Fusarium head blight (FHB) caused by Fusarium graminearum is a destructive disease of wheat and barley worldwide. In a previous study of systematic characterization of protein kinase genes in F. graminearum, mutants of three putative components of the osmoregulation MAP kinase pathway were found to have distinct colony morphology and hyphal growth defects on PDA plates. Because the osmoregulation pathway is not known to regulate aerial hyphal growth and branching, in this study we further characterized the functions of the FgHog1 pathway in growth, pathogenesis, and development. The Fghog1, Fgpbs2, and Fgssk2 mutants were all reduced in growth rate, aerial hyphal growth, and hyphal branching angle. These mutants were not only hypersensitive to osmotic stress but also had increased sensitivity to oxidative, cytoplasm membrane, and cell wall stresses. The activation of FgHog1 was blocked in the Fgpbs2 and Fgssk2 mutants, indicating the sequential activation of FgSsk2-FgPbs2-FgHog1 cascade. Interestingly, the FgHog1 MAPK pathway mutants appeared to be sensitive to certain compounds present in PDA. They were female sterile but retained male fertility. We also used the metabolomics profiling approach to identify compatible solutes that were accumulated in the wild type but not in the Fghog1 deletion mutant. Overall, our results indicate that the FgSsk2-FgPbs2-FgHog1 MAPK cascade is important for regulating hyphal growth, branching, plant infection, and hyperosmotic and general stress responses in F. graminearum.

Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M. oryzae. CAP1 may also play a role in feedback inhibition of Ras2 signaling when Pmk1 is activated.

Author Summary

In Magnaporthe oryzae, cAMP signaling is known to play an important role in surface recognition and plant penetration. The Mac1 adenylate cyclase is essential for plant infection. To better understand Mac1 activation mechanisms, in this study we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting protein is the adenylate cyclase associated protein (CAP) encoded by the CAP1 gene. Results from our study indicated that Cap1 is important for Mac1 activation and plant infection in M. oryzae. The Δcap1 mutant was defective in germ tube growth and appressorium formation and failed to cause typical blast lesions. Like LifeAct, Cap1 localized to apical patches in vegetative hyphae but not in invasive hyphae. The P2 proline-rich region was important for Cap1 localization but the actin-binding domain played a role in feedback inhibition of Ras signaling. To our knowledge, functional characterization of CAP genes has not been reported in filamentous fungi. Our results indicate that CAP1 is important for regulating adenylate cyclase activities, appressorium morphogenesis, and plant infection. Further characterization of CAP1 will be important to better understand the interaction between cAMP signaling and the PMK1 pathway in M. oryzae.

Arginine methylation of non-histone proteins by protein arginine methyltransferase (PRMT) has been shown to be important for various biological processes from yeast to human. Although PRMT genes are well conserved in fungi, none of them have been functionally characterized in plant pathogenic ascomycetes. In this study, we identified and characterized all of the four predicted PRMT genes in Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant had pleiotropic defects. AMT1 is a predicted type I PRMT gene that is orthologous to HMT1 in Saccharomyces cerevisiae. The Δamt1 mutant was slightly reduced in vegetative growth but normal in asexual and sexual reproduction. It had increased sensitivities to oxidative and membrane stresses. DON mycotoxin production and virulence on flowering wheat heads also were reduced in the Δamt1 mutant. The introduction of the wild-type AMT1 allele fully complemented the defects of the Δamt1 mutant and Amt1-GFP fusion proteins mainly localized to the nucleus. Hrp1 and Nab2 are two hnRNPs in yeast that are methylated by Hmt1 for nuclear export. In F. graminearum, AMT1 is required for the nuclear export of FgHrp1 but not FgNab2, indicating that yeast and F. graminearum differ in the methylation and nucleo-cytoplasmic transport of hnRNP components. Because AMT2 also is a predicted type I PRMT with limited homology to yeast HMT1, we generated the Δamt1 Δamt2 double mutants. The Δamt1 single and Δamt1 Δamt2 double mutants had similar defects in all the phenotypes assayed, including reduced vegetative growth and virulence. Overall, data from this systematic analysis of PRMT genes suggest that AMT1, like its ortholog in yeast, is the predominant PRMT gene in F. graminearum and plays a role in hyphal growth, stress responses, and plant infection.

During the process of matrix vesicle (MV)-mediated initiation of mineralisation, chondrocytes and osteoblasts mineralise the extracellular matrix by promoting the seeding of basic calcium phosphate crystals of hydroxyapatite (HA) along the collagen fibrils. This orchestrated process is carefully regulated by the balanced action of propagators and inhibitors of calcification. The primary antagonistic regulators of extracellular matrix mineralisation are phosphate (Pi) and inorganic pyrophosphate (PPi). Studies in mouse models and in humans have established critical roles for Pi/PPi homeostasis in biomineralisation. In this review, we present the regulators of Pi/PPi, as derived from animal models, and discuss their clinical relevance to physiological and pathological mineralisation.

The formation of appressoria, specialized plant penetration structures of Magnaporthe oryzae, is regulated by the MST11-MST7-PMK1 MAP kinase cascade. One of its downstream transcription factor, MST12, is important for penetration and invasive growth but dispensable for appressorium formation. To identify additional downstream targets that are regulated by Pmk1, in this study we performed phosphorylation assays with a protein microarray composed of 573 M. oryzae transcription factor (TF) genes. Three of the TF genes phosphorylated by Pmk1 in vitro were further analyzed by coimmunoprecipitation assays. One of them, MoSFL1, was found to interact with Pmk1 in vivo. Like other Sfl1 orthologs, the MoSfl1 protein has the HSF-like domain. When expressed in yeast, MoSFL1 functionally complemented the flocculation defects of the sfl1 mutant. In M. oryzae, deletion of MoSFl1 resulted in a significant reduction in virulence on rice and barley seedlings. Consistent with this observation, the Mosfl1 mutant was defective in invasive growth in penetration assays with rice leaf sheaths. In comparison with that of vegetative hyphae, the expression level of MoSFL1 was increased in appressoria and infected rice leaves. The Mosfl1 mutant also had increased sensitivity to elevated temperatures. In CM cultures of the Mosfl1 and pmk1 mutants grown at 30°C, the production of aerial hyphae and melanization were reduced but their growth rate was not altered. When assayed by qRT-PCR, the transcription levels of the MoHSP30 and MoHSP98 genes were reduced 10- and 3-fold, respectively, in the Mosfl1 mutant. SFL1 orthologs are conserved in filamentous ascomycetes but none of them have been functionally characterized in non-Saccharomycetales fungi. MoSfl1 has one putative MAPK docking site and three putative MAPK phosphorylation sites. Therefore, it may be functionally related to Pmk1 in the regulation of invasive growth and stress responses in M. oryzae.

Secreted protein, acidic and rich in cysteine (SPARC) has been found to be involved in various stages of tumor progression such as migration, invasion, extracellular matrix deposition and angiogenesis. To obtain an insight into the role of SPARC in the progression of laryngeal and hypopharyngeal carcinoma, we investigated SPARC transcription levels and promoter methylation in carcinoma cell lines and primary tumors. Reverse transcription-PCR showed that SPARC was silenced in laryngeal and hypopharyngeal carcinoma cell lines, in which aberrant promoter methylation was detected. Hypermethylation of SPARC was detected in 56.1% (23/41) of laryngeal carcinoma and 70.0% (7/10) of hypopharyngeal carcinoma biopsies, but only in 11.1% (1/9) of normal epithelial specimens by a methylation-specific PCR assay. Bisulphite genomic sequencing indicated that CpG sites in the SPARC promoter were heavily methylated in cell lines and primary tumors. Moreover, pharmacological demethylation treatment rescued SPARC expression with 5-aza-2′-deoxycytidine (5-aza-dC) in the laryngeal carcinoma cell lines. SPARC promoter hypermethylation was significantly correlated with lymph node metastasis (p<0.01). Our findings suggest that hypermethylation of SPARC is a frequent and tumor-specific event in laryngeal and hypopharyngeal carcinomas and may serve as a biomolecular marker for diagnosis and prognosis.

Surface recognition and penetration are among the most critical plant infection processes in foliar pathogens. In Magnaporthe oryzae, the Pmk1 MAP kinase regulates appressorium formation and penetration. Its orthologs also are known to be required for various plant infection processes in other phytopathogenic fungi. Although a number of upstream components of this important pathway have been characterized, the upstream sensors for surface signals have not been well characterized. Pmk1 is orthologous to Kss1 in yeast that functions downstream from Msb2 and Sho1 for filamentous growth. Because of the conserved nature of the Pmk1 and Kss1 pathways and reduced expression of MoMSB2 in the pmk1 mutant, in this study we functionally characterized the MoMSB2 and MoSHO1 genes. Whereas the Momsb2 mutant was significantly reduced in appressorium formation and virulence, the Mosho1 mutant was only slightly reduced. The Mosho1 Momsb2 double mutant rarely formed appressoria on artificial hydrophobic surfaces, had a reduced Pmk1 phosphorylation level, and was nonresponsive to cutin monomers. However, it still formed appressoria and caused rare, restricted lesions on rice leaves. On artificial hydrophilic surfaces, leaf surface waxes and primary alcohols-but not paraffin waxes and alkanes- stimulated appressorium formation in the Mosho1 Momsb2 mutant, but more efficiently in the Momsb2 mutant. Furthermore, expression of a dominant active MST7 allele partially suppressed the defects of the Momsb2 mutant. These results indicate that, besides surface hydrophobicity and cutin monomers, primary alcohols, a major component of epicuticular leaf waxes in grasses, are recognized by M. oryzae as signals for appressorium formation. Our data also suggest that MoMsb2 and MoSho1 may have overlapping functions in recognizing various surface signals for Pmk1 activation and appressorium formation. While MoMsb2 is critical for sensing surface hydrophobicity and cutin monomers, MoSho1 may play a more important role in recognizing rice leaf waxes.

Author Summary

The rice blast fungus is a major pathogen of rice and a model for studying fungal-plant interactions. Like many other fungal pathogens, it can recognize physical and chemical signals present on the rice leaf surface and form a highly specialized infection structure known as appressorium. A well conserved signal transduction pathway involving the protein kinase gene PMK1 is known to regulate appressorium formation and plant penetration in this pathogen. However, it is not clear about the sensor genes that are involved in recognizing various plant surface signals. In this study we functionally characterize two putative sensor genes called MoMSB2 and MoSHO1. Genetic and biochemical analyses indicated that these two genes have overlapping functions in recognizing different physical and chemical signals present on the rice leaf surface for the activation of the Pmk1 pathway and appressorium formation. We found that primary alcohols, a major component of leaf waxes in grasses, can be recognized by the rice blast fungus as chemical cues. While MoMSB2 is critical for sensing hydrophobicity and precursors of cutin molecules of rice leaves, MoSHO1 appears to be more important than MoMSB2 for recognizing wax components.

Background:

Paeonia sinjiangensis K.Y. Pan is a perennial herb belonging to the ranunculaceae family and it is one of the most important crude drugs in traditional Chinese medicine. In this article, Paeonia sinjiangensis K.Y. Pan rich in polysaccharide is used as an experimental material.

Materials and Method:

Study the effects of proportion, temperature, times and time taken for the extraction yield of polysaccharide through a single-factor exploration. Then, through an orthogonal experiment (L9(3)4), it was investigated to get the best extraction conditions.

Results:

The results showed that the ratio of solvent to raw material, number of extractions and duration of extraction were the main variables that influenced the yields of extracts. The separation procedure of precipitation with alcohol and the purification from the removing proteins were deeply analyzed. Meanwhile the contents of polysaccharide were determined by anthrone colorimetry.

Conclusion:

The highest yield was obtained when the ratio of solvent to raw material, number of extractions, and duration of extraction were 8:1, 2, and 1.5 h, respectively. The content of soluble polysaccharide is 51.57%.

Objective:

This paper investigates the total polyphenolic and flavonoid content as well as the antioxidant activity of Ziziphora clinopodioides Lam. extracts of different polarity.

Materials and Methods:

The total polyphenolic content was analysed using the Folin–Ciocalteu method. Total flavonoid content analysis was performed using the colorimetric method.

Results:

The total polyphenolic content of Z. clinopodioides is concentrated in parts of ethyl acetate (19.27%), chloroform (4.99%) and n-butanol extracts (3.94%) containing a small amount of the total polyphenolic content. The petroleum ether (0.23%) and ethanol extracts (1.64%) contain almost no polyphenolic content. The total flavonoid content of Z. clinopodioides is concentrated in parts of ethyl acetate (65.61%), chloroform (14.36%) and n-butanol extracts (10.76%) containing a small amount of the total polyphenolic content. The Z. clinopodioides Lam. ethyl acetate extract exhibits a good antioxidant activity.

Conclusion:

Ethyl acetate extracts contain a large number of polyphenolic compounds (19.27%) and flavonoids (65.61%) owing to good antioxidant capacity.

Background

Epigenetic silencing of tumor suppressor genes play important roles in NPC tumorgenesis. Tissue factor pathway inhibitor-2 (TFPI-2), is a protease inhibitor. Recently, TFPI-2 was suggested to be a tumor suppressor gene involved in tumorigenesis and metastasis in some cancers. In this study, we investigated whether TFPI-2 was inactivated epigenetically in nasopharyngeal carcinoma (NPC).

Methods

Transcriptional expression levels of TFPI-2 was evaluated by RT-PCR. Methylation status were investigated by methylation specific PCR and bisulfate genomic sequencing. The role of TFPI-2 as a tumor suppressor gene in NPC was addressed by re-introducing TFPI-2 expression into the NPC cell line CNE2.

Results

TFPI-2 mRNA transcription was inactivated in NPC cell lines. TFPI-2 was aberrantly methylated in 66.7% (4/6) NPC cell lines and 88.6% (62/70) of NPC primary tumors, but not in normal nasopharyngeal epithelia. TFPI-2 expression could be restored in NPC cells after demethylation treatment. Ectopic expression of TFPI-2 in NPC cells induced apoptosis and inhibited cell proliferation, colony formation and cell migration.

Conclusions

Epigenetic inactivation of TFPI-2 by promoter hypermethylation is a frequent and tumor specific event in NPC. TFPI-2 might be considering as a putative tumor suppressor gene in NPC.

Background:

It is reported that the plant Hyssopus cuspidatus Boriss from Xinjiang has great value. This article deals with the detailed pharmacognostic evaluation of the crude drug H. cuspidatus Boriss.

Materials and Methods:

The essential oil of H. cuspidatus Boriss from Xinjiang, China, was extracted by the method of hydrodistillation and the chemical composition of the essential oil was analyzed by gas chromatography-mass spectrometry (GC–MS).

Results:

The yield of essential oil based on the dry weight of the plant was 0.6%(w/w). Fifty compounds accounting for 99.42% of the total oil were identified. The major components were oxygenated terpenes (66.33%), monoterpenes (26.14%), oxygenated sesquiterpenes (1.25%), and octane (1.85%).

Conclusion:

Oxygenated terpenes were the main group of the compounds. The physicochemical parameters presented in this article may be proposed as parameters to establish the authenticity of H. cuspidatus Boriss and can possibly aid pharmacognostic and taxonomic species identification.

A rapid, effective, binary reverse phase rapid resolution liquid chromatographic method has been developed for the determination of Paeoniflorin extracted from Paeonia sinjiang K. Y. Pan. with short run time. RRLC separation was achieved by using Agilent (Zorbas XDB-C18 4.6 mm × 50 mm, 1.8 μm) column with mobile phase complosed of methanol and 0.05 mol/l potassium phosphate monobasic. Flow rate was at 1.0 ml/min. Retention time was at about 1.16 min. Recovery rate was 99.8% and relative standard deviation of three replicate samples tested was 1.8%. RRLC method has been applied to both in vitro studies of paeoniflorin formulation and drug estimation in biological samples.