Calcineurin Subunits A and B Interact to Regulate Growth and Asexual and Sexual Development in Neurospora crassa

Calcineurin is a calcium/calmodulin dependent protein phosphatase in eukaryotes that consists of a catalytic subunit A and a regulatory subunit B. Previous studies in the filamentous fungus Neurospora crassa had suggested that the catalytic subunit of calcineurin might be an essential protein. We generated N. crassa strains expressing the A (cna-1) and B (cnb-1) subunit genes under the regulation of P_tcu-1, a copper-responsive promoter. In these strains, addition of bathocuproinedisulfonic acid (BCS), a copper chelator, results in induction of cna-1 and cnb-1, while excess Cu²⁺ represses gene expression. Through analysis of these strains under repressing and inducing conditions, we found that the calcineurin is required for normal growth, asexual development and female fertility in N. crassa. Moreover, we isolated and analyzed cnb-1 mutant alleles generated by repeat-induced point mutation (RIP), with the results further supporting roles for calcineurin in growth and fertility in N. crassa. We demonstrated a direct interaction between the CNA-1 and CNB-1 proteins using an assay system developed to study protein-protein interactions in N. crassa.     

The Wor1-like Protein Fgp1 Regulates Pathogenicity,Toxin Synthesis and Reproduction in the Phytopathogenic Fungus Fusarium graminearum

WOR1 is a gene for a conserved fungal regulatory protein controlling the dimorphic switch and pathogenicity determents in Candida albicans and its ortholog in the plant pathogen Fusarium oxysporum, called SGE1, is required for pathogenicity and expression of key plant effector proteins. F. graminearum, an important pathogen of cereals, is not known to employ switching and no effector proteins from F. graminearum have been found to date that are required for infection. In this study, the potential role of the WOR1-like gene in pathogenesis was tested in this toxigenic fungus. Deletion of the WOR1 ortholog (called FGP1) in F. graminearum results in greatly reduced pathogenicity and loss of trichothecene toxin accumulation in infected wheat plants and in vitro. The loss of toxin accumulation alone may be sufficient to explain the loss of pathogenicity to wheat. Under toxin-inducing conditions, expression of genes for trichothecene biosynthesis and many other genes are not detected or detected at lower levels in Δfgp1 strains. FGP1 is also involved in the developmental processes of conidium formation and sexual reproduction and modulates a morphological change that accompanies mycotoxin production in vitro. The Wor1-like proteins in Fusarium species have highly conserved N-terminal regions and remarkably divergent C-termini. Interchanging the N- and C- terminal portions of proteins from F. oxysporum and F. graminearum resulted in partial to complete loss of function. Wor1-like proteins are conserved but have evolved to regulate pathogenicity in a range of fungi, likely by adaptations to the C-terminal portion of the protein.     

黄瓜转新型抗菌蛋白基因GNK2-1及其抗枯萎病的研究

GNK2-1为一种来自银杏(Ginkgo biloba)种仁的新型抗真菌蛋白, 具有较强的真菌抗性且性质稳定。序列分析表明,其结构与所有已知的抗真菌蛋白不同, 而与富含半胱氨酸的植物类受体激酶的胞外结构域相似。为探索GNK2-1基因在黄瓜(Cucumis sativus)抗病反应中的作用, 利用基因重组技术构建了GNK2-1的高效组成型表达载体, 并利用根癌农杆菌(Agrobacterium tumefaciens)介导转入黄瓜栽培品种农城3号(Cucumis sativus ‘Nongcheng No.3’)基因组中。通过对获得的抗性植株进行PCR、RT-PCR和Western blot检测分析, 结果表明GNK2-1基因可在T₀代转基因植株中转录表达, 并能在T₁代转基因黄瓜中稳定遗传。离体枯萎病抗性鉴定结果表明, 转GNK2-1基因的黄瓜对枯萎病的抗性增强, GNK2-1可以作为黄瓜抗病性改良的潜在基因资源。     

Pterostilbene Acts through Metastasis-Associated Protein 1 to Inhibit Tumor Growth,Progression and Metastasis in Prostate Cancer

The development of natural product agents with targeted strategies holds promise for enhanced anticancer therapy with reduced drug-associated side effects. Resveratrol found in red wine, has anticancer activity in various tumor types. We reported earlier on a new molecular target of resveratrol, the metastasis-associated protein 1 (MTA1), which is a part of nucleosome remodeling and deacetylation (NuRD) co-repressor complex that mediates gene silencing. We identified resveratrol as a regulator of MTA1/NuRD complex and re-activator of p53 acetylation in prostate cancer (PCa). In the current study, we addressed whether resveratrol analogues also possess the ability to inhibit MTA1 and to reverse p53 deacetylation. We demonstrated that pterostilbene (PTER), found in blueberries, had greater increase in MTA1-mediated p53 acetylation, confirming superior potency over resveratrol as dietary epigenetic agent. In orthotopic PCa xenografts, resveratrol and PTER significantly inhibited tumor growth, progression, local invasion and spontaneous metastasis. Furthermore, MTA1-knockdown sensitized cells to these agents resulting in additional reduction of tumor progression and metastasis. The reduction was dependent on MTA1 signaling showing increased p53 acetylation, higher apoptotic index and less angiogenesis in vivo in all xenografts treated with the compounds, and particularly with PTER. Altogether, our results indicate MTA1 as a major contributor in prostate tumor malignant progression, and support the use of strategies targeting MTA1. Our strong pre-clinical data indicate PTER as a potent, selective and pharmacologically safe natural product that may be tested in advanced PCa.     

Myosin VIIA defects, which underlie the Usher 1B syndrome in humans, lead to deafness in Drosophila

In vertebrates, auditory and vestibular transduction occurs on apical projections (stereocilia) of specialized cells (hair cells). Mutations in myosin VIIA (myoVIIA), an unconventional myosin, lead to deafness and balance anomalies in humans, mice, and zebrafish; individuals are deaf, and stereocilia are disorganized. The exact mechanism through which myoVIIA mutations result in these inner-ear anomalies is unknown. Proposed inner-ear functions for myoVIIA include anchoring transduction channels to the stereocilia membrane, trafficking stereocilia linking components, and anchoring hair cells by associating with adherens junctions. The Drosophila myoVIIA homolog is crinkled (ck). The Drosophila auditory organ, Johnston's organ (JO), is developmentally and functionally related to the vertebrate inner ear. Both derive from modified epithelial cells specified by atonal and spalt homolog expression, and both transduce acoustic mechanical energy (and references therein). Here, we show that loss of ck/myoVIIA function leads to complete deafness in Drosophila by disrupting the integrity of the scolopidia that transduce auditory signals. We demonstrate that ck/myoVIIA functions to organize the auditory organ, that it is functionally required in neuronal and support cells, that it is not required for TRPV channel localization, and that it is not essential for scolopidial-cell-junction integrity.     

FigA,a Putative Homolog of Low-Affinity Calcium System Member Fig1 in Saccharomyces cerevisiae,Is Involved in Growth and Asexual and Sexual Development in Aspergillus nidulans

Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca²⁺ rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.     

Effect of 5-Azacytidine on the Light-Sensitive Formation of Sexual and Asexual Reproductive Structures in wc-1 and wc-2 Mutants of Neurospora crassa

Under nitrogen starvation conditions, illumination by blue light of wc-1 and wc-2 mutants of the ascomycete Neurospora crassa failed to stimulate the formation of protoperithecia and inhibit condition (contrary to what was observed in the mycelium of the wild-type fungus). The data obtained indicate that wc-1 and wc-2 genes of N. crassa are involved in the light-dependent formation of protoperithecia and conidia. The effects of 5-azacytidine (an inhibitor of DNA methylation) under the same experimental conditions suggest that the balance between the formation of sexual and asexual reproductive structures, maintained in N. crassa, depends on genome methylation processes sensitive to the action of light, which is mediated by the photoreceptor complex of WC proteins.     

Chemokine-Like Receptor 1 Deficiency Does Not Affect the Development of Insulin Resistance and Nonalcoholic Fatty Liver Disease in Mice

The adipokine chemerin and its receptor, chemokine-like receptor 1 (Cmklr1), are associated with insulin resistance and nonalcoholic fatty liver disease (NAFLD), which covers a broad spectrum of liver diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). It is possible that chemerin and/or Cmklr1 exert their effects on these disorders through inflammation, but so far the data have been controversial. To gain further insight into this matter, we studied the effect of whole-body Cmklr1 deficiency on insulin resistance and NAFLD. In view of the primary role of macrophages in hepatic inflammation, we also transplanted bone marrow from Cmklr1 knock-out (Cmklr1^-/-) mice and wild type (WT) mice into low-density lipoprotein receptor knock-out (Ldlr^-/-) mice, a mouse model for NASH. All mice were fed a high fat, high cholesterol diet containing 21% fat from milk butter and 0.2% cholesterol for 12 weeks. Insulin resistance was assessed by an oral glucose tolerance test, an insulin tolerance test, and by measurement of plasma glucose and insulin levels. Liver pathology was determined by measuring hepatic inflammation, fibrosis, lipid accumulation and the NAFLD activity score (NAS). Whole-body Cmklr1 deficiency did not affect body weight gain or food intake. In addition, we observed no differences between WT and Cmklr1^-/- mice for hepatic inflammatory and fibrotic gene expression, immune cell infiltration, lipid accumulation or NAS. In line with this, we detected no differences in insulin resistance. In concordance with whole-body Cmklr1 deficiency, the absence of Cmklr1 in bone marrow-derived cells in Ldlr^-/- mice did not affect their insulin resistance or liver pathology. Our results indicate that Cmklr1 is not involved in the pathogenesis of insulin resistance or NAFLD. Thus, we recommend that the associations reported between Cmklr1 and insulin resistance or NAFLD should be interpreted with caution.     

Screening survey of co-production of fusaric acid,fusarin C,and fumonisins B1, B2 and B3 by Fusarium strains grown in maize grains

Fusarium species isolated from Belgian maize were screened for their ability to produce fusarin C, fusaric acid, fumonisins B₁ (FB₁), FB₂ and FB₃ in maize grains. First, cultivation of Fusarium species in Myro liquid medium allowed overcoming the shortage of the standard of fusarin C on the market. All Fusarium verticillioides produced much higher contents of mycotoxins in Myro compared to Fusarium graminearum or Fusarium venenatum. The optimization of the LC-MS/MS method resulted in low limits of detection and quantification for fusarin C, fusaric acid, FB₁, FB₂ and FB₃ determination in maize grains. Its application for screening the potential toxin production ability evidenced that the concentrations of the analytes were significantly increased at various levels when F. verticillioides strains were cultivated in maize grains and reached 441 mg kg^?1 for fusaric acid, 74 mg kg^?1 for fusarin C, 1,301 mg kg^?1 for FB₁, 367 mg kg^?1 for FB₂ and 753 mg kg^?1 for FB₃.     

Host Protein BSL1 Associates with Phytophthora infestans RXLR Effector AVR2 and the Solanum demissum Immune Receptor R2 to Mediate Disease Resistance

Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor.     

蛋白质酪氨酸磷酸酶1B（PTP1B）与2型糖尿病及肥胖的关系

蛋白质酪氨酸磷酸酶1B（PTP1B）是一种在体内广泛表达的胞内蛋白质酪氨酸磷酸酶,在调节胰岛素敏感性和能量代谢的过程中起着重要作用。通过抑制PTP1B可增加胰岛素和瘦蛋白（leptin）的活性, 为寻找2型糖尿病、肥胖的治疗提供了光明前景。