Isolation of the transposable element hupfer from the entomopathogenic fungus Beauveria bassiana by insertion mutagenesis of the nitrate reductase structural gene

A transposable element has been isolated from the entomopathogenic fungus Beauveria bassiana by trapping it in the nitrate reductase structural gene, which has been cloned from this species. The element had inserted in the first exon of the nia gene and appeared to have duplicated the sequence TA at the site of insertion. It was 3336?bp long with 30-bp imperfect, inverted, terminal repeats. The element, called hupfer, contained an open reading frame encoding a 321-amino acid protein similar to the IS630- or mariner-Tc1-like transposases, and a residual sequence of about 2?kb which was not significantly similar to any published sequence. There are fewer than five copies of this transposable element present per genome in the fungus.     

The isolation of Ant1, a transposable element from Aspergillus niger

A transposable element has been isolated from the industrially important fungus Aspergillus niger (strain N402). The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. It had inserted at a TA site and appeared to have duplicated the target site upon insertion. The isolated element was found to be 4798 by in length and contained 37-bp inverted, imperfect, terminal repeats (ITRs). The sequence of the central region of the element revealed an open reading frame (designated ORF1) which showed similarity, at the amino acid level, to the transposase of the Tc1/mariner class of DNA transposons. Another sequence within the central region of the element showed similarity to the 3 coding and downstream untranslated region of the amyA gene of A. niger. Sequence homology and structural features indicate that this element, which has been named Ant1 (A. niger transposon 1), is related to the Tc1/mariner group of DNA transposons. Ant1 is apparently present as a single copy in strain N402 of A. niger.     

Three highly divergent subfamilies of the impala transposable element coexist in the genome of the fungus Fusarium oxysporum

The transposable element impala is a member of the widespread superfamily of Tc1-mariner transposons, identified in the genome of the plant pathogenic fungus Fusarium oxysporum. This element is present in a low copy number and is actively transposed in the F.␣oxysporum strain F24 that is pathogenic for melons. The structure of the impala family was investigated by cloning and sequencing all the genomic copies. The analysis revealed that this family is composed of full-length and truncated copies. Four copies contained a long open reading frame that could potentially encode a transposase of 340 amino acids. The presence of conserved functional domains (a nuclear localisation signal, a catalytic DDE domain and a DNA-binding domain) suggests that these four copies may be autonomous elements. Sequence comparisons and phylogenetic analysis of the impala copies defined three subfamilies, which differ by a high level of nucleotide polymorphism (around 20%). The coexistence of these divergent subfamilies in the same genome may indicate that the impala family is of ancient origin and/or that it arose by successive horizontal transmission events. Received: 2 December 1997 / Accepted: 28 April 1998     

Hemocytic defense response to the entomopathogenic fungus Beauveria bassiana in the migratory grasshopper Melanoplus sanguinipes

Hemocytic defense response of the migratory grasshopper, Melanoplus sanguinipes (Fab.), to conidia of the white muscardine fungus, Beauveria bassiana (Bals.) Vuill., was studied using phase-contrast photomicroscopy, hemocyte counting and hemocyte aggregation or nodule formation. Grasshoppers were injected with an aqueous suspension of conidia. Adherence of B. bassiana conidia to granulocytes occurred within 10 min and the conidia were encapsulated by these hemocytes 6 h postinjection. Hemocytopenia was accompanied by nodule formation after injection of B. bassiana conidia into grasshoppers. The conidia germinated within the nodules and grew into hyphal forms within the hemolymph 12 to 24 h postinjection. We conclude that B. bassiana competes well with nodule formation by hemocytes of M. sanguinipes and often escapes complete encapsulation.

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Résumé Le mécanisme de défense du hémocyte de la sauterelle migratrice Melanoplus sanguinipes envers le champignon pathogène Beauveria bassiana a été etudié a l'aide du photomicroscope a contrase de phase, par dénombrement des hemocytes, ainsi que des nodules formées par l'agrégation des hémocytes. L'adhérence des conidies de B. bassiana aux hemocytes a été observée 10 min après l'injection et leur encapsulement après 1 h. Une baisse des taux d'hémocytes a fait suite a la formation de nodules apres l'injection de conidies dans les sauterelles. Après le déclin initial du taux des hémocytes, une hausse s'est produite dans les sauterelles auquelles on a injecté 10⁶ conidies. Les conidies ont germé dans les nodules et la croissance du mycélium s'est produite dans l'hémolymphe 24 h après avoir injecté. Cette étude a revelé que M. sanguinipes peut exercer temporairement un mécanisme de défense cellulaire contre des conidies fongiques a une concentration de 10⁶ conidies.

     

球孢白僵菌种内比较线粒体基因组学分析

【目的】明确球孢白僵菌种内线粒体基因组的分化程度。【方法】从GenBank下载已知的球孢白僵菌6个菌株线粒体基因组序列,详细分析基因组的组成结构,比较外显子区、内含子区和基因间区的碱基变异情况,分析菌株间的系统发育关系。【结果】球孢白僵菌不同菌株的线粒体基因组大小为28.8–32.3 kb,都有14个常见的核心蛋白编码基因、2个rRNA基因和25个tRNA基因,具有很强的共线性关系。但是,不同菌株含有的线粒体内含子数目存在差异(2–5个/菌株),在cox1、cox2和nad1基因中表现出内含子插入/缺失多态性,这是导致线粒体基因组大小变化的主要因素。对外显子、内含子和基因间区的碱基变异情况进行分析,发现内含子和基因间区相对变异较大,而外显子区相对变异较小。系统发育分析发现,这些球孢白僵菌菌株以很高的支持度聚在一起,具有相同内含子分布规律的菌株也具有较近的聚类关系。【结论】本研究首次报道球孢白僵菌因内含子数目不同、插入缺失突变和单核苷酸变异等在线粒体基因组上表现出较大程度的遗传分化,为认识真菌种内线粒体基因组分化提供了新的证据。     

Regulation of the transposable element mariner

The mariner/ Tc1 superfamily of transposable elements is widely distributed in animal genomes and is especially prevalent in insects. Their wide distribution results from their ability to be disseminated among hosts by horizontal transmission and also by their ability to persist in genomes through multiple speciation events. Although a great deal is known about the molecular mechanisms of transposition and excision, very little is known about the mechanisms by which transposition is controlled within genomes. The issue of mariner/Tc1 regulation is critical in view of the great interest in these elements as vectors for germline transformation of insect pests and vectors of human disease. Several potentially important regulatory mechanisms have been identified in studies of genetically engineered mariner elements. One mechanism is overproduction inhibition, in which excessive wild-type transposase reduces the rate of excision of a target element. A second mechanism is mediated by certain mutant transposase proteins, which antagonize the activity of the wild-type transposase. The latter process may help explain why the vast majority of MLEs in nature undergo ‘vertical inactivation’ by multiple mutations and, eventually, stochastic loss. Another potential mechanism of regulation may result from transposase titration by defective elements that retain their DNA binding sites and ability to transpose. There is also evidence that some mariner/Tc1 elements can be mobilized in a type of hybrid dysgenesis. This revised version was published online in August 2006 with corrections to the Cover Date.     

The Arabidopsis transposable element Tag1 is widely distributed among Arabidopsis ecotypes

Tag1 is an autonomous transposable element (3.3 kb in length) first identified as an insertion in the CHL1 (NRT1) gene of Arabidopsis thaliana. Tag1 has been found in the Landsberg erecta ecotype of A. thaliana but not in Columbia or WS. In this paper, 41 additional ecotypes were examined for the presence of Tag1. Using an internal Tag1 fragment as probe, we found that DNA from 19 of the 41 ecotypes strongly hybridized to Tag1. Almost all of the Tag1-containing ecotypes had only one or two copies of Tag1 per haploid genome, as determined by Southern blot analysis. The only exception, Bf-1 from Bretagny-sur-Orge, France, had four copies. Two ecotypes, Di-G and S96, gave identical Southern blot patterns to that of Landsberg erecta and were subsequently shown to contain Tag1 at the same two positions found in Landsberg erecta (loci designated as Tag1-2 and Tag1-3). Two other ecotypes, Ag-0 and Lo-1, had a Tag1 element located at Tag1-2 but not at Tag1-3. The distance between these two loci was determined to be 0.37 cM. Analysis of DNA from two related species, A. griffithiana and A. pumila, showed that both species contain sequences that hybridize to Tag1 and that could be amplified with an oligonucleotide specific to the terminal inverted repeats of Tag1. These results show that Tag1 and related elements are present, and may be useful for insertional mutagenesis, in many A. thaliana ecotypes and several Arabidopsis species. Received: 18 August 1997 / Accepted: 9 October 1997     

Isolation of the facA (acetyl-CoA synthetase) gene of Phycomyces blakesleeanus

A 5.6 kb DNA fragment from the fungus Phycomyces blakesleeanus has been cloned and sequenced. The fragment contains a gene that probably codes for the enzyme acetyl-coenzyme A synthetase (facA). The amino acid sequence deduced for the P. blakesleeanns protein is highly homologous to those of acetyl-coA-synthetases from other organisms. When placed under the control of a constitutive promoter from Aspergillus nidulans, the cloned gene complemented a facA mutation of this organism. In P. blakesleeanns, the expression of facA is induced by acetate.     

NirA1基因对球孢白僵菌生长、抗逆及毒力的影响

[目的] 探究参与硝酸盐同化的一个特异转录因子NirA1对球孢白僵菌（Beauveria bassiana）生长、抗逆及毒力的影响。[方法] 利用RT-PCR检测球孢白僵菌NirA1基因在不同培养基中的表达特征。通过构建敲除突变株ΔNirA1、超量表达OENirA1及回复互补菌株ComNirA1,解析NirA1在球孢白僵菌生长发育、逆境胁迫反应和致病昆虫中的功能。[结果] NirA1基因在营养较贫瘠的CZB培养基中表达量高于丰富培养基PDB和SDB。敲除NirA1导致了菌株在人工培养基上的生长缓慢,对NaNO₂和Urea的利用效率降低。RT-PCR显示,ΔNirA1中硝酸盐转运蛋白基因NrtA的表达较野生型显著下调。在CZA、PDA和1/4 SDAY培养基中,其分生孢子产量分别较野生型降低了21.6%、16.2%和25.6%。逆境胁迫分析发现,在高温（32℃）,高渗（1 mol/L NaCl）和H₂O₂处理后,ΔNirA1菌落生长抑制率较野生型分别降低29.0%、25.2%及49.0%;但在添加SDS和刚果红（CR）的处理中,突变体菌落的生长抑制率分别较野生型升高34.1%和96.2%。因此,缺失NirA后,菌株对SDS和CR更加敏感,但对高温、H₂O₂和NaCl的耐受性提高。以3龄的大蜡螟为试虫的毒力测定表明,敲除NirA1基因导致菌株的毒力提高,半致死时间较野生型提前17.4%。[结论] NirA1在球孢白僵菌生长过程中参与了氮源的利用,并在球孢白僵菌的菌落生长、分生孢子生产、胁迫反应和致病宿主过程中发挥了重要作用。     

The transposable element impala, a fungal member of the Tc1-mariner superfamily

A new transposable element has been isolated from an unstable niaD mutant of the fungus Fusarium oxysporum. This element, called impala, is 1280 nucleotides long and has inverted repeats of 27 bp. Impala inserts into a TA site and leaves behind a footprint when it excises. The inserted element, impala-160, is cis-active, but is probably trans-defective owing to several stop codons and frameshifts. Similarities exist between the inverted repeats of impala and those of transposons belonging to the widely dispersed mariner and Tc1 families. Moreover, translation of the open reading frame revealed three regions showing high similarities with Tc1 from Caenorhabditis elegans and with the mariner element of Drosophila mauritiana. The overall comparison shows that impala occupies an intermediate position between the mariner and Tcl-like elements, suggesting that all these elements belong to the same superfamily. The degree of relatedness observed between these elements, described in different kingdoms, raises the question of their origin and evolution.     

Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3 Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Evaluation of the fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes), a potential biological control agent of Lutzomyia longipalpis (Diptera, Psychodidae)

Visceral leishmaniasis is a zoonosis whose primary vector in Brazil is the sandfly Lutzomyia longipalpis Lutz & Neiva. Presently, efforts to control the vector have not been effective in reducing the prevalence of disease. A possible alternative to current strategies is the biological control of the vector using entomopathogenic fungi. This study evaluates the effects of the fungus, Beauveria bassiana (Bals.) Vuilleman, in different developmental stages of L. longipalpis. Five concentrations of the fungus were utilized ranging from 10⁴ to 10⁸ conidia/ml, with appropriate controls. The unhatched eggs, larvae and dead adults exposed to B. bassiana were sown to reisolate the fungus. The fungus was subsequently identified by polymerase chain reaction (PCR) and DNA sequencing. Exposure to B. bassiana reduced the number of eggs that hatched by 59% (P < 0.01). The longevity of infected adults was 5 days, significantly lower than that of the negative control which was 7 days (P < 0.001). The longevity of the adult sandfly exposed to the positive chemical (pyrethroid, cypermetherin) control was less than 1 day. The effects of fungal infection on the hatching of eggs laid by infected females were also significant and dose-dependent (P < 0.05). With respect to fungal post-infection growth parameters, only germination and sporulation were significantly higher than the fungi before infection (P < 0.001). The identity of the reisolated fungus was confirmed by automated DNA sequencing post-passage in all insect stages. These data show that B. bassiana has good pathogenic potential, primarily on L. longipalpis larvae and adults. Consequently, the use of this fungus in sandfly control programs has potential in reducing the use of chemical insecticides, resulting in benefits to humans and the environment.     

球孢白僵菌Hog1 MAPK同源基因BbHog1的克隆及特征分析

根据几种丝状真菌Hog1 MAPK的保守氨基酸序列设计简并引物,从昆虫病原真菌球孢白僵菌中扩增出MAPK同源基因的部分片段,然后利用YADE法延伸该片段的上、下游邻接序列,获得MAPK编码基因的全长序列,命名为BbHog1。序列分析表明,该基因编码358个氨基酸的多肽,推测分子量为40.99kDa,等电点为5.49。BbHog1含有MAPK保守的蛋白激酶激活域(TGY),序列与粗糙脉孢霉os-2(AF297032)、烟曲霉OSM1(XM_747571)、隐球酵母HOG1(AF243531)和酿酒酵母Hog1(Z73285)等Hog1 MAPK高度同源,相似性分别为94%、89%、83%和80%。系统聚类结果表明,BbHog1与酵母Hog1 MAPK同源。Southern杂交表明,BbHog1在球孢白僵菌基因组中以单拷贝形式存在。Northern分析表明,BbHog1在高渗、亚高温和营养胁迫等条件下的表达明显升高。由此推测,BbHog1基因可能与球孢白僵菌对逆境胁迫的适应性调节密切相关。     

Intragenic location of an unstable insertion element within gene b5 of the fungus Ascobolus immersus

Summary A fine structure map of gene b5 has been established in Ascobolus immersus and the unstable mutant site b5-301 (phenotype: ascospore coloration) has been found to map within this gene. This map was constructed using seven b5 mutants induced by ICR170 and is based on the additivity of recombinant frequencies and confirmed by three point tests. The unstable site 301 is located between the induced sites. In particular, mutant 249 is located to the left of site 301, whereas sites 601 and 754 are located to the right.Previous studies showed that the inducing gene of mutant b5-301 reversions are either closely linked to the b5 locus or within it in certain strains. The study of asci resulting from reciprocal recombination between unstable mutant site and several induced mutant sites showed that neither crossovers located on the left nor the right of site 301, separate the unstable site from the inducing gene. Thus, the inducing gene was found to map within gene b5 as did the inducible site.These results constitute a genetic argument showing the presence of an insertion element. In this case, the insertion structure contains at least the integration site (inducible site) and the inducing gene which allows the excision.     

Infection of Blissus antillus (Hemiptera: Lygaeidae) Eggs by the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana

This study determined the pathogenicity and virulence of Beauveria bassiana and Metarhizium anisopliae to eggs of the chinch bug Blissus antillus (Hemiptera: Lygaeidae). Eggs were inoculated under laboratory conditions by immersion in concentrations of 1 × 10⁴ and 5 × 10⁶ conidia/ml. Inoculated eggs were kept under controlled conditions. Evaluations were carried out daily for 20 days. M. anisopliae isolates were highly virulent to eggs, even at 1 × 10⁴ conidia/ml. All B. bassiana isolates tested were considered to be of low virulence or avirulent. The most virulent isolate tested was ESALQ 818 (M. anisopliae), which caused 96.7% infection, when eggs were immersed in suspensions of 1 × 10⁴ conidia/ml. Conidial production on infected eggs was observed to be highest for M. anisopliae isolate CG144, with a mean value of 11.6 × 10⁵ conidia/ml/egg. Infection of Blissus eggs oviposited on plant stems was greater when M. anisopliae isolate CG144 was formulated in mineral oil (63.5% mortality) than when formulated in Tween 80 (27.1% mortality).