Colloid Chitin Azure Is a Dispersible, Low-Cost Substrate for Chitinase Measurements in a Sensitive, Fast, Reproducible Assay

Shen, Chia–Rui; Chen, Yu‐Sheng; Yang, Ching-Jen; Chen, Jeen-Kuan; Liu, Chao-Lin

doi:10.1177/1087057109355057

Cited by 50 publications

(51 citation statements)

References 19 publications

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“…The mixture was centrifuged, and the absorbance of the supernatant was measured using a colorimeter at 550 nm. One unit of chitinase activity was defined as the release of 1 mol GlcNAc per gram of protein in 30 min at 28°C (29). Protease activity was assayed using the substrate N-succinyl-(Ala) 2 -Pro-Phe-p-nitroanilide (Sigma) as described previously (30).…”

mentioning

confidence: 99%

MrSkn7 Controls Sporulation, Cell Wall Integrity, Autolysis, and Virulence in Metarhizium robertsii

Shang

Chen

et al. 2015

Eukaryot Cell

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Two-component signaling pathways generally include sensor histidine kinases and response regulators. We identified an ortholog of the response regulator protein Skn7 in the insect-pathogenic fungus Metarhizium robertsii, which we named MrSkn7. Gene deletion assays and functional characterizations indicated that MrSkn7 functions as a transcription factor. The MrSkn7 null mutant of M. robertsii lost the ability to sporulate and had defects in cell wall biosynthesis but was not sensitive to oxidative and osmotic stresses compared to the wild type. However, the mutant was able to produce spores under salt stress. Insect bioassays using these spores showed that the virulence of the mutant was significantly impaired compared to that of the wild type due to the failures to form the infection structure appressorium and evade host immunity. In particular, deletion of MrSkn7 triggered cell autolysis with typical features such as cell vacuolization, downregulation of repressor genes, and upregulation of autolysis-related genes such as extracellular chitinases and proteases. Promoter binding assays confirmed that MrSkn7 could directly or indirectly control different putative target genes. Taken together, the results of this study help us understand the functional divergence of Skn7 orthologs as well as the mechanisms underlying the development and control of virulence in insect-pathogenic fungi. Both prokaryotes and eukaryotes have developed complicated systems to sense and adapt to changing environments. In bacteria, sensing and processing of environmental stimuli largely rely on two-component signal (TCS) transduction pathways that consist of a membrane-bound histidine kinase receptor and a corresponding response regulator (RR) (1, 2). Similar TCS systems are also present in fungi, and the mechanisms underlying their function have been well studied in the budding yeast Saccharomyces cerevisiae (3, 4). The TCS pathway consists of a sensor kinase, Sln1, and two response regulators, Ssk1 and Skn7 (5, 6). The cytosolic Ssk1 controls the Hog1 mitogen-activated protein kinase pathway, while the highly conserved Skn7 functions as a stress response transcription factor that consists of an N-terminal HSF (heat shock factor)-type DNA binding domain and a C-terminal RR receiver domain (5). The orthologs of Skn7 in yeasts and different filamentous fungi generally contribute to cell wall integrity, sporulation, osmotic stress, and oxidative stress (5). Interestingly, deletion of MoSkn7 (Magnaporthe oryzae Skn7) in M. oryzae (7), FgSkn7 (Fusarium graminearum Skn7) in F. graminearum (8), and BcSkn7 (Botrytis cinerea Skn7) in B. cinerea (9) did not impair fungal virulence and thereby the ability to infect their respective plant hosts. In contrast, AaSkn7 (Alternaria alternata Skn7) is required by A. alternata to infect citrus (10). In mammalian pathogens, the virulence of Candida albicans and Cryptococcus neoformans were attenuated by the deletion of CaSkn7 (C. albicans Skn7) (11) and CnSkn7 (C. neoformans Skn7) (12), respectively, compa...

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confidence: 99%

MrSkn7 Controls Sporulation, Cell Wall Integrity, Autolysis, and Virulence in Metarhizium robertsii

Shang

Chen

et al. 2015

Eukaryot Cell

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“…Chitinoclastic activity has been described previously in several microorganisms, including Vibrio furnissii [5], Vibrio carchariae [80], Amycolatopsis orientalis or Kitasatospora sp. [81], Serratia marcescens [82], Escherichia coli [83] and Streptomyces coelicolor [84]. The chitinoclastic cascade in Vibrio has been characterized [6] and found to involve 10 genes that are implicated in chitin catabolism.…”

Section: Discussionmentioning

confidence: 99%

Short-Chain Chitin Oligomers: Promoters of Plant Growth

et al. 2017

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Chitin is the second most abundant biopolymer in nature after cellulose, and it forms an integral part of insect exoskeletons, crustacean shells, krill and the cell walls of fungal spores, where it is present as a high-molecular-weight molecule. In this study, we showed that a chitin oligosaccharide of lower molecular weight (tetramer) induced genes in Arabidopsis that are principally related to vegetative growth, development and carbon and nitrogen metabolism. Based on plant responses to this chitin tetramer, a low-molecular-weight chitin mix (CHL) enriched to 92% with dimers (2mer), trimers (3mer) and tetramers (4mer) was produced for potential use in biotechnological processes. Compared with untreated plants, CHL-treated plants had increased in vitro fresh weight (10%), radicle length (25%) and total carbon and nitrogen content (6% and 8%, respectively). Our data show that low-molecular-weight forms of chitin might play a role in nature as bio-stimulators of plant growth, and they are also a known direct source of carbon and nitrogen for soil biomass. The biochemical properties of the CHL mix might make it useful as a non-contaminating bio-stimulant of plant growth and a soil restorer for greenhouses and fields.

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“…Also, Siderophore Siderophore [28] and chitinase [29] production were evaluated. Finally, to test for thermic tolerance, isolates were placed on PDA, and grown at 30 ± 1°C, 40 ± 1°C, 50 ± 1°C and 60 ± 1°C, for 5 days in full darkness.…”

Section: Biocontrol Potential Evaluationmentioning

confidence: 99%

“…To evaluate the produced conidia in each experimental unit, a 1 mL aliquot was taken into a microcentrifuge tube and counting was performed directly using an improved Neubauer Chamber (Housser Scientific®) as described before [15,[25][26][27][28][29][30][31]. Concentration values are expressed in conidia.mL -1 .…”

Section: Evaluation Of Conidia and Biomass Productionmentioning

confidence: 99%

Isolation of Trichoderma Spp. from Desert Soil, Biocontrol Potential Evaluation and Liquid Culture Production of Conidia Using Agricultural Fertilizers

AH¹,

G²

2016

J Fertil Pestic

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Colloid Chitin Azure Is a Dispersible, Low-Cost Substrate for Chitinase Measurements in a Sensitive, Fast, Reproducible Assay

Cited by 50 publications

References 19 publications

MrSkn7 Controls Sporulation, Cell Wall Integrity, Autolysis, and Virulence in Metarhizium robertsii

MrSkn7 Controls Sporulation, Cell Wall Integrity, Autolysis, and Virulence in Metarhizium robertsii

Short-Chain Chitin Oligomers: Promoters of Plant Growth

Isolation of Trichoderma Spp. from Desert Soil, Biocontrol Potential Evaluation and Liquid Culture Production of Conidia Using Agricultural Fertilizers

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