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Fulltext Fatty Acid Synthase Beta Dehydratase in the Lipid Biosynthesis Pathway Is Required for Conidiogenesis, Pigmentation and Appressorium Formation in Magnaporthe oryzae S6

Sangappillai V, Nadarajah K

Int J Mol Sci, 2020 Sep 30;21(19).
PMID: 33007862 DOI: 10.3390/ijms21197224

Lipid biosynthesis produces glycerol, which is important in fueling turgor pressure necessary for germination and penetration of plant host by fungi. As the relationship between pathogenicity and the lipid biosynthetic pathway is not fully understood, we have elucidated the role of the fatty acid synthase beta subunit dehydratase (FAS1) gene in lipid biosynthesis. The FAS1 gene was silenced through homologous double crossover in Magnaporthe oryzae strain S6 to study the effect on lipid biosynthesis. The vegetative growth of Δfas1 mutants show the highest drop on oleic acid (between 10 and 50%), while the mycelial dry weight of mutants dropped significantly on all media. Conidiation of FAS1 mutants show a ~10- and ~5-fold reduction on oatmeal and Potato Dextrose Agar (PDA), respectively. Mutants formed mycelium that were mildly pigmented, indicating that the deletion of FAS1 may have affected melanin biosynthesis. Biochemical and gene expression studies concluded that the fatty acid degradation pathway might have been interrupted by FAS1 deletion. FAS1 mutants showed no enzyme activity on glucose or olive oil, suggesting that the mutants may lack functional peroxisomes and be defective in β-oxidation of fatty acids, hence explaining the reduced lipid deposits in the spores.

Matched MeSH terms: Ascomycota/genetics*
Fulltext Functional expression of a novel α-amylase from Antarctic psychrotolerant fungus for baking industry and its magnetic immobilization

He L, Mao Y, Zhang L, Wang H, Alias SA, Gao B, et al.

BMC Biotechnol, 2017 02 28;17(1):22.
PMID: 28245836 DOI: 10.1186/s12896-017-0343-8

BACKGROUND: α-Amylase plays a pivotal role in a broad range of industrial processes. To meet increasing demands of biocatalytic tasks, considerable efforts have been made to isolate enzymes produced by extremophiles. However, the relevant data of α-amylases from cold-adapted fungi are still insufficient. In addition, bread quality presents a particular interest due to its high consummation. Thus developing amylases to improve textural properties could combine health benefits with good sensory properties. Furthermore, iron oxide nanoparticles provide an economical and convenient method for separation of biomacromolecules. In order to maximize the catalytic efficiency of α-amylase and support further applications, a comprehensive characterization of magnetic immobilization of α-amylase is crucial and needed.
RESULTS: A novel α-amylase (AmyA1) containing an open reading frame of 1482 bp was cloned from Antarctic psychrotolerant fungus G. pannorum and then expressed in the newly constructed Aspergillus oryzae system. The purified recombinant AmyA1 was approximate 52 kDa. AmyA1 was optimally active at pH 5.0 and 40 °C, and retained over 20% of maximal activity at 0-20 °C. The K m and V max values toward soluble starch were 2.51 mg/mL and 8.24 × 10-2 mg/(mL min) respectively, with specific activity of 12.8 × 103 U/mg. AmyA1 presented broad substrate specificity, and the main hydrolysis products were glucose, maltose, and maltotetraose. The influence of AmyA1 on the quality of bread was further investigated. The application study shows a 26% increase in specific volume, 14.5% increase in cohesiveness and 14.1% decrease in gumminess in comparison with the control. AmyA1 was immobilized on magnetic nanoparticles and characterized. The immobilized enzyme showed improved thermostability and enhanced pH tolerance under neutral conditions. Also, magnetically immobilized AmyA1 can be easily recovered and reused for maximum utilization.
CONCLUSIONS: A novel α-amylase (AmyA1) from Antarctic psychrotolerant fungus was cloned, heterologous expression in Aspergillus oryzae, and characterized. The detailed report of the enzymatic properties of AmyA1 gives new insights into fungal cold-adapted amylase. Application study showed potential value of AmyA1 in the food and starch fields. In addition, AmyA1 was immobilized on magnetic nanoparticles and characterized. The improved stability and longer service life of AmyA1 could potentially benefit industrial applications.

Matched MeSH terms: Ascomycota/genetics
Morphological and molecular characterization, sexual reproduction, and pathogenicity of Setosphaeria rostrata isolates from rice leaf spot

Kusai NA, Azmi MM, Zainudin NA, Yusof MT, Razak AA

Mycologia, 2016 09;108(5):905-914.
PMID: 27474518

Setosphaeria rostrata, a common plant pathogen causing leaf spot disease, affects a wide range of plant species, mainly grasses. Fungi were isolated from brown spots on rice leaves throughout Peninsular Malaysia, and 45 isolates were identified as Setosphaeria rostrata The isolates were then characterized using morphological and molecular approaches. The mating type was determined using PCR amplification of the mating type alleles, and isolates of opposite mating types were crossed to examine sexual reproduction. Based on nuclear ribosomal DNA ITS1-5.8S-ITS2 region (ITS) and beta-tubulin (BT2) sequences, two phylogenetic trees were constructed using the maximum likelihood method; S. rostrata was clustered in one well-supported clade. Pathogenicity tests showed that S. rostrata isolates are pathogenic, suggesting that it is the cause of the symptoms. Mating-type analyses indicated that three isolates carried the MAT1-1 allele, and the other 42 isolates carried MAT1-2 After isolates with opposite mating types were crossed on Sach's medium and incubated for 3 wk, six crosses produced pseudothecia that contained eight mature ascospores, and 12 other crosses produced numerous pseudothecia with no ascospores. To our knowledge, this is the first report on S. rostrata isolated from leaf spots on rice.

Matched MeSH terms: Ascomycota/genetics
Emergence of Aureobasidium pullulans as human fungal pathogen and molecular assay for future medical diagnosis

Chan GF, Puad MS, Chin CF, Rashid NA

Folia Microbiol (Praha), 2011 Sep;56(5):459-67.
PMID: 21909832 DOI: 10.1007/s12223-011-0070-9

Despite the great importance of Aureobasidium pullulans in biotechnology, the fungus had emerged as an opportunistic human pathogen, especially among immunocompromised patients. Clinical detection of this rare human fungal pathogen presently relies on morphology diagnosis which may be misleading. Thus, a sensitive and accurate quantitative molecular assay for A. pullulans remains lacking. In this study, we presented the microscopy observations of A. pullulans that reveals the phenotypic plasticity of the fungus. A. pullulans-specific primers and molecular beacon probes were designed based on the fungal 18S ribosomal RNA (rRNA) gene. Comparison of two probes with varied quencher chemistry, namely BHQ-1 and Tamra, revealed high amplification efficiency of 104% and 108%, respectively. The optimized quantitative real-time PCR (qPCR) assays could detect and quantify up to 1 pg concentration of A. pullulans DNA. Both assays displayed satisfactory performance parameters at fast thermal cycling mode. The molecular assay has great potential as a molecular diagnosis tool for early detection of fungal infection caused by A. pullulans, which merits future study in clinical diagnosis.

Matched MeSH terms: Ascomycota/genetics
Fulltext Comparative chemical screening and genetic analysis reveal tentoxin as a new virulence factor in Cochliobolus miyabeanus, the causal agent of brown spot disease on rice

De Bruyne L, Van Poucke C, Di Mavungu DJ, Zainudin NA, Vanhaecke L, De Vleesschauwer D, et al.

Mol Plant Pathol, 2016 Aug;17(6):805-17.
PMID: 26456797 DOI: 10.1111/mpp.12329

Brown spot disease, caused by Cochliobolus miyabeanus, is currently considered to be one of the most important yield reducers of rice (Oryza sativa L.). Despite its agricultural importance, little is known about the virulence mechanisms deployed by the fungus. Therefore, we set out to identify novel virulence factors with a role in disease development. This article reports, for the first time, the production of tentoxin by C. miyabeanus as a virulence factor during brown spot disease and the identification of the non-ribosomal protein synthetase (NRPS) CmNps3, responsible for tentoxin biosynthesis. We compared the chemical compounds produced by C. miyabeanus strains differing in virulence ability using ultra-high-performance liquid chromatography (UHPLC) coupled to high-resolution Orbitrap mass spectrometry (HRMS). The production of tentoxin by a highly virulent strain was revealed by principal component analysis of the detected ions and confirmed by UHPLC coupled to tandem-quadrupole mass spectrometry (MS/MS). The corresponding NRPS was identified by in silico genome analysis and confirmed by gene deletion. Infection tests with wild-type and Cmnps3 mutants showed that tentoxin acts as a virulence factor and is correlated with chlorosis development during the second phase of infection. Although rice has previously been classified as a tentoxin-insensitive plant species, our data demonstrate that tentoxin production by C. miyabeanus affects symptom development.

Matched MeSH terms: Ascomycota/genetics*