Characterization of the mating-type genes in Leptographium procerum and Leptographium profanum

Leptographium procerum and the closely related species Leptographium profanum, are ascomycetes associated with root-infesting beetles on pines and hardwood trees, respectively. Both species occur in North America where they are apparently native. L. procerum has also been found in Europe, China New Zealand, and South Africa where it has most probably been introduced. As is true for many other Leptographium species, sexual states have never been observed in L. procerum or L. profanum. The objectives of this study were to clone and characterize the mating type loci of these fungi, and to develop markers to determine the mating types of individual isolates. To achieve this, a partial sequence of MAT1-2-1 was amplified using degenerate primers targeting the high mobility group (HMG) sequence. A complete MAT1-2 idiomorph of L. profanum was subsequently obtained by screening a genomic library using the HMG sequence as a probe. Long range PCR was used to amplify the complete MAT1-1 idiomorph of L. profanum and both the MAT1-1 and MAT1-2 idiomorphs of L. procerum. Characterization of the MAT idiomorphs suggests that the MAT genes are fully functional and that individuals of both these species are self-sterile in nature with a heterothallic mating system. Mating type markers were developed and tested on a population of L. procerum isolates from the USA, the assumed center of origin for this species. The results suggest that cryptic sexual reproduction is occurring or has recently taken place within this population.     

rDNA-targeted PCR primers and FISH probe in the detection of Ophiocordyceps sinensis hyphae and conidia

Ophiocordyceps sinensis (Berk.) Sung, Sung, Hywel-Jones & Spatafora (syn. Cordyceps sinensis) one of the entomopathogenic fungi, is a rare Traditional Chinese Medicine (TCM) found in the Qinghai-Tibetan Plateau. Polymerase Chain Reaction (PCR) and Fluorescence in situ hybridization (FISH) methods are necessary to identify the mycelia or spores of O. sinensis from its habitat and to monitor its dispersal, colonization and infectivity. To develop both primers and probe specific to O. sinensis, ribosomal DNA (rDNA) amplified with universal primers from O. sinensis genomic DNA and seven closely related fungi were sequenced. According to these sequences, the upper and lower primers (OsT-F and OsT-R) were designed within internal transcribed spacer region 1 (ITS1) and ITS2 and flanked by universal primers ITS5 and ITS4, respectively. The designed primers were used for general PCR, touchdown PCR, or both together with the universal primers for nested-touchdown PCR. The results showed that only the extracted DNA of O. sinensis was specifically amplified. The sensitivity of nested-touchdown PCR with extracted DNA of O. sinensis is as low as 10⁻¹⁴ g (10 fg) and at least 1000 times higher than the other PCR methods. In addition, Cy5-labeled probe (OsLSU) for cytoplasmic LSU rRNA was hybridized with the ascospores of O. sinensis. It showed a strong red fluorescence throughout the whole cell but did not cross-react with other entomopathogenic fungi. Taken together, these methods were useful for studying the biology and ecology of O. sinensis.     

Evaluation of the potential for sexual reproduction in field populations of Cercospora beticola from USA

Cercospora leaf spot, caused by the hemibiotrophic fungal pathogen Cercospora beticola, is the most economically damaging foliar disease of sugarbeet worldwide. Although most C. beticola populations display characteristics reminiscent of sexual recombination, no teleomorph has been described. To assess whether populations in northern United States have characteristics consistent with sexual reproduction, 1024 isolates collected over a 3-y period were analyzed for frequency and distribution of mating type genes. After clone correction, an approximately equal distribution of mating types was found for each sampling year. Mating type frequency was also assessed in individual lesions. Lesions always consisted of isolates with a single mating type and microsatellite haplotype, but both mating types and up to five microsatellite haplotypes could be found on an individual leaf. The MAT1-1-1 and MAT1-2-1 genes were sequenced from 28 MAT1-1 and 28 MAT1-2 isolates, respectively. Three MAT1-1-1 nucleotide haplotypes were identified that encoded a single amino acid sequence. For MAT1-2-1, five nucleotide haplotypes were identified that encoded four protein variants. MAT1-1-1 and MAT1-2-1 gene expression analyses were conducted on plants inoculated with either or both mating types. MAT1-1-1 expression remained low, but MAT1-2-1 spiked during late stages of colonization. A segment of the MAT1-2-1 coding sequence was also found in MAT1-1 isolates. Taken together, these results suggest that C. beticola has the potential for sexual reproduction.     

Both mating types in the heterothallic fungus Ophiostoma quercus contain MAT1-1 and MAT1-2 genes

In heterothallic Ascomycota, two opposite but distinct mating types control all sexual processes. Using mating crosses, mating types were assigned to ten isolates of the heterothallic fungal species Ophiostoma quercus. Primers were subsequently designed to target the MAT1-1-1, MAT1-1-3 (of the mating type 1 idiomorph), and MAT1-2-1 (of the mating type 2 idiomorph) genes in these isolates. Results showed that all isolates contained the full gene sequence for the MAT1-2-1 gene. In addition, fragments of the MAT1-1-1 and MAT1-1-3 genes were sequenced from all isolates. These results were unexpected, as each isolate from a heterothallic species would typically contain only one of the two possible MAT idiomorphs.     

Analysis of the <Emphasis Type="Italic">MAT1-2-1</Emphasis> gene of <Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis>

A single MAT1-2-1 gene was identified from a mating pair of the filamentous ascomycete Colletotrichum lindemuthianum. The MAT1-2-1 genes from both mating partners carried an open reading frame (ORF) of 870 bp encoding a putative protein of 290 amino acids that includes the highly conserved high mobility group (HMG) domain typical of the fungal MAT1-2-1 genes. Three introns were confirmed within the C. lindemuthianum ORF, two of which were found to be conserved relative to a previously reported MAT1-2-1 gene from C. gloeosporioides. The amino acid sequence of the HMG domain from C. lindemuthianum MAT1-2-1 was also compared with those from other ascomycetes. These results suggest that although the MAT1-2-1 genes are highly conserved among ascomycetes, the mechanism which defines mating partners in the genus Colletotrichum is distinct to the idiomorph system described for other members of this phylum.     

Mating type gene analyses in the genus Diplodia: From cryptic sex to cryptic species

Cryptic species are common in Diplodia, a genus that includes some well-known and economically important plant pathogens. Thus, species delimitation has been based on the phylogenetic species recognition approach using multigene genealogies. We assessed the potential of mating type (MAT) genes sequences as phylogenetic markers for species delimitation in the genus Diplodia. A PCR-based mating type diagnostic assay was developed that allowed amplification and sequencing of the MAT1-1-1 and MAT1-2-1 genes, and determination of the mating strategies used by different species. All species tested were shown to be heterothallic. Phylogenetic analyses were performed on both MAT genes and also, for comparative purposes, on concatenated sequences of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α) and beta-tubulin (tub2). Individual phylogenies based on MAT genes clearly differentiated all species analysed and agree with the results obtained with the commonly used multilocus phylogenetic analysis approach. However, MAT genes genealogies were superior to multigene genealogies in resolving closely related cryptic species. The phylogenetic informativeness of each locus was evaluated revealing that MAT genes were the most informative loci followed by tef1-α. Hence, MAT genes can be successfully used to establish species boundaries in the genus Diplodia.     

Molecular phylogeny of the Pycnonotus sinensis and Pycnonotus taivanus in Taiwan based on sequence variations of nuclear CHD and mitochondrial cytochrome b genes

Sequences of the partial 293-bp nuclear Z-chromosome-linked chromo-helicase binding protein (CHD-Z) and 729-bp mitochondrial cytochrome b (cyt b) genes were obtained from two species of the same family Pycnonotidae of Pycnonotus sinensis (P. sinensis, Chinese Bulbul) and Pycnonotus taivanus (P. taivanus, Taiwan Bulbul) distributed in Taiwan. A panel of 10 individuals (n = 5 for each) with unknown relationship was used to characterize single nucleotide polymorphisms (SNPs) of these genes. We identified 2 and 10 SNPs in CHD-Z and cyt b loci, respectively. Frequency of SNPs was 10 per every 1465- and 729-bp on average. Pairwise nucleotide divergences of CHD-Z and cyt b genes among 10 specimens ranged from 0 to 0.0034 and 0 to 0.0055, respectively. Phylogenetic analysis suggested that the P. taivanus group assignment based on the CHD-Z and cyt b sequences is obviously very similar to P. sinensis.     

Identification of the Mating-Type (MAT) Locus That Controls Sexual Reproduction of Blastomyces dermatitidis

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.     

Mating behaviour and chemical communication in the invasive Chinese mitten crab Eriocheir sinensis

Mating in the Chinese mitten crab (Eriocheir sinensis) was examined; in particular the nature of mating and the role of sex pheromones. A semi-lunar periodicity (16.8 days and 14.5 days, respectively) was observed in the mating frequencies in two consecutive breeding seasons (2001-2002 and 2002-2003). This semi-lunar rhythm coincided with spring tides (full and new moon), and activity peaked in November. Observation of the progression of specific behaviour types in mating and non-mating pairs revealed that pairs which would go on to complete mating progressed from fighting to mating behaviour significantly faster than non-mating pairs. These findings indicate that mate recognition occurs only after physical contact. Reproductively active pairs (ascertained from mating experiments) were then used for several bioassays aiming to assess under which conditions pheromones may be released by females. Firstly, male E. sinensis were exposed to female smell in an actograph experiment and secondly, male antennule flick rate was recorded before and after exposure to the urine of a sexually active female. In both cases no change in male E. sinensis behaviour was observed. Both experiments used females which had not had immediately prior exposure to males. However, in further experiments using water where mating had occurred, a significant response in antennule flick rate was triggered. Finally, a sponge assay was used in order to test the male attraction to a sponge injected with a water sample of varying concentrations (0.5×, 1×, 3×, 4.5×, 9×) of female smell. These samples were conditioned using a female immediately following a mating attempt. Males tried to grasp the sponge at 3× increased concentrations or higher. In conclusion, this study found no indication that E. sinensis females release a distance pheromone, but instead that mate recognition occurs after physical contact between male and female, most likely via a contact pheromone.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7

A multiplex PCR procedure that detects six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in Escherichia coli O157:H7 was developed. Analyses of the available sequences of the six major virulence genes and the published primers allowed us to develop the six-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting six bands for fliC, stx1, stx2, eae, rfbE, and hlyA were even and distinct with product sizes of 949, 655, 477, 375, 296, and 199 bp, respectively. The procedure was validated with a total of 221 E. coli strains that included 4 ATCC, 84 cattle, and 57 human E. coli O157:H7 strains as well as 76 non-O157 cattle and human E. coli strains. The results of all 221 strains were similar to the results generated by established multiplex PCR methods that involved two separate reactions to detect five virulence genes (stx1, stx2, eae, fliC, and hlyA). Specificity of the O antigen was indicated by amplification of only O157, and not O25, O26, O55, O78, O103, O111, O127, and O145 E. coli serotypes. Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 10⁴ CFU/g (10 cells/reaction) of E. coli O157. After a 6-h enrichment of E. coli O157-spiked samples, a sensitivity level of 10 CFU/g was achieved.     

The ascl1a and dlx genes have a regulatory role in the development of GABAergic interneurons in the zebrafish diencephalon

During development of the mouse forebrain interneurons, the Dlx genes play a key role in a gene regulatory network (GRN) that leads to the GABAergic phenotype. Here, we have examined the regulatory relationships between the ascl1a, dlx, and gad1b genes in the zebrafish forebrain. Expression of ascl1a overlaps with dlx1a in the telencephalon and diencephalon during early forebrain development. The loss of Ascl1a function results in a loss of dlx expression, and subsequent losses of dlx5a and gad1b expression in the diencephalic prethalamus and hypothalamus. Loss of Dlx1a and Dlx2a function, and, to a lesser extent, of Dlx5a and Dlx6a, impairs gad1b expression in the prethalamus and hypothalamus. We conclude that dlx1a/2a act downstream of ascl1a but upstream of dlx5a/dlx6a and gad1b to activate GABAergic specification. This pathway is conserved in the diencephalon, but has diverged between mammals and teleosts in the telencephalon.     

Characterization and expression of cry4Cb1 and cry30Ga1 from Bacillus thuringiensis strain HS18-1

We characterized a novel Bacillus thuringiensis isolate native to China (HS18-1) that shows a spherical crystal harboring two major proteins of about 70 and 130 kDa, and contains three novel cry genes (cry4Cb1, cry30Ga1, cry54-type). Furthermore, the cry4Cb1 and cry30Ga1 genes were expressed in Escherichia coli BL21 (DE3): pLysS. Insecticidal activity tests showed that the cry4Cb1 protein exhibited larvicidal activity against Aedes aegypti (Diptera) and the cry30Ga1 protein was toxic to both A. aegypti and P. xylostella (Lepidoptera).     

Effects of the mermithid nematode Ovomermis sinensis on the hemocytes of its host Helicoverpa armigera

Little is known about the mechanism by which mermithid nematodes avoid encapsulation responses of insect hosts. In this study, we investigated the influence of the mermithid nematode Ovomermis sinensis on host Helicoverpa armigera hemocyte number, encapsulation activity, spreading behavior and cytoskeleton. Parasitism by O. sinensis caused a significant increase in the total hemocyte counts (THC) and plasmatocyte numbers of H. armigera. However, in vivo encapsulation assays revealed that hemocyte encapsulation abilities of H. armigera were suppressed by O. sinensis. Moreover, parasitism by O. sinensis changed the spreading behavior and cytoskeletons of the host hemocytes. The results suggested that O. sinensis could actively suppress the hemocyte immune response of its host, possibly by destroying the host hemocyte cytoskeleton. This is the first report of a possible mechanism by which mermithid nematodes suppress encapsulation responses of insect hosts.     

br regulates the expression of the ecdysone biosynthesis gene npc1

The growth and metamorphosis of insects are regulated by ecdysteroid hormones produced in the ring gland. Ecdysone biosynthesis-related genes are both highly and specifically expressed in the ring gland. However, the intrinsic regulation of ecdysone biosynthesis has received little attention. Here we used the Drosophila npc1 gene to study the mechanism of ring gland-specific gene expression. npc1 is important for sterol trafficking in the ring gland during ecdysone biosynthesis. We have identified a conserved ring gland-specific cis-regulatory element (RSE) in the npc1 promoter using promoter fusion reporter analysis. Furthermore, genetic loss-of-function analysis and in vitro electrophoretic mobility shift assays revealed that the ecdysone early response gene broad complex (br) is a vital factor in the positive regulation of npc1 ring gland expression. Moreover, br also affects the ring gland expression of many other ecdysone biosynthetic genes as well as torso and InR, two key factors in the regulation of ecdysone biosynthesis. These results imply that ecdysone could potentially act through its early response gene br to achieve positive feedback regulation of ecdysone biosynthesis during development.     

Functional divergence of two duplicated D-lineage MADS-box genes BdMADS2 and BdMADS4 from Brachypodium distachyon

MADS-box genes are core members of the ABCDE model for flower development where D-lineage genes play essential roles in ovule identity determination. We report here the cloning and functional characterization of two duplicated MADS-box genes, BdMADS2 and BdMADS4 from Brachypodium distachyon, the model plant of temperate grasses. BdMADS2 and BdMADS4 were highly similar to grass D-lineage MADS-box genes on the protein level and they fell in a distinctive clade on the phylogenetic tree, with conserved intron/exon structures to their rice and maize orthologues. Quantitative real time PCR revealed comparable expression levels were detected in all floral organs of Brachypodium for both genes, except for the carpel where the expression level of BdMADS2 was five times higher than that of BdMADS4. Over expression of these two genes in Arabidopsis caused curly rosette leaves, small sepals and petals, and early flowering. However, BdMADS4 showed stronger phenotypic effects than BdMADS2, suggesting functional divergence between the two genes. Cis-regulatory element prediction showed that the promoter region (including the first intron) of BdMADS4 possesses much less class I BPC protein binding motifs than that of BdMADS2 which may be responsible for the specific expression in carpels. Yeast two-hybrid assays showed that both BdMADS2 and BdMADS4 can interact with BdSEP3, but BdMADS2 can additionally interact with the putative APETALA1 orthologue (BdAP1), suggesting a deviation in their protein interaction patterns. Taken together, our data demonstrate a significant divergence between the two Brachypodium D-lineage MADS-box genes and provide evidences for their sub-functionalization.     

A PCR-based method for mating type determination inCochliobolus heterostrophus

A rapid and accurate method based on multiplex PCR using three different primers was developed for determining mating type inCochliobolus heterostrophus. The primers of MAT113 and MAT123 were uniquely derived fromMAT1-1 andMAT1-2, respectively, and the primer MATcon5 is conserved in the flanking regions of the idiomorphs. The amplification product was estimated to be 702 bp (MAT1-1) or 547 bp (MAT1-2). Crossing experiments confirmed the accuracy of this method, which requires less time than the conventional determination of mating type.