Sulfonylurea resistance as a new selectable marker for the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is a filamentous ascomycete that is pathogenic towards a broad host range of insect targets and is increasingly serving as a model for examining fungal development and host-pathogen interactions. B. bassiana displays a prohibitive level of resistance against many current fungal and/or yeast selection markers including hygromycin, neomycin, and zeocin. A genetic transformation system for B. bassiana based upon the use of a sulfonylurea resistance cassette derived from the Magnaporthe grisea, acetolactate synthase gene (sur) was developed. The transformation frequency ranged from 100–150 transformants per microgram DNA/10⁸ cells and Southern blot analysis indicated that the plasmid vector was randomly integrated into the genome of B. bassiana. In addition, a construct bearing the sur gene and the enhanced green fluorescent protein gene egfp as a visual marker was used to successfully transform B. bassiana. Over 95% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. The described transformation method increases opportunities for the genetic manipulation of B. bassiana.     

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.     

广东省柑橘炭疽病病原菌的形态与分子鉴定

炭疽病是柑橘的主要真菌性病害之一。2007年春,广东省德庆县名优柑橘品种贡柑炭疽病暴发流行。为了明确该县及广东省其他地区柑橘炭疽病菌的种类,为防治提供依据,对采集自广东省6个地区柑橘属10个栽培品种上的炭疽病样本进行病原菌分离,共获得柑橘炭疽病菌单孢菌株75株,对其中10株代表性的菌株进行了种类鉴定。通过培养性状和形态学特征观测、核糖体DNA(rDNA)内转录间区(ITS)序列分析、ITS区特异性引物PCR检测和系统发育关系比较等方面的研究,结果表明:10个柑橘炭疽病菌菌株均为盘长孢状刺盘孢Colletotrichum gloeosporioides,未发现国际上其他国家报道的严重危害柑橘花器和幼果部位的柑橘花后落果病病原菌——尖刺盘孢C.acutatum。     

Direct transformation of a clinical isolate of Candida parapsilosis using a dominant selection marker

Candida parapsilosis is a human pathogenic fungus with increasing importance, particularly in nosocomial infections. For detailed molecular genetic explorations of prototrophic clinical isolates of C. parapsilosis, we developed an efficient transformation system based on a dominant selectable marker. The gene encoding resistance to mycophenolic acid (MPA) was used for selection in yeast transformation. C. parapsilosis cells were transformed with a plasmid vector containing the Candida albicans inosine monophosphate dehydrogenase gene (IMH3) responsible for mycophenolic acid resistance. Transformation was carried out both by electroporation and by the lithium acetate (LiAc) method. The LiAc method resulted in very poor transformation efficiency, while the modified electroporation method yielded a high number of mitotically stable transformants exhibiting unambiguous MPA resistance. Two hundred transformants were analysed for the presence of the C. albicans IMH3(r) gene by polymerase chain reaction. Integration of single or multiple plasmid copies into the genomic DNA of C. parapsilosis was determined by Southern hybridization. To our knowledge, the present study is the first report about a method based on a dominant selectable marker for the transformation of a prototrophic, clinical isolate of C. parapsilosis. The described technique may prove to be an efficient tool for the examination of the biology and virulence of this pathogenic yeast.     

Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per μg of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per μg of vector DNA. However, a 380-fold improvement (3,417 transformants per μg of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.     

Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility

A diverse collection of isolates identified as Colletotrichum acutatum, including a range of fruit-rot and foliar pathogens, was examined for mtDNA RFLPs and RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase (GS) gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene. RFLPs of mtDNA, RFLPs of the 900-bp GS intron and sequence analysis of each intron identified the same seven distinct molecular groups, or clades, within C. acutatum sensu lato. Sequence analysis produced highly concordant tree topologies with definitive phylogenetic relationships within and between the clades. The clades might represent phylogenetically distinct species within C. acutatum sensu lato. Mating tests also were conducted to assess sexual compatibility with tester isolates known to outcross to form the teleomorph Glomerella acutata. Mating compatibility was identified within one clade, C, and between two phylogenetically distinct clades, C and J4. The C clade represented isolates from a wide range of hosts and geographic origins. J4 clade contained isolates from Australia or New Zealand recovered from fruit rot and pine seedlings with terminal crook disease. That isolates in two phylogenetically distinct clades were capable of mating suggests that genetic isolation occurred before reproductive isolation. No other isolates were sexually compatible with the mating testers, which also were in groups C and J4. Certain clades identified by mtDNA and intron analysis (D1, J3 and J6) appeared to represent relatively host-limited populations. Other clades (C1, F1 and J4) contained isolates from a wide range of hosts. Isolates described as C. acutatum f. sp. pineum were clearly polyphyletic.     

潮霉素B抗性为选择标记的整合型表达载体的构建及在米根霉中的遗传转化

为了建立适合米根霉的遗传转化体系,应用重叠延伸PCR的方法构建了以潮霉素B抗性为选择标记的单交换整合型表达载体p BS-hygro-ldh A;分别采用PEG/Ca Cl2介导的原生质体转化、原生质体电转化及萌发孢子电转化的方法将表达载体p BS-hygro-ldh A转化入米根霉AS 3.819菌株中,并研究了菌丝酶解时间、孢子萌发时间以及电转化电场强度对于转化效率的影响;通过荧光定量PCR(q PCR)对米根霉转化子基因组中质粒整合拷贝数进行了检测,并研究了其对米根霉转化子抗性稳定性的影响。实验结果表明成功获得整合了表达载体p BS-hygro-ldh A的米根霉转化子。菌丝酶解140 min产生的原生质体其再生率和转化率最高,原生质体电转化最佳电场强度为13 k V/cm,孢子萌发2.5 h转化率最高,萌发孢子电转化最佳电场强度为14 k V/cm。萌发孢子电转化方法转化率要高于原生质体转化的方法。荧光定量PCR检测结果表明,在一定范围内,高质粒整合拷贝数的米根霉转化子比较稳定。研究建立了用于工业米根霉菌株的遗传转化体系,为米根霉代谢调控研究以及菌种改造工作提供了基础与支持。     

Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus Rosellinia necatrix

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25 degrees C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern blot analysis. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system f or insertional mutagenesis in R necatrix and provide a simple and reliable method for genetic manipulation.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens.

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

Transformation frequencies are enhanced and vector DNA is targeted during retransformation of Leptosphaeria maculans, a fungal plant pathogen.

Summary Leptosphaeria maculans, a fungal pathogen of Brassica spp., was successfully transformed with the vector pAN8-1, encoding phleomycin resistance. Protoplasts of a vigorous Phleo^r transformant were then retransformed using the partially homologous vector, pAN7-1 which encodes hygromycin B resistance. Retransformation of this strain to hygromycin resistance occurred at frequencies that were consistently twofold higher than with the original recipient strain. Linearised pAN7-1 DNA transformed phleomycin-resistant protoplasts at higher frequencies still. All the transformants that were tested retained a phleomycin-resistant phenotype (20/20). Molecular analysis of five transformants generated with circular pAN7-1 DNA indicated that in four cases the pAN7-1 vector had integrated into pAN8-1 sequences. These results suggest that transformation frequencies in L. maculans are limited by the ability of vector DNA to integrate into the genome. Hence, construction of strains with target sites for integration may prove to be a generally useful method for improving transformation frequencies of poorly characterised filamentous fungi, particularly when using heterologous vectors. This would greatly facilitate the identification of genes by transfer of gene libraries and the standardisation of chromosomal location effects in studies of expression of nested promoter deletions.     

Genetic transformation and genes for resistance to abiotic and biotic stresses in Citrus and its related genera

Citrus is the most important tree fruit crop in the world. However, citrus production is affected by both biotic and abiotic stresses, including drought, extreme temperature, salinity, citrus canker, citrus tristeza virus, and Huanglongbing (or citrus greening), among others. These stresses can severely influence growth and development of both rootstocks and/or scions of citrus trees, thus reducing both fruit production and fruit quality. Modern advances in the tools of plant biotechnology and advances in genomics play important roles in understanding how citrus crops can cope with diseases and adverse environmental conditions. Within the last decades, much progress has been made in identifying and cloning of genes involved in resistance to biotic and abiotic stresses as well in genetic transformation of Citrus and its related genera, such as Poncirus trifoliata and Fortunella spp. In this review, we will provide information on advances and insights on genetic transformation protocols as well as availability of characterized genes involved in resistance to both abiotic and biotic stresses. This will be followed with a discussion on perspectives of future developments in this field.     

Characterization of the membrane sensor PenJ for β-lactam antibodies from Bacillus licheniformis by amino acid substitution

Abstract The beta scanner and pattern matching software integrated in the automated microbiology identification system (AMBIS) were used to assess the relatedness of isolates of Colletotrichum acutatum, C. kahawae, C. fragariae, C. gloeosporioides and C. musae based on mitochondrial DNA restriction fragment length polymorphisms. The dendrograms generated reflected the intra-specific variation and inter-specific relationships of these species as determined by other previously reported methods. The adaptability of AMBIS as a rapid method for assessing genetic relatedness of fungal species based on DNA polymorphisms is discussed.     

Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli

A new, heterologous, dominant marker for selection of Aspergillus transformants is described. This marker is based on the Escherichia coli hygromycin B (HmB) phosphotransferase gene (hph). Expression of the hph gene is controlled by A. nidulans gpd and trpC expression signals. An Aspergillus transformation vector was constructed which contains this marker and confers HmB resistance to Aspergillus species. With both A. niger and A. nidulans, transformation frequencies of 5-20 transformants per micrograms vector DNA were obtained. Cotransformation with other vectors was shown to be very efficient in both species, when selection for HmB resistance was applied.     

A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.     

Transforming the sapstaining fungus Ophiostoma piceae with Agrobacterium tumefaciens

A transformation protocol mediated by Agrobacterium tumefaciens is described for the sapstaining fungus Ophiostoma piceae. We compared transformants obtained from Agrobacterium with those obtained from yeast-like cells made into spheroplasts and treated with CaCl2. For all putative transformants analyzed, Southern hybridization confirmed that the hygromycin resistance gene had been integrated into the genomic DNA. While all transformants obtained from the treated spheroplasts had multiple copy vector insertion, 85% of the Agrobacterium-mediated transformants had single copy vector insertion.     

柑桔基因转化新方法的研究

尽管应用基因转化进行果树品种改良已日益引起重视,但是在受体的应用和转化方法上还存在着诸多困难。一方面,大多数果树尚不能从细胞或原生质体再生成完整植株,即使少数已可以再生的果树树种,也并非众多品种都能再生成功,而是存在着明显的基因型差异性。同时,还有再生植株童期过长的问题。另一方面,目前在植物基因转化中常用的两种方法即DNA直接吸入法和农杆菌介导的载体法,若以细胞或原生质体为受体,不仅存在再生困难的问题,而且再生过程费时长;若以叶盘、愈伤组织或珠心组织等为受体,既需要在转化后除去农杆菌,又需要排除转化与非转化组织的嵌合性。这些因素都大大地限制了基因转化在果树中的应用。因此,根据多年生果树的生长特点,建立一种适用的基因转化技术已成当务之急。本文采用农杆菌介导的附体腋芽转化-离体扩繁鉴定的方法,成功地将GUS基因转入沙田柚。结果证明这是一种简单、快速、高效的基因转化方法。     

柑桔遗传转化技术的研究进展

柑桔是当今世界种植面积最大的果树。遗传转化技术的发展为柑桔育种提供了一条全新的途径。该文就柑桔遗传转化的研究进展 ,包括外源DNA的直接转化与农杆菌介导的转化 ,作一简要的综述 ,并对当前研究中存在的问题及今后的研究方向作了进一步的探讨。