Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi
TL;DR: The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi.
Abstract: The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Here, we present a USER Friendly cloning based technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium tumefaciens and protoplast based transformation technologies. The system has been tested by the construction of vectors for targeted replacement of 17 genes and overexpression of 12 genes in Fusarium graminearum. The results show that four fragment vectors can be constructed in a single cloning step with an average efficiency of 84% for gene replacement and 80% for targeted overexpression. The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi.
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TL;DR: It is demonstrated that Ave1 activates Ve1-mediated resistance and markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis, and that Verticillium acquired Ave1 from plants through horizontal gene transfer.
Abstract: Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum, but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. lycopersici. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens.
461 citations
Cites methods from "Efficient four fragment cloning for..."
...dahliae were generated by cloning of the Ave1 flanking sequences in pRF-HU2 (51)....
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TL;DR: It is shown that extensive chromosomal rearrangements in the strictly asexual plant pathogenic fungus Verticillium dahliae establish highly dynamic lineage-specific genomic regions that act as a source for genetic variation to mediate aggressiveness.
Abstract: Sexual recombination drives genetic diversity in eukaryotic genomes and fosters adaptation to novel environmental challenges. Although strictly asexual microorganisms are often considered as evolutionary dead ends, they comprise many devastating plant pathogens. Presently, it remains unknown how such asexual pathogens generate the genetic variation that is required for quick adaptation and evolution in the arms race with their hosts. Here, we show that extensive chromosomal rearrangements in the strictly asexual plant pathogenic fungus Verticillium dahliae establish highly dynamic lineage-specific (LS) genomic regions that act as a source for genetic variation to mediate aggressiveness. We show that such LS regions are greatly enriched for in planta-expressed effector genes encoding secreted proteins that enable host colonization. The LS regions occur at the flanks of chromosomal breakpoints and are enriched for retrotransposons and other repetitive sequence elements. Our results suggest that asexual pathogens may evolve by prompting chromosomal rearrangements, enabling rapid development of novel effector genes. Likely, chromosomal reshuffling can act as a general mechanism for adaptation in asexually propagating organisms.
288 citations
Cites methods from "Efficient four fragment cloning for..."
...PCR products were subsequently cloned into pRF-HU2 (Frandsen et al. 2008)....
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TL;DR: The complete genome of P. digitatum, the first of a phytopathogenic Penicillium species, is a valuable tool for understanding the virulence mechanisms and host-specificity of this economically important pest.
Abstract: Penicillium digitatum is a fungal necrotroph causing a common citrus postharvest disease known as green mold. In order to gain insight into the genetic bases of its virulence mechanisms and its high degree of host-specificity, the genomes of two P. digitatum strains that differ in their antifungal resistance traits have been sequenced and compared with those of 28 other Pezizomycotina. The two sequenced genomes are highly similar, but important differences between them include the presence of a unique gene cluster in the resistant strain, and mutations previously shown to confer fungicide resistance. The two strains, which were isolated in Spain, and another isolated in China have identical mitochondrial genome sequences suggesting a recent worldwide expansion of the species. Comparison with the closely-related but non-phytopathogenic P. chrysogenum reveals a much smaller gene content in P. digitatum, consistent with a more specialized lifestyle. We show that large regions of the P. chrysogenum genome, including entire supercontigs, are absent from P. digitatum, and that this is the result of large gene family expansions rather than acquisition through horizontal gene transfer. Our analysis of the P. digitatum genome is indicative of heterothallic sexual reproduction and reveals the molecular basis for the inability of this species to assimilate nitrate or produce the metabolites patulin and penicillin. Finally, we identify the predicted secretome, which provides a first approximation to the protein repertoire used during invasive growth. The complete genome of P. digitatum, the first of a phytopathogenic Penicillium species, is a valuable tool for understanding the virulence mechanisms and host-specificity of this economically important pest.
202 citations
Cites background or methods from "Efficient four fragment cloning for..."
...The two flanking fragments were introduced into pRFHU2 following the USER protocol described by [87] and the resulting plasmid was introduced into E....
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...This plasmid derives from plasmid pRFHU2 [87] and contains the flanking regions of the two genes surrounding the hygromycin resistant cassette in the T-DNA region of the plasmid....
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TL;DR: Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection and it was demonstrated that neither patulin nor citrinin are required by P. expandum to successfully infect apples.
Abstract: The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples. Li et al. ( MPMI-12-14-0398-FI ) reported similar results and conclusions in their recently accepted paper.
179 citations
Cites methods from "Efficient four fragment cloning for..."
...Both DNA fragments, corresponding to promoter and terminator regions, and the digested pRFHU2 vector (Frandsen et al. 2008) were mixed together and were treated with the USER enzyme (New England Biolabs) to obtain plasmids pRFHU2-patK, pRFHU2-patL, pRFHU2-patN, and pRFHU2-pksCT....
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TL;DR: This chapter presents a general protocol for converting any vector into a USER-compatible vector, together with protocols for both USER cloning and USER fusion.
Abstract: The explosive development of the field of molecular biology has led to the need for simpler and more efficient cloning techniques. These requirements are elegantly met by the ligation-free cloning technique called USER cloning. USER cloning is suitable not only for everyday and high-throughput cloning but also for the one-step construction of complex DNA constructs, which can be achieved in a variant called USER fusion. In this chapter, we present a general protocol for converting any vector into a USER-compatible vector, together with protocols for both USER cloning and USER fusion.
162 citations
Cites background from "Efficient four fragment cloning for..."
...the number of nts lying between the restriction and nicking site may be increased and/or varied, which might be beneficial when having more than one USER cassette in a single vector and attempting simultaneous PCR product insertion into the different sites (13)....
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References
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TL;DR: Replacement of aurR1 with hygB shows that it encodes a positively acting transcription factor that is required for the full expression of PKS12, aurJ, aurF, gip1 and FG02329.1, which belong to the gene cluster, and bio‐ and chemoinformatics combined with chemical analysis of replacement mutants indicate a five‐step enzyme catalysed pathway for the biosynthesis of aurofusarin.
Abstract: Fungal polyketide biosynthesis typically involves multiple enzymatic steps and the encoding genes are often found in gene clusters. A gene cluster containing PKS12, the polyketide synthase gene responsible for the synthesis of the pigment aurofusarin, was analysed by gene replacement using Agrobacterium tumefaciens-mediated transformation to determine the biosynthesis pathway of aurofusarin. Replacement of aurR1 with hygB shows that it encodes a positively acting transcription factor that is required for the full expression of PKS12, aurJ, aurF, gip1 and FG02329.1, which belong to the gene cluster. AurR1 and PKS12 deletion mutants are unable to produce aurofusarin and rubrofusarin. Bio- and chemoinformatics combined with chemical analysis of replacement mutants (DeltaaurJ, DeltaaurF, Deltagip1, DeltaaurO and DeltaPKS12) indicate a five-step enzyme catalysed pathway for the biosynthesis of aurofusarin, with rubrofusarin as an intermediate. This links the biosynthesis of naphthopyrones and naphthoquinones together. Replacement of the putative transcription factor aurR2 results in an increased level of rubrofusarin relative to aurofusarin. Gip1, a putative laccase, is proposed to be responsible for the dimerization of two oxidized rubrofusarin molecules resulting in the formation of aurofusarin.
167 citations
"Efficient four fragment cloning for..." refers methods in this paper
...tumefaciens mediated transformation, PCR based screening methods [28,15] and quantitative Real-Time PCR to determine the gene copy number [29] can be perfected to give a realistic high throughput system for large scale functional studies of genes in filamentous fungi....
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...The HRS's are identical to the sequences surrounding the target locus in the genome and are typically amplified by PCR. Homologous recombination between the vector DNA and the genome results in a replacement of the target DNA with the selection marker....
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...We have shown that ATMT is an excellent method for site-directed genome modifications in F. graminearum, with 200 transformants per 106 spores, with 60% targeted integrations, using 2 kb HRS's [15]....
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...graminearum, with 200 transformants per 106 spores, with 60% targeted integrations, using 2 kb HRS's [15]....
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...This requires an efficient and reliable method for directional four fragment cloning, allowing fusion of the two HRS's on each side of the selection marker gene and to the vector backbone....
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TL;DR: It is shown that a gene cluster, including the F. graminearum PKS12 gene, is responsible for the biosynthesis of aurofusarin, and targeted mutagenesis of F. pseudograminearums showed an albino phenotype.
166 citations
"Efficient four fragment cloning for..." refers methods in this paper
...The gDNA was purified following the procedure described by Malz [23]....
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TL;DR: It is shown that endo VIII removes from double-stranded oligodeoxyribonucleotides the stable oxidative products of cytosine, 5-Hydroxycytosine and 5-hydroxyuracil, in addition to amino acid sequence homology.
158 citations
"Efficient four fragment cloning for..." refers background in this paper
...The presence of an abasic site in the DNA permit the DNA glycosylase-lyase Endo VIII to break the phosphodiester backbone at both the 3' and 5' sides of the abasic position, resulting in a single strand break [31]....
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TL;DR: Gene disruption and fermentation studies in F. sporotrichioides indicate that TRI13 is required for the addition of the C-4 oxygen of T-2 toxin, but that TRI14 is not required for trichothecene biosynthesis.
154 citations
"Efficient four fragment cloning for..." refers methods in this paper
...graminearum, the PH-1 wild type strain was grown in liquid YPG medium [22] for three days at 25°C in darkness with stir at 150 rpm....
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TL;DR: This review compares the available commercial and open-source recombinational cloning methods with regard to their use in creating comprehensive open reading frame (ORF) clone collections with an emphasis on the properties requisite to use in a high-throughput setting.
Abstract: The creation of genome-scale clone resources is a difficult and costly process, making it essential to maximize the efficiency of each step of clone creation. In this review, we compare the available commercial and open-source recombinational cloning methods with regard to their use in creating comprehensive open reading frame (ORF) clone collections with an emphasis on the properties requisite to use in a high-throughput setting. The most efficient strategy to the creation of ORF clone resources is to build a master clone collection that serves as a quality validated source for producing collections of expression clones. We examine the methods for recombinational cloning available for both the creation of master clones and their conversion into expression clones. Alternative approaches to creating clones involving mixing of cloning methods, including gap-repair cloning, are also explored.
134 citations
"Efficient four fragment cloning for..." refers methods in this paper
...Examples are the Xi-cloning, InFusion cloning, Ligase independent cloning (LIC-PCR), Recombinational cloning and USER Friendly cloning techniques [16-20]....
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