• BMB Reports
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• v.36 no.6
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• pp.603-607
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• 2003

Abstract Plant lipid transfer proteins (LTPs) are a class of proteins whose functions are still unknown. Some are proposed to have antimicrobial activities. To understand whether LTP110, a rice LTP that we previously identified from rice leaves, plays a role in the protection function against some serious rice pathogens, we investigated the antifungal and antibacterial properties of LTP110. A cDNA sequence, encoding the mature peptide of LTP110, was cloned into the Impact-CN prokaryotic expression system. The purified protein was used for an in vitro inhibition test against rice pathogens, Pyricularia oryzae and Xanthomonas oryzae. The results showed that LTP110 inhibited the germination of Pyricularia oryzae spores, and its inhibitory activity decreased in the presence of a divalent cation. This suggests that the antifungal activity is affected by ions in the media; LTP110 only slightly inhibited the growth of Xanthomonas oryzae. However, the addition of LTP110 to cultured Chinese hamster ovarian cells did not retard growth, suggesting that the toxicity of LTP110 is only restricted to some cell types. Its antimicrobial activity is potentially due to interactions between LTP and microbe-specific structures.

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1223-1229
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• 2009

To construct a new recombinant strain of Bacillus velezensis that has antifungal and insecticidal activity via the expression of the insecticidal Bacillus thuringiensis crystal protein, a B. thuringiensis expression vector (pHT1K-1Ac) was generated that contained the B. thuringiensis cry1Ac gene under the control of its endogenous promoter in a minimal E. coli-B. thuringiensis shuttle vector (pHT1K). This vector was introduced into a B. velezensis isolate that showed high antifungal activities against several plant diseases, including rice blast (Magnaporthe grisea), rice sheath blight (Rhizotonia solani), tomato gray mold (Botrytis cinerea), tomato late blight (Phytophthora infestans), and wheat leaf rust (Puccinia recondita), by electroporation. The recombinant B. velezensis strain was confirmed by PCR using cry1Ac-specific primers. Additionally, the recombinant strain produced a protein approximately 130 kDa in size and parasporal inclusion bodies similar to B. thuringiensis. The in vivo antifungal activity assay demonstrated that the activity of the recombinant B. velezensis strain was maintained at the same level as that of wild-type B. velezensis. Furthermore, it exhibited high insecticidal activity against a lepidopteran pest, Plutella xylostella, although its activity was lower than that of a recombinant B. thuringiensis strain, whereas wild-type B. velezensis strain did not show any insecticidal activity. These results suggest that this recombinant B. velezensis strain can be used to control harmful insect pests and fungal diseases simultaneously in one crop.

• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.81-88
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• 1990

For the selection of powerful antagonistic bacterium for biological control of soilborne Fusarium solani causing root rot of many important crops, the best YPL-1 strain was selected among 300 strains of bacteria isolated from rhizosphere in ginseng root rot-suppressive soil. The strain was identified to be a species to Pseudomonas stutzeri. With in vitro fungal inhibition tests, antagonistic substance of P. stutzeri YPL-1 against F. solani was presumed to be heat unstable, macromolecular substances such as protein. Also, it was shown that antifungal activity of P. stutzeri YPL-1 increased in proportion to its chitinase production. P. stutzeri YPL-M122 (chi-, lam -) which was deprived of the productivity of chitinase and laminarinase by NTG mutagenesis had lost antifungal activity, completely. And P. stutzeri YPL-MI53 (chi-) had only 4.1% of its antifungal activity. P. stutzeri YPL-1 was not able to produce any extracellular siderophore in iron-deficent minimal medium. It is confident that the antifungal mechanism of P. stutzeri YPL-1 for biocontrol of F. solani depends on lysis rather than antibiosis :the mechanism of lysis appears to involve enzymatic degradation of the cell will components of F. solani by hydrolytic enzymes of more chitinase and less laminarinase.

• Korean Journal of Microbiology
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• v.47 no.4
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• pp.295-301
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• 2011

Aquaporin is a water channel protein, which is classified as Major Intrinsic Protein (MIP), found in almost all organisms from bacteria to human. To date, more than 200 members of this family were identified. There are two major categories of MIP channels, orthodox aquaporins and aquaglyceroporins, which facilitate the diffusion across biological membranes of water or glycerol and other uncharged compounds, respectively. The full genome sequencing of various fungal species revealed 3 to 5 aquaporins in their genome. Although some functions of aquaporins found in yeast were characterized, however, no functional characteristics were studied so far in filamentous fungi, including Aspergillus sp. In this study, one orthodox aquaporin homolog gene, aqpA, and four aquaglyceroporin homologs, aqpB-E, in a model filamentous fungus Aspergillus nidulans were identified and the function of the aqpA gene was characterized. Knock-out of the aqpA gene didn't show any obvious phenotypic change under the osmotic stress, indicating that the function of the gene does not involved in the osmotic stress response or the function could be redundant. However, the mutant showed antifungal susceptibility resistance phenotype, suggesting that the function of the aqpA gene could be involved in sensing the antifungal substances rather than the osmotic stress response.

• Biomolecules & Therapeutics
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• v.29 no.2
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• pp.234-247
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• 2021

We used a heterozygous gene deletion library of fission yeasts comprising all essential and non-essential genes for a microarray screening of target genes of the antifungal terbinafine, which inhibits ergosterol synthesis via the Erg1 enzyme. We identified 14 heterozygous strains corresponding to 10 non-essential [7 ribosomal-protein (RP) coding genes, spt7, spt20, and elp2] and 4 essential genes (tif302, rpl2501, rpl31, and erg1). Expectedly, their erg1 mRNA and protein levels had decreased compared to the control strain SP286. When we studied the action mechanism of the non-essential target genes using cognate haploid deletion strains, knockout of SAGA-subunit genes caused a down-regulation in erg1 transcription compared to the control strain ED668. However, knockout of RP genes conferred no susceptibility to ergosterol-targeting antifungals. Surprisingly, the RP genes participated in the erg1 transcription as components of repressor complexes as observed in a comparison analysis of the experimental ratio of erg1 mRNA. To understand the action mechanism of the interaction between the drug and the novel essential target genes, we performed isobologram assays with terbinafine and econazole (or cycloheximide). Terbinafine susceptibility of the tif302 heterozygous strain was attributed to both decreased erg1 mRNA levels and inhibition of translation. Moreover, Tif302 was required for efficacy of both terbinafine and cycloheximide. Based on a molecular modeling analysis, terbinafine could directly bind to Tif302 in yeasts, suggesting Tif302 as a potential off-target of terbinafine. In conclusion, this genome-wide screening system can be harnessed for the identification and characterization of target genes under any condition of interest.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1999.04a
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• pp.39.2-39
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• 1999

• Polymer(Korea)
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• v.33 no.4
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• pp.389-395
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• 2009

To develop the carrier of hydrophobic antifungal agents based on low molecular weight water-soluble chitosan (LMWSC), LMWSC was chemically modified with deoxycholic acid (DA) which is one of the bile acid as a hydrophobic group. The nanoparticles (WSCDA) using DA conjugated LMWSC were characterized using dynamic light scattering (DLS) and transmittance electron microscope (TEM). The particle size of WSCDA ranged from 250 to 350 nm and increased with the number of DA substitution. The loaded itraconazole as an antifungal agent WSCDA nanoparticles (WSCDA-ITCN) were prepared by solvent evaporation method. The drug content and the loading efficiency were investigated approximately $9{\sim}10%$ and $61{\sim}68%$ by UV spectrophotometer, respectively. The release of drug from nanoparticles was slow and showed sustained release characteristics. Based on the results of release study that the higher DA contents in WSCDA, the slower the releasing rate, the WSCDA-ITCN could be used as an excellent antifungal agent.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2005.09a
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• pp.139-143
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• 2005

Cytochrome P450 14${\alpha}$-sterol demethylase enzyme (CYP51) is the target a of azole type antifungals. The azole blocks the ergosterol synthesis and thereby inhibits fungal growth. A three-dimensional (3D) homology model of CYP51 from Candida albicans was constructed based on the X-ray crystal structure of CYP51 from Mycobacterium tuberculosis. Using this model, the binding modes for the substrate (24-methylene-24, 25-dihydrolanosterol) and the known inhibitors (fluconazole, voriconazole, oxiconazole, miconazole) were predicted from docking. Virtual screening was performed employing Structure Based Focusing (SBF). In this procedure, the pharmacophore models for database search were generated from the protein-ligands interactions each other. The initial structure-based virtual screening selected 15 compounds from a commercial available 3D database of approximately 50,000 molecule library, Being evaluated by a cell-based assay, 5 compounds were further identified as the potent inhibitors of Candida albicans CYP51 (CACYP51) with low minimal inhibitory concentration (MIC) range. BMD-09-01${\sim}$BMD-09-04 MIC range was 0.5 ${\mu}$g/ml and BMD-09-05 was 1 ${\mu}$g/ml. These new inhibitors provide a basis for some non-azole antifungal rational design of new, and more efficacious antifungal agents.

• Mycobiology
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• v.48 no.1
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• pp.37-43
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• 2020

A serious leaf disease caused by Colletotrichum dematium was found during the cultivation of Sarcandra glabra in Jingxi, Rong'an, and Donglan Counties in Guangxi Province, which inflicted huge losses to plant productivity. Biological control gradually became an effective control method for plant pathogens. Many studies showed that the application of actinomycetes in biological control has been effective. Therefore, it may be of great significance to study the application of actinomycetes on controlling the diseases caused by S. glabra. Strains of antifungal actinomycetes capable of inhibiting C. dematium were identified, isolated and screened from healthy plants tissues and the rhizospheres in soils containing S. glabra. In this study, 15 actinomycetes strains were isolated and among these, strains JT-2F, DT-3F, and JJ-3F, appeared to show antagonistic effects against anthracnose of S. glabra. The strains JT-2F and DT-3F were isolated from soil, while JJ-3F was isolated from plant stems. The antagonism rate of strain JT-2F was 86.75%, which was the highest value among the three strains. Additionally, the JT-2F strain also had the strongest antagonistic activity when the antagonistic activities were tested against seven plant pathogens. Strain JT-2F is able to produce proteases and cellulase to degrade the protein and cellulose components of cell walls of C. dematium, respectively. This results in mycelia damage which leads to inhibition of the growth of C. dematium. Strain JT-2F was identified as Streptomyces tsukiyonensis based on morphological traits and 16S rDNA sequence analysis.

• KSBB Journal
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• v.29 no.3
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• pp.131-138
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• 2014

In this study, we isolated two novel chitinase producing bacterial strains from yellow loess samples collected from Jullanamdo province. The chitinase producing bacteria were isolated based on the zone size of clearance in the chitin agar plates. Both of them were gram positive, rod ($2{\sim}3{\times}0.3{\sim}0.4{\mu}m$), spore-forming, and motility positive. They were facultative anaerobic, catalase positive and hydrolyzed starch, gelatin, and casein. From the 16s rRNA gene sequence analysis, the isolates were labeled as Bacillus licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02. The isolates showed higher extracellular chitinase activities than B. licheniformis ATCC 14580 as a control. The optimum temperature and pH for chitinase production were $40^{\circ}C$ and pH 7.0, respectively. Response Surface Methodology (RSM) was used to optimize the culture medium for efficient production of the chitinase. Under this optimal condition, 1.5 times higher chitinase activity of B. licheniformis KYLS-CU02 was obtained. Extracellular chitinases of the two isolates were purified through ammonium sulfate precipitation and anion-exchange DEAE-cellulose column chromatography. The specific activities of purified chitinase from B. licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02 were 7.65 and 5.21 U/mg protein, respectively. The molecular weights of the two purified chitinases were 59 kDa. Further, the purified chitinase of B. licheniformis KYLS-CU01 showed high antifungal activity against Fusarium sp.. In conclusion, these two bacterial isolates can be used as a biopesticide to control pathogenic fungi.