The peroxins BcPex8, BcPex10, and BcPex12 are required for the development and pathogenicity of Botrytis cinerea.

Peroxisomes have been proved playing roles in infection of several plant pathogens. Although the contribution of a portion of peroxins in pathogenicity was demonstrated, most of them are undocumented in fungi, especially, Botrytis cinerea. The homologs of Pex8, Pex10, and Pex12 in B. cinerea were functionally characterized in this work using gene disruption strategies. Compared with the wild-type strain (WT), the Δbcpex8, Δbcpex10, and Δbcpex12 mutants exhibited significant reduction in melanin production, fatty acid utilization, and decreased tolerance to high osmotic pressure and reactive oxygen species (ROS). The mycelial growth and conidiation of were significantly inhibited in Δbcpex8, Δbcpex10, and Δbcpex12 strains. The mycelial growth rates of Δbcpex8, Δbcpex10, and Δbcpex12 were reduced by 32, 35, and 34%, respectively, compared with WT and ectopic transformant (ET), and the conidiation was reduced by approximately 89, 27, and 88%, respectively. The conidial germination, germ tube elongation, and the formation of initiate infection structures (IFSs) were also reduced by the deletion of the genes. The pathogenicity was tested on the leaves of tobacco and strawberry, and fruits of tomato. On the leaves of tobacco and strawberry, the Δbcpex8, Δbcpex10, and Δbcpex12 mutants could not induce necrotic lesions, and the lesions on tomato fruits infected with the mutants were significantly reduced than those of the wide type. The results indicated that BcPEX8, BcPEX10, and BcPEX12 are indispensable for the development and pathogenicity of B. cinerea.

MAT Loci Play Crucial Roles in Sexual Development but Are Dispensable for Asexual Reproduction and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae.

Magnaporthe oryzae, a fungal pathogen that causes rice blast, which is the most destructive disease of rice worldwide, has the potential to perform both asexual and sexual reproduction. MAT loci, consisting of MAT genes, were deemed to determine the mating types of M. oryzae strains. However, investigation was rarely performed on the development and molecular mechanisms of the sexual reproduction of the fungus. In the present work, we analyzed the roles of two MAT loci and five individual MAT genes in the sex determination, sexual development and pathogenicity of M. oryzae. Both of the MAT1-1 and MAT1-2 loci are required for sex determination and the development of sexual structures. MAT1-1-1, MAT1-1-3 and MAT1-2-1 genes are crucial for the formation of perithecium. MAT1-1-2 impacts the generation of asci and ascospores, while MAT1-2-2 is dispensable for sexual development. A GFP fusion experiment indicated that the protein of MAT1-1-3 is distributed in the nucleus. However, all of the MAT loci or MAT genes are dispensable for vegetative growth, asexual reproduction, pathogenicity and pathogenicity-related developments of the fungus, suggesting that sexual reproduction is regulated relatively independently in the development of the fungus. The data and methods of this work may be helpful to further understand the life cycle and the variation of the fungus.

Correction: A handy method to remove bacterial contamination from fungal cultures.

[This corrects the article DOI: 10.1371/journal.pone.0224635.].

A handy method to remove bacterial contamination from fungal cultures.

Contamination control and removal are very important technical aspects of microbiological research. Bacterial contamination is very common in fungal cultures. Currently, the commonly used approach for inhibiting bacteria is antibiotic treatment; however, there are drawbacks to using antibiotics, including incomplete removal, limited antibacterial spectra, tendency toward recontamination, effects to fungal strains, and potential risks to the environment. Therefore, in the present work, we developed a new method for bacterial removal from fungi cultured on solid medium, the Cabin-Sequestering (CS) method, based on the different culture characteristics between fungi and bacteria. First, 3-5 mm round or square holes (the "cabin") are excavated on a solid medium plate. The fungal strain containing possible bacterial contamination is inoculated into the cabin. The cabin is then covered with a sterilized coverslip, followed by incubation at the appropriate temperature. After 7-10 days of culturing, fungal hyphae grow out along the edge of the coverslip; however, the contaminating bacteria cannot pass through the space formed between the medium and the coverslip and, thus, remain in the cabin. The newly grown fungal hyphae around the coverslip are re-inoculated into fresh culture plates, where they form bacteria-free fungal colonies. The CS method is easy handling, with a short experimental cycle and rare recontamination. When necessary, it can also be used in combination with antibiotics in bacterial removal operations.

Pex13 and Pex14, the key components of the peroxisomal docking complex, are required for peroxisome formation, host infection and pathogenicity-related morphogenesis in Magnaporthe oryzae.

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfill multiple important metabolisms. Pex13 and Pex14 are key components of the peroxisomal docking complex in yeasts and mammals. In the present work, we functionally characterized the homologues of Pex13 and Pex14 (Mopex13 and Mopex14) in the rice blast fungus Magnaporthe oryzae. Mopex13 and Mopex14 were peroxisomal membrane distributed and were both essential for the maintenance of Mopex14/17 on the peroxisomal membrane. Mopex13 and Mopex14 interacted with each other, and with Mopex14/17 and peroxisomal matrix protein receptors. Disruption of Mopex13 and Mopex14 resulted in a cytoplasmic distribution of peroxisomal matrix proteins and the Woronin body protein Hex1. In the ultrastructure of Δmopex13 and Δmopex14 cells, peroxisomes were detected on fewer occasions, and the Woronin bodies and related structures were dramatically affected. The Δmopex13 and Δmopex14 mutants were reduced in vegetative growth, conidial generation and mycelial melanization, in addition, Δmopex13 showed reduced conidial germination and appressorial formation and abnomal appressorial morphology. Both Δmopex13 and Δmopex14 were deficient in appressorial turgor and nonpathogenic to their hosts. The infection failures in Δmopex13 and Δmopex14 were also due to their reduced ability to degrade fatty acids and to endure reactive oxygen species and cell wall-disrupting compounds. Additionally, Mopex13 and Mopex14 were required for the sexual reproduction of the fungus. These data indicate that Mopex13 and Mopex14, as key components of the peroxisomal docking complex, are indispensable for peroxisomal biogenesis, fungal development and pathogenicity in the rice blast fungus.

The functions of PEX genes in peroxisome biogenesis and pathogenicity in phytopathogenic fungi.

Peroxisomes are cellular organelles present ubiquitously in eukaryotic cells and are involved in ß-oxidation, glyoxylate cycle and a variety of biochemical metabolisms. Recently peroxisomes have been demonstrated to play vital roles in the host infection processes by plant fungal pathogens. The biogenesis of peroxisomes requires a category of proteins named peroxins, which are encoded by the PEX genes. So far, more than 10 PEX genes were isolated in phytopathogenic fungi, and significant research efforts are focused on the mechanism of peroxisome formation and the roles of peroxisome in the development and pathogenicity of fungal pathogens. In this review, we summarize the latest advances in peroxisome biogenesis and functions in pathogenic fungi, including the roles of PEXs in life cycle of peroxisome, peroxisome related metabolisms, and fungal development, infection and pathogenicity, in order to provide references for future studies in plant pathogenic fungi and the control of disease.

Synthesis, characterization, and antibacterial activity of cross-linked chitosan-glutaraldehyde.

This present study deals with synthesis, characterization and antibacterial activity of cross-linked chitosan-glutaraldehyde. Results from this study indicated that cross-linked chitosan-glutaraldehyde markedly inhibited the growth of antibiotic-resistant Burkholderia cepacia complex regardless of bacterial species and incubation time while bacterial growth was unaffected by solid chitosan. Furthermore, high temperature treated cross-linked chitosan-glutaraldehyde showed strong antibacterial activity against the selected strain 0901 although the inhibitory effects varied with different temperatures. In addition, physical-chemical and structural characterization revealed that the cross-linking of chitosan with glutaraldehyde resulted in a rougher surface morphology, a characteristic Fourier transform infrared (FTIR) band at 1559 cm⁻¹, a specific X-ray diffraction peak centered at 2θ = 15°, a lower contents of carbon, hydrogen and nitrogen, and a higher stability of glucose units compared to chitosan based on scanning electron microscopic observation, FTIR spectra, X-ray diffraction pattern, as well as elemental and thermo gravimetric analysis. Overall, this study indicated that cross-linked chitosan-glutaraldehyde is promising to be developed as a new antibacterial drug.

Characterization of physiological response and identification of associated genes under heat stress in rice seedlings.

Global warming, which is caused by greenhouse gas emissions, makes food crops more vulnerable to heat stress. Understanding the heat stress-related mechanisms in crops and classifying heat stress-related genes can increase our knowledge in heat-resistant molecular biology and propel developments in molecular design breeding, which can help rice cope with unfavorable temperatures. In this study, we carried out a physiological analysis of rice plants after heat stress. The results show a dramatic increase in malondialdehyde contents and SOD activities. We successfully isolated 11 heat-related rice genes with known function annotation through DNSH, which is an improved SSH method for screening long cDNA fragments. The reanalysis of microarray data from public database revealed that all these genes displayed various expression patterns after heat stress, drought, cold and salt. Quantitative real-time reverse transcription PCR was also performed to validate the expression of these genes after heat stress. The expressions in 10 genes were all significantly changed except for contig 77, which is a CBL-interacting protein kinase. Several reports have been published about the members of the same gene family.

[Bioinformatic research of the family of PEX11, peroxisome proliferous factor in fungus].

The family members of PEX11 are key factors involved in regulation of peroxisome proliferation. Sixty-six PEX11p candidates of PEX11 gene family from 26 representative fungal species were obtained and analyzed by bioinformatic strategies. In most filamentous fungi, 2 or 3 potential PEX11ps were found, in contrast with 1 or 2 in yeast species. Compared with other fungal species, the Ascomycetes tend to have more PEX11ps, and even 5 in several individuals. The data of phylogenetic analysis and protein structure indicated that all of the PEX11ps were divided into 3 groups: I, II, and III. The members of group I and group III existed in most species, while those in group II were found only in Pezizomycotina. By MEME analysis, 5-6 conserved motifs were found in each PEX11ps. Among them,motif 8 in C-terminal had the most conservation, indicating that this motif probably plays a key role in maintaining the proper function of PEX11p.

[Progress on avirulence genes of the rice blast fungus Magnaporthe grisea].

Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most devastating diseases of rice. The specific interaction between rice and M. grisea is an important model system for studying the host-pathogen interaction mechanisms. In this article, we summarized recent research progresses on avirulence genes, which are the most important effectors in M. grisea with the focus on chromosome mapping, cloning method, functional analysis, and evolution study of avirulence genes, and the possible hotspot of the research on avirulence genes in the future was also been discussed. This knowledge may shed a light on the molecular mechanism underlying avirulence genes function and the possible interaction relationship between the host and the pathogen.

Antibacterial activity and mechanism of action of chitosan solutions against apricot fruit rot pathogen Burkholderia seminalis.

The in vitro antibacterial activity and mechanism of action of two kinds of acid-soluble chitosan and one water-soluble chitosan against apricot fruit rot pathogen Burkholderia seminalis was examined in this study. Results showed that water-soluble chitosan displayed limited antibacterial activity at four tested concentrations. However, two kinds of acid-soluble chitosan solution at 2.0 mg/mL had strong antibacterial activity against B. seminalis although weak antibacterial activity was observed at a concentration lower than 1 mg/mL. The antibacterial activity of acid-soluble chitosan may be due to membrane disruption, cell lysis, abnormal osmotic pressure, and additional chitosan coating around the bacteria based on integrity of cell membranes test, out membrane permeability assays and transmission electron microscopy observation. In addition, biofilm biomass were markedly reduced after treating with two kinds of acid-soluble chitosan at concentrations of 2.0 and 1.0 mg/mL for 3 and 12 h, indicating the importance of biofilm formation in the antibacterial mechanism of chitosan. Overall, the results clearly indicated that two kinds of acid-soluble chitosan had a potential to control the contamination of apricot fruits caused by B. seminalis.

Fluorescent co-localization of PTS1 and PTS2 and its application in analysis of the gene function and the peroxisomal dynamic in Magnaporthe oryzae.

The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals (PTS1 and PTS2) in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein (GFP)-PTS1, GFP-PTS2 and red fluorescence protein (RFP)-PTS1, were constructed and introduced into Magnaporthe oryzae Guy11 cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTS1 machinery was revealed by comparing the fluorescence backgrounds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into Deltamgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement of peroxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTS1 and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes.

[Strategies of targeted gene replacement in filamentous fungi].

Targeted gene replacement (TGR) is an important technique for gene function analysis. With the development of genome sequencing and transformation, TGR has been applied widely to filamentous fungi, and many new systems and approaches have been established. In this paper, strategies involved in TGR in filamentous fungi were reviewed, including transformation systems, targeting vector construction, mutant selection and so on. Compared with TGR, RNAi and other techniques of gene function analysis were introduced and reviewed as well.

[Association of haplotype formed on HLA-DRB1 and HLA-DQA1 alleles with outcomes of hepatitis B virus infection].

OBJECTIVE: To assess the association of haplotype of HLA-DRB1 and HLA-DQA1 alleles with outcomes of hepatitis B virus infection in Han population of north China. METHODS: Two hundred and seven chronic hepatitis B (HB) patients, two hundred and twelve chronic asymptomatic hepatitis B virus (HBV) carriers (HBV carrier) and one hundred and forty-eight self-limited HBV infection were investigated for HLA-DRB1 and HLA-DQA1 alleles by sequence specific-polymerase chain reaction (PCR-SSP). RESULTS: The frequency of DRB1*04-DQA1*0301 haplotype was 10.03% in self-limited HBV infection subjects, significantly higher than that in chronic HB patients (3.66%) (P=0.0005)ûthe frequency of DRB1*15/*16-DQA1*0102 haplotype was 6.80% in self-limited HBV infection subjects, significantly higher than 1.94% in chronic HB patients (P=0.0012) and 1.65% in asymptomatic HBV carriers (P=0.0004)ûwhile the frequency of DRB1*04-DQA1*0302 haplotype was 3.10% in chronic HB patients, higher than that in self-limited HBV infection subjects (0.39%) (P=0.0077). CONCLUSION: Individuals with different haplotypes composed of HLA-DRB1 and HLA-DQA1 might have different outcomes of HBV infection.

[Association of polymorphisms of human leucocyte antigen -DRB1 and -DQA1 allele with outcomes of hepatitis B virus infection in Han population of north China].

OBJECTIVE: To assess the association of polymorphisms of human leucocyte antigen (HLA) -DRB1 and -DQA1 region allele with outcomes of hepatitis B virus (HBV) infection in Han population of north China. METHODS: A total of 207 chronic hepatitis B (HB) patients, 212 chronic asymptomatic HBV carriers (HBV carrier), and 148 self-limited HBV infection were recruited to examine the association between gene polymorphisms and outcomes of HBV infection. Polymerase chain reaction-sequence specific primers (PCR-SSP) technique was used to genotype HLA-DRB1 and HLA-DQA1 loci. RESULTS: The frequency of HLA-DQA1 * 0301 in chronic HB patients (14.81%) was significantly lower than those in HBV carriers (25.24%) and self-limited HBV infection subjects (25.00%) (Pc = 0.002; Pc = 0.007). The frequency of HLA-DQA1 * 0102 in self-limited HBV infection subjects (8.78%) was significantly higher than those in chronic HB patients (2.18%) and HBV carriers (1.89%) (Pc = 0.000; P = 0.000). In addition, the frequency of HLA-DQA1 * 0302 in self-limited HBV infection subjects (4.05%) was significantly lower than that in chronic HB patients (11.41%) (Pc = 0.005). HLA-DQA1 * 0302 was demonstrated to be risk factors of chronic HBV (OR = 3.913, P = 0.0006), while HLA-DQA1* 0102 and HLA-DQA1 * 0301 to be protective factors against chronic HBV (OR = 0.200, P = 0.0004; OR = 0.258, P = 0.0000) after age, sex, smoking and drinking were adjusted by logistic regression analysis. There were positive interactions between drinking and HLA-DQA1 * 0102 [interaction index (II) = 1.49] or HLA-DQA1 * 0302 (II = 12.12). There were negative interactions between drinking and HLA-DQA1 * 0301 (II = 0.78) CONCLUSIONS: The subjects with HLA-DQA1 * 0302 allele have an increased risk to chronic HB infection compared with other subjects without this allele, while HLA-DQA1 * 0301 and HLA-DQA1 * 0102 are associated with HBV clearness. Gene-environment interaction can affect the outcomes of HBV infection.

Association of -238G/A polymorphism of tumor necrosis factor-alpha gene promoter region with outcomes of hepatitis B virus infection in Chinese Han population.

AIM: To clarify whether -238G/A polymorphism of tumor necrosis factor-alpha (TNF-alpha) gene promoter region was associated with outcomes of hepatitis B virus (HBV) infection in Han population of northern China, and to analyze the gene-environment interaction between -238G/A polymorphism and cigarette smoking or alcohol consumption. METHODS: A case-control study was conducted to analyze the association of TNF-alpha gene promoter polymorphism with HBV infection outcomes. A total of 207 patients with chronic hepatitis B (HB) and 148 cases of self-limited HBV infection from Ditan Hospital and Shunyi District Hospital in Beijing, respectively were recruited. History of smoking and alcohol drinking was inquired by a questionnaire. The -238G/A polymorphism of TNF-alpha gene promoter was genotyped by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP). RESULTS: The frequencies of GG and GA genotypes were 98.07% and 1.93% in chronic HB patients and 93.24% and 6.76% in self-limited HBV infection individuals, respectively (chi(2)=5.30, P=0.02). The frequency of G allele was significantly higher in patients with chronic HB that in individuals with self-limited HBV infection (99.03% vs 96.62%, chi(2)=5.20, P=0.02). Only modestly increased risk of onset of chronic HB was found in smokers (OR=1.40, 95% CI: 0.87-2.28, P=0.14) and drinkers (OR=1.26, 95%CI: 0.78-2.05, P=0.32). There was a positive interaction between genotype GG and cigarette smoking with an interaction index (II) of 2.95, or alcohol consumption with an II of 1.64. CONCLUSION: The -238G/A polymorphism of TNF-alpha gene promoter region is independently associated with different outcomes of HBV infection.