Genome Sequence Resources for Four Phytopathogenic Fungi from the Colletotrichum orbiculare Species Complex.

Colletotrichum orbiculare species complex fungi are hemibiotrophic plant pathogens that cause anthracnose of field crops and weeds. Members of this group have genomes that are remarkably expanded relative to other Colletotrichum fungi and compartmentalized into AT-rich, gene-poor and GC-rich, gene-rich regions. Here, we present an updated version of the C. orbiculare genome, as well as draft genomes of three other members from the C. orbiculare species complex: the alfalfa pathogen C. trifolii, the prickly mallow pathogen C. sidae, and the burweed pathogen C. spinosum. The data reported here will be important for comparative genomics analyses to identify factors that play a role in the evolution and maintenance of the expanded, compartmentalized genomes of these fungi, which may contribute to their pathogenicity.

Recognition of pathogen-derived sphingolipids in Arabidopsis.

In plants, many invading microbial pathogens are recognized by cell-surface pattern recognition receptors, which induce defense responses. Here, we show that the ceramide Phytophthora infestans-ceramide D (Pi-Cer D) from the plant pathogenic oomycete P. infestans triggers defense responses in Arabidopsis. Pi-Cer D is cleaved by an Arabidopsis apoplastic ceramidase, NEUTRAL CERAMIDASE 2 (NCER2), and the resulting 9-methyl-branched sphingoid base is recognized by a plasma membrane lectin receptor-like kinase, RESISTANT TO DFPM-INHIBITION OF ABSCISIC ACID SIGNALING 2 (RDA2). 9-Methyl-branched sphingoid base is specific to microbes and induces plant immune responses by physically interacting with RDA2. Loss of RDA2 or NCER2 function compromised Arabidopsis resistance against an oomycete pathogen. Thus, we elucidated the recognition mechanisms of pathogen-derived lipid molecules in plants.

Correlation with larval body size of mRNA levels of growth hormone, growth hormone receptor I and insulin-like growth factor I in larval torafugu Takifugu rubripes.

The full-length of insulin-like growth factor (IGF) complementary (c)DNAs encoded by igf-I and igf-II from torafugu pufferfish Takifugu rubripes were cloned in the present study. The deduced amino acid sequences of the two genes showed c. 80% identity each with those of Igf-I and Igf-II from other teleosts, respectively. Two growth hormone (GH) receptors, ghr1 and ghr2, were also cloned in silico using the T. rubripes Fugu genome database. The transcripts of T. rubripes igf-I were detected in slow muscle, heart, skin, gill, liver and intestine but not in fast muscle, spleen and testis of adult fish, whereas those of igf-II were found in all tissues examined. Subsequently, the accumulated messenger (m)RNA levels of igf-I and igf-II were investigated in an F(2) population derived from a male of an apparent fast-growing T. rubripes strain and a wild female T. rubripes together with those of other growth-related genes encoding Gh, Ghr1 and Ghr2, and with those of prolactin (Prl) and leptin (Lep) previously reported. The accumulated mRNA levels of igf-I, gh and ghr1 were significantly correlated to growth rate at larval stages in the population, but not for those of igf-II, prl, ghr2 and lep. Although it is unclear whether or not this phenotype is directly related to the heredity of the fast-growing strain, the findings suggest that the expression of igf-I, gh and ghr1 is involved in the regulation of growth rate at larval stages in T. rubripes.

Colletotrichum shisoi sp. nov., an anthracnose pathogen of Perilla frutescens in Japan: molecular phylogenetic, morphological and genomic evidence.

Species of the fungal genus Colletotrichum are among the most devastating pathogens of agricultural crops in the world. Based on DNA sequence data (ITS, GAPDH, CHS-1, ACT, TUB2) and morphology, we revealed Colletotrichum isolates infecting the oil crop Perilla frutescens, commonly known as shiso, to represent a previously unknown species of the C. destructivum species complex and described it as C. shisoi. We found that C. shisoi appears to be able to adopt a hemibiotrophic lifestyle, characterised by the formation of biotrophic hyphae followed by severe necrotic lesions on P. frutescens, but is less virulent on Arabidopsis, compared to its close relative C. higginsianum which also belongs to the C. destructivum species complex. The genome of C. shisoi was sequenced, annotated and its predicted proteome compared with four other Colletotrichum species. The predicted proteomes of C. shisoi and C. higginsianum, share many candidate effectors, which are small, secreted proteins that may contribute to infection. Interestingly, C. destructivum species complex-specific secreted proteins showed evidence of increased diversifying selection which may be related to their host specificities.

Signal transduction in plant immunity.

Significant recent advances in the understanding of plant defense mechanisms include the isolation and characterization of resistance genes against bacterial, fungal and viral pathogens, the identification of genes involved in cell death, and the demonstration of the involvement of reactive oxygen species and salicylic acid in the signal-transduction pathways for expression of induced resistance.

Draft Genome Assembly of Colletotrichum chlorophyti, a Pathogen of Herbaceous Plants.

Colletotrichum chlorophyti is a fungal pathogen that infects various herbaceous plants, including crops such as legumes, tomato, and soybean. Here, we present the genome of C. chlorophyti NTL11, isolated from tomato. Analysis of this genome will allow a clearer understanding of the molecular mechanisms underlying fungal host range and pathogenicity.

The U-box protein family in plants.

The U-box is a highly conserved domain recently identified at the C terminus of yeast UFD2, an E4 ubiquitination factor. In yeast, UFD2 is the only U-box-containing protein, but there are two UFD2 homologs and several other proteins containing a U-box domain in humans. Intriguingly, a database search revealed 37 predicted proteins containing a U-box in Arabidopsis. The plant U-box (PUB) proteins form five distinct subclasses, suggesting that they play diverse roles. The ARC1 gene from Brassica, required for self-incompatibility, is currently the only PUB gene functionally characterized. Here, we discuss the characteristics and possible functions of the PUB gene family.

Evidence for direct action of estradiol on growth hormone-releasing factor (GRF) in rat hypothalamus: localization of [3H]estradiol in GRF neurons.

Sex steroids have been shown to influence the secretion of GH. There appears to be no good evidence of the effect of estradiol on the anterior pituitary, while the central site of estradiol action on the regulation of GH secretion is not known. The present investigation was carried out to determine whether some of the GH-releasing factor (GRF) neurons and somatostatin (SRIF) neurons in the hypothalamus and GH cells in the pituitary contain estradiol receptors. Colocalization of [3H]estradiol and antibodies to GRF or SRIF in brain and antibodies to GH in pituitary was studied to show interrelationships between estrogen target cells and peptidergic cells. Eight female Sprague-Dawley rats were ovariectomized, each rat was treated with colchicine, and 24-48 h later the animals were given an iv injection of [2,4,6,7,16,17-3H]estradiol (SA, 166 Ci/mM) at a dose of 0.5 micrograms/100 g BW. One hour after the injection, the rats were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The hypothalami from the perfused rats and the pituitaries from unperfused rats were frozen in isopentane precooled in liquid nitrogen (-190 C) and processed for autoradiography. The brain autoradiograms were immunostained for GRF, SRIF, and tyrosine hydroxylase [TH; an enzyme for the synthesis of dopamine (DA)], and the pituitary autoradiograms were immunostained for GH by the avidin-biotin peroxidase method. The majority of GRF-containing neurons were found in the arcuate nucleus, with some scattered cells in the lateral region of the ventromedial nucleus and the basal lateral hypothalamus. In the central portion of the arcuate nucleus, 20-30% of GRF-containing neurons showed nuclear concentration of [3H]estradiol. In the anterior portion of the hypothalamus, 10-15% of immunoreactive GRF-containing neurons were labeled with [3H]estradiol. In the lateral basal hypothalamus and the lateral region to the ventromedial nucleus, a few GRF neurons showed nuclear concentration of radioactivity. In contrast, a few SRIF cells in hypothalamic periventricular nucleus showed nuclear labeling with [3H]estradiol. Dual immunostaining with GRF and TH antibodies revealed that the estradiol-labeled GRF neurons did not contain TH immunoreactivity. In addition, 80-90% of GH cells in the anterior pituitary showed nuclear concentration of [3H]estradiol. The present studies demonstrate for the first time that certain populations of GRF neurons are targets for estradiol and indicate that estradiol acts directly on certain hypothalamic GRF neurons. The results suggest that estradiol may have a role in the regulation of GH secretion by modulating GRF release and acting directly on the somatotrophs.

Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens.

The virB operon of the Agrobacterium tumefaciens Ti plasmid encodes 11 proteins. Specific antisera to VirB2, VirB3 and VirB9 were used to locate these virulence proteins in the A. tumefaciens cell. Immunoblot analysis located VirB2 protein to the inner and outer membranes; VirB3 and VirB9 were likewise associated with both membranes, but mainly in the outer membrane. VirB2 is processed from a 12.3-kDa protein into a 7.2-kDa polypeptide. Such sized protein results from cleavage at residue Ala47, upstream of which two additional alanine residues Ala45-Ala46 are contained and bearing resemblance to a signal peptide peptidase-I cleavage sequence. VirB2 and VirB3 sequences are strikingly similar to the pilin biosynthetic proteins TraA and TraL encoded by the tra operon of F and R1-19 plasmids. Since traA encodes a propilin that is cleaved into a 7.2-kDa conjugative pilin product and since this cleavage site is present in both TraA and VirB2, we propose that virB2 encodes a pilin-like protein which together with VirB3 and VirB9 as well as other VirB proteins may be used for interkingdom T-DNA transfer between bacteria and plants.

Huge ovarian endometrial cyst: a case report.

We are reporting a rare case of a huge ovarian endometrioma, one as large as an adult head, and the difficulty of differential diagnosis regarding this ovarian tumor.

Regulators of cell death in disease resistance.

Cell death and disease resistance are intimately connected in plants. Plant disease resistance genes (R genes) are key components in pathogen perception and have a potential to activate cell death pathways. Analysis of R proteins suggests common molecular mechanisms for pathogen recognition and signal emission whereas the subsequent signalling unexpectedly involves a network of pathways of parallel, branching and converging action. Disease resistance signalling mutants have revealed novel types of regulatory proteins whose biochemical functions are still unknown. Accumulation of small molecules such as salicylic acid, reactive oxygen intermediates, and nitric oxide amplifies resistance responses and directs cells to initiate cell death programs. Genetic analyses of lesion mimic mutants provide a glimpse of how cell death thresholds are set via an interplay of positive and negative regulatory components.

The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein.

The process of T-DNA transfer from Agrobacterium tumefaciens to plant cells is thought to involve passage of a DNA-protein complex through a specialized structure in the bacterial membrane. The virB operon of A. tumefaciens encodes 11 proteins, of which 9 are known to be located in the membranes and 10 have been shown to be essential for virulence. Sequence comparisons between proteins encoded by the virB operon and those encoded by operons from conjugative plasmids indicated that VirB proteins may form a structure similar to a conjugative pilus. Here, we examine the effects of mutations in virB4 on the accumulation and localization of other VirB proteins. VirB4 shares amino acid sequence similarity with the TraC protein of plasmid F, which is essential for pilus formation in Escherichia coli, and with the PtlC protein of Bordetella pertussis, which is required for toxin secretion. Polar and nonpolar virB4 mutants were examined, and all were shown to be unable to accumulate VirB3 protein to wild-type levels. A low level of VirB3 protein which was present in induced NT1RE cells harboring virB4 nonpolar mutant pBM1130 was found to associate with the inner membrane fraction only, whereas in wild-type cells VirB3 associated with both inner and outer membranes. The results indicate that for VirB3 to accumulate in the outer membrane, VirB4 must also be present, and it is possible that one role of VirB4 is in the correct assembly of a VirB protein membrane structure.

Chromosome landing at the barley Rar1 locus.

The barley Rar1 gene is an essential component of the race-specific, Mla-12-specified powdery mildew resistance reaction. As part of a map-based cloning strategy designed to isolate Rar1, five barley yeast artificial chromosomes (YACs) have been identified, ranging in size from 300 to 1100 kb. PCR-based YAC end-specific markers have been established and were employed to construct a local YAC contig. Four out of five YAC clones were found to be non-colinear with the source DNA. High-resolution genetic mapping of the YAC ends demonstrated that the set of five overlapping YAC clones encompasses the barley Rar1 gene.

Characterization of the virB operon of an Agrobacterium tumefaciens Ti plasmid: nucleotide sequence and protein analysis.

The virulence regulon of the Agrobacterium tumefaciens TiC58 plasmid is composed of six operons, virA, virB, virG, virC, virD and virE, which direct the transfer of T-DNA into plant cells. The 9.5 kbp virB operon is the largest of these operons and its entire nucleotide sequence was determined and found to contain eleven open reading frames (ORFs). Gene fusions of each VirB ORF to T7 phi 10 were made and overexpressed in Escherichia coli to confirm that they encode proteins of predicted size. Hydrophobic analysis of these peptide sequences revealed nine proteins that contain hydrophobic spanning regions including signal-peptide-like sequences. These data suggest that the majority of VirB proteins may associate with bacterial cell membranes, while the two additional proteins possess a potential ATP-binding site. Strong homologies in amino acid sequences were observed between nopaline- and octopine-type plasmids. Specific differences in amino acid sequence encoded by VirB ORFs of nopaline and octopine Ti plasmid and a functional role of the gene products are discussed.

A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion.

Organisms with large genomes contain vast amounts of repetitive DNA sequences, much of which is composed of retrotransposons. Amplification of retrotransposons has been postulated to be a major mechanism increasing genome size and leading to "genomic obesity." To gain insights into the relation between retrotransposons and genome expansion in a large genome, we have studied a 66-kb contiguous sequence at the Rar1 locus of barley in detail. Three genes were identified in the 66-kb contig, clustered within an interval of 18 kb. Inspection of sequences flanking the gene space unveiled four novel retroelements, designated Nikita, Sukkula, Sabrina, and BAGY-2 and several units of the known BARE-1 element. The retroelements identified are responsible for at least 15 integration events, predominantly arranged as multiple nested insertions. Strikingly, most of the retroelements exist as solo LTRs (Long Terminal Repeats), indicating that unequal crossing over and/or intrachromosomal recombination between LTRs is a common feature in barley. Our data suggest that intraelement recombination events deleted most of the original retrotransposon sequences, thereby providing a possible mechanism to counteract retroelement-driven genome expansion.

An inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes.

The 9.5 kb virB operon is the largest of the six major operons in the Ti plasmid vir region. This operon contains eleven genes, the largest of which is virB4. This gene encodes an 84 kDa protein whose function has not been identified. Its roles in conferring virulence on Agrobacterium tumefaciens and in the T-DNA transfer process were determined by generating non-polar mutants by using the Tn5pvirB transposon in which the virB promoter is transcribed downstream of its position of insertion. Several independent mutants were isolated and each insertion site in virB4 was confirmed by nucleotide sequence analysis. These mutants were tested for T-DNA transfer ability by agroinfection and for tumorigenicity by inoculation in Brassica and Datura. All mutants were agroinfection- and tumorigenicity-negative. These data strongly suggest that virB4 is essential for both the interkingdom transfer of the T-DNA and virulence. Furthermore, by using anti-VirB4 serum, the protein product of virB4 was localized to the inner-membrane fraction of A. tumefaciens. Purified VirB4 protein hydrolyses ATP and this activity was quenched by the anti-VirB4 serum. The energy generated by VirB4 ATPase therefore may be used to transfer T-DNA or to assemble the T-DNA transfer apparatus on the bacterial membrane. Protein sequence analyses revealed striking similarities between VirB4 protein and the proteins required for conjugative transfer, which include TraC, TrwK, and TrbE of plasmids F, R388, and RP4, respectively. These findings suggest that VirB proteins play a direct role in the assembly of a conjugative transfer apparatus required for the transfer of the T-DNA from A. tumefaciens to plant cells.

Mechanisms involved in the pituitary desensitization induced by gonadotropin-releasing hormone agonists.

OBJECTIVE: We investigated the mechanisms of desensitization induced by gonadotropin-releasing hormone agonist in the pituitary. STUDY DESIGN: Effects of gonadotropin-releasing hormone agonist on the pituitary were studied in vitro and in vivo in the rat. In the clinical study serum luteinizing hormone was measured by radioimmunoassay with a polyclonal luteinizing hormone antibody (luteinizing hormone-radioimmunoassay) and by immunoradiometric assay with monoclonal luteinizing hormone antibodies (luteinizing hormone-immunoradiometric assay) during gonadotropin-releasing hormone agonist treatment. RESULTS: In the in vitro study bead-attached pituitary cells that were desensitized with a continuous infusion of 10(-7)mol/L gonadotropin-releasing hormone responded to 50 mmol/L K+. In the in vivo study gonadotropin-releasing hormone binding sites and rat luteinizing hormone beta-messenger ribonucleic acid in the pituitary decreased during gonadotropin-releasing hormone agonist treatment, but serum levels of rat luteinizing hormone did not decrease. In addition, a disparity between luteinizing hormone-radioimmunoassay and luteinizing hormone-immunoradiometric assay was demonstrated during gonadotropin-releasing hormone agonist treatment. CONCLUSION: Pituitary desensitization in response to gonadotropin-releasing hormone agonist may not be wholly receptor mediated and a nonreceptor process may be involved.

VirB2 is a processed pilin-like protein encoded by the Agrobacterium tumefaciens Ti plasmid.

The mechanism of DNA transmission between distinct organisms has remained a subject of long-standing interest. Agrobacterium tumefaciens mediates the transfer of plant oncogenes in the form of a 25-kb T-DNA sector of a resident Ti plasmid. A growing body of evidence leading to the elucidation of the mechanism involved in T-DNA transfer comes from studies on the vir genes contained in six major operons that are required for the T-DNA transfer process. Recent comparative amino acid sequence studies of the products of these vir genes have revealed interesting similarities between Tra proteins of Escherichia coli F factor, which are involved in the biosynthesis and assembly of a conjugative pilus, and VirB proteins encoded by genes of the virB operon of A. tumefaciens pTiC58. We have previously identified VirB2 as a pilin-like protein with processing features similar to those of TraA of the F plasmid and have shown that VirB2 is required for the biosynthesis of pilin on a flagella-free Agrobacterium strain. In the present work, VirB2 is found to be processed and localized primarily to the cytoplasmic membrane in E. coli. Cleavage of VirB2 was predicted previously to occur between alanine and glutamine in the sequence -Pro-Ala-Ala-Ala-Glu-Ser-. This peptidase cleavage sequence was mutated by an amino acid substitution for one of the alanine residues (D for A at position 45 [A45D]), by deletion of the three adjacent alanines, and by a frameshift mutation 22 bp upstream of the predicted Ala-Glu cleavage site. With the exception of the frameshift mutation, the alanine mutations do not prevent VirB2 processing in E. coli, while in A. tumefaciens they result in VirB2 instability, since no holo- or processed protein is detectable. All of the above mutations abolish virulence. The frameshift mutation abolishes processing in both organisms. These results indicate that VirB2 is processed into a 7.2-kDa structural protein. The cleavage site in E. coli appears to differ from that predicted in A. tumefaciens. Yet, the cleavage sites are relatively close to each other since the final cleavage products are similar in size and are produced irrespective of the length of the amino-terminal portion of the holoprotein. As we observed previously, the similarity between the processing of VirB2 in A. tumefaciens and the processing of the propilin TraA of the F plasmid now extends to E. coli.

Increases in ovarian GnRH receptors by following GnRH treatment.

Using a gonadotropin-releasing hormone (GnRH) analog, [D-Ser (tBu6)] des-Gly10-GnRH-N-ethlamide (GnRHa) as a ligand the binding capacity of the rat ovary to GnRH during sexual maturation and the mechanism regulating GnRH binding capacity were examined. Specific high affinity binding sites for GnRH were observed in the ovary and the Kd values for the granulosa cells and the residual tissue were similar to those of whole ovary. During sexual maturation, the GnRH binding capacity of the ovary rose from 7 days of age to a peak of 28 days and declined during the prepubertal period. The treatment with PMSG decreased GnRH binding capacity in the residual tissue as well as in the whole ovary but did not change the binding capacity in the granulosa cells in diethylstilbestrol (DES) primed hypophysectomized rats. Repeated injections of GnRH caused a significant increase in the number of GnRH receptors of the ovary in PMSG treated DES primed hypophysectomized rats but not in the saline treated rat. The granulosa cells exhibited increases in GnRH binding capacity following repeated administrations of GnRH more than the residual tissue did. In GnRH treated DES primed hypophysectomized rats, increasing doses of PMSG increased the binding capacity in the granulosa cells but decreased the binding capacity in the residual tissue. From these findings, GnRH in combination with PMSG seems to have stimulatory effects on GnRH binding capacity and to increase the sensitivity to GnRH in the granulosa cells.