A newly constructed Agrobacterium-mediated transformation system revealed the influence of nitrogen sources on the function of the LaeA regulator in Penicillium chrysogenum

Penicillium chrysogenum is not only an industrially important filamentous fungus for penicillin production, but it also represents as a promising cell factory for production of natural products. Development of efficient transformation systems with suitable selection markers is essential for genetic manipulations in P. chrysogenum. In this study, we have constructed a new and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system with two different selection markers conferring the resistance to nourseothricin and phleomycin for P. chrysogenum. Under the optimized conditions for co-cultivation at 22 °C for 60 h with acetosyringone concentration of 200 μM, the transformation efficiency of the ATMT system could reach 5009 ± 96 transformants per 10⁶ spores. The obtained transformants could be exploited as the T-DNA insertion mutants for screening genes involved in morphogenesis and secondary metabolism. Especially, the constructed ATMT system was applied successfully to generate a knockout mutant of the laeA regulatory gene and relevant complementation strains in a wild strain of P. chrysogenum. Our results indicated that the LaeA regulator controls growth, sporulation, osmotic stress response and antibiotic production in P. chrysogenum, but its function is reliant on nitrogen sources. Furthermore, we showed that the laeA orthologous genes from the citrus postharvest pathogen P. digitatum and from the industrial fungus Aspergillus niger could recover the phenotypic defects in the P. chrysogenum laeA deletion mutant. Conclusively, this work provides a new ATMT system, which can be employed for T-DNA insertional mutagenesis, heterologous gene expression or for molecular inspections of potential genes related to secondary metabolism in P. chrysogenum.     

Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border (RB) end of the T-DNA is largely preserved whereas the left border (LB) end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61 % of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67 % of T-DNA integrations are integrations at a single chromosomal site and 31 % of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma.     

Agrobacterium tumefaciens-Mediated Transformation of the Lichen Fungus,Umbilicaria muehlenbergii

Transformation-mediated mutagenesis in both targeted and random manners has been widely applied to decipher gene function in diverse fungi. However, a transformation system has not yet been established for lichen fungi, severely limiting our ability to study their biology and mechanism underpinning symbiosis via gene manipulation. Here, we report the first successful transformation of the lichen fungus, Umbilicaria muehlenbergii, via the use of Agrobacterium tumefaciens. We generated a total of 918 transformants employing a binary vector that carries the hygromycin B phosphotransferase gene as a selection marker and the enhanced green fluorescent protein gene for labeling transformants. Randomly selected transformants appeared mitotically stable, based on their maintenance of hygromycin B resistance after five generations of growth without selection. Genomic Southern blot showed that 88% of 784 transformants contained a single T-DNA insert in their genome. A number of putative mutants affected in colony color, size, and/or morphology were found among these transformants, supporting the utility of Agrobacterium tumefaciens-mediated transformation (ATMT) for random insertional mutagenesis of U. muehlenbergii. This ATMT approach potentially offers a systematic gene functional study with genome sequences of U. muehlenbergii that is currently underway.     

Agrobacterium-Mediated Transformation and Insertional Mutagenesis in <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> for Investigating Varied Pathogenicity Lifestyles

Colletotrichum acutatum is a cosmopolitan pathogen causing economically important diseases known as anthracnose on a wide range of hosts. This fungus exhibits varied pathogenicity lifestyles and the tools essential to understand the molecular mechanisms are still being developed. The transformation methods currently available for this species for gene discovery and functional analysis involve protoplast transformation and are laborious and inefficient. We have developed a protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of C. acutatum. Using this protocol we were able to transform C. acutatum isolates belonging to different genetic groups and originating from different hosts. The transformation efficiency was up to 156 transformants per 10⁴ conidia, with >70% transformants showing single location/single copy integration of T-DNA. Binary vector pBHt2-GFP was constructed, enabling green fluorescence protein tagging of C. acutatum strains, which will be a useful tool for epidemiology and histopathology studies. The ATMT protocol developed was used to identify putative pathogenicity mutants, suggesting the applicability of this technique for rapid generation of a large panel of insertional mutants of C. acutatum leading to the identification of the genes associated with the varied lifestyles.     

Agrobacterium tumefaciens-Mediated Transformation of Aspergillus fumigatus: an Efficient Tool for Insertional Mutagenesis and Targeted Gene Disruption

Agrobacterium tumefaciens was used to transform Aspergillus fumigatus by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of A. fumigatus conidia and Agrobacterium (1:10 ratio) for 48 h at 24°C resulted in high frequencies of transformation (>100 transformants/10⁷ conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose alb1, a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of Δalb1 mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing alb1 disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in A. fumigatus.     

Development of an activation tagging system for the basidiomycetous medicinal fungus Antrodia cinnamomea

This study describes the development of an efficient and reliable activation tagging system for the medicinal fungus Antrodia cinnamomea. For successful Agrobacterium tumefaciens-mediated transformation, different parameters were considered. The Agrobacterium concentration of 5 × 10⁸ cfu ml⁻¹, 1 mm acetosyringone, 25-d-old mycelia at 0.2 g ml⁻¹, and co-culture period of 6 d were found to be the most optimal conditions for enhancing the transformation efficiency. The mitotic stability of transferred DNA (T-DNA) was demonstrated by growing eight randomly selected putative transformants in malt extract agar medium for five subcultures. Insertion of T-DNA into the genome of transformants was confirmed by PCR and Southern hybridization. Results showed that 88 % of the mutants contained a single T-DNA insertion. Two of the mutants were observed with different triterpenoid profiles compared with the untransformed cultures. Our results suggest a new functional genomics approach to tag the triterpenoid biosynthesis genes in A. cinnamomea.     

Towards a novel efficient T-DNA-based mutagenesis and screening system using green fluorescent protein as a vital reporter in the industrially important fungus <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Agrobacterium-mediated T-DNA transfer has been proven to be an efficient strategy for insertional mutagenesis and elucidation of gene function in filamentous fungi. The implementation of large-scale T-DNA insertional mutagenesis requires the development of high-efficient transformation and high-throughput screening procedures. Here, using green fluorescent protein (GFP) as a vital marker, a highly efficient T-DNA-based mutagenesis and screening system was developed in Trichoderma reesei. The uridine auxotrophic T. reesei M23 as the host was transformed with A. tumefaciens EH105 strain harboring a binary vector pC-OEP, which beared the pyrG gene for primary selection on minimal medium without uridine and the egfp gene for fluorescence-based rapid screening of the mitotically stable transformants. The efficiency of transformation was up to 10–20 transformants per 10⁵ target conidia. Microscopic examination revealed strong GFP expression and fluorescence emission in conidia, growing hyphae and mycelia. An effective and convenient screening procedure using 96-well plates and multilabel counter for fluorescence intensity counting was developed to rapidly identify the T-DNA tagged T. reesei mutants. Furthermore, the presence of T-DNA integration at random sites in the genome was confirmed by Southern blot analysis. This report of the T-DNA-based mutagenesis and rapid screening system using GFP as a vital reporter provides a promising strategy to speeding up the genome-scale T-DNA insertional mutagenesis and functional genomics analysis of this cellulolytic fungus T. reesei.     

An efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for aflatoxin generation fungus <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ～ 60 positive transformants per 10⁶ conidia using our protocol. A small-scale insertional mutant library (～ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.     

稻瘟病菌T-DNA插入方法优化及其突变体分析

优化了农杆菌介导转化稻瘟病菌获得T-DNA插入突变的条件,包括选择转化子的潮霉素B用量,抑制农杆菌的抗生素头孢噻肟钠和羧苄青霉素的配比,不同转化阶段培养基的选择等。转化1×10⁶个孢子平均可获得约500个左右的转化子,PCR和TAILPCR检测表明约85%转化子中含T-DNA插入。对1520个突变体进行形态变异观察,发现菌落颜色突变的有15个;随机取58个突变体进行比较,发现产孢量减少的4个,孢子萌发率降低的8个,附着胞形成率降低的9个;还获得对水稻品种C101LAC（Pi-1）和751127（Pi-9）致病的突变体,为进一步克隆相应的无毒基因奠定了基础。     

Identification of Pathogenicity-Related Genes in the Vascular Wilt Fungus <Emphasis Type="Italic">Verticillium dahliae</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated T-DNA Insertional Mutagenesis

Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.     

Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N

The development of an efficient transformation system is required to alter the expression of symbiosis-regulated genes and to develop insertional mutagenesis in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Vegetative mycelium of this fungus was transformed by Agrobacterium tumefaciens-mediated gene transfer. The selection marker was the hygromycin resistance gene of Escherichia coli (hph) under the control of the gpd promoter from Agaricus bisporus and the CaMV 35S terminator as part of the T-DNA. PCR amplification of hph and Southern blot analyses showed that the genome of the hygromycin-resistant transformants contained the cassette. The latter proved mostly single copy and random integration of part of the transgene into the fungal genome. A. tumefaciens-mediated gene transfer should facilitate future development of insertional mutagenesis, targeted gene disruption and RNA interference technology in L. bicolor.     

A Simplified and efficient method for transformation and gene tagging of Ustilago maydis using frozen cells

Ustilago maydis is an important model fungal organism for diverse studies. Little improvement has been made in the method for its transformation since the PEG-mediated transfection of spheroplasts that was reported more than 20 years ago. We have constructed binary T-DNA vectors carrying Hygromycin and Nourseothricin resistance gene cassettes and have developed a highly efficient method for transformation of this fungus based on Agrobacterium tumefaciens-mediated transformation (ATMT). Through a series of optimization, at least 1 × 10⁴ Hygromycin B resistant colony forming units (CFU) have been achieved on each 90 mm agar plate using 10⁶ sporidia. Optimal pH value for ATMT is approximately 5.6. Approximately 96% Hygromycin B-resistant transformants contain a single-copy T-DNA inserted into the nuclear genome. Analysis of 204 T-DNA flanking sequences showed that 15.2% of them were found in the coding sequences and a further 37.25% within 0.5 kb from the coding sequences at the 5′ UTR or promoter regions. In addition, a method for preparation and preservation of transformation-ready T-DNA donor and receptor cells has been developed allowing gene tagging experiments to be performed on-demand. An initial screening of 5000 mutants resulted in the identification of a putative farnesyl transferase beta subunit and a PRE6 homologue as new players of sexual mating in U. maydis.     

Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin

An endolichenic fungus Xylaria grammica {"type":"entrez-nucleotide","attrs":{"text":"EL000614","term_id":"124922317"}}EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.     

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。     

落叶型大丽轮枝菌T-DNA插入突变体库的构建

采用ATMT技术建立大丽轮枝菌落叶型菌株XJ2008菌株的T-DNA插入突变体文库,共获得6 043个突变体。从中随机挑选104个突变体,以野生型XJ2008菌株为参照,评价其致病性、菌落生长速率、分生孢子及微菌核的产生能力等。结果表明,有12.5%的突变体丧失产孢能力,4.8%的突变体的生长速率显著减慢,8.7%的突变体的生长速率显著加快,12.5%的突变体丧失产生微菌核的能力,47.1%的突变体的致病性显著低于野生型菌株XJ2008,且突变体2-736、2-740、2-745的病情指数分别约为野生型菌株XJ2008的0.184、0.168和0.197倍。该突变体库突变体遗传稳定性好,性状多样性丰富。     

Generation and identification of DNA sequence flanking T-DNA integration site of Trichoderma atroviride mutants with high dichlorvos-degrading capacity

A protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of biocontrol fungus Trichoderma atroviride strain T23 was developed to construct mutants with improved dichlorvos-degradation ability. A transformation frequency of 5 × 10⁻⁶ was achieved. Among 110 genetically stable T-DNA transformants of T. atroviride T23, two transformants, AMT-12 and AMT-28, confirmed by Southern blot analysis to have single-copy inserts of T-DNA, showed an increase in dichlorvos-degradation ability of more than 10% compared to that of the wild type, exhibited similar tolerance to the pesticide, but lower spore formation ability. Five transformants exhibited a reduction in degradation of more than 70%, exhibited wild-type spore formation, and tolerated up to 800 μg/mL of dichlorvos. The left-flanking sequence of the insertion site in AMT-12 was cloned as a 1845-bp fragment and shown to have 89% identity to the DNA from T. atroviride IMI 206040; however, the involvement of this DNA in dichlorvos degradation remains still to be determined. This study can promote both a more efficient isolation of DNA sequence flanking T-DNA integration site in T. atroviride mutants and a more rational utilization of these transformants in dichlorvos degradation.     

Genome-wide analysis of T-DNA integration into the chromosomes of Magnaporthe oryzae

Agrobacterium tumefaciens-mediated transformation (ATMT) has become a prevalent tool for functional genomics of fungi, but our understanding of T-DNA integration into the fungal genome remains limited relative to that in plants. Using a model plant-pathogenic fungus, Magnaporthe oryzae, here we report the most comprehensive analysis of T-DNA integration events in fungi and the development of an informatics infrastructure, termed a T-DNA analysis platform (TAP). We identified a total of 1110 T-DNA-tagged locations (TTLs) and processed the resulting data via TAP. Analysis of the TTLs showed that T-DNA integration was biased among chromosomes and preferred the promoter region of genes. In addition, irregular patterns of T-DNA integration, such as chromosomal rearrangement and readthrough of plasmid vectors, were also observed, showing that T-DNA integration patterns into the fungal genome are as diverse as those of their plant counterparts. However, overall the observed junction structures between T-DNA borders and flanking genomic DNA sequences revealed that T-DNA integration into the fungal genome was more canonical than those observed in plants. Our results support the potential of ATMT as a tool for functional genomics of fungi and show that the TAP is an effective informatics platform for handling data from large-scale insertional mutagenesis.     

Agrobacterium tumefaciens-Mediated Transformation of the Causative Agent of Valsa canker of Apple Tree Valsa mali var. mali

Valsa mali var. mali (Vmm), which is the causative agent of Valsa canker of apple tree, causes heavy damage to apple production in eastern Asia. In this article, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of Vmm and expression of gfp (green fluorescent protein) in this fungus. The transformation system was optimized to a transformation efficiency of approximately 150 transformants/10⁶ conidia, and a library containing over 4,000 transformants was generated. The tested transformants were mitotically stable. One hundred percent hph (hygromycin B phosphotransferase) integration into Vmm was identified by PCR and five single-copy integration of T-DNA was detected in the eighteen transformants by Southern blot. To our knowledge, this is the first report of ATMT of Vmm. Furthermore, this library has been used to identify genes involved in the virulence of the pathogen, and the transformation system may also be useful to the transformation of other species of the genus Valsa.