DelsGate, a robust and rapid gene deletion construction method.

With the increasing availability of fungal genome sequences there is great demand for fast, simple high-throughput methods to generate constructs for gene deletion. Here we describe a method that combines PCR and Gateway cloning technology together with use of the I-SceI homing endonuclease to generate precise deletion constructs in a very simple, universal and robust manner in just 2 days. These constructs are then used to produce deletion mutants in the organism of interest following applicable methods for that species. In establishing this protocol we determined empirically that 1 kb was a suitable flank length to facilitate homologous recombination in our species of interest, Ustilago maydis. The method, which we have named DelsGate (Deletions via Gateway), consists of standard PCR of only the 5' and 3' 1 kb gene flanks directly followed by in vitro Gateway cloning and final generation of the circular deletion construct by in vivo recombination in Escherichia coli. For use in DelsGate we have modified a Gateway cloning vector to include selectable markers for transformation of Ascomycetes and the Basidiomycete fungus U. maydis which causes corn smut disease. We have tested the reproducibility of the DelsGate approach by generating deletion constructs for 12 U. maydis genes. Although not tested here, the PCR and transformation steps of DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species. DelsGate has the potential to be universal for all organisms with efficient transformation and homologous recombination systems.     

DelsGate, a robust and rapid gene deletion construction method

With the increasing availability of fungal genome sequences there is great demand for fast, simple high-throughput methods to generate constructs for gene deletion. Here we describe a method that combines PCR and Gateway cloning technology together with use of the I-SceI homing endonuclease to generate precise deletion constructs in a very simple, universal and robust manner in just 2 days. These constructs are then used to produce deletion mutants in the organism of interest following applicable methods for that species. In establishing this protocol we determined empirically that 1 kb was a suitable flank length to facilitate homologous recombination in our species of interest, Ustilago maydis. The method, which we have named DelsGate (Deletions via Gateway), consists of standard PCR of only the 5' and 3' 1 kb gene flanks directly followed by in vitro Gateway cloning and final generation of the circular deletion construct by in vivo recombination in Escherichia coli. For use in DelsGate we have modified a Gateway cloning vector to include selectable markers for transformation of Ascomycetes and the Basidiomycete fungus U. maydis which causes corn smut disease. We have tested the reproducibility of the DelsGate approach by generating deletion constructs for 12 U. maydis genes. Although not tested here, the PCR and transformation steps of DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species. DelsGate has the potential to be universal for all organisms with efficient transformation and homologous recombination systems.     

PCR ligation mutagenesis in transformable streptococci: application and efficiency

PCR ligation mutagenesis is a novel technique that can easily be adapted for many gene modification purposes. Successful application of this versatile technique involves sequence identification of the target gene region, creation of a mutagenic construct consisting of two gene-flanking proximal sequences specifically ligated to a selectable marker, and incorporation of this construct into the genome via genetic transformation and homologous recombination. In this study, we demonstrate the use of PCR, followed by restriction digestion and re-ligation to generate transforming constructs for the rapid deletion of open reading frames in transformable streptococci. Moreover, we characterized the dependence of transformation efficiency for mutant generation on the length of the homologous regions harbored by the mutagenic construct. Our results indicated that PCR ligation mutagenesis could be reliably employed for the systematic generation of gene deletion mutants in both highly transformable Streptococcus mutans and S. pneumoniae. Evaluation of the method showed a strong influence of the length of homologous flanking region on integration efficiency.     

带有报告基因的板栗疫病菌寄生隐赤壳分泌表达突变体库的构建及分泌缺陷突变体的筛选

丝状真菌分泌蛋白与其致病性密切相关,目前对于病原真菌的蛋白胞外分泌途径及其调控机制的报道不多。为建立一个方便高效的真菌分泌蛋白调控途径的遗传研究体系,本研究以植物病原丝状真菌——板栗疫病菌寄生隐赤壳Cryphonectria parasitica为对象,选取分泌表达量最高的两个分泌蛋白的信号肽SP1和SP2,分别构建带有GUS报告基因的分泌蛋白表达载体pCPXBle-SP1-GUS和pCPXBle-SP2-GUS并用于转化板栗疫病菌。选择高效分泌GUS蛋白的转化株SP1-9为出发菌株,利用农杆菌介导的遗传转化技术构建了T-DNA插入突变体库,从576个突变体中筛选到2株GUS分泌表达明显降低的突变体。本研究成功构建了可用于研究丝状真菌蛋白分泌的遗传研究体系,并筛选获得了分泌蛋白缺陷突变体,为深入研究丝状真菌分泌途径及其调控机制奠定了基础。     

Development of new tools for studying gene function in fungi based on the Gateway system

Genomic information of many fungi has been released but large scale functional genomic studies are still limited by a lack of high-throughput methods. The low rates of homologous recombination and low rates of transformation are limiting steps in filamentous fungi, but the molecular tools are also lagging behind. In this paper we describe two new high-throughput functional genomic tools for filamentous fungi that are based on the Gateway technology. One system is the Gateway RNAi vector for fungi that allows gene silencing in a high-throughput manner. The other system is a high-throughput deletion construct system. These systems were tested using the PAC1 gene of Colletotrichum gloeosporioides. Using these types of approaches, large scale functional genomics experiments can be performed in filamentous fungi.     

根癌农杆菌介导真菌遗传转化的研究及应用

根癌农杆菌介导转化法（Agrobacterium tumefaciens-mediated transformation,ATMT）具有转化效率高、遗传稳定、适用范围广等诸多优点,已成为真菌遗传转化研究中的强有力手段,在真菌基因资源开发、真菌性疾病研究和外源蛋白表达研究中发挥巨大作用。本文概述了根癌农杆菌转化法在真菌转化中的研究进展、技术优缺点、转化机制、实验方法和应用现状,着重介绍影响其转化效率的因素并对优化方法进行探讨,展望了该技术在真菌基因资源发掘、基因编辑等方面的应用前景,为今后真菌的遗传转化研究提供参考。     

Targeted gene deletion in Aspergillus fumigatus using microbial machinery and a recyclable marker

The emerging invasive fungal pathogen Aspergillus fumigatus causes very serious infections among immunocompromised patient populations. While the genome of this pathogen has been sequenced, a major barrier to better understanding the complex biology of this eukaryotic organism is a lack of tools for efficient genetic manipulation. To improve upon this, we have generated a new gene deletion system for A. fumigatus using yeast recombinational cloning and Agrobacterium tumefaciens mediated transformation (ATMT) employing a recyclable marker system. This system reduced the time for generating a gene deletion strain in our hands by two-thirds (12 weeks to 3 weeks) using minimal human labor, and we demonstrate that it can be used to efficiently generate multiple gene deletions within a single strain.     

Rapid one-step construction of hairpin RNA

Hairpin RNA (hpRNA) is commonly used for gene-function exploration and gene engineering. In this study, a novel method was developed to construct intron-containing hairpin RNA (ihpRNA) rapidly and efficiently based on Overlap Extension PCR (OE-PCR). This method, Mixed One-step OE-PCR (MOOE-PCR), can amplify two inverted repeats of DNA fragments and a spliceable intron in parallel, and then assemble them to generate ihpRNA constructs in the same tube without the purification of intermediate products. This method required a PCR process of 38-40 cycles and ordinary PCR reagents. A total of 10 ihpRNA constructs were amplified successfully using this method, with the stems ranging from 50 bp to 484 bp in length. Our results suggest that this novel method is a useful strategy for constructing ihpRNA.     

Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans

A method to rapidly generate gene replacement constructs by fusion PCR is described for Aspergillus nidulans. The utility of the approach is demonstrated by green fluorescent protein (GFP) tagging of A. nidulans ndc80 to visualize centromeres through the cell cycle. The methodology makes possible large-scale GFP tagging, promoter swapping, and deletion analysis of A. nidulans.     

有害疣孢霉Hypomyces perniciosus遗传转化体系的建立及其突变体库的构建

有害疣孢霉Hypomyces perniciosus是引起双孢蘑菇Agaricus bisporus湿泡病的病原真菌,目前其致病分子机理尚不清楚,而高效稳定的遗传转化体系和突变体库构建是挖掘和研究病原菌致病基因的基础和有效手段。因此,本实验以高致病力的有害疣孢霉菌株WH001为研究对象,采用冻融法将双元载体pBHt1转入农杆菌AGL-1中,建立并优化根癌农杆菌介导的遗传转化体系,并利用其构建T-DNA插入突变体库。结果表明有害疣孢霉菌株WH001的潮霉素（Hygromycin,Hyg）耐受浓度为250ng/L,当农杆菌侵染液浓度OD₆₀₀=1,侵染时间为30min,乙酰丁香酮（Acetosyringone,AS）浓度为1.5mg/mL,共培养时间为3d时,转化体系效率最高。然后利用该优化体系构建有害疣孢霉的突变体库,通过PCR检测和形态学鉴定获得若干表型发生改变、稳定遗传的T-DNA插入突变体,与原菌种WH001相比,突变体在菌丝形态、生长速率、色素分泌和致病力等方面发生改变。本研究为进一步挖掘有害疣孢霉未知基因功能、解析生物学性状、探讨致病分子机制奠定基础。     

A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs

Alternaria brassicicola causes black spot disease of cultivated Brassicas and has been used consistently as a necrotrophic fungal pathogen for studies with Arabidopsis. In A. brassicicola, mutant generation has been the most rate-limiting step for the functional analysis of individual genes due to low efficiency of both transformation and targeted integration. To improve the targeted gene disruption efficiency as well as to expedite gene disruption construct production, we used a short linear construct with minimal elements, an antibiotic resistance selectable marker gene, and a 250- to 600-bp-long partial target gene. The linear minimal element (LME) constructs consistently produced stable transformants for diverse categories of genes. Typically, 100% of the transformants were targeted gene disruption mutants when using the LME constructs, compared with inconsistent transformation and usually less than 10% targeted gene disruption with circular plasmid disruption constructs. Each mutant displayed a unique molecular signature thought to originate from endogenous exonuclease activities in fungal cells. Our data suggests that a DNA double-stranded break repair mechanism (DSBR) functions to increase targeting efficiency. This method is advantageous for high throughput gene disruption, overexpression, and reporter gene introduction within target genes, especially for asexual filamentous fungi where genetic approaches are unfavorable.     

Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei

Penicillium marneffei is an opportunistic pathogen of humans and displays a temperature dependent dimorphic transition. Like many fungi, exogenous DNA introduced by DNA mediated transformation is integrated randomly into the genome resulting in inefficient gene deletion and position-specific effects. To enhance successful gene targeting, the consequences of perturbing components of the non-homologous end joining recombination pathway have been examined. The deletion of the KU70 and LIG4 orthologs, pkuA and ligD, respectively, dramatically enhanced the observed homologous recombination frequency leading to efficient gene deletion. While ΔpkuA was associated with reduced genetic stability over-time, ΔligD represents a suitable recipient strain for downstream applications and combined with a modified Gateway? system for the rapid generation of gene deletion constructs, this represents an efficient pipeline for characterizing gene function in P. marneffei.     

橡胶树胶孢炭疽菌T-DNA插入突变体库构建及其致病缺陷转化子筛选

【目的】进一步研究橡胶树胶孢炭疽菌致病分子机理。【方法】通过含ILV1基因(具氯嘧磺隆抗性)的pSULF.gfp双元载体农杆菌AGL-1介导进行橡胶树胶孢炭疽菌遗传转化,利用氯嘧磺隆抗性标记筛选转化子,对转化子PCR验证及荧光显微观察;采用离体古铜期橡胶树叶无伤接种法进行致病性缺陷转化子筛选,并对转化子进行遗传稳定性检测。【结果】获得含3 721个转化子的T-DNA插入突变体库,转化效率为150 400个转化子/106孢子,从3 721个转化子中筛选得到致病性缺陷转化子25个;随机选取20个转化子进行遗传稳定性测定,在不含氯嘧磺隆PDA平板上继代培养10次后仍保持氯嘧磺隆抗性,且表型稳定,表明插入外源基因能够稳定遗传。【结论】可以利用根癌农杆菌介导橡胶孢炭疽菌转化,构建橡胶树胶孢炭疽菌T-DNA插入突变体库,筛选致病缺陷突变菌,为进一步研究该菌致病相关基因提供材料。     

根癌农杆菌介导丝状真菌遗传转化的研究进展

根癌农杆菌介导的丝状真菌遗传转化是近年建立起来的一种新方法,该方法和以往的真菌转化体系相比具有转化方法简单、材料易得、效率高以及转化子中T-DNA单拷贝插入比例高等特点。就根癌农杆菌转化的丝状真菌种类、转化的具体过程以及影响转化效率的因素等方面进行了综述,并展望了该方法的应用前景。     

Constructs and methods for high-throughput gene silencing in plants

Gene silencing can be achieved by transformation of plants with constructs that express self-complementary (termed hairpin) RNA containing sequences homologous to the target genes. The DNA sequences encoding the self-complementary regions of hairpin (hp) RNA constructs form an inverted repeat. The inverted repeat can be stabilized in bacteria through separation of the self-complementary regions by a "spacer" region. When the spacer sequence encodes an intron, the efficiency of gene silencing is very high. There are at least three ways in which hpRNA constructs can be made. The construct may be generated from standard binary plant transformation vectors in which the hairpin-encoding region is generated de novo for each gene. Alternatively, generic gene-silencing vectors such as the pHANNIBAL and the pHELLSGATE series can be used. They simply require the insertion of PCR products, derived from the target gene, into the vectors by conventional cloning or by using the Gateway directed recombination system. In this article, we describe and evaluate the advantages of these vectors and then provide the protocols for their efficient use.     

Agrobacterium tumefaciens-mediated transformation in Colletotrichum falcatum and C. acutatum

Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming an effective system as an insertional mutagenesis tool in filamentous fungi. We developed and optimized ATMT for two Colletotrichum species, C. falcatum and C. acutatum, which are the causal agents of sugarcane red rot and pepper anthracnose, respectively. A. tumefaciens strain SK1044, carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein (GFP) gene, was used to transform the conidia of these two Colletotrichum species. Transformation efficiency was correlated with cocultivation time and bacterial cell concentration and was higher in C. falcatum than in C. acutatum. Southern blot analysis indicated that about 65% of the transformants had a single copy of the T-DNA in both C. falcatum and C. acutatum and that T-DNA integrated randomly in both fungal genomes. T-DNA insertions were identified in transformants through thermal asymmetrical interlaced PCR (TAIL-PCR) followed by sequencing. Our results suggested that ATMT can be used as a molecular tool to identify and characterize pathogenicity-related genes in these two economically important Colletotrichum species.     

Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae

Autophagy is a widely conserved intracellular process for degradation and recycling of proteins,organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar in...     

A PCR-based system for highly efficient generation of gene replacement mutants in<Emphasis Type="Italic"> Ustilago maydis</Emphasis>

Ustilago maydis, the causative agent of corn smut disease, is one of the most versatile model systems for the study of plant pathogenic fungi. With the availability of the complete genomic sequence there is an increasing need to improve techniques for the generation of deletion mutants in order to elucidate the functions of unknown genes. Here a method is presented which allows one to generate constructs for gene replacement without the need for cloning. The 5 and 3-regions of the target gene are first amplified by PCR, and subsequently ligated directionally to a marker cassette via two distinct Sfi I sites, providing the flanking homologies needed for homologous recombination in U. maydis. Then the ligation product is used as a template for the amplification of the deletion construct, which can be used directly for transformation of U. maydis. The use of the fragments generated by PCR drastically increases the frequency of homologous recombination when compared to the linearized plasmids routinely used for gene replacement in U. maydis.Communicated by G. Jürgens     

Efficient and high-throughput vector construction and Agrobacterium-mediated transformation of Arabidopsis thaliana suspension-cultured cells for functional genomics

We established a large-scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.