Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxysporum f. sp. melonis

Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for pathogenicity of Fusarium oxysporum f. sp. melonis. Of the 1,129 REMI transformants tested, 13 showed reduced pathogenicity on susceptible melon cultivars. One of the mutants, FMMP95-1, was an arginine auxotroph. Structural analysis of the tagged site in FMMP95-1 identified a gene, designated ARG1, which possibly encodes argininosuccinate lyase, catalyzing the last step for arginine biosynthesis. Complementation of FMMP95-1 with the ARG1 gene caused a recovery in pathogenicity, indicating that arginine auxotrophic mutation causes reduced pathogenicity in this pathogen.     

Transformation of Corynespora cassiicola by Agrobacterium tumefaciens

The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon (Cucumis melo), and cucumber (Cucumis sativus); the most economically important cucurbit crops grown in China. An understanding of the molecular mechanisms underlying the pathogenicity of C. cassiicola is essential for the development of new strategies to control this disease-causing fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) might be useful to obtain transformants of C. cassiicola, for the ultimate identification of genes involved in pathogenicity. In the present work, we established and optimized an ATMT protocol using A. tumefaciens strain AGL-1 carrying the vector pATMT1 for C. cassiicola. Efficiency of ATMT was 102–148 transformants per 10⁶ conidia and successive subculturing of transformants on non-selective and selective media demonstrated that the integrated transfer (T)-DNA was stably inherited in C. cassiicola transformants. The integration of the hygromycin B phosphotransferase (hph) gene into C. cassiicola was validated by PCR and Southern blot analyses, which revealed that nearly 90 % of the transformants contained single-copy T-DNA. The transformants with altered phenotypes were characterized. Three of these transformants completely lost pathogenicity and other three showed strongly impaired pathogenicity relative to the Cc-GX strain on muskmelon leaves. These results strongly suggest that ATMT may be used as a molecular tool for identifying genes relevant to pathogenicity in the fungus C. cassiicola, an emerging threat to several agronomically important plants in China.     

Transformation of Coniothyrium minitans, a parasite of Sclerotinia sclerotiorum, with Agrobacterium tumefaciens

Coniothyrium minitans is a potential biological control agent of the plant pathogenic fungus Sclerotinia sclerotiorum. In this research, T-DNA insertional transformation of strain ZS-1 of C. minitans mediated by Agrobacterium tumefaciens was obtained, with optimization of spore maturity for transformation. After confirmation by PCR, transformants were subjected to Southern blot analysis, and results showed that more than 82.7% of transformants had single T-DNA insertions, and 12.1% of transformants had two copies T-DNA insertions. The genomic DNA segments of transformants flanking the T-DNA could be amplified from both borders with TAIL-PCR. Four types of mutants were screened and identified from the T-DNA insertional library, which comprised sporulation deficient mutants, pathogenicity deficient mutants, pigment change mutants and antibiotic deficient mutant, and some of the mutants were described; the number and frequency of each type of mutant from the library were calculated, and the frequency of each type is 3.27 x 10(-3), 1.0 x 10(-4), 1.4 x 10(-4), 2.5 x 10(-4), respectively. The successful creation of the T-DNA insertional transformation library may help us to unravel the interaction between a parasite and its host at a molecular level, to clarify the differentiation and development of this fungus, and to analyze and clone functional genes from the biocontrol microorganism in tripartite associations.     

Transformation and mutagenesis of the nematode-trapping fungus Monacrosporium sphaeroides by restriction enzyme-mediated integration (REMI)

In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants microg(-1) per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. 90% of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.     

Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola

We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations with linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme-recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.     

A critical assessment of Agrobacterium tumefaciens-mediated transformation as a tool for pathogenicity gene discovery in the phytopathogenic fungus Leptosphaeria maculans

We evaluated the usefulness and robustness of Agrobacterium tumefaciens-mediated transformation (ATMT) as a high-throughput transformation tool for pathogenicity gene discovery in the filamentous phytopathogen Leptosphaeria maculans. Thermal asymmetric interlaced polymerase chain reaction allowed us to amplify the left border (LB) flanking sequence in 135 of 400 transformants analysed, and indicated a high level of preservation of the T-DNA LB. In addition, T-DNA preferentially integrated as a single copy in gene-rich regions of the fungal genome, with a probable bias towards intergenic and/or regulatory regions. A total of 53 transformants out of 1388 (3.8%) showed reproducible pathogenicity defects when inoculated on cotyledons of Brassica napus, with diverse altered phenotypes. Co-segregation of the altered phenotype with the T-DNA integration was observed for 6 of 12 transformants crossed. If extrapolated to the whole collection, this indicates that 1.9% of the collection actually corresponds to tagged pathogenicity mutants. The preferential insertion into gene-rich regions along with the high ratio of tagged mutants renders ATMT a tool of choice for large-scale gene discovery in L. maculans.     

Enhanced frequency of Beauveria bassiana blastospore transformation by restriction enzyme-mediated integration and electroporation

The techniques of restriction enzyme-mediated integration (REMI) and electroporation (EP) were applied for the first time to improving the blastospore transformation of fungal biocontrol agent Beauveria bassiana for higher frequency. The blastospores from < or =24 h incubation in glucose-mineral medium after shaking conidia for 48 h in Subouraud dextrose broth were found most competent for integrating 1 microg plasmid DNA vectoring the phosphinothricin (PPT) resistance gene bar in 360 microL reaction system containing 100 U HindIII or XbaI. Such blastospores were also most suitable for EP transformation at the optimized field strength of 10 kV cm(-1). The optimized REMI and EP generated averagely 39 and 53 transformants microg(-1) plasmid DNA whereas polyethylene glycol (PEG) integration yielded only 22. All transformants grew well on Czapek's agar containing 400 microg PPT mL(-1) after three rounds of cultivation on the same agar excluding PPT but their parental strain showed no resistance. The target gene inserted into the genomes of 10 transformants randomly taken from REMI or EP transformation was consistently detected by both PCR and Southern blotting. Compared to the PEG integration, REMI and EP enhanced the frequency of the blastospore transformation by 73 and 137%, respectively.     

Identifying pathogenicity genes in the rubber tree anthracnose fungus Colletotrichum gloeosporioides through random insertional mutagenesis

To gain more insight into the molecular mechanisms of Colletotrichum gloeosporioides pathogenesis, Agrobacterium tumefaciens-mediated transformation (ATMT) was used to identify mutants of C. gloeosporioides impaired in pathogenicity. An ATMT library of 4128 C. gloeosporioides transformants was generated. Transformants were screened for defects in pathogenicity with a detached copper brown leaf assay. 32 mutants showing reproducible pathogenicity defects were obtained. Southern blot analysis showed 60.4% of the transformants had single-site T-DNA integrations. 16 Genomic sequences flanking T-DNA were recovered from mutants by thermal asymmetric interlaced PCR, and were used to isolate the tagged genes from the genome sequence of wild-type C. gloeosporioides by Basic Local Alignment Search Tool searches against the local genome database of the wild-type C. gloeosporioides. One potential pathogenicity genes encoded calcium-translocating P-type ATPase. Six potential pathogenicity genes had no known homologs in filamentous fungi and were likely to be novel fungal virulence factors. Two putative genes encoded Glycosyltransferase family 28 domain-containing protein and Mov34/MPN/PAD-1 family protein, respectively. Five potential pathogenicity genes had putative function matched with putative protein of other Colletotrichum species. Two known C. gloeosporioides pathogenicity genes were also identified, the encoding Glomerella cingulata hard-surface induced protein and C. gloeosporioides regulatory subunit of protein kinase A gene involved in cAMP-dependent PKA signal transduction pathway.     

Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation

A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function.     

Transformation of taxol-producing endophytic fungi by restriction enzyme-mediated integration (REMI)

The REMI method was used to introduce the plasmid pV2 harboring the hygromycin B phosphotransferase (hph) gene controlled by the Aspergillus nidulans trpC promoter and the trpC terminator into a taxol-producing endophytic fungus BT2. REMI transformation yielded stable transformants capable of continuing to grow on PDA medium containing 125 mug mL(-1) hygromycin B. The transformation efficiency was about 5-6 transformants mug(-1) plasmid DNA. The presence of hph gene in transformants was confirmed by PCR and Southern blot analyses. To the authors' knowledge, this is the first report on the transformation of taxol-producing endophytic fungi by the REMI technique. This study provides an effective approach for improving taxol production of endophytic fungi by the genetic engineering of taxol biosynthetic pathway genes in the future.     

Degradation of cyanide by Trichoderma mutants constructed by restriction enzyme mediated integration (REMI)

REMI technique was used to construct mutants with improved cyanide-degradation ability from biocontrol fungus Trichoderma koningii strain T30. The plasmid pV2 transformation was confirmed by PCR and Southern blot analysis. Out of 21 transformants, 15 single-copied transformants (71.4%) were found. To compare enzyme activities of rhodanese and cyanide hydratase, T. atroviride T23, T. harzianum T21 and their transformants constructed by REMI previously were also included. Transformants TkB6 (0.173 micromols thiocyanate formed min(-1)mg protein(-1)) from T30 and TaK1 (0.174 micromols thiocyanate formed min(-1)mg protein(-1)) from T23 showed higher rhodanese activity than other transformants and their wild strains. TkA9 (5.53 micromols formamide formed h(-1)mg protein(-1)) from T30 and Th64 (5.35 micromols formamide formed h(-1)mg protein(-1)) from T21 had higher cyanide hydratase activity than other transformants and their wild strains.     

捕食线虫真菌捕食器相关基因标记Ⅰ.REMI转化捕食线虫真菌及转化子特征分析

采用REMI技术转化了少孢节丛孢、指状节丛孢和贵州节丛孢3种捕食线虫真菌,并对转化条件、转化子的形态特征、胞外蛋白酶的分泌差异、抗性稳定性进行了测定,分析了转化子对线虫的致病性和对土壤抑菌作用的忍耐性。     

捕食线虫真菌捕食器相关基因标记Ⅰ.——REMI转化捕食线虫真菌及转化子特征分析

采用REMI技术转化了少孢节丛孢、指状节丛孢和贵州节丛孢3种捕食线虫真菌,并对转化条件、转化子的形态特征、胞外蛋白酶的分泌差异、抗性稳定性进行了测定,分析了转化子对线虫的致病性和对土壤抑菌作用的忍耐性。     

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in medicinal fungus Cordyceps militaris

Cordyceps militaris is an insect-born fungus with various biological and pharmacological activities. The mutant library of C. militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation (ATMT), for the ultimate identification of genes involved in isolate degeneration during fruiting body production. Successful transformation of C.?militaris JM4 by A. tumefaciens AGL-1 carrying vector pATMT1 was performed, with efficiency in the range of 30-600 transformants per 1×10(5) conidia. Acetosyringone (AS) supplement in C. militaris ATMT was not necessary during either precultivation or cocultivation. The transformation procedure was optimised based on the ratios between donor A. tumefaciens and recipient conidia, and pH value of cocultivation media. The integration of the hyg gene into C. militaris genome was determined by PCR and Southern blot analysis, suggesting that 67-88% resulting transformants in cultivation conditions with or without AS were inserted by T-DNA and 55-80% were single-copy. Special mutants with altered phenotypes and growth potentials were characterised. The efficient TAIL-PCR approach was established for identifying T-DNA flanking sequences from C. militaris mutants. The successful construction of the mutant library indicated the usefulness of this approach for functional genetic analysis in this important fungus.     

Screening and identification of insertion mutants from Bipolaris eleusines by mutagenesis based on restriction enzyme-mediated integration

Ophiobolin A is sesterterpenoid-type phytotoxin and may be an important candidate for development of new crop protection and pharmaceutical products. The restriction enzyme-mediated integration (REMI) method was used to introduce the plasmid pSH75 into the ophiobolin A-producing filamentous fungus Bipolaris eleusines. A total of 323 stable transformants were obtained, all of which were capable of growing on potato-dextrose agar medium containing 200?μg?mL(-1) hygromycin B. The transformation frequency was about 4-5 transformants?μg(-1) plasmid DNA. An ophibolin A-deficient transformant (B014) was assessed and the presence of the hph gene in this transformant was confirmed by PCR. The cell-free cultural filtrates of this transformant showed significantly less inhibition on mycelial growth of the fungal pathogen Rhizoctoni solani but little effect on barnyard grass as opposed to that of the wild-type B.?eleusines. There was no detectable amount of ophiobolin A in B014 samples measured with HPLC. This research suggests REMI as a potential approach for improving the production of ophiobolin A by B.?eleusines via genetic engineering to upregulate certain genes responsible for desired biosynthetic pathways.     

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。     

Deletions in the gibberellin biosynthesis gene cluster of Gibberella fujikuroi by restriction enzyme-mediated integration and conventional transformation-mediated mutagenesis.

We induced mutants of Gibberella fujikuroi deficient in gibberellin (GA) biosynthesis by transformation-mediated mutagenesis with the vector pAN7-1. We recovered 24 GA-defective mutants in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme by restriction enzyme-mediated integration (REMI) significantly increased the transformation rate and the rate of single-copy integration events. Of 1,600 REMI transformants, two produced no GAs. Both mutants had multiple copies of the vector pAN7-1 and one had a Southern blot pattern similar to those of the 24 conventionally transformed GA-deficient mutants. Biochemical analysis of the two REMI mutants confirmed that they cannot produce ent-kaurene, the first specific intermediate of the GA pathway. Feeding the radioactively labelled precursors ent-kaurene and GA12-aldehyde followed by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. Southern blot analysis and pulsed-field gel electrophoresis of the transformants using the bifunctional ent-copalyl diphosphate/ent-kaurene synthase gene (cps/ks) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants obtained by conventional insertional transformation. We conclude that transformation procedures with and without the addition of restriction enzymes can lead to insertion-mediated mutations and to deletions and chromosome translocations.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants. Received: 6 March 1998 / Accepted: 25 May 1998     

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.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.