scholarly journals Enhanced Resistance To Fungal Pathogens in Transgenic Peanut (Arachis Hypogaea L.) Cultivar L14 by Overexpression of Gene encoding Chitinase 42 kDa from Trichoderma Asperellum SH16

2021 ◽  
Author(s):  
Phung Thi Bich Hoa ◽  
Nguyen Hoang Tue ◽  
Huynh Thi Quynh Trang ◽  
Hoang Anh Thu ◽  
Le Ngoc Huyen Nhung ◽  
...  
Keyword(s):  
Arachis Hypogaea ◽  
Fungal Pathogens ◽  
Pathogenic Fungus ◽  
Chitinase Activity ◽  
Trichoderma Asperellum ◽  
Wild Type ◽  
Synthetic Genes ◽  
Chitinase Genes ◽  
Transgenic Peanut

Abstract This study reports the expression of 42 kDa chitinase genes from Trichoderma asperellum SH16 in peanut (Arachis hypogaea) roots under the regulation of tissue-specific Asy promoter through Agrobacterium tumefaciens-mediated transformation. The 42 kDa chitinase genes, including one wild-type sequence (Chi42) and two synthetic sequences (syncodChi42-1 and syncodChi42-2) which were optimized for codon usage for plant expression, were incorporated into the peanut genome and successfully expressed in their roots. The investigation revealed that the enzyme chitinase from two synthetic genes had higher activity than that from the wild-type gene, about 901 U/mg (140 U/mL) and 1124 U/mg (197 U/mL) vs about 508 U/mg (87 U/mL). Transgenic peanut roots also exhibited extracellular chitinase activity which was driven by signal peptide of rice amylase 3D gene against the pathogenic fungus Sclerotium rolfsii under in vitro conditions. The higher chitinase activity of two synthetic genes in peanut roots promises potential applications in the field of transgenic crops against phytopathogenic fungi.

Transfer of a plant chitinase gene into a nitrogen-fixing Azospirillum and study of its expression

2004 ◽  
Vol 50 (7) ◽  
pp. 509-513 ◽  
Author(s):  
Jayaraman Jayaraj ◽  
Subbaratnam Muthukrishnan ◽  
George H Liang
Keyword(s):  
Azospirillum Brasilense ◽  
Pathogenic Fungus ◽  
Chitinase Activity ◽  
Chitinase Gene ◽  
Cell Protein ◽  
Broad Host Range ◽  
Nitrogen Fixing ◽  
Rice Chitinase ◽  
Chitinase Genes

Azospirillum is used extensively in rice and other cereal crops as a biofertilizer. There is a substantial opportunity to improve the efficiency of this bacterium through the transfer of genes of agricultural importance from other organisms. Chitinases are antifungal proteins, and expression of chitinase genes in Azospirillum would help to develop strains with potential antifungal activities. So far there are no reports about transfer of plant genes into Azospirillum and their expression. The present study was aimed at expressing an antifungal gene (a rice chitinase) of plant origin in Azospirillum brasilense. A rice chitinase cDNA (RC 7) that codes for a 35 kDa protein was subcloned into a broad host range plasmid pDSK519 under the control of LacZ promoter. The plasmid was mobilized into the nitrogen-fixing bacterium, Azospirillum brasilense strain SP51eFL1, through biparental mating. The conjugation frequency was in the range of 35–40 × 10–6. The transconjugants grew in nitrogen-free media and fixed gaseous nitrogen in vitro. However, their growth and nitrogen-fixing ability were slightly less than those of the wild-type. Expression of the protein was demonstrated through western blotting of the total cell protein, which detected a 35 kDa band that was immuno-reactive to a barley chitinase antibody. The cell lysates also hydrolyzed various chitin substrates, which resulted in release of free sugars demonstrating the chitinase activity of transconjugants. The expressed protein also had antifungal activity as demonstrated by inhibition of growth of the plant pathogenic fungus, Rhizoctonia solani.Key words: Azospirillum-transformation, rice chitinase gene, protein expression, chitinase activity.

scholarly journals Structure and inhibition of Cryptococcus neoformans sterylglucosidase to develop antifungal agents

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nivea Pereira de Sa ◽  
Adam Taouil ◽  
Jinwoo Kim ◽  
Timothy Clement ◽  
Reece M. Hoffmann ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Rational Design ◽  
Antifungal Agents ◽  
Pathogenic Fungus ◽  
Pathogenic Fungi ◽  
Growth Defect ◽  
Wild Type ◽  
Secondary Infections ◽  
Heavy Burden

AbstractPathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3β-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3β-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1’s substrate specificity and enable the rational design of antifungal agents targeting Sgl1.

scholarly journals THEORETICAL APPROACH ON TARGETING PLANT FUNGAL PATHOGENIC PROTEINS AGAINST NATURALLY ISOLATED COMPOUNDS FROM CHITINIPHILUS SHINANONENSIS

2019 ◽  
pp. 138-142
Author(s):  
KRITHIKA S ◽  
CHELLARAM C
Keyword(s):  
Drug Targets ◽  
Fungal Pathogens ◽  
Interaction Analysis ◽  
Pathogenic Fungus ◽  
Three Dimensional ◽  
Data Bank ◽  
Asiatic Acid ◽  
Literature Study

Objective: The objective of this study was to find the potency and bioefficacy of Asiatic acid and triterpene against four different plant fungal pathogens using a structure-based drug designing approach. Methods: The pathogenic fungus which causes a dreadful effect on plants is reviewed from literature study, and its three-dimensional structures are retrieved from the protein data bank database. On the other hand, ligands are prepared. Finally, prepared fungal drug targets are docked with naturally isolated compounds using AutoDock tools. Results: Both compounds Asiatic acid and triterpene structures are complementary to bind at the active site of four different drug targets. Comparatively, it is more favorable for Avr2 effector protein from Fusarium oxysporum with Ki value of 126.60 μM, 1.76 μM, and dock score value of −5.32 kcal/mol and −7.85 kcal/mol for Asiatic acid and triterpene, respectively. Thus, interaction analysis was carried out only for these protein-ligand complexes. Conclusion: The computational biology study states that these two compounds can be the lead candidate for treating disease caused by plant fungal pathogen F. oxysporum. However, further study has to be done in vitro and in vivo to prove its same efficacy.

scholarly journals Identification of ENA1 as a Virulence Gene of the Human Pathogenic Fungus Cryptococcus neoformans through Signature-Tagged Insertional Mutagenesis

2009 ◽  
Vol 8 (3) ◽  
pp. 315-326 ◽  
Author(s):  
Alexander Idnurm ◽  
Felicia J. Walton ◽  
Anna Floyd ◽  
Jennifer L. Reedy ◽  
Joseph Heitman
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogens ◽  
Insertional Mutagenesis ◽  
Pathogenic Fungus ◽  
Mammalian Host ◽  
New Genes ◽  
Strain Collection ◽  
Mutant Strains ◽  
Human Pathogenic Fungus

ABSTRACT A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nutrient limitation. A screen of 10% of the strain collection in mice identified an avirulent mutant strain with an insertion in the ENA1 gene, which is predicted to encode a fungus-specific sodium or potassium P-type ATPase. The results of the deletion of the gene and complementation experiments confirmed its key role in mammalian virulence. ena1 mutant strains exhibited no change in sensitivity to high salt concentrations but were sensitive to alkaline pH conditions, providing evidence that the fungus may have to survive at elevated pH during infection of the mammalian host. The mutation of the well-characterized virulence factor calcineurin (CNA1) also rendered C. neoformans strains sensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutant strains were upregulated in response to high pH, and cna1 ena1 double mutant strains exhibited increased sensitivity to elevated pH, indicating that at least two pathways in the fungus mediate survival under alkaline conditions. Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals.

Independent regulation of chitin synthase and chitinase activity in Candida albicans and Saccharomyces cerevisiae

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 921-928 ◽  
Author(s):  
Serena Selvaggini ◽  
Carol A. Munro ◽  
Serge Paschoud ◽  
Dominique Sanglard ◽  
Neil A. R. Gow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
De Novo ◽  
Gene Families ◽  
Chitinase Activity ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Growing Cell ◽  
Chitinase Genes

Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C. albicans during yeast–hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast–hypha morphogenesis in C. albicans.

scholarly journals The Chitinolytic Activity of Listeria monocytogenes EGD Is Regulated by Carbohydrates but Also by the Virulence Regulator PrfA

2010 ◽  
Vol 76 (19) ◽  
pp. 6470-6476 ◽  
Author(s):  
M. H. Larsen ◽  
J. J. Leisner ◽  
H. Ingmer
Keyword(s):  
Listeria Monocytogenes ◽  
Virulence Gene ◽  
Chitinase Activity ◽  
Exponential Growth Phase ◽  
Wild Type ◽  
Chitinolytic Activity ◽  
Carbohydrate Polymers ◽  
Terrestrial Environments ◽  
In The Wild

ABSTRACT Chitin, an insoluble polymer of N-acetyl-d-glucosamine (GlcNAc), is one of the most abundant carbohydrate polymers in marine and terrestrial environments. Chitin hydrolysis by Listeria monocytogenes depends on two chitinase-encoding genes, chiA and chiB, and the aim of this study was to investigate their regulation. Chitin induces the expression of both chitinases in late exponential growth phase, and chiA but not chiB is furthermore induced by the monomer GlcNAc. Furthermore, their expression is subjected to catabolite control. Chitinases expressed by bacterial pathogens have proven to be important not only for nutrient acquisition and environmental survival but also for infecting animals and humans. Interestingly, the central L. monocytogenes virulence gene regulator, PrfA, is required for the chitinolytic phenotype, as chitinase activity was significantly reduced in prfA mutant cells compared to its level in wild-type cells. In agreement with this, Northern blot analysis showed that the amounts of chiA and chiB transcripts upon induction by chitin were significantly lower in the prfA mutant than in the wild type. The chitinolytic activity and chiA and chiB expression were reduced in the absence of the sigB gene, indicating that σB is also important for the production of chitinases. The chiA, chiB, and chiA chiB mutants were not impaired for in vitro adhesion and invasion in epithelial cell lines, but the chiA chiB double mutant showed less survival ability in a chitin-enriched medium. The regulation of chitinolytic activity in L. monocytogenes is complex, and taken together, the results indicate that the biological role of this activity may not be limited to the external environment.

scholarly journals Unveiling the function and regulation control of the DUF3129 family proteins in fungal infection of hosts

2019 ◽  
Vol 374 (1767) ◽  
pp. 20180321 ◽  
Author(s):  
Wei Huang ◽  
Song Hong ◽  
Guirong Tang ◽  
Yuzhen Lu ◽  
Chengshu Wang
Keyword(s):  
Lipid Droplets ◽  
Fungal Pathogens ◽  
Pathogenic Fungus ◽  
Fungal Species ◽  
Wild Type ◽  
Metarhizium Robertsii ◽  
Host Interactions ◽  
Eukaryotic Proteins ◽  
Insect Pathogens ◽  
Mutant Cells

Many prokaryotic and eukaryotic proteins contain domains of unknown function (DUFs). A DUF3129 family of proteins is widely encoded in the genomes of fungal pathogens. A few studies in plant and insect pathogens indicated that the DUF3129 genes are required for fungal penetration of host cuticles with an unclear mechanism. We found that a varied number of DUF3129 proteins is present in different fungal species and the proteins are evolutionarily diverged from each other at the inter- and intra-specific levels. By using the insect pathogenic fungus Metarhizium robertsii as a model, we performed experiments and found that the seven DUF3129 proteins encoded by this fungus are localized to cellular lipid droplets (LDs). Individual deletion of these genes did not affect fungal formation of the infection structure appressoria and the accumulation of LDs in fungal conidia. When compared with the wild-type (WT) strain, insect bioassays revealed that the virulence of most null mutants were significantly impaired during topical infection but not during injection of insects. Carbon starvation and the subsequent Western blot analysis indicated that the LD-specific perilipin protein was completely degraded in the WT cells whereas varied levels of perilipin could be detected in the mutant cells, which signified that depletion of LD content was delayed in mutant cells, and DUF3129 proteins are therefore involved in LD degradation. We also provided biochemical evidence that these DUF3129 genes are transcriptionally regulated by a yeast Ste12-like transcription factor. The findings of this study not only unveil the function of DUF3129 proteins but also better understand the diverse mechanism of fungus–host interactions. This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management’.

scholarly journals Inactivation of the Lipoxygenase ZmLOX3 Increases Susceptibility of Maize to Aspergillus spp.

2009 ◽  
Vol 22 (2) ◽  
pp. 222-231 ◽  
Author(s):  
Xiquan Gao ◽  
Marion Brodhagen ◽  
Tom Isakeit ◽  
Sigal Horowitz Brown ◽  
Cornelia Göbel ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Jasmonic Acid ◽  
Fungal Pathogens ◽  
Wild Type ◽  
Maize Line ◽  
Plant Host ◽  
Fungal Development ◽  
Fatty Acid Hydroperoxides

Plant and fungal lipoxygenases (LOX) catalyze the oxidation of polyunsaturated fatty acids, creating fatty-acid hydroperoxides (oxylipins). Fungal oxylipins are required for normal fungal development and secondary metabolism, and plant host–derived oxylipins interfere with these processes in fungi, presumably by signal mimicry. The maize LOX gene ZmLOX3 has been implicated previously in seed-Aspergillus interactions, so we tested the interactions of a mutant maize line (lox3-4, in which ZmLOX3 is disrupted) with the mycotoxigenic seed-infecting fungi Aspergillus flavus and Aspergillus nidulans. The lox3-4 mutant was more susceptible than wild-type maize to both Aspergillus species. All strains of A. flavus and A. nidulans produced more conidia and aflatoxin (or the precursor sterigmatocystin) on lox3-4 kernels than on wild-type kernels, in vitro and under field conditions. Although oxylipins did not differ detectably between A. flavus–infected kernels of the lox3-4 and wild-type (WT) maize, oxylipin precursors (free fatty acids) and a downstream metabolite (jasmonic acid) accumulated to greater levels in lox3-4 than in WT kernels. The increased resistance of the lox3-4 mutant to other fungal pathogens (Fusarium, Colletotrichum, Cochliobolus, and Exserohilum spp.) is in sharp contrast to results described herein for Aspergillus spp., suggesting that outcomes of LOX-governed host-pathogen interactions are pathogen-specific.

The URNA Genes of Herpesvirus Saimiri (Strain C488) Are Dispensable for Transformation of Human T Cells In Vitro

1999 ◽  
Vol 73 (12) ◽  
pp. 10551-10555 ◽  
Author(s):  
Armin Ensser ◽  
André Pfinder ◽  
Ingrid Müller-Fleckenstein ◽  
Bernhard Fleckenstein
Keyword(s):  
Cell Culture ◽  
Herpesvirus Saimiri ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Human T Cell ◽  
Human T Cells ◽  
Small Nuclear Rnas ◽  
T Cell Lines

ABSTRACT The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.

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