scholarly journals Targeted Disruption of Scytalone Dehydratase Gene Using Agrobacterium tumefaciens-Mediated Transformation Leads to Altered Melanin Production in Ascochyta lentis

2020 ◽  
Vol 6 (4) ◽  
pp. 314
Author(s):  
Johannes W. Debler ◽  
Bernadette M. Henares
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fungal Pathogens ◽  
Molecular Mechanisms ◽  
Ascochyta Blight ◽  
Gene Replacement ◽  
Melanin Production ◽  
Replacement Method ◽  
Ascochyta Lentis ◽  
Targeted Gene Replacement ◽  
Scytalone Dehydratase

Sustainable crop production is constantly challenged by the rapid evolution of fungal pathogens equipped with an array of host infection strategies and survival mechanisms. One of the devastating fungal pathogens that infect lentil is the ascomycete Ascochyta lentis which causes black spot or ascochyta blight (AB) on all above ground parts of the plant. In order to explore the mechanisms involved in the pathogenicity of A. lentis, we developed a targeted gene replacement method using Agrobacterium tumefaciens mediated transformation (ATMT) to study and characterize gene function. In this study, we investigated the role of scytalone dehydratase (SCD) in the synthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in AlKewell. Two SCD genes have been identified in AlKewell, AlSCD1 and AlSCD2. Phylogenetic analysis revealed that AlSCD1 clustered with the previously characterized fungal SCDs; thus, AlSCD1 was disrupted using the targeted gene replacement vector, pTAR-hyg-SCD1. The vector was constructed in a single step process using Gibson Assembly, which facilitated an easy and seamless assembly of multiple inserts. The resulting AlKewell scd1::hyg transformants appeared light brown/brownish-pink in contrast to the dark brown pycnidia of the WT strain and ectopic transformant, indicating an altered DHN-melanin production. Disruption of AlSCD1 gene did not result in a change in the virulence profile of AlKewell towards susceptible and resistant lentil varieties. This is the first report of a targeted gene manipulation in A. lentis which serves as a foundation for the functional gene characterization to provide a better understanding of molecular mechanisms involved in pathogen diversity and host specificity.

scholarly journals Agrobacterium tumefaciens-mediated transformation and expression of GFP in Ascochyta lentis to characterize ascochyta blight disease progression in lentil

PLoS ONE ◽  
2019 ◽  
Vol 14 (10) ◽  
pp. e0223419 ◽  
Author(s):  
Bernadette M. Henares ◽  
Johannes W. Debler ◽  
Lina M. Farfan-Caceres ◽  
Christina R. Grime ◽  
Robert C. Lee
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Disease Progression ◽  
Ascochyta Blight ◽  
Ascochyta Lentis ◽  
Blight Disease

scholarly journals The T788G Mutation in thecyp51CGene Confers Voriconazole Resistance in Aspergillus flavus Causing Aspergillosis

2012 ◽  
Vol 56 (5) ◽  
pp. 2598-2603 ◽  
Author(s):  
Wei Liu ◽  
Yi Sun ◽  
Wei Chen ◽  
Weixia Liu ◽  
Zhe Wan ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Resistant Strain ◽  
Antifungal Susceptibility ◽  
Gene Replacement ◽  
Content Type ◽  
Replacement Method ◽  
First Choice ◽  
The One ◽  
A Site ◽  
First Time

ABSTRACTWith voriconazole (VRC) being approved as the first choice in treating invasive aspergillosis (IA) and its increasing use in treatment, a VRC-resistant strain ofAspergillus flavus, the second leading cause of IA afterAspergillus fumigatus, has emerged. The VRC-resistant strain ofA. flavuswas isolated for the first time from the surgical lung specimen of an IA patient with no response to VRC therapy. In order to ascertain the mechanism of VRC resistance, the azole target enzyme genes in this strain ofA. flavuswere cloned and sequenced, and 4 mutations generating amino acid residue substitutions were found in thecyp51Cgene. To further determine the role of this mutated gene for VRC resistance inA. flavus, anAgrobacterium tumefaciens-mediated gene replacement approach was applied. Consequently, the mutatedcyp51Cgene from thisA. flavusstrain was proven to confer the VRC resistance. Finally, to discern the one out of the four mutations in thecyp51Cgene that is responsible for contributing to VRC resistance, a site-directed gene mutagenesis procedure combined with a gene replacement method was performed. As a result, the T788G missense mutation in thecyp51Cgene was identified as responsible for VRC resistance inA. flavus. These findings indicated that the detection of this mutation inA. flavuscould serve as an indicator for physicians to avoid the use of VRC during IA treatment. Further comprehensive surveillance for antifungal susceptibility, as well as intensive study on the mechanism of azole resistance inA. flavuscausing IA, would be required to fully understand this mechanism.

scholarly journals A Homeobox Gene Is Essential for Conidiogenesis of the Rice Blast Fungus Magnaporthe oryzae

2010 ◽  
Vol 23 (4) ◽  
pp. 366-375 ◽  
Author(s):  
Wende Liu ◽  
Shiyong Xie ◽  
Xinhua Zhao ◽  
Xin Chen ◽  
Wenhui Zheng ◽  
...  
Keyword(s):  
Magnaporthe Oryzae ◽  
Rice Blast ◽  
Homeobox Gene ◽  
Gene Replacement ◽  
Fungal Genome ◽  
Protein Signaling ◽  
Homeodomain Proteins ◽  
Conidial Production ◽  
Targeted Gene Replacement ◽  
Severity Of The Disease

Magnaporthe oryzae starts its infection by the attachment of pyriform conidia on rice tissues, and severity of the disease epidemic is proportional to the quantity of conidia produced in the rice blast lesions. However, the mechanism of conidial production is not well understood. Homeodomain proteins play critical roles in regulating various growth and developmental processes in fungi and other eukaryotes. Through targeted gene replacement, we find that deletion of HTF1, one of seven homeobox genes in the fungal genome, does not affect mycelial growth but causes total defect of conidial production. Further observation revealed that the Δhtf1 mutant produces significantly more conidiophores, which curve slightly near the tip but could not develop sterigmata-like structures. Although the Δhtf1 mutant fails to form conidia, it could still develop melanized appressoria from hyphal tips and infect plants. The expression level of HTF1 is significantly reduced in the Δmgb1 G-β and ΔcpkA deletion mutant, and the ACR1 but not CON7 gene that encodes transcription factor required for normal conidiogenesis is significantly downregulated in the Δhtf1 mutant. These data suggest that the HTF1 gene is essential for conidiogenesis, and may be functionally related to the trimeric G-protein signaling and other transcriptional regulators that are known to be important for conidiation in M. oryzae.

scholarly journals Comparative Proteomics Reveals the Potential Targets of BcNoxR, a Putative Regulatory Subunit of NADPH Oxidase of Botrytis cinerea

2016 ◽  
Vol 29 (12) ◽  
pp. 990-1003 ◽  
Author(s):  
Hua Li ◽  
Zhanquan Zhang ◽  
Chang He ◽  
Guozheng Qin ◽  
Shiping Tian
Keyword(s):  
Nadph Oxidase ◽  
Botrytis Cinerea ◽  
Proteomic Analysis ◽  
Fungal Pathogens ◽  
Molecular Mechanisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Tomato Fruit ◽  
Polymerase Chain Reaction Analysis ◽  
Regulatory Subunit ◽  
Wild Type

The NADPH oxidase (NOX) complex has been shown to play a crucial role in stress response and in the virulence of various fungal pathogens. The underlying molecular mechanisms of NOX, however, remain largely unknown. In the present study, a comparative proteomic analysis compared changes in protein abundance in wild-type Botrytis cinerea and ΔbcnoxR mutants in which the regulatory subunit of NOX was deleted. The ΔbcnoxR mutants exhibited reduced growth, sporulation, and impaired virulence. A total of 60 proteins, representing 49 individual genes, were identified in ΔbcnoxR mutants that exhibited significant differences in abundance relative to wild-type. Reverse transcription-quantitative polymerase chain reaction analysis demonstrated that the differences in transcript levels for 36 of the genes encoding the identified proteins were in agreement with the proteomic analysis, while the remainder exhibited reverse levels. Functional analysis of four proteins that decreased abundance in the ΔbcnoxR mutants indicated that 6-phosphogluconate dehydrogenase (BcPGD) played a role in the growth and sporulation of B. cinerea. The Δbcpgd mutants also displayed impaired virulence on various hosts, such as apple, strawberry, and tomato fruit. These results suggest that NOX can influence the expression of BcPGD, which has an impact on growth, sporulation, and virulence of B. cinerea.

scholarly journals The collagen receptor uPARAP/Endo180 regulates collectins through unique structural elements in its FNII domain

2020 ◽  
Vol 295 (27) ◽  
pp. 9157-9170 ◽  
Author(s):  
Kirstine Sandal Nørregaard ◽  
Oliver Krigslund ◽  
Niels Behrendt ◽  
Lars H. Engelholm ◽  
Henrik Jessen Jürgensen
Keyword(s):  
Fungal Pathogens ◽  
Molecular Mechanisms ◽  
Mannose Receptor ◽  
Surfactant Protein ◽  
Surfactant Protein D ◽  
Cellular Regulation ◽  
Urokinase Plasminogen Activator Receptor ◽  
Receptor Associated Protein ◽  
Additional Level ◽  
Collagen Receptor

C-type lectins that contain collagen-like domains are known as collectins. These proteins are present both in the circulation and in extravascular compartments and are central players of the innate immune system, contributing to first-line defenses against viral, bacterial, and fungal pathogens. The collectins mannose-binding lectin (MBL) and surfactant protein D (SP-D) are regulated by tissue fibroblasts at extravascular sites via an endocytic mechanism governed by urokinase plasminogen activator receptor–associated protein (uPARAP or Endo180), which is also a collagen receptor. Here, we investigated the molecular mechanisms that drive the uPARAP-mediated cellular uptake of MBL and SP-D. We found that the uptake depends on residues within a protruding loop in the fibronectin type-II (FNII) domain of uPARAP that are also critical for collagen uptake. Importantly, however, we also identified FNII domain residues having an exclusive role in collectin uptake. We noted that these residues are absent in the related collagen receptor, the mannose receptor (MR or CD206), which consistently does not interact with collectins. We also show that the second C-type lectin-like domain (CTLD2) is critical for the uptake of SP-D, but not MBL, indicating an additional level of complexity in the interactions between collectins and uPARAP. Finally, we demonstrate that the same molecular mechanisms enable uPARAP to engage MBL immobilized on the surface of pathogens, thereby expanding the potential biological implications of this interaction. Our study reveals molecular details of the receptor-mediated cellular regulation of collectins and offers critical clues for future investigations into collectin biology and pathology.

Resistance to antifungal therapies

2017 ◽  
Vol 61 (1) ◽  
pp. 157-166 ◽  
Author(s):  
Rajendra Prasad ◽  
Atanu Banerjee ◽  
Abdul Haseeb Shah
Keyword(s):  
Fungal Pathogens ◽  
Molecular Mechanisms ◽  
Fungal Infections ◽  
Therapeutic Strategies ◽  
Antifungal Resistance ◽  
Antifungal Drugs ◽  
Therapeutic Interventions ◽  
Medical Professionals ◽  
Comprehensive Understanding ◽  
Novel Targets

The evolution of antifungal resistance among fungal pathogens has rendered the limited arsenal of antifungal drugs futile. Considering the recent rise in the number of nosocomial fungal infections in immunocompromised patients, the emerging clinical multidrug resistance (MDR) has become a matter of grave concern for medical professionals. Despite advances in therapeutic interventions, it has not yet been possible to devise convincing strategies to combat antifungal resistance. Comprehensive understanding of the molecular mechanisms of antifungal resistance is essential for identification of novel targets that do not promote or delay emergence of drug resistance. The present study discusses features and limitations of the currently available antifungals, mechanisms of antifungal resistance and highlights the emerging therapeutic strategies that could be deployed to combat MDR.

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