scholarly journals The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR/Cas9 and Cre/loxP systems

2021 ◽  
Author(s):  
Kohji Yamada ◽  
Toya Yamamoto ◽  
Kanon Uwasa ◽  
Keishi Osakabe ◽  
Yoshitaka Takano
Keyword(s):  
Gene Targeting ◽  
Gene Disruption ◽  
Fungal Growth ◽  
Inducible Promoter ◽  
Phytopathogenic Fungi ◽  
Colletotrichum Orbiculare ◽  
Selective Marker ◽  
Fungal Virulence ◽  
Knockout Mutants ◽  
Low Efficiency

Phytopathogenic fungi belonging to the Colletotrichum genus cause devastating damage for many plant species. Among them, Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to efficiently generate multiple knockout mutants of C. orbiculare. We first found that CRISPR/Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR/Cas9 vector, more than 90 % of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre/loxP marker recycling system for C. orbiculare because limited availability of desired selective markers hampers sequential gene disruption. In this system, integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling reuse of the same selective marker for the next transformation. Using our CRISPR/Cas9 and Cre/loxP systems, we attempted to identify functional sugar transporters in C. orbiculare. Multiple disruptions of putative quinate transporter genes restrict fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. Our analyses revealed that quinate acquisition is dispensable during fungal infection because this mutant displayed normal virulence to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants established in this study, we inferred that repetitive rounds of gene disruption using CRISPR/Cas9 and Cre/loxP systems do not cause negative effects for fungal virulence and growth. Therefore, these systems will be powerful tools to perform systematic gene targeting approach for C. orbiculare.

scholarly journals Double Gamers—Can Modified Natural Regulators of Higher Plants Act as Antagonists against Phytopathogens? The Case of Jasmonic Acid Derivatives

2020 ◽  
Vol 21 (22) ◽  
pp. 8681
Author(s):  
Nicolò Orsoni ◽  
Francesca Degola ◽  
Luca Nerva ◽  
Franco Bisceglie ◽  
Giorgio Spadola ◽  
...  
Keyword(s):  
Jasmonic Acid ◽  
Plant Pathogens ◽  
Higher Plants ◽  
Fungal Growth ◽  
Pathogenic Fungi ◽  
Inhibitory Effect ◽  
Fungal Species ◽  
Phytopathogenic Fungi ◽  
Phaeomoniella Chlamydospora ◽  
Horticultural Crops

As key players in biotic stress response of plants, jasmonic acid (JA) and its derivatives cover a specific and prominent role in pathogens-mediated signaling and hence are promising candidates for a sustainable management of phytopathogenic fungi. Recently, JA directed antimicrobial effects on plant pathogens has been suggested, supporting the theory of oxylipins as double gamers in plant-pathogen interaction. Based on these premises, six derivatives (dihydrojasmone and cis-jasmone, two thiosemicarbazonic derivatives and their corresponding complexes with copper) have been evaluated against 13 fungal species affecting various economically important herbaceous and woody crops, such as cereals, grapes and horticultural crops: Phaeoacremonium minimum, Neofusicoccum parvum, Phaeomoniella chlamydospora, Fomitiporia mediterranea, Fusarium poae, F. culmorum, F. graminearum, F. oxysporum f. sp. lactucae,F. sporotrichioides, Aspergillus flavus, Rhizoctonia solani,Sclerotinia spp. and Verticillium dahliae. The biological activity of these compounds was assessed in terms of growth inhibition and, for the two mycotoxigenic species A. flavus and F. sporotrichioides, also in terms of toxin containment. As expected, the inhibitory effect of molecules greatly varied amongst both genera and species; cis-jasmone thiosemicarbazone in particular has shown the wider range of effectiveness. However, our results show that thiosemicarbazones derivatives are more effective than the parent ketones in limiting fungal growth and mycotoxins production, supporting possible applications for the control of pathogenic fungi.

scholarly journals Novel Bifunctional Inhibitor of Xylanase and Aspartic Protease: Implications for Inhibition of Fungal Growth

2001 ◽  
Vol 45 (7) ◽  
pp. 2008-2017 ◽  
Author(s):  
Chandravanu Dash ◽  
Absar Ahmad ◽  
Devyani Nath ◽  
Mala Rao
Keyword(s):  
Growth Inhibition ◽  
Fungal Growth ◽  
Phytopathogenic Fungi ◽  
Aspartic Protease ◽  
Cellular Growth ◽  
Alternative Mechanism ◽  
Trinitrobenzenesulfonic Acid ◽  
Woodward’S Reagent K ◽  
Bifunctional Inhibitor ◽  
Fungal Growth Inhibition

ABSTRACT A novel bifunctional inhibitor (ATBI) from an extremophilicBacillus sp. exhibiting an activity against phytopathogenic fungi, including Alternaria, Aspergillus, Curvularia, Colletotricum, Fusarium, and Phomopsis species, and the saprophytic fungus Trichoderma sp. has been investigated. The 50% inhibitory concentrations of ATBI ranged from 0.30 to 5.9 μg/ml, whereas the MIC varied from 0.60 to 3.5 μg/ml for the fungal growth inhibition. The negative charge and the absence of periodic secondary structure in ATBI suggested an alternative mechanism for fungal growth inhibition. Rescue of fungal growth inhibition by the hydrolytic products of xylanase and aspartic protease indicated the involvement of these enzymes in cellular growth. The chemical modification of Asp or Glu or Lys residues of ATBI by 2,4,6-trinitrobenzenesulfonic acid and Woodward's reagent K, respectively, abolished its antifungal activity. In addition, ATBI also inhibited xylanase and aspartic protease competitively, withKi values 1.75 and 3.25 μM, respectively. Our discovery led us to envisage a paradigm shift in the concept of fungal growth inhibition for the role of antixylanolytic activity. Here we report for the first time a novel class of antifungal peptide, exhibiting bifunctional inhibitory activity.

scholarly journals Three Related Enzymes in Candida albicans Achieve Arginine- and Agmatine-Dependent Metabolism That Is Essential for Growth and Fungal Virulence

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Katja Schaefer ◽  
Jeanette Wagener ◽  
Ryan M. Ames ◽  
Stella Christou ◽  
Donna M. MacCallum ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Galleria Mellonella ◽  
Fungal Growth ◽  
Open Reading Frames ◽  
Mammalian Host ◽  
No Production ◽  
Triple Mutant ◽  
Fungal Virulence ◽  
Content Type

ABSTRACT Amino acid metabolism is crucial for fungal growth and development. Ureohydrolases produce amines when acting on l-arginine, agmatine, and guanidinobutyrate (GB), and these enzymes generate ornithine (by arginase), putrescine (by agmatinase), or GABA (by 4-guanidinobutyrase or GBase). Candida albicans can metabolize and grow on arginine, agmatine, or guanidinobutyrate as the sole nitrogen source. Three related C. albicans genes whose sequences suggested that they were putative arginase or arginase-like genes were examined for their role in these metabolic pathways. Of these, Car1 encoded the only bona fide arginase, whereas we provide evidence that the other two open reading frames, orf19.5862 and orf19.3418, encode agmatinase and guanidinobutyrase (Gbase), respectively. Analysis of strains with single and multiple mutations suggested the presence of arginase-dependent and arginase-independent routes for polyamine production. CAR1 played a role in hyphal morphogenesis in response to arginine, and the virulence of a triple mutant was reduced in both Galleria mellonella and Mus musculus infection models. In the bloodstream, arginine is an essential amino acid that is required by phagocytes to synthesize nitric oxide (NO). However, none of the single or multiple mutants affected host NO production, suggesting that they did not influence the oxidative burst of phagocytes. IMPORTANCE We show that the C. albicans ureohydrolases arginase (Car1), agmatinase (Agt1), and guanidinobutyrase (Gbu1) can orchestrate an arginase-independent route for polyamine production and that this is important for C. albicans growth and survival in microenvironments of the mammalian host.

scholarly journals In vitro fungitoxic potential of Lippia gracilis (Schauer) essential oil against phytopathogens

2020 ◽  
pp. 667-674
Author(s):  
Kevison Romulo da Silva França ◽  
Flavia Mota de Figueredo Alves ◽  
Tiago Silva Lima ◽  
Alda Leaby dos Santos Xavier ◽  
Plínio Tércio Medeiros de Azevedo ◽  
...  
Keyword(s):  
Essential Oil ◽  
Mycelial Growth ◽  
Fungal Growth ◽  
Fungal Species ◽  
Phytopathogenic Fungi ◽  
Negative Control ◽  
Growth Speed ◽  
Randomized Design

This study evaluates the in vitro effects of Lippia gracilis essential oil on the mycelial growth of phytopathogenic fungi. Experiments were carried out using a completely randomized design to assess the effects of eight treatments. Five replicates were evaluated for each experimental group. The essential oil was incorporated into the potato dextrose culture medium and poured into Petri dishes. Treatments were comprised of different concentrations of the oil (0.0125, 0.025, 0.05, 0.1, and 0.2%), a negative control (0.0%), and two positive controls (commercial fungicides). The plates were inoculated with fungi including Colletotrichum gloeosporioides, C. musae, C. fructicola, C. asianum, Alternaria alternata, A. brassicicola, Fusarium solani, F. oxysporum f. sp. cubense, and Lasiodiplodia theobromae and were incubated for seven days at 27 ± 2°C. The following variables were measured to verify the differences observed among treatments: percentage of mycelial growth inhibition and index of mycelial growth speed. All concentrations of L. gracilis oil inhibited the mycelial growth of the fungal species evaluated. The complete inhibition was observed between concentrations of 0.0125 and 0.1%. Treatment with oil inhibited fungal growth with similar, or even greater, efficiency than commercial fungicides.. We recommend the development of in vivo tests to verify whether L. gracilis essential oil can protect against fungal disease in live plants.

scholarly journals Structure-guided approaches to targeting stress responses in human fungal pathogens

2020 ◽  
Vol 295 (42) ◽  
pp. 14458-14472
Author(s):  
Emmanuelle V. LeBlanc ◽  
Elizabeth J. Polvi ◽  
Amanda O. Veri ◽  
Gilbert G. Privé ◽  
Leah E. Cowen
Keyword(s):  
Stress Response ◽  
Stress Responses ◽  
Fungal Pathogens ◽  
Fungal Infections ◽  
Fungal Growth ◽  
Small Gtpase ◽  
Ecological Niches ◽  
Attractive Alternative ◽  
Response Regulators ◽  
Fungal Virulence

Fungi inhabit extraordinarily diverse ecological niches, including the human body. Invasive fungal infections have a devastating impact on human health worldwide, killing ∼1.5 million individuals annually. The majority of these deaths are attributable to species of Candida, Cryptococcus, and Aspergillus. Treating fungal infections is challenging, in part due to the emergence of resistance to our limited arsenal of antifungal agents, necessitating the development of novel therapeutic options. Whereas conventional antifungal strategies target proteins or cellular components essential for fungal growth, an attractive alternative strategy involves targeting proteins that regulate fungal virulence or antifungal drug resistance, such as regulators of fungal stress responses. Stress response networks enable fungi to adapt, grow, and cause disease in humans and include regulators that are highly conserved across eukaryotes as well as those that are fungal-specific. This review highlights recent developments in elucidating crystal structures of fungal stress response regulators and emphasizes how this knowledge can guide the design of fungal-selective inhibitors. We focus on the progress that has been made with highly conserved regulators, including the molecular chaperone Hsp90, the protein phosphatase calcineurin, and the small GTPase Ras1, as well as with divergent stress response regulators, including the cell wall kinase Yck2 and trehalose synthases. Exploring structures of these important fungal stress regulators will accelerate the design of selective antifungals that can be deployed to combat life-threatening fungal diseases.

Secondary metabolites from endophytic Streptomyces aureofaciens CMUAc130 and their antifungal activity

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1691-1695 ◽  
Author(s):  
Thongchai Taechowisan ◽  
Chunhua Lu ◽  
Yuemao Shen ◽  
Saisamorn Lumyong
Keyword(s):  
Culture Filtrate ◽  
Fungal Growth ◽  
Zingiber Officinale ◽  
Phytopathogenic Fungi ◽  
Active Ingredients ◽  
Streptomyces Aureofaciens ◽  
Mass Spectral ◽  
Causative Agents ◽  
Fungal Growth Inhibition

Streptomyces aureofaciens CMUAc130 was isolated from the root tissue of Zingiber officinale Rosc. (Zingiberaceae). It was an antagonist of Colletotrichum musae and Fusarium oxysporum, the causative agents of anthracnose of banana and wilt of wheat, respectively. Evidence for the in vitro antibiosis of S. aureofaciens CMUAc130 was demonstrated by the zone of fungal-growth inhibition. Microscopic observations showed thickness and bulbous structures at the edges of the inhibited fungal hyphae. The culture filtrate and crude extract from this strain were all inhibitory to tested phytopathogenic fungi. The major active ingredients from the culture filtrate of S. aureofaciens CMUAc130 were purified by silica gel-column chromatography and identified to be (i) 5,7-dimethoxy-4-p-methoxylphenylcoumarin and (ii) 5,7-dimethoxy-4-phenylcoumarin by NMR and mass-spectral data, respectively. Bioassay studies showed that compounds (i) and (ii) had antifungal activities against tested fungi, and their MICs were found to be 120 and 150 μg ml−1, respectively. This is the first report of compounds (i) and (ii) from micro-organisms as active ingredients for the control of phytopathogenic fungi.

scholarly journals Reverse Genetics for Functional Genomics of Phytopathogenic Fungi and Oomycetes

2009 ◽  
Vol 2009 ◽  
pp. 1-11 ◽  
Author(s):  
Vijai Bhadauria ◽  
Sabine Banniza ◽  
Yangdou Wei ◽  
You-Liang Peng
Keyword(s):  
Functional Genomics ◽  
Gene Disruption ◽  
Insertional Mutagenesis ◽  
Reverse Genetics ◽  
Phytopathogenic Fungi ◽  
Sequence Information ◽  
Biological Functions ◽  
Targeted Gene Disruption ◽  
Local Lesions ◽  
Genome Sequence Information

Sequencing of over 40 fungal and oomycete genomes has been completed. The next major challenge in modern fungal/oomycete biology is now to translate this plethora of genome sequence information into biological functions. Reverse genetics has emerged as a seminal tool for functional genomics investigations. Techniques utilized for reverse genetics like targeted gene disruption/replacement, gene silencing, insertional mutagenesis, and targeting induced local lesions in genomes will contribute greatly to the understanding of gene function of fungal and oomycete pathogens. This paper provides an overview on high-throughput reverse genetics approaches to decode fungal/oomycete genomes.

Cytological responses induced by five phytopathogenic fungi in a nonhost plant, Phaseolus vulgaris

1986 ◽  
Vol 64 (3) ◽  
pp. 648-657 ◽  
Author(s):  
Myriam R. Fernandez ◽  
Michèle C. Heath
Keyword(s):  
Phenolic Compounds ◽  
Phaseolus Vulgaris ◽  
Fungal Growth ◽  
Phytopathogenic Fungi ◽  
French Bean ◽  
Mesophyll Cells ◽  
Fresh Tissue ◽  
Susceptible Host ◽  
Nonhost Plant ◽  
Blue Reaction

Responses of the French bean plant (Phaseolus vulgaris L. cv. Pinto) to the nonpathogenic fungi Helminthosporium maydis Nisikado and Miyake (perfect stage Cochliobolus heterostrophus (Drechsler) Drechsler), Stemphylium sarcinaeforme (Cav.) Wiltish., S. botryosum Wallr., Cladosporium fulvum Cooke, and Uromyces vignae Barcl. were examined by cytological and histochemical techniques. All fungi penetrated bean leaves through stomata, and elicited similar modifications in guard and mesophyll cells. Responses involving cell contents were collapse, browning, autofluorescence, and a toluidine blue reaction indicative of the presence of phenolic compounds. Responses associated with cell walls were yellowing, autofluorescence, staining for phenolic compounds, and deposition of callose and of noncallosic, refractive material. In some of the interactions, decolorization and clearing of the tissue for light microscopy induced changes in the frequencies of autofluorescence of cell contents and walls when compared with those observed in fresh tissue. Despite the general similarity of responses elicited by each fungus, striking variations were found among fungi in the percentage of infection sites showing a particular response. For some responses, this variation seemed to be related to the type of interaction each of the fungi has with its susceptible host. It is suggested that even in nonhost plants, the nature and activities of the fungus determine the frequency and extent of the plant's responses. Also, it seems unlikely that all of these responses are involved in the cessation of fungal growth.

scholarly journals Gene Targeting in Gram-Negative Bacteria by Use of a Mobile Group II Intron (“Targetron”) Expressed from a Broad-Host-Range Vector

2007 ◽  
Vol 73 (8) ◽  
pp. 2735-2743 ◽  
Author(s):  
Jun Yao ◽  
Alan M. Lambowitz
Keyword(s):  
Host Range ◽  
Gene Targeting ◽  
Inducible Promoter ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Efficient Gene ◽  
Range Vector ◽  
Group Ii ◽  
Broad Host Range Vector

ABSTRACT Mobile group II introns (“targetrons”) can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via an m-toluic acid-inducible promoter from a broad-host-range vector containing an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.

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