Showing papers in "Current Genetics in 2014"

Intronic and plasmid-derived regions contribute to the large mitochondrial genome sizes of Agaricomycetes

Abstract: Sizes of mitochondrial genomes vary extensively between fungal species although they typically contain a conserved set of core genes We have characterised the mitochondrial genome of the conifer root rot pathogen Heterobasidion irregulare and compared the size, gene content and structure of 20 Basidiomycete mitochondrial genomes The mitochondrial genome of H irregulare was 114, 193 bp and contained a core set of 15 protein coding genes, two rRNA genes and 26 tRNA genes In addition, we found six non-conserved open reading frames (ORFs) and four putative plasmid genes clustered in three separate regions together with 24 introns and 14 intronic homing endonuclease genes, unequally spread across seven of the core genes The size differences among the 20 Basidiomycetes can largely be explained by length variation of intergenic regions and introns The Agaricomycetes contained the nine largest mitochondrial genomes in the Basidiomycete group and Agaricomycete genomes are significantly (p < 0001) larger than the other Basidiomycetes A feature of the Agaricomycete mitochondrial genomes in this study was the simultaneous occurrence of putative plasmid genes and non-conserved ORFs, with Cantharellus cibarius as only exception, where no non-conserved ORF was identified This indicates a mitochondrial plasmid origin of the non-conserved ORFs or increased mitochondrial genome dynamics of species harbouring mitochondrial plasmids We hypothesise that two independent factors are the driving forces for large mitochondrial genomes: the homing endonuclease genes in introns and integration of plasmid DNA

An optimized transformation protocol for Lipomyces starkeyi

Abstract: We report the development of an efficient genetic transformation system for Lipomyces starkeyi based on a modified lithium acetate transformation protocol. L. starkeyi is a highly lipogenic yeast that grows on a wide range of substrates. The initial transformation rate for this species was extremely low, and required very high concentrations of DNA. A systematic approach for optimizing the protocol resulted in an increase in the transformation efficiency by four orders of magnitude. Important parameters included cell density, the duration of incubation and recovery periods, the heat shock temperature, and the concentration of lithium acetate and carrier DNA within the transformation mixture. We have achieved efficiencies in excess of 8,000 transformants/µg DNA, which now make it possible to screen libraries in the metabolic engineering of this yeast. Metabolic engineering based on this transformation system could improve lipogenesis and enable formation of higher value products.

Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae

Abstract: The development of highly efficient genome engineering reagents is of paramount importance to launch the next wave of biotechnology. TAL effectors have been developed as an adaptable DNA binding scaffold that can be engineered to bind to any user-defined sequence. Thus, TAL-based DNA binding modules have been used to generate chimeric proteins for a variety of targeted genome modifications across eukaryotic species. For example, TAL effectors fused to the catalytic domain of FokI endonuclease (TALENs) were used to generate site-specific double strand breaks (DSBs), the repair of which can be harnessed to dictate user-desired, genome-editing outcomes. To cleave DNA, FokI endonuclease must dimerize which can be achieved using a pair of TALENs that bind to the DNA targeted in a tail-to-tail orientation with proper spacing allowing the dimer formation. Because TALENs binding to DNA are dependent on their repeat sequences and nucleotides binding specificities, homodimers and heterodimers binding can be formed. In the present study, we used several TALEN monomers with increased repeats binding degeneracy to allow homodimer formation at increased number of genomic loci. We assessed their binding specificities and genome modification activities. Our results indicate that homodimeric TALENs could be used to modify the yeast genome in a site-specific manner and their binding to the promoter regions might modulate the expression of target genes. Taken together, our data indicate that homodimeric TALENs could be used to achieve different engineering possibilities of biotechnological applications and that their transcriptional modulations need to be considered when analyzing their phenotypic effects.

Identification and functional analysis of AG1-IA specific genes of Rhizoctonia solani

Abstract: Rhizoctonia solani is an important necrotrophic fungal pathogen which causes disease on diverse plant species. It has been classified into 14 genetically distinct anastomosis groups (AGs), however, very little is known about their genomic diversity. AG1-IA causes sheath blight disease in rice and controlling this disease remains a challenge for sustainable rice cultivation. Recently the draft genome sequences of AG1-IA (rice isolate) and AG1-IB (lettuce isolate) had become publicly available. In this study, using comparative genomics, we report identification of 3,942 R. solani genes that are uniquely present in AG1-IA. Many of these genes encode important biological, molecular functions and exhibit dynamic expression during in-planta growth of the pathogen in rice. Based upon sequence similarity with genes that are required for plant and human/zoonotic diseases, we identified several putative virulence/pathogenicity determinants amongst AG1-IA specific genes. While studying the expression of 19 randomly selected genes, we identified three genes highly up-regulated during in-planta growth. The detailed in silico characterization of these genes and extent of their up-regulation in different rice genotypes, having variable degree of disease susceptibility, suggests their importance in rice–Rhizoctonia interactions. In summary, the present study reports identification, functional characterization of AG1-IA specific genes and predicts important virulence determinants that might enable the pathogen to grow inside hostile plant environment. Further characterization of these genes would shed useful insights about the pathogenicity mechanism of AG1-IA on rice.

Evidence for somatic transcription of male-transmitted mitochondrial genome in the DUI species Ruditapes philippinarum (Bivalvia: Veneridae)

Abstract: In species with doubly uniparental inheritance (DUI), males are heteroplasmic for two sex-linked mitochondrial genomes (M- and F-mtDNA). While a role of M-mtDNA in male gametogenesis and sperm function is evident, there is an ongoing debate on whether it is transcribed or not in male soma. In this work we report a qPCR analysis in the DUI species Ruditapes philippinarum, showing that M-mtDNA is transcribed in somatic tissues. We observed a correlation between DNA copy numbers of the two analyzed genes, cytochrome b and a novel male-specific mitochondrial gene thought to be involved in DUI (orf21), and between their transcription levels. No correlation between a transcript and its DNA copy number was found, supporting the existence of complex regulatory mechanisms of mitochondrial transcription. We found the highest amount of mtDNA and mtRNA in gonads, likely due to the intense cell proliferation and high energy request for gametogenesis, while the observed variation among specimens is probably related to their different stages of gonad development. Finally, orf21 showed a highly variable transcription in advanced stages of gametogenesis. We hypothesize a differential storage of orf21 transcripts in spermatozoa, representing different paternal contributions to progeny, possibly leading to different developmental outcomes. A transcriptional activity does not necessarily imply the translation of M-mtDNA genes, and studies on mitochondrial proteins and their localization are needed to definitively assess the functioning of male-transmitted mitochondria in male soma. All that considered, the male soma of DUI species may represent an intriguing experimental model to study cytoplasmic genetic conflicts.

Activation of signaling pathways related to cell wall integrity and multidrug resistance by organic solvent in Saccharomyces cerevisiae.

Abstract: Organic solvents are toxic to living cells. In eukaryotes, cells with organic solvent tolerance have only been found in Saccharomyces cerevisiae. Although several factors contributing to organic solvent tolerance have been identified in previous studies, the mechanism of how yeast cells naturally respond to organic solvent stress is not known. We demonstrated that the pleiotropic drug resistance (PDR) pathway contributed to response to organic solvent stress. Activation of the PDR pathway by mutations in the transcription factors Pdr1p and Pdr3p led to organic solvent tolerance. Exposure to organic solvents also induced transcription levels of PDR5, which encodes a major drug efflux pump. Overproduction of Pdr5p improved organic solvent tolerance, presumably by exporting organic solvents out of the cell. In addition, we showed that the cell wall integrity (CWI) pathway was induced in response to organic solvents to upregulate genes encoding the cell wall-related proteins Wsc3p and Ynl190wp. WSC3 and YNL190W were upregulated independently of the PDR pathway. Among the components of the CWI pathway, the cell surface sensors (Wsc3p and Mid2p) and the transcription factors (Swi4p and Swi6p) appeared to be particularly involved in the response to organic solvents. Our findings indicate that S. cerevisiae activates two different signaling pathways, the PDR pathway and the CWI pathway, to cope with stresses from organic solvents.

Rapid, efficient and precise allele replacement in the fission yeast Schizosaccharomyces pombe

Abstract: Gene targeting provides a powerful tool to modify endogenous loci to contain specific mutations, insertions and deletions. Precise allele replacement, with no other chromosomal changes (e.g., insertion of selectable markers or heterologous promoters), maintains physiologically relevant context. Established methods for precise allele replacement in fission yeast employ two successive rounds of transformation and homologous recombination and require genotyping at each step. The relative efficiency of homologous recombination is low and a high rate of false positives during the second round of gene targeting further complicates matters. We report that pop-in, pop-out allele replacement circumvents these problems. We present data for 39 different allele replacements, involving simple and complex modifications at seven different target loci, that illustrate the power and utility of the approach. We also developed and validated a rapid, efficient process for precise allele replacement that requires only one round each of transformation and genotyping. We show that this process can be applied in population scale to an individual target locus, without genotyping, to identify clones with an altered phenotype (targeted forward genetics). It is therefore suitable for saturating, in situ, locus-specific mutation screens (e.g., of essential or non-essential genes and regulatory DNA elements) within normal chromosomal context.

The complete mitochondrial genome sequence of Brassica oleracea and analysis of coexisting mitotypes.

Abstract: The complete mitochondrial genome sequences of Brassica species have provided insight into inter- and intraspecific variation of plant mitochondrial genomes. However, the size of mitochondrial genome sequenced for Brassica oleracea hitherto does not match to its physical mapping data. This fact led us to investigate B. oleracea mitochondrial genome in detail. Here we report novel B. oleracea mitochondrial genome, derived from var. capitata, a cabbage cultivar ‘‘Fujiwase’’. The genome was assembled into a 219,952-bp circular sequence that is comparable to the mitochondrial genomes of other Brassica species (ca. 220–232 kb). This genome contained 34 protein-coding genes, 3 rRNA genes and 17 tRNA genes. Due to absence of a large repeat (140 kb), the mitochondrial genome of ‘‘Fujiwase’’ is clearly smaller than the previously reported mitochondrial genome of B. oleracea accession ‘‘08C717’’ (360 kb). In both mitotypes, all genes were identical, except cox2-2, which was present only in the Fujiwase type. At least two rearrangement events via large and small repeat sequences have contributed to the structural differences between the two mitotypes. PCR-based marker analysis revealed that the Fujiwase type is predominant, whereas the 08C717 type coexists at low frequency in all B. oleracea cultivars examined. Intraspecific variations in the mitochondrial genome in B. oleracea may occur because of heteroplasmy, coexistence of different mitotypes within an individual, and substoichiometric shifting. Our data indicate that the Fujiwase-type genome should be used as the representative genome of B. oleracea.

The heterogeneity of the rDNA-ITS sequence and its phylogeny in Rhizoctonia cerealis, the cause of sharp eyespot in wheat

Abstract: The sequence heterogeneity of the ribosomal internal transcribed spacer (ITS) region was investigated for Rhizoctonia cerealis isolates from the anastomosis group AG-DI. Although sequence variability of the ITS has been reported in a few multinucleate R. solani isolates, it has very rarely been reported in binucleate Rhizoctonia spp. isolates and has never been described in R. cerealis, the pathogen of wheat sharp eyespot. In this study, the ITS regions of 15 R. cerealis isolates were cloned and sequenced. The results revealed more than one different ITS sequence within each isolate. This is the first evidence of ITS sequence heterogeneity in R. cerealis. Based on these ITS sequences, different sequences of one isolate did not cluster in one clade, but all of the sequences of the 15 isolates were clustered in the anastomosis subgroup AG-DI, suggesting that the heterogeneity of the ITS did not affect the molecular identification of their anastomosis group. Haplotype analyses indicated that there might be three evolutionary origins of R. cerealis, or a recombination event could be the cause of different ITS sequences in one genome. This study demonstrates the variability and the evolution of Rhizoctonia, especially binucleate R. cerealis. These findings will help design disease control strategies.

Genetic differentiation and spatial structure of Geosmithia morbida, the causal agent of thousand cankers disease in black walnut (Juglans nigra).

Abstract: The main objectives of this study were to evaluate genetic composition of Geosmithia morbida populations in the native range of black walnut and provide a better understanding regarding demography of the pathogen. The fungus G. morbida, and the walnut twig beetle, Pityophthorus juglandis, have been associated with a disease complex of black walnut (Juglans nigra) known as thousand cankers disease (TCD). The disease is manifested as branch dieback and canopy loss, eventually resulting in tree death. In 2010, the disease was detected in black walnut in Tennessee, and subsequently in Virginia and Pennsylvania in 2011 and North Carolina in 2012. These were the first incidences of TCD east of Colorado, where the disease has been established for more than a decade on indigenous walnut species. A genetic diversity and population structure study of 62 G. morbida isolates from Tennessee, Pennsylvania, North Carolina and Oregon was completed using 15 polymorphic microsatellite loci. The results revealed high haploid genetic diversity among seven G. morbida populations with evidence of gene flow, and significant differentiation among two identified genetic clusters. There was a significant correlation between geographic and genetic distance. Understanding the genetic composition and demography of G. morbida can provide valuable insight into recognizing factors affecting the persistence and spread of an invasive pathogen, disease progression, and future infestation predictions. Overall, these data support the hypotheses of two separate, highly diverse pathogen introductions into the native range of black walnut.

Tissue-specific mitochondrial heteroplasmy at position 16,093 within the same individual

Abstract: Human mitochondrial DNA (mtDNA) research has entered a massively parallel sequencing (MPS) era, providing deep insight into mtDNA genomics and molecular diagnostics. Analysis can simultaneously include coding and control regions, many samples can be studied in parallel, and even minor heteroplasmic changes can be detected. We investigated heteroplasmy using 16 different tissues from three unrelated males aged 40–54 years at the time of death. mtDNA was enriched using two independent overlapping long-range PCR amplicons and analysed by employing illumina paired-end sequencing. Point mutation heteroplasmy at position 16,093 (m.16093T > C) in the non-coding regulatory region showed great variability among one of the studied individuals; heteroplasmy extended from 5.1 % in red bone marrow to 62.0 % in the bladder. Red (5.1 %) and yellow bone marrow (8.9 %) clustered into one group and two arteries and two aortas from different locations into another (31.2–50.9 %), giving an ontogenetic explanation for the formation of somatic mitochondrial heteroplasmy. Our results demonstrate that multi-tissue screening using MPS provides surprising data even when there is a limited number (3) of study subjects and they give reason to speculate that mtDNA heteroplasmic frequency, distribution, and even its possible role in complex diseases or phenotypes seem to be underestimated.

Mitochondrial genome of the basidiomycetous yeast Jaminaea angkorensis.

Abstract: Jaminaea angkorensis is an anamorphic basidiomycetous yeast species originally isolated from decaying leaves in Cambodia. Taxonomically, J. angkorensis is affiliated with Microstromatales (Exobasidiomycetes, Ustilaginomycotina, Basidiomycota) and represents a basal phylogenetic lineage of this fungal order. To perform a comparative analysis of J. angkorensis with other basidiomycetes, we determined and analyzed its complete mitochondrial DNA sequence. The mitochondrial genome is represented by 29,999 base pairs long, circular DNA containing 32 % guanine and cytosine residues. Its genetic organization is relatively compact and comprises typical genes for 15 conserved proteins involved in oxidative phosphorylation (atp6, 8, and 9; cob; cox1, 2, and 3; and nad1, 2, 3, 4, 4L, 5, and 6) and translation (rps3), two ribosomal RNAs (rnl and rns) and twenty-two transfer RNAs (trnA-Y). Although the gene content is similar to other basidiomycetes, the gene orders in the examined species exhibit only a limited synteny, reflecting their phylogenetic distances and extensive genome rearrangements. In addition, a comparative analysis of basidiomycete mitochondrial genomes indicates that stop-to-tryptophan reassignment of the UGA codon was accompanied by structural alterations of tRNA-Trp(CCA). These results provide an insight into the evolution of the genetic code in fungal mitochondria.

Mutation of essential Hsp90 co-chaperones SGT1 or CNS1 renders yeast hypersensitive to overexpression of other co-chaperones

Abstract: The essential molecular chaperone Hsp90 functions with over ten co-chaperones in Saccharomyces cerevisiae, but the in vivo roles of many of these co-chaperones are poorly understood. Two of these co-chaperones, Cdc37 and Sgt1, target specific types of clients to Hsp90 for folding. Other co-chaperones have general roles in supporting Hsp90 function, but the degree of overlapping or competing functions is unclear. None of the chaperones, when overexpressed, were able to rescue the lethality of an SGT1 disruption strain. However, overexpression of SBA1, PPT1, AHA1 or HCH1 caused varying levels of growth defects in an sgt1-K360E strain. Negative effects of CPR6 overexpression were similarly observed in cells expressing the temperature-sensitive mutation cns1-G90D. In all cases, alterations within co-chaperones designed to disrupt Hsp90 interaction relieved the negative growth defects. Sgt1-K360E and Cns1-G90D were previously shown to exhibit reduced Hsp90 interaction. Our results indicate that overexpression of other co-chaperones further disrupts the essential functions of Cns1 and Sgt1. However, the specificity of the negative effects indicates that only a subset of co-chaperones competes with Sgt1 or Cns1 for binding to Hsp90. This provides new evidence that co-chaperones selectively compete for binding to subpopulations of cellular Hsp90 and suggest that changes in the relative levels of co-chaperones may have dramatic effects on Hsp90 function.

Antifungal activity of plant defensin AFP1 in Brassica juncea involves the recognition of the methyl residue in glucosylceramide of target pathogen Candida albicans

Abstract: An antifungal defensin, AFP1, of Brassica juncea inhibits the growth of various microorganisms. The molecular details of this inhibition remain largely unknown. Herein, we reveal that a specific structure of fungal sphingolipid glucosylceramide (GlcCer) is critical for the sensitivity of Candida albicans cells to AFP1. Our results revealed that AFP1 induces plasma membrane permeabilization and the production of reactive oxygen species (ROS) in wild-type C. albicans cells, but not in cells lacking the ninth methyl residue of the GlcCer sphingoid base moiety, which is a characteristic feature of fungi. AFP1-induced ROS production is responsible for its antifungal activity, with a consequent loss of yeast cell viability. These findings suggest that AFP1 specifically recognizes the structural difference of GlcCer for targeting of the fungal pathogens.

Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR

Abstract: Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the β-actin gene (actb), β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR.

Mitochondrial D-loop and Cytochrome Oxidase C subunit I polymorphisms among the breast cancer patients of Mizoram, Northeast India

Abstract: Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations as it is prone to oxidative stress. The aim of the present study is to assess the novel mutations in mitochondrial genes from blood samples among the breast cancer patients from a less studied Northeast Indian population. D, B, L haplogroups were observed in the cancer samples and a total of 44 mtDNA D-loop sequence variations at 42 distinct nucleotide positions were found. All the sequence variations were transitional substitutions and 6 were heteroplasmic states, except for a cytosine copy number change (9C/8C) at np 303e309 in three samples examined. A total of 88 Cytochrome Oxidase C subunit I (COXI) sequence differences with respect to the Revised Cambridge Reference Sequence (rCRS) were identified including 20 missense variants with 100 % sample mutation frequency. All 20 missense mutations are highly conserved with a Cumulate Index of 100 %. Among 88 COXI mutations, 24 (13 were Non-Synonymous and 11 were Synonymous) were not previously reported (novel mutation) in the literature or the public mtDNA mutation databases. Analysis of three-dimensional structure of COXI open reading frame (ORF) predicted the effect of one single codon (96R > C, 217T > I, 224-225GG > EE and 227D > T) mutations located in the signal peptide binding position. Analysis of mitochondrial DNA mutations, as a viable alternative, has the advantage of being capable of detecting inherent risk factors for breast cancer development.

A spindle pole antigen gene MoSPA2 is important for polar cell growth of vegetative hyphae and conidia, but is dispensable for pathogenicity in Magnaporthe oryzae

Abstract: Spa2 is an important component of the multiprotein complex polarisome, which is involved in the establishment, maintenance, termination of polarized cell growth and is important for defining tip growth of filamentous fungi. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that formed smaller colony and conidia compared with the wild type. In the mutant, a spindle pole antigen gene MoSPA2 was disrupted by the integration of an exogenous plasmid. Targeted gene deletion and complementation assays demonstrated the gene disruption was responsible for the defects of the insertional mutant. Interestingly, the MoSpa2-GFP fusion protein was found to accumulate as a spot at hyphal tips, septa of hyphae and conidial tip cells where germ tubes are usually produced, but not in appressoria, infection hyphae or at the septa of conidia. Furthermore, the deletion mutants of MoSPA2 exhibited slower hyphal tip growth, more hyphal branches, and smaller size of conidial tip cells. However, MoSPA2 is not required for plant infection. These results indicate that MoSPA2 is required for vegetative hyphal growth and maintaining conidium morphology and that spotted accumulation of MoSpa2 is important for its functions during cell polar growth.

Selection and characterization of promoters based on genomic approach for the molecular breeding of oleaginous fungus Mortierella alpina 1S-4

Abstract: To express a foreign gene effectively, a good expression system is required. In this study, we investigated various promoters as useful tools for gene manipulation in oleaginous fungus Mortierella alpina 1S-4. We selected and cloned the promoter regions of 28 genes in M. alpina 1S-4 on the basis of expression sequence tag abundance data. The activity of each promoter was evaluated using the β-glucuronidase (GUS) reporter gene. Eight of these promoters were shown to enhance GUS expression more efficiently than a histone promoter, which is conventionally used for the gene manipulation in M. alpina. Especially, the predicted protein 3 and the predicted protein 6 promoters demonstrated approximately fivefold higher activity than the histone promoter. The activity of some promoters changed along with the cultivation phase of M. alpina 1S-4. Seven promoters with constitutive or time-dependent, high-level expression activity were selected, and deletion analysis was carried out to determine the promoter regions required to retain activity. This is the first report of comprehensive promoter analysis based on a genomic approach for M. alpina. The promoters described here will be useful tools for gene manipulation in this strain.

Characterization of galactose-dependent promoters from an oleaginous fungus Mortierella alpina 1S-4

Abstract: An inducible promoter is a useful tool for the controlled expression of a given gene. In this report, we describe galactose-dependent promoters for potential use in an oleaginous fungus Mortierella alpina. We cloned the putative promoter regions of two genes encoding galactose metabolic enzymes, GAL1 and GAL10, from the genome of M. alpina 1S-4. The β-glucuronidase (GUS) reporter gene assay in M. alpina 1S-4 revealed that regulation of these promoters was dependent on the presence of galactose in the medium both with and without other sugars. With the GAL10 promoter, an approximately 50-fold increase of GUS activity was demonstrated by addition of galactose into the culture media at any cultivation phase. The 5′ deletion analysis of the GAL10 promoter revealed that a promoter region of over 2,000 bp length was required for its high-level activity and sufficient inducible response. Significantly, this is the first report of inducible promoters of zygomycetes. The GAL10 promoter will be a valuable tool for gene manipulation in M. alpina 1S-4.

Characterization of mating type genes supports the hypothesis that Stagonosporopsis chrysanthemi is homothallic and provides evidence that Stagonosporopsis tanaceti is heterothallic

Abstract: To understand the organization of the mating type locus of Stagonosporopsis tanaceti and Stagonosporopsis chrysanthemi, and its potential role in the epidemiology of ray blight of pyrethrum and chrysanthemum, respectively, the mating type (MAT) locus of these species was cloned and characterized using PCR-based techniques. The complete MAT locus of each species was cloned and annotated including complete and/or partial hypothetical genes flanking the idiomorphs. Analysis of the MAT locus organization indicated that S. chrysanthemi is likely homothallic with both MAT1-2-1 and MAT1-1-1 co-located within the idiomorph, and this was supported by production of the teleomorph in cultures of single-conidial-derived isolates. Sequencing of the MAT locus and flanking genes of S. tanaceti demonstrated that only a single MAT gene, MAT1-1-1, was located within this idiomorph and suggesting that S. tanaceti is heterothallic. MAT-specific PCR primers were developed and used to determine mating type of isolates sampled from diseased pyrethrum fields in Australia. These results indicated that only one mating type of S. tanaceti was present in Tasmania, Australia. The absence of a second mating type suggests that this species does not reproduce sexually in Tasmania, Australia and that ascospores are unlikely to be a source of inoculum for ray blight of pyrethrum. The MAT-specific PCR assay will be a valuable tool to distinguish mating types present among isolates of S. tanaceti, to monitor populations of S. tanaceti for the introduction of a second mating type and to differentiate S. tanaceti from S. chrysanthemi.

Efficient and versatile transformation systems in entomopathogenic fungus Lecanicillium species.

Abstract: Lack of genetic tools in entomopathogenic fungi, especially those for targeted homologous recombination, hindered the advance in this field. To facilitate the genetic study, we constructed a transformation system in entomopathogenic fungus Lecanicillium sp. strain HF627 using the uridine auxotrophic pyrG mutant strain as host and endogenous pyrG as marker. pUC19 harboring endogenous pyrG successfully restored the uridine auxotrophy of the host strain, and the integration of the vector DNA was confirmed by Southern hybridization. An autonomously replicating vector harboring an AMA1 sequence was constructed and applied to the constructed transformation system, which improved the transformation efficiency 16.7-fold. Southern hybridization revealed replication of the AMA1-harboring vector with an average copy number of 2.4. A ku80 knock-out strain was created to improve the efficiency of gene targeting. Deletion of the pyrG locus, which is homologous to the marker gene, from the ku80 knock-out strain achieved a targeting efficiency of 62.5 % against both trp1 and his3; the levels of these genes were 3.2- and 5-fold higher, respectively, than the ku80-intact strain. With the pyrG-deleted and ku80-inactivated strain constructed in this study, transformation and targeted homologous recombination were highly enhanced, by which genetic analysis in Lecanicillium spp. will be performed quickly and efficiently.

Analysis of simple sequence repeats in the Gaeumannomyces graminis var. tritici genome and the development of microsatellite markers

Abstract: Understanding the genetic structure of Gaeumannomyces graminis var. tritici is essential for the establishment of efficient disease control strategies. It is becoming clear that microsatellites, or simple sequence repeats (SSRs), play an important role in genome organization and phenotypic diversity, and are a large source of genetic markers for population genetics and meiotic maps. In this study, we examined the G. graminis var. tritici genome (1) to analyze its pattern of SSRs, (2) to compare it with other plant pathogenic filamentous fungi, such as Magnaporthe oryzae and M. poae, and (3) to identify new polymorphic SSR markers for genetic diversity. The G. graminis var. tritici genome was rich in SSRs; a total 13,650 SSRs have been identified with mononucleotides being the most common motifs. In coding regions, the densities of tri- and hexanucleotides were significantly higher than in noncoding regions. The di-, tri-, tetra, penta, and hexanucleotide repeats in the G. graminis var. tritici genome were more abundant than the same repeats in M. oryzae and M. poae. From 115 devised primers, 39 SSRs are polymorphic with G. graminis var. tritici isolates, and 8 primers were randomly selected to analyze 116 isolates from China. The number of alleles varied from 2 to 7 and the expected heterozygosity (He) from 0.499 to 0.837. In conclusion, SSRs developed in this study were highly polymorphic, and our analysis indicated that G. graminis var. tritici is a species with high genetic diversity. The results provide a pioneering report for several applications, such as the assessment of population structure and genetic diversity of G. graminis var. tritici.

Functional characterization of sucrose non-fermenting 1 protein kinase complex genes in the Ascomycete Fusarium graminearum

Abstract: Sucrose non-fermenting 1 (SNF1) protein kinase complex is a heterotrimer that functions in energy homeostasis in eukaryotes by regulating transcription of glucose-repressible genes. Our previous study revealed that SNF1 of the homothallic ascomycete fungus Fusarium graminearum plays important roles in vegetative growth, sexual development, and virulence. In this study, we further identified the components of the SNF1 complex in F. graminearum and characterized their functions. We found that the SNF1 complex in F. graminearum consists of one alpha subunit (FgSNF1), one beta subunit (FgGAL83), and one gamma subunit (FgSNF4). Deletion of Fggal83 and Fgsnf4 resulted in alleviated phenotype changes in vegetative growth and sexual development as compared to those of the Fgsnf1 deletion mutant. However, all of the single, double, and triple deletion mutants among Fgsnf1, Fggal83, and Fgsnf4 had similar levels of decreased virulence. In addition, there was no synergistic effect of the mutant (single, double, or triple deletions of SNF1 complex component genes) phenotypes except for sucrose utilization. In this study, we revealed that FgSNF1 is mainly required for SNF1 complex functions, and the other two SNF1 complex components have adjunctive roles with FgSNF1 in sexual development and vegetative growth but have a major role in virulence in F. graminearum.

The mutagenic footprint of low-fidelity Pol I ColE1 plasmid replication in E. coli reveals an extensive interplay between Pol I and Pol III

Abstract: ColE1 plasmid replication is unidirectional and requires two DNA polymerases: DNA polymerase I (Pol I) and DNA polymerase III (Pol III). Pol I initiates leading-strand synthesis by extending an RNA primer, allowing the Pol III holoenzyme to assemble and finish replication of both strands. The goal of the present work is to study the interplay between Pol I and Pol III during ColE1 plasmid replication, to gain new insights into Pol I function in vivo. Our approach consists of using mutations generated by a low-fidelity mutant of Pol I (LF-Pol I) during replication of a ColE1 plasmid as a footprint for Pol I replication. This approach allowed mapping areas of Pol I replication on the plasmid with high resolution. In addition, we were able to approximate the strandedness of Pol I mutations throughout the plasmid, allowing us to estimate the spectrum of the LF-Pol I in vivo. Our study produced the following three mechanistic insights: (1) we identified the likely location of the polymerase switch at ~200 bp downstream of replication initiation; (2) we found evidence suggesting that Pol I can replicate both strands, supporting earlier studies indicating a functional redundancy between Pol I and Pol III (3) we found evidence pointing to a specific role of Pol I during termination of lagging-strand replication. In addition, we illustrate how our strand-specific footprinting approach can be used to dissect factors modulating Pol I fidelity in vivo.

Multiple independent insertions of 5S rRNA genes in the spliced-leader gene family of trypanosome species

Abstract: Analyses of the 5S rRNA genes found in the spliced-leader (SL) gene repeat units of numerous trypanosome species suggest that such linkages were not inherited from a common ancestor, but were the result of independent 5S rRNA gene insertions. In trypanosomes, 5S rRNA genes are found either in the tandemly repeated units coding for SL genes or in independent tandemly repeated units. Given that trypanosome species where 5S rRNA genes are within the tandemly repeated units coding for SL genes are phylogenetically related, one might hypothesize that this arrangement is the result of an ancestral insertion of 5S rRNA genes into the tandemly repeated SL gene family of trypanosomes. Here, we use the types of 5S rRNA genes found associated with SL genes, the flanking regions of the inserted 5S rRNA genes and the position of these insertions to show that most of the 5S rRNA genes found within SL gene repeat units of trypanosome species were not acquired from a common ancestor but are the results of independent insertions. These multiple 5S rRNA genes insertion events in trypanosomes are likely the result of frequent founder events in different hosts and/or geographical locations in species having short generation times.

“Cryptic” group-I introns in the nuclear SSU-rRNA gene of Verticillium dahliae

Abstract: Group-I introns are widespread—though irregularly distributed—in eukaryotic organisms, and they have been extensively used for discrimination and phylogenetic analyses. Within the Verticillium genus, which comprises important phytopathogenic fungi, a group-I intron was previously identified in the SSU-rRNA (18S) gene of only V. longisporum. In this work, we aimed at elucidating the SSU-located intron distribution in V. dahliae and other Verticillium species, and the assessment of heterogeneity regarding intron content among rDNA repeats of fungal strains. Using conserved PCR primers for the amplification of the SSU gene, a structurally similar novel intron (sub-group IC1) was detected in only a few V. dahliae isolates. However, when intron-specific primers were used for the screening of a diverse collection of Verticillium isolates that originally failed to produce intron-containing SSU amplicons, most were found to contain one or both intron types, at variable rDNA repeat numbers. This marked heterogeneity was confirmed with qRT-PCR by testing rDNA copy numbers (varying from 39 to 70 copies per haploid genome) and intron copy ratios in selected isolates. Our results demonstrate that (a) IC1 group-I introns are not specific to V. longisporum within the Verticillium genus, (b) V. dahliae isolates of vegetative compatibility groups (VCGs) 4A and 6, which bear the novel intron at most of their rDNA repeats, are closely related, and (c) there is considerable intra-genomic heterogeneity for the presence or absence of introns among the ribosomal repeats. These findings underline that distributions of introns in the highly heterogeneous repetitive rDNA complex should always be verified with sensitive methods to avoid misleading conclusions for the phylogeny of fungi and other organisms.

The expression of PHO92 is regulated by Gcr1, and Pho92 is involved in glucose metabolism in Saccharomyces cerevisiae

Abstract: Ydr374c (Pho92) contains a YTH domain in its C-terminal region and is a human YTHDF2 homologue. Previously, we reported that Pho92 regulates phosphate metabolism by regulating PHO4 mRNA stability. In this study, we found that growth of the ∆pho92 strain on SG media was slower than that of the wild type and that PHO92 expression was up-regulated by non-fermentable carbon sources, such as ethanol and glycerol, but not by fermentable carbon sources. Furthermore, two conserved Gcr1-binding regions were identified in the upstream, untranslated region of PHO92. Gcr1 is an important factor involved in the coordinated regulation of glycolytic gene expression. Mutation of two Gcr1-binding sites of the PHO92 upstream region resulted in a growth defect on SD media. Finally, mutagenesis of the Gcr1-binding sites of the PHO92 upstream region and deletion of GCR1 resulted in up-regulation of PHO92, and this resulted from inhibition of PHO4 mRNA degradation. Based on these results, we suggest that Gcr1 regulates the expression of PHO92, and Pho92 is involved in glucose metabolism.

Gene expression and metabolite changes during Tuber magnatum fruiting body storage.

Abstract: The aim of this study was to investigate the impact of different 4 °C post-harvest storage periods on the quality of the white truffle Tuber magnatum. The expression of selected genes and the profiles of non-volatile metabolites have been analyzed. The up-regulation of genes related to cell wall metabolism and to a putative laccase points to cell wall modifications and browning events during cold storage. Time course RT-qPCR experiments have demonstrated that such transcription events probably depend on the ripening status, since this is delayed in partially ripe fruiting bodies. Changes in the concentrations of linoleate-derived metabolites occur during the first 3 days of considered cold storage, while the other metabolites, such as the amino acids, do not change. Taken together, the results demonstrate that complex molecular events occur in white truffles in the post-harvest period and before they are used as fresh products.

Application of a peptide-based assay to characterize inhibitors targeting protein kinases from yeast

Abstract: Chemical molecules that inhibit protein kinase activity are important tools to assess the functions of protein kinases in living cells. To develop, test and characterize novel inhibitors, a convenient and reproducible kinase assay is of importance. Here, we applied a biotinylated peptide-based method to assess adenosine triphosphate-competitive inhibitors that target the yeast kinases Hog1, Elm1 and Elm1-as. The peptide substrates contained 13 amino acids, encompassing the consensus sequence surrounding the phosphorylation site. To test whether the lack of distal sites affects inhibitor efficacy, we compared the peptide-based assay with an assay using full-length protein as substrate. Similar inhibitor efficiencies were obtained irrespective of whether peptide or full-length protein was used as kinase substrates. Thus, we demonstrate that the peptide substrates used previously (Diner et al. in PLoS One 6(5):e20012, 2011) give accurate results compared with protein substrates. We also show that the peptide-based method is suitable for selectivity assays and for inhibitor screening. The use of biotinylated peptide substrates provides a simple and reliable assay for protein kinase inhibitor characterization. The utility of this approach is discussed.

A modular plasmid system for protein co-localization and bimolecular fluorescence complementation in filamentous fungi.

Abstract: To elucidate the function of a protein, it is crucial to know its subcellular location and its interaction partners. Common approaches to resolve those questions rely on the genetic tagging of the gene-of-interest (GOI) with fluorescent reporters. To determine the location of a tagged protein, it may be co-localized with tagged marker proteins. The interaction of two proteins under investigation is often analysed by tagging both with the C- and N-terminal halves of a fluorescent protein. In fungi, the tagged GOI are commonly introduced by serial transformation with plasmids harbouring a single tagged GOI and subsequent selection of suitable strains. In this study, a plasmid system is presented that allows the tagging of several GOI on a single plasmid. This novel double tagging plasmid system (DTPS) allows a much faster and less laborious generation of double-labelled fungal strains when compared with conventional approaches. The DTPS also enables the combination of as many tagged GOI as desired and a simple exchange of existing tags. Furthermore, new tags can be introduced smoothly into the system. In conclusion, the DTPS allows an efficient tagging of GOI with a high degree of flexibility and therefore accelerates functional analysis of proteins in vivo.