Mutational analysis of the glycosylphosphatidylinositol (GPI) anchor pathway demonstrates that GPI-anchored proteins are required for cell wall biogenesis and normal hyphal growth in Neurospora crassa

Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal "cell-within-a-cell" phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa.     

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.     

Overexpression of a 3-ketoacyl-CoA thiolase in Leptosphaeria maculans causes reduced pathogenicity on Brassica napus

Agrobacterium tumefaciens-mediated random mutagenesis was used to generate insertional mutants of the fungus Leptosphaeria maculans. Of 91 transformants screened, only one (A3) produced lesions of reduced size on cotyledons of canola (Brassica napus). Genes flanking the T-DNA insertion had the best matches to an alcohol dehydrogenase class 4 (ADH4)-like gene (Adh4L) and a 3-ketoacyl-CoA thiolase gene (Thiol) and were expressed in mutant A3 in vitro and in planta at significantly higher levels than in the wild type. This is the first report of a T-DNA insertion in fungi causing increased gene expression. Transformants of the wild-type isolate expressing both Adh4L and Thiol under the control of a heterologous promoter had similar pathogenicity to mutant A3. Ectopic expression of only thiolase resulted in loss of pathogenicity, suggesting that thiolase overexpression was primarily responsible for the reduced pathogenicity of the A3 isolate. The thiolase gene encoded a functional protein, as shown by assays in which a nontoxic substrate (2, 4 dichlorophenoxybutyric acid) was converted to a toxic product. The use of a translational fusion with a reporter gene showed thiolase expressed in organelles that are most likely peroxisomes.     

Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages

Glycosylphosphatidylinositol (GPI)-anchored proteins are involved in cell wall integrity and cell-cell interactions. We disrupted the Candida albicans homologue of the Saccharomyces cerevisiae GPI7/LAS21 gene, which encodes a GPI anchor-modifying activity. In the mutant and on solid media, the yeast-to-hyphae transition was blocked, whereas chlamydospore formation was enhanced. However, the morphogenetic switch was normal in liquid medium. Abnormal budding patterns, cytokinesis and cell shape were observed in both liquid and solid media. The cell wall structure was also modified in the mutants, as shown by hypersensitivity to Calcofluor white. In vitro and in vivo assays revealed that the mutant interacted with its host in a modified way, resulting in reduced virulence in mice and reduced survival in the gastrointestinal environment of mice. The mitogen-activated protein (MAP) kinase pathway of macrophages was downregulated by the wild-type cells but not by the DeltaCagpi7 null strains. In agreement with this abnormal behaviour, mutant cells were more sensitive to the lytic action of macrophages. Our results indicate that a functional GPI anchor is required for full hyphal formation in C. albicans, and that perturbation of the GPI biosynthesis results in hypersensitivity to host defences.     

Analysis of loss of pathogenicity mutants reveals that repeat-induced point mutations can occur in the Dothideomycete Leptosphaeria maculans

Restriction enzyme mediated insertional mutagenesis using a plasmid, pUCATPH, that confers hygromycin resistance, generated loss-of-pathogenicity mutants of Leptosphaeria maculans, the fungus that causes blackleg disease of Brassica napus. Of 516 L. maculans transformants analysed, 12 were pathogenicity mutants. When eight of these mutants were crossed to an isolate that attacks B. napus, cosegregation of pUCATPH sequences and loss of pathogenicity was not observed, suggesting that these mutations were not linked to plasmid sequences. In seven of eight crosses analysed, progeny with the hygromycin resistance gene were hygromycin-sensitive. Sequence analysis of an amplified fragment of pUCATPH in six clones derived from one 'silenced' progeny showed mutation of GC to AT on one DNA strand, reminiscent of repeat-induced point mutation (RIP) in Neurospora crassa. One loss-of-pathogenicity mutant had pUCATPH inserted in the promoter of a gene with an open reading frame of 529 amino acids that had no database match. Reintroduction of a wild-type copy of the gene to this mutant restored the ability to form lesions on cotyledons of B. napus.     

The localization change of Ybr078w/Ecm33, a yeast GPI-associated protein,from the plasma membrane to the cell wall,affecting the cellular function

The YBR078W/ECM33 gene of Saccharomyces cerevisiae encodes a glycosylphosphatidylinositol (GPI)-attached protein and its disruptant strain exhibited a temperature-sensitive (ts) growth defect. A HA-tagged Ybr078w protein, which complemented the ts growth phenotype of the ybr078wdelta strain, was predominantly located on the plasma membrane by GPI anchoring. To examine the requirement of the GPI anchoring on the plasma membrane for the function, the omega-minus region of Ybr078w was replaced with those of Ydr534c/Fit1 and Ynl327w/Egt2, which are known as GPI-dependent cell wall proteins. The replacement induced the change in localization of the mutant proteins from the plasma membrane to the cell wall and the mutant proteins lost the function to complement the ts cell growth defect of the ybr078wdelta strain. In addition, a similar result was obtained in a mutant protein, where the authentic SKKSK sequence at the omega-5 to omega-1 site of Ybr078w was replaced with a synthetic ISSYS sequence. It is concluded that the GPI anchoring on the plasma membrane is required for the Ybr078w function.     

Glycosylphosphatidylinositol (GPI) anchor is required in Aspergillus fumigatus for morphogenesis and virulence

In yeast, glycosylphosphatidylinositol (GPI) is essential for viability and plays an important role in biosynthesis and organization of cell wall. Initiation of the GPI anchor biosynthesis is catalysed by the GPI-N-acetylglucosaminyltransferase complex (GPI-GnT). The GPI3 (SPT14) gene is thought to encode the catalytic subunit of GPI-GnT complex. In contrast to Saccharomyces cerevisiae, little is known about the GPI biosynthesis in filamentous fungi. In this study, the afpig-a gene was identified as the homologue of the GPI3/pig-A gene in Aspergillus fumigatus, an opportunistic fungal pathogen. By replacement of the afpig-a gene with a pyrG gene, we obtained the null mutants. Although the Deltaafpig-a mutant exhibited a significant increased cell lysis instead of temperature-sensitive or conditional lethal phenotype associated to the GPI3 mutant of yeast, they could survive at temperatures from 30 degrees C to 50 degrees C. The analysis of the mutants showed that a completely blocking of the GPI anchor synthesis in A. fumigatus led to cell wall defect, abnormal hyphal growth, rapid conidial germination and aberrant conidiation. In vivo assays revealed that the mutant exhibited a reduced virulence in immunocompromised mice. The GPI anchor was not essential for viability, but required for the cell wall integrity, morphogenesis and virulence in A. fumigatus.     

Disruption of Botrytis cinerea class I chitin synthase gene Bcchs1 results in cell wall weakening and reduced virulence

To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed chitin synthase mutants. A 620 bp class I chitin synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent chitin synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.     

Pathways of infection of Brassica napus roots by Leptosphaeria maculans

Infection of Brassica napus cotyledons and leaves by germinating ascospores of Leptosphaeria maculans leads to production of leaf lesions followed by stem cankers (blackleg). Leptosphaeria maculans also causes root rot but the pathway of infection has not been described. An L. maculans isolate expressing green fluorescent protein (GFP) was applied to the petiole of B. napus plants. Hyphal growth was followed by fluorescence microscopy and by culturing of sections of plant tissue on growth media. Leptosphaeria maculans grew within stem and hypocotyl tissue during the vegetative stages of plant growth, and proliferated into the roots within xylem vessels at the onset of flowering. Hyphae grew in all tissues in the stem and hypocotyl, but were restricted mainly to xylem tissue in the root. Leptosphaeria maculans also infected intact roots when inoculum was applied directly to them and hyphae entered at sites of lateral root emergence. Hyphal entry may occur at other sites but the mechanism is uncertain as penetration structures were not observed. Infection of B. napus roots by L. maculans can occur via above- and below-ground sources of inoculum, but the relative importance of the infection pathways under field conditions is unknown.     

Characterisation of an Arabidopsis-Leptosphaeria maculans pathosystem: resistance partially requires camalexin biosynthesis and is independent of salicylic acid, ethylene and jasmonic acid signalling

Out of 168 Arabidopsis accessions screened with isolates of Leptosphaeria maculans, one (An-1) showed clear disease symptoms. In order to identify additional components involved in containment of L. maculans in Arabidopsis, a screen for L. maculans-susceptible (lms) mutants was performed. Eleven lms mutants were isolated, which displayed differential susceptibility responses to L. maculans. lms1 was crossed with Columbia (Col-0) and Ws-0, and mapping data for both populations showed the highest linkage to a region on chromosome 2. Reduced levels of PR-1 and PDF1.2 expression were found in lms1 compared to wild-type plants 48 h after pathogen inoculation. In contrast, the lms1 mutant displayed upregulation of either marker gene upon chemical treatment, possibly as an effect of an altered ethylene (ET) response. To assess the contribution of different defence pathways, genotypes implicated in salicylic acid (SA) signalling plants expressing the bacterial salicylate hydroxylase (nahG) gene, non-expressor of PR1 (npr1)-1 and phytoalexin-deficient (pad4-1), jasmonic acid (JA) signalling (coronatine insensitive (coi)1-16, enhanced disease susceptibility (eds)8-1 and jasmonic acid resistant (jar)1-1) and ET signalling (eds4-1, ethylene insensitive (ein)2, ein3-1 and ethylene resistant (etr)1-1) were screened. All the genotypes screened were as resistant as wild-type plants, demonstrating the dispensability of the pathways in L. maculans resistance. When mutants implicated in cell death responses were assayed, responsive to antagonist 1 (ran1)-1 exhibited a weak susceptible phenotype, whereas accelerated cell death (acd)1-20 showed a rapid lesion development. Camalexin is only partially responsible for L. maculans containment in Arabidopsis, as pad3-1 and enhanced susceptibility to Alternaria (esa)1 clearly showed a susceptible response while wild-type levels of camalexin were present in An-1 and lms1. The data presented point to the existence of multiple defence mechanisms controlling the containment of L. maculans in Arabidopsis.     

Mannosyltransferase is required for cell wall biosynthesis, morphology and control of asexual development in Neurospora crassa

Two Neurospora mutants with a phenotype that includes a tight colonial growth pattern, an inability to form conidia and an inability to form protoperithecia have been isolated and characterized. The relevant mutations were mapped to the same locus on the sequenced Neurospora genome. The mutations responsible for the mutant phenotype then were identified by examining likely candidate genes from the mutant genomes at the mapped locus with PCR amplification and a sequencing assay. The results demonstrate that a map and sequence strategy is a feasible way to identify mutant genes in Neurospora. The gene responsible for the phenotype is a putative alpha-1,2-mannosyltransferase gene. The mutant cell wall has an altered composition demonstrating that the gene functions in cell wall biosynthesis. The results demonstrate that the mnt-1 gene is required for normal cell wall biosynthesis, morphology and for the regulation of asexual development.     

Genetic dissection of a complex neurological mutant, polyhomeotic, in Drosophila

Null mutations at the polyhomeotic locus of Drosophila produce a complex phenotype during embryogenesis, which includes death of the ventral epidermis, misregulation of homeotic and segmentation gene expression, and global misrouting of CNS axons. It is shown here, through the use of mosaic analyses, double mutant combinations, and in vitro culture experiments, that all aspects of the phenotype with the exception of the axonal phenotype are cell autonomous. The changes in homeotic and segmentation gene expression in the CNS are not caused by death of the ventral epidermis, but are cell autonomous effects which most likely cause changes in neuronal cell identity. The axonal phenotype associated with ph mutations is also independent of epidermal cell death, but may be due to the nonautonomous effects of altered neuronal identities or to death or transformation of some as yet unidentified cell type. Despite the apparent autonomy of the ph mutation, mutant neurons can influence the development of adjacent wild-type neurons, presumably by depriving them of their normal fasciculation partners.     

Deletion of the GPIdeAc gene alters the location and fate of glycosylphosphatidylinositol precursors in Trypanosoma brucei

Glycosylphosphatidylinositol (GPI) membrane anchors are ubiquitous among the eukaryotes. In most organisms, the pathway of GPI biosynthesis involves inositol acylation and inositol deacylation as discrete steps at the beginning and end of the pathway, respectively. The bloodstream form of the protozoan parasite Trypanosoma brucei is unusual in that these reactions occur on multiple GPI intermediates and that it can express side chains of up to six galactose residues on its mature GPI anchors. An inositol deacylase gene, T. brucei GPIdeAc, has been identified. A null mutant was created and shown to be capable of expressing normal mature GPI anchors on its variant surface glycoprotein. Here, we show that the null mutant synthesizes galactosylated forms of the mature GPI precursor, glycolipid A, at an accelerated rate (2.8-fold compared to wild type). These free GPIs accumulate at the cell surface as metabolic end products. Using continuous and pulse-chase labeling experiments, we show that there are two pools of glycolipid A. Only one pool is competent for transfer to nascent variant surface glycoprotein and represents 38% of glycolipid A in wild-type cells. This pool rises to 75% of glycolipid A in the GPIdeAc null mutant. We present a model for the pathway of GPI biosynthesis in T. brucei that helps to explain the complex phenotype of the GPIdeAc null mutant.     

Brassinin oxidase, a fungal detoxifying enzyme to overcome a plant defense -- purification, characterization and inhibition

Blackleg fungi [Leptosphaeria maculans (asexual stage Phoma lingam) and Leptosphaeria biglobosa] are devastating plant pathogens with well-established stratagems to invade crucifers, including the production of enzymes that detoxify plant defenses such as phytoalexins. The significant roles of brassinin, both as a potent crucifer phytoalexin and a biosynthetic precursor of several other plant defenses, make it critical to plant fitness. Brassinin oxidase, a detoxifying enzyme produced by L. maculans both in vitro and in planta, catalyzes the detoxification of brassinin by the unusual oxidative transformation of a dithiocarbamate to an aldehyde. Purified brassinin oxidase has an apparent molecular mass of 57 kDa, is approximately 20% glycosylated, and accepts a wide range of cofactors, including quinones and flavins. Purified brassinin oxidase was used to screen a library of brassinin analogues and crucifer phytoalexins for potential inhibitory activity. Unexpectedly, it was determined that the crucifer phytoalexins camalexin and cyclobrassinin are competitive inhibitors of brassinin oxidase. This discovery suggests that camalexin could protect crucifers from attacks by L. maculans because camalexin is not metabolized by this pathogen and is a strong mycelial growth inhibitor.     

灰葡萄孢分生孢子产生相关基因的克隆及功能分析

[目的]克隆灰葡萄孢分生孢子产生相关基因,并研究其功能,为进一步研究灰葡萄孢分生孢子产生机理和灰葡萄孢侵染及致病机理奠定基础.[方法]通过筛选灰葡萄孢ATMT突变体库,获得一株不能产生分生孢子的突变菌株BCt78,采用PCR和Southern Blotting技术,对突变菌株BCt78进行分子鉴定.利用TAIL-PCR技术获得T-DNA插入位点的侧翼序列;将所获得侧翼序列与灰葡萄孢基因组数据库中的已知基因序列进行BLAST分析,推测出T-DNA的插入位点;通过PCR进一步验证T-DNA的插入位点,利用RT-PCR技术确定突变基因;最后对突变菌株的菌落形态、生长速度、胞壁降解酶活力、粗毒素的生物活性、对番茄叶片的致病能力及部分致病相关基因的表达情况进行研究.[结果]TAIL-PCR结果证实T-DNA插入到灰葡萄孢BCIG 12707.1基因的ATG起始密码子区;RT-PCR结果证实突变基因为BCIG_12707.1,该基因DNA全长为135 bp,编码一个44个氨基酸的假定蛋白(Hypothetical protein).突变菌株在PDA培养基上菌落呈灰白色,生长速度减慢,不能产生分生孢子及菌核;对番茄叶片的致病性增强,且胞壁降解酶(PG、PMG和Cx)活力增强;突变菌株中参与细胞壁降解的角质酶基因cutA和多聚半乳糖醛酸酶基因Bepg1,信号转导途径基因(PKA1、PKA2、Bac、Bmp3),产毒素基因BcBOT2(Sesquiterpene synthase),漆酶基因Lac1,跨膜蛋白基因Btp1表达都增强.[结论]BC1G_ 12707.1基因在灰葡萄孢分生孢子产生、菌核形成及致病力等方面起重要作用.