scholarly journals Living Colors in the Gray Mold Pathogen Botrytis cinerea: Codon-Optimized Genes Encoding Green Fluorescent Protein and mCherry, Which Exhibit Bright Fluorescence

2011 ◽  
Vol 77 (9) ◽  
pp. 2887-2897 ◽  
Author(s):  
Michaela Leroch ◽  
Dennis Mernke ◽  
Dieter Koppenhoefer ◽  
Prisca Schneider ◽  
Andreas Mosbach ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Botrytis Cinerea ◽  
Fluorescent Protein ◽  
Gc Content ◽  
Gray Mold ◽  
Mrna Levels ◽  
Content Type ◽  
Mold Fungus ◽  
Encoding Gene ◽  
Green Fluorescent

ABSTRACTThe green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungusBotrytis cinereabecause of low fluorescence intensity. The codon usage ofB. cinereagenes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of theB. cinereaenhanced GFP (eGFP)-encoding gene (Bcgfp) for improved expression inB. cinerea. Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bcgfpwas used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bcgfpfusions for quantitative evaluation of various toxic compounds as inducers of theatrBgene encoding an ABC-type drug efflux transporter ofB. cinerea. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence inB. cinerea.

scholarly journals Novel Hypovirulence-Associated RNA Mycovirus in the Plant-Pathogenic Fungus Botrytis cinerea: Molecular and Biological Characterization

2015 ◽  
Vol 81 (7) ◽  
pp. 2299-2310 ◽  
Author(s):  
Lin Yu ◽  
Wen Sang ◽  
Ming-De Wu ◽  
Jing Zhang ◽  
Long Yang ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Fluorescent Protein ◽  
Rna Virus ◽  
Pathogenic Fungus ◽  
Ornamental Plants ◽  
Gray Mold ◽  
Open Reading Frames ◽  
Content Type ◽  
Molecular Features ◽  
Green Fluorescent

ABSTRACTBotrytis cinereais a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 ofB. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A −1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The −1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the constructdsRed-KNOT-eGFPinEscherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to otherB. cinereastrains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence inB. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1.

scholarly journals Gray Mold Populations in German Strawberry Fields Are Resistant to Multiple Fungicides and Dominated by a Novel Clade Closely Related to Botrytis cinerea

2012 ◽  
Vol 79 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Michaela Leroch ◽  
Cecilia Plesken ◽  
Roland W. S. Weber ◽  
Frank Kauff ◽  
Gabriel Scalliet ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Host Preference ◽  
Gray Mold ◽  
Vegetable Production ◽  
Multiple Gene ◽  
Content Type ◽  
Chemical Treatments ◽  
Mold Fungus ◽  
Encoding Gene ◽  
Specific Species

ABSTRACT The gray mold fungus Botrytis cinerea is a major threat to fruit and vegetable production. Strawberry fields usually receive several fungicide treatments against Botrytis per season. Gray mold isolates from several German strawberry-growing regions were analyzed to determine their sensitivity against botryticides. Fungicide resistance was commonly observed, with many isolates possessing resistance to multiple (up to six) fungicides. A stronger variant of the previously described multidrug resistance (MDR) phenotype MDR1, called MDR1h, was found to be widely distributed, conferring increased partial resistance to two important botryticides, cyprodinil and fludioxonil. A 3-bp deletion mutation in a transcription factor-encoding gene, mrr1 , was found to be correlated with MDR1h. All MDR1h isolates and the majority of isolates with resistance to multiple fungicides were found to be genetically distinct. Multiple-gene sequencing confirmed that they belong to a novel clade, called Botrytis group S, which is closely related to B. cinerea and the host-specific species B. fabae . Isolates of Botrytis group S genotypes were found to be widespread in all German strawberry-growing regions but almost absent from vineyards. Our data indicate a clear subdivision of gray mold populations, which are differentially distributed according to their host preference and adaptation to chemical treatments.

scholarly journals The Autophagy Gene BcATG8 Regulates the Vegetative Differentiation and Pathogenicity of Botrytis cinerea

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Weichao Ren ◽  
Na Liu ◽  
Chengwei Sang ◽  
Dongya Shi ◽  
Mingguo Zhou ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Plant Pathogen ◽  
Fluorescent Protein ◽  
Degradation Process ◽  
Gray Mold ◽  
Broad Host Range ◽  
Physical Interaction ◽  
Content Type ◽  
Autophagy Gene ◽  
Mold Fungus

ABSTRACT Autophagy is a conserved degradation process that maintains intracellular homeostasis to ensure normal cell differentiation and development in eukaryotes. ATG8 is one of the key molecular components of the autophagy pathway. In this study, we identified and characterized BcATG8 , a homologue of Saccharomyces cerevisiae (yeast) ATG8 in the necrotrophic plant pathogen Botrytis cinerea . Yeast complementation experiments demonstrated that BcATG8 can functionally complement the defects of the yeast ATG8 null mutant. Direct physical interaction between BcAtg8 and BcAtg4 was detected in the yeast two-hybrid system. Subcellular localization assays showed that green fluorescent protein-tagged BcAtg8 (GFP-BcAtg8) localized in the cytoplasm as preautophagosomal structures (PAS) under general conditions but mainly accumulated in the lumen of vacuoles in the case of autophagy induction. Deletion of BcATG8 (Δ BcAtg8 mutant) blocked autophagy and significantly impaired mycelial growth, conidiation, sclerotial formation, and virulence. In addition, the conidia of the Δ BcAtg8 mutant contained fewer lipid droplets (LDs), and quantitative real-time PCR (qRT-PCR) assays revealed that the basal expression levels of the LD metabolism-related genes in the mutant were significantly different from those in the wild-type (WT) strain. All of these phenotypic defects were restored by gene complementation. These results indicate that BcATG8 is essential for autophagy to regulate fungal development, pathogenesis, and lipid metabolism in B. cinerea . IMPORTANCE The gray mold fungus Botrytis cinerea is an economically important plant pathogen with a broad host range. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. Exploring the fundamental biology of B. cinerea can provide the theoretical basis for sustainable and long-term disease management. Autophagy is an intracellular process for degradation and recycling of cytosolic materials in eukaryotes and is now known to be vital for fungal life. Here, we report studies of the biological role of the autophagy gene BcATG8 in B. cinerea . The results suggest that autophagy plays a crucial role in vegetative differentiation and virulence of B. cinerea .

scholarly journals Genetic Tools To Enhance the Study of Gene Function and Regulation in Staphylococcus aureus

2013 ◽  
Vol 79 (7) ◽  
pp. 2218-2224 ◽  
Author(s):  
Jeffrey L. Bose ◽  
Paul D. Fey ◽  
Kenneth W. Bayles
Keyword(s):  
Staphylococcus Aureus ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Exchange System ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Genetic Tools ◽  
Allelic Exchange ◽  
Green Fluorescent

ABSTRACTThebursa aurealistransposon has been used to create transposon insertion libraries ofBacillus anthracisandStaphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessentialS. aureusgenes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study ofS. aureus.

Relative Strengths and Regulation of Different Promoter-Associated Sequences for Various blaSHV Genes and Their Relationships to β-Lactam Resistance

2017 ◽  
Vol 27 (2) ◽  
pp. 91-101 ◽  
Author(s):  
Yao Zhai ◽  
Zhao Zhang ◽  
Zhanwei Wang ◽  
Yusheng Chen ◽  
Qi Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Mrna Expression ◽  
Inhibitory Concentration ◽  
Fluorescent Protein ◽  
Mrna Levels ◽  
Associated Sequences ◽  
Regulation Function ◽  
Green Fluorescent ◽  
Mrna Expression Levels

<b><i>Aims:</i></b> This work investigated the relative strengths of different <i>bla</i><sub>SHV</sub> promoter-associated sequences and their regulation function in <i>bla</i><sub>SHV</sub> expression and β-lactam resistance. <b><i>Methods:</i></b> Recombinant plasmids with the promoter-associated sequences (P-W, P-S, P-IS, and P-WPD), <i>tac</i> promoter, and combined fragments of promoter and <i>bla</i><sub>SHV</sub> were separately constructed and transformed into <i>Escherichia coli</i> DH5α. The relative strengths of the promoters indicated by the intensities of green fluorescent protein and the mRNA expression levels of <i>bla</i><sub>SHV</sub> were compared. The minimum inhibitory concentration and extended spectrum β-lactamase phenotypes were evaluated. <b><i>Results:</i></b> The relative strengths were ranked as P-<i>tac</i> > P-WPD > P-IS > P-S > P-W. The mRNA expression and β-lactam resistance levels of the different promoter-associated sequence groups were generally consistent with the strength rank, but the extent of <i>gfp</i> and <i>bla</i><sub>SHV</sub> mRNA levels varied significantly in each group. The β-lactam resistance levels were inconsistent with the strength rank in certain <i>bla</i><sub>SHV</sub> groups. In relation to the different promoter-associated sequences,<i> bla</i><sub>SHV-ESBLs</sub> displayed significantly different change modes of β-lactam resistance compared with <i>bla</i><sub>SHV-non-ESBLs</sub>. <b><i>Conclusion:</i></b> The mRNA expression and β-lactam resistance of the <i>bla</i><sub>SHV</sub> showed consistencies and inconsistencies with the strengths of the promoter-associated sequences. The mechanisms accounting for these discrepancies need further investigation.

scholarly journals Spread from the Sink to the Patient: In Situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli To Model Bacterial Dispersion from Hand-Washing Sink-Trap Reservoirs

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Shireen Kotay ◽  
Weidong Chai ◽  
William Guilford ◽  
Katie Barry ◽  
Amy J. Mathers
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Hand Washing ◽  
Resistant Bacteria ◽  
Droplet Dispersion ◽  
Content Type ◽  
E Coli ◽  
Mode Of Transmission ◽  
Green Fluorescent

ABSTRACT There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients.

scholarly journals PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

2013 ◽  
Vol 80 (4) ◽  
pp. 1477-1481 ◽  
Author(s):  
Karina Klevanskaa ◽  
Nadja Bier ◽  
Kerstin Stingl ◽  
Eckhard Strauch ◽  
Stefan Hertwig
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent Protein ◽  
Vibrio Vulnificus ◽  
Shuttle Vector ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent

ABSTRACTAn efficient electroporation procedure forVibrio vulnificuswas designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 106transformants per μg DNA were achieved. The vector stably replicated in bothV. vulnificusandEscherichia coliand was also successfully introduced intoVibrio parahaemolyticusandVibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and thevvhBAhemolysin operon were inserted into the vector and functionally expressed inVibrioandE. coli.

scholarly journals The Genetic Transformation ofChlamydia pneumoniae

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Kensuke Shima ◽  
Maximilian Wanker ◽  
Rachel J. Skilton ◽  
Lesley T. Cutcliffe ◽  
Christiane Schnee ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
Shuttle Vector ◽  
Vector System ◽  
Species Barrier ◽  
Content Type ◽  
Green Fluorescent ◽  
Pathogenic Gene ◽  
Plasmid Backbone

ABSTRACTWe demonstrate the genetic transformation ofChlamydia pneumoniaeusing a plasmid shuttle vector system which generates stable transformants. The equineC. pneumoniaeN16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearingC. pneumoniaetransformants. Using the pRSGFPCAT-Cpn plasmid construct, expression of RSGFP in koala isolateC. pneumoniaeLPCoLN was demonstrated. Furthermore, we discovered that the human cardiovascular isolateC. pneumoniaeCV-6 and the human community-acquired pneumonia-associatedC. pneumoniaeIOL-207 could also be transformed with pRSGFPCAT-Cpn. In previous studies, it was shown thatChlamydiaspp. cannot be transformed when the plasmid shuttle vector is constructed from a different plasmid backbone to the homologous species. Accordingly, we confirmed that pRSGFPCAT-Cpn could not cross the species barrier in plasmid-bearing and plasmid-freeC. trachomatis,C. muridarum,C. caviae,C. pecorum, andC. abortus. However, contrary to our expectation, pRSGFPCAT-Cpn did transformC. felis. Furthermore, pRSGFPCAT-Cpn did not recombine with the wild-type plasmid ofC. felis. Taken together, we provide for the first time an easy-to-handle transformation protocol forC. pneumoniaethat results in stable transformants. In addition, the vector can cross the species barrier toC. felis, indicating the potential of horizontal pathogenic gene transfer via a plasmid.IMPORTANCEThe absence of tools for the genetic manipulation ofC. pneumoniaehas hampered research into all aspects of its biology. In this study, we established a novel reproducible method forC. pneumoniaetransformation based on a plasmid shuttle vector system. We constructed aC. pneumoniaeplasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase inC. pneumoniae.C. pneumoniaetransformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation inC. pneumoniaeusing pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions inC. pneumoniaebiology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species.

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