector gene, named OhEF 2, was cloned and transformed into Arabidopsis, and the transgenic plants of OhEF 2gene in ArabidopsisCol-0 background were constructed and inoculated with O. heveae.Inoculation assay showed that OhEF 2obviously enhanced the susceptibility of Arabidopsisto O. heveae. However, OhEF 2did not increase the virulence of Pseudomonas syringaeDC 3000, suggesting that OhEF 2probably plays a role only in the process of powdery mildew infection. Further study showed that OhEF 2decreased the callose deposition and PR1gene expression triggered by O. heveaein Arabidopsis, which lays a foundation for the future study of OhEF 2virulence mechanisms in host plants.
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Key words:
- Oidium heveae/
- effector protein/
- OhEF 2/
- virulence mechanism
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图 2橡胶树白粉菌效应蛋白OhEF 2拟南芥转基因植株的构建
A.效应蛋白OhEF 2转基因植株表型观察;B.效应蛋白OhEF 2转基因植株蛋白表达分析。Bar=1 cm。
Fig. 2The construction ofOidium heveaeeffector OhEF 2 transgenic plants inArabidopsisCol-0 background
A. The phenotype observation of effector OhEF-2 transgenic plants; B. Protein detection of effector OhEF-2 transgenic plants. Bar=1 cm.
图 3效应蛋白OhEF 2正调控拟南芥对橡胶树白粉菌的感病性
A.橡胶树白粉菌细胞进入率测定结果;B.橡胶树白粉菌菌丝生长测定结果;C.分生孢子计数结果。**:P<0.01。
Fig. 3Effector protein OhEF 2 positively regulates the susceptibility ofArabidopsistoOidium heveae
A. Quantitative assessment of host cell entry rates; B. Quantitative analysis of hyphal growth ofO. heveae; C. The numbers of conidiospores per colony were counted at 10 dpi. **:P<0.01.
图 4OhEF 2转基因植株叶片发病症状及显微观察结果
A. 叶片症状观察结果,Bar=5 mm;B. 显微观察结果,Bar=200 μm。
Fig. 4Symptoms and light microscopy of leaves of OhEF2 transgenic plants inoculated withOidium heveae
A. Symptoms of WT Col-0/ Empty vector and OhEF 2 transgenic plants at 10 dpi, Bar=5 mm; B. Light microscopy images, Bar=200 μm.
图 6效应蛋白OhEF 2抑制橡胶树白粉菌在拟南芥上激发的胼胝体沉积
A. 胼胝体计数结果;B. 叶片显微观察结果。**:P<0.01。
Fig. 6Effector protein OhEF 2 inhibits the callose deposition triggered byOidium heveaeinArabidopsis
A. Average number of callose deposits per microscope field of 0.1 mm2on Col-0 and OhEF 2 transgenic plants; B. Light microscopy images. Bar=200 μm. **:P<0.01.
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