Optimization of transgene-mediated silencing in Phytophthora infestans and its association with small-interfering RNAs

Methods for silencing genes in Phytophthora transformants have been demonstrated previously, but wide variation in effectiveness was reported in different studies. To optimize this important tool for functional genomics, we compared the abilities of sense, antisense, and hairpin transgenes introduced by protoplast, electroporation, and bombardment methods to silence the inf1 elicitin gene in Phytophthora infestans. A hairpin construct induced silencing three times more often than sense or antisense vectors, and protoplast transformation twice as much as electroporation. Using hairpins introduced into protoplasts, 61% of strains were silenced, and transgene copy number was positively correlated with silencing. The utility of bombardment was reduced by the occurrence of heterokaryons containing silenced and non-silenced nuclei, but silenced strains were obtainable from about 20% of primary transformants by single-nuclear purification. Most inf1-deficient strains were fully silenced, however some exhibited partial suppression. These produced inf1-derived RNAs of about 21-nt which correspond to both the sense and antisense strands of inf1, implicating an RNAi-like mechanism in silencing.     

转录后基因沉默系统研究烟草rbcS基因功能

初步建立了利用病毒载体诱导转录后基因沉默系统研究烟草(Nicotiana benthamiana)1,5-二磷酸核酮糖羧化酶／加氧酶小亚基(Ribulose-1,5-bisphosphate carboxylase／oxylase small subunit,rbcS)基因功能的模式。用携带与1,5-二磷酸核酮糖羧化酶／加氧酶小亚基基因同源的cDNA片段的烟草脆裂病毒载体(pTV．rbcS)侵染烟草(Nicotiana benthamiana),诱导内源rbcS基因沉默并在此基础上建立了研究rbcS基因功能的模式：初步进行了rbcS基因沉默后的表型分析、转录水平分析、蛋白质表达水平分析以及利用HPLC方法定量分析rbcS基因沉默后的光合色素变化。结果表明：病毒诱导基因沉默瞬时表达体系中烟草最佳侵染时期为苗龄21-24d,用于侵染的重组农杆菌的最佳浓度的OD值为1～1．5;烟草Rubisco小亚基的表达量可能调节Rubisco大亚基的表达量;烟草rbcS基因与光合作用中的光能收集无关。对rbcS基因沉默的烟草叶片及对照烟草叶片的部分重要光合作用指标分析表明,运用烟草脆裂病毒载体诱导转录后基因沉默系统研究烟草rbcS基因功能具有可行性,为进一步深入研究rbcS基因功能奠定了基础。     

辣椒CaWRKY14转录因子的生物学特性研究

WRKY转录因子是植物响应病原菌胁迫最重要的转录因子之一,且参与抗病反应及信号传导通路的调控。为研究辣椒WRKY基因的生物学特征,以辣椒高抗疫病材料CM334为试材,克隆获得响应疫霉菌诱导的转录因子CaWRKY14。生物信息学分析表明,该基因DNA全长2 530 bp,cDNA全长1 662 bp,含有5个内含子,编码553个氨基酸,含有1个WRKY保守结构域,属于Group Ⅱ(b)。实时荧光定量表达分析表明,CaWRKY14不仅受ABA和疫霉菌胁迫诱导表达,且表达量分别在12 h和24 h时达到峰值,分别是对照的8.54和8.04倍,同时也受高盐、热激和干旱胁迫诱导。利用VIGS技术对CaWRKY14转录因子进行沉默后发现,抗病材料CM334接种疫霉菌后趋于发病。研究表明,CaWRKY14基因在辣椒响应疫霉菌胁迫进程中可能发挥着重要作用。     

Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules

We investigated the efficacy of self-complementary hairpin RNA (hpRNA) expression to induce RNA silencing in the roots and nodules of model legume Lotus japonicus, using hairy root transformation mediated by Agrobacterium rhizogenes. Transgenic lines that express beta-glucuronidase (GUS) by constitutive or nodule-specific promoters were supertransformed by infection of A. rhizogenes harboring constructs for the expression of hpRNAs with sequences complementary to the GUS coding region. GUS activity in more than 60% of the hairy roots was decreased or silenced almost completely. Silencing of the GUS gene was also observed in symbiotic nodules formed on hairy roots in both early and late stages of nodule organogenesis. These results indicate that transient RNA silencing by hairy root transformation provides a powerful tool for loss-of-function analyses of genes that function in roots and root nodules.     

A novel Phytophthora infestans haustorium-specific membrane protein is required for infection of potato

Phytophthora infestans causes late-blight, a devastating and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration, followed by infection vesicles, and haustoria to establish a biotrophic phase of interaction. Here we report the cloning, from a suppression subtractive hybridization library, of a P. infestans gene called Pihmp1 encoding a putative glycosylated protein with four closely spaced trans-membrane helices. Pihmp1 expression is upregulated in germinating cysts and in germinating cysts with appressoria, and significantly upregulated throughout infection of potato. Transient gene silencing of Pihmp1 led to loss of pathogenicity and indicated involvement of this gene in the penetration and early infection processes of P. infestans. P. infestans transformants expressing a Pihmp1::monomeric red fluorescent protein (mRFP) fusion demonstrated that Pihmp1 was translated in germinating sporangia, germinating cysts and appressoria, accumulated in the appressorium, and was located at the haustorial membrane during infection. Furthermore, we discovered that haustorial structures are formed over a 3 h period, maturing for up to 12 h, and that their formation is initiated only at sites on the surface of intercellular hyphae where Pihmp1::mRFP is localized. We propose that Pihmp1 is an integral membrane protein that provides physical stability to the plasma membrane of P. infestans infection structures. We have provided the first evidence that the surface of oomycete haustoria possess proteins specific to these biotrophic structures, and that formation of biotrophic structures (infection vesicles and haustoria) is essential to successful host colonization by P. infestans.