scholarly journals The Fusarium Circinatum Gene Fcrho1, Encoding a Putative Rho1 GTPase, Is Involved in Vegetative Growth but Dispensable for Pathogenic Development

Forests ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 684
Author(s):  
E. Muñoz-Adalia ◽  
M. Cañizares ◽  
Mercedes Fernández ◽  
Julio Diez ◽  
M. García-Pedrajas
Keyword(s):  
Deletion Mutants ◽  
Fusarium Circinatum ◽  
Northern Spain ◽  
Pine Seedlings ◽  
Virulence Assay ◽  
Rate Characteristic ◽  
Hyphal Morphology ◽  
Forest Diseases

Fusarium circinatum is the causal agent of pine pitch canker (PPC), one of the most devastating forest diseases worldwide. This fungus causes severe damping-off in pine seedlings and growth reduction, wilting and the development of cankers in pine forests and plantations. A draft of the complete genome sequence of this phytopathogen was recently made available. This information was used to annotate in silico the gene Fcrho1 as a putative homolog of Rho1 GTPase genes. In this study, we generated Fcrho1 deletion mutants in two F. circinatum wildtype strains isolated from damaged trees in northern Spain. For that, we used a modified version of the OSCAR methodology, an approach not previously used in F. circinatum that allows the generation of deletion constructs in a single cloning step. The conidiation and spore germination of the resulting deletion mutants were not affected, neither the hyphal morphology. However, the mutant strains showed significantly reduced growth in vitro and more foamy macroscopic hyphal morphology than their corresponding ectopic and wildtype strains. Finally, an in vivo virulence assay showed that the reduced in vitro growth rate characteristic to the deletion mutants does not impact their pathogenicity.

scholarly journals A Comparative Study of the Pathogenicity of Fusarium circinatum and other Fusarium Species in Polish Provenances of P. sylvestris L.

Forests ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 560 ◽  
Author(s):  
Kateryna Davydenko ◽  
Justyna Nowakowska ◽  
Tomasz Kaluski ◽  
Magdalena Gawlak ◽  
Katarzyna Sadowska ◽  
...  
Keyword(s):  
Scots Pine ◽  
Fungal Pathogen ◽  
Fusarium Species ◽  
Pitch Canker ◽  
Fusarium Circinatum ◽  
Northern Spain ◽  
Fusarium Spp ◽  
Pine Seedlings ◽  
Unintentional Introduction ◽  
Different Levels

The fungal pathogen Fusarium circinatum is the causal agent of Pine Pitch Canker (PPC), a disease which seriously affects different species of pine in forests and nurseries worldwide. In Europe, the fungus affects pines in northern Spain and Portugal, and it has also been detected in France and Italy. Here, we report the findings of the first trial investigating the susceptibility of Polish provenances of Scots pine, Pinus sylvestris L., to infection by F. circinatum. In a greenhouse experiment, 16 Polish provenances of Scots pine were artificially inoculated with F. circinatum and with six other Fusarium species known to infect pine seedlings in nurseries. All pines proved highly susceptible to PPC and displayed different levels of susceptibility to the other Fusarium spp. tested. The findings obtained indicate the potentially strong threat of establishment of an invasive pathogen such as F. circinatum following unintentional introduction into Poland.

scholarly journals Evidence for Two CRIB Domains in Phospholipase D2 (PLD2) That the Enzyme Uses to Specifically Bind to the Small GTPase Rac2

2011 ◽  
Vol 286 (18) ◽  
pp. 16308-16320 ◽  
Author(s):  
Hong-Juan Peng ◽  
Karen M. Henkels ◽  
Madhu Mahankali ◽  
Mary C. Dinauer ◽  
Julian Gomez-Cambronero
Keyword(s):  
Specific Binding ◽  
Strong Association ◽  
Living Cells ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Deletion Mutants ◽  
Ph Domain ◽  
Phospholipase D2

Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named “CRIB-1” and “CRIB-2” in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent Kd of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a “termination signal” leading to PLD2 inactivation.

scholarly journals Characterization of the interaction domains of Ure2p, a prion-like protein of yeast

1999 ◽  
Vol 338 (2) ◽  
pp. 403-407 ◽  
Author(s):  
Eric FERNANDEZ-BELLOT ◽  
Elisabeth GUILLEMET ◽  
Agnès BAUDIN-BAILLIEU ◽  
Sébastien GAUMER ◽  
Anton A. KOMAR ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Deletion Mutants ◽  
Loss Of Function ◽  
Hybrid Analysis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Interaction Domains ◽  
Two Hybrid

In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic element [URE3] behaves as a prion. A hypothesis has been put forward which states that [URE3] arises spontaneously from its cellular isoform Ure2p (the product of the URE2 gene), and propagates through interactions of the N-terminal domain of the protein, thus leading to its aggregation and loss of function. In the present study, various N- and C-terminal deletion mutants of Ure2p were constructed and their cross-interactions were tested in vitro and in vivo using affinity binding and a two-hybrid analysis. We show that the self-interaction of the protein is mediated by at least two domains, corresponding to the first third of the protein (the so-called prion-forming domain) and the C-terminal catalytic domain.

scholarly journals Intravital Imaging of Candida albicans Identifies Differential In Vitro and In Vivo Filamentation Phenotypes for Transcription Factor Deletion Mutants

mSphere ◽  
2021 ◽  
Author(s):  
Rohan S. Wakade ◽  
Manning Huang ◽  
Aaron P. Mitchell ◽  
Melanie Wellington ◽  
Damian J. Krysan
Keyword(s):  
Candida Albicans ◽  
Fungal Infections ◽  
Deletion Mutants ◽  
Filamentous Form ◽  
Content Type ◽  
Yeast Form ◽  
Common Causes ◽  
Do So

Candida albicans is one of the most common causes of fungal infections in humans. C. albicans undergoes a transition from a round yeast form to a filamentous form during infection, which is critical for its ability to cause disease. Although this transition has been studied in the laboratory for years, methods to do so in an animal model of infection have been limited.

scholarly journals Preliminary assessment of pathogenicity of Fusarium circinatum on germlings of Pinus sylvestris in Ukraine

2019 ◽  
pp. 117-123
Author(s):  
K. V. DAVYDENKO
Keyword(s):  
Pinus Sylvestris ◽  
Pathogenicity Test ◽  
Pitch Canker ◽  
Fusarium Circinatum ◽  
Preliminary Assessment ◽  
Pine Seedlings ◽  
The World ◽  
High Level ◽  
Near Future

Fusarium circinatum is the causal agent of Pine Pitch Canker (PPC) of pines and established in the Iberian Peninsula in Europe only. However, it is hypothesized that its range could expand through the Europe in the near future. The disease is very harmful to forests and nurseries all around the world. Despite the aggressiveness of this fungus, no trials on the susceptibility of Ukrainian Scots pine provenances from Pinus sylvestris to F. circinatum have been performed. The first preliminary pathogenicity test was carried out in vitro and revealed the strong probability of high level of pine seedlings susceptibility to pitch canker.

scholarly journals 4165 Mechanisms of Prophage-Mediated Virulence Driving Community-Acquired MRSA Contagion

2020 ◽  
Vol 4 (s1) ◽  
pp. 11-12
Author(s):  
Robert James Ulrich ◽  
Irnov Irnov ◽  
William Sause ◽  
Magdelena Podkowik ◽  
Victor Torres ◽  
...  
Keyword(s):  
Candidate Genes ◽  
Bacterial Species ◽  
Bacterial Virulence ◽  
Infection Model ◽  
Deletion Mutants ◽  
Toxin Gene ◽  
Healthy Children ◽  
Mosaic Block

OBJECTIVES/GOALS: We recently identified a CA-MRSA strain in Brooklyn, New York (USA300-BKV) causing an outbreak of severe skin infections in predominantly healthy children. The evolution of USA300-BKV included acquisition of a novel prophage, and our objective is to identify the prophage-encoded gene(s) and mechanism responsible for increased bacterial virulence. METHODS/STUDY POPULATION: We deleted candidate genes from a novel mosaic block of phage-encoded genes in USA300-BKV that have been shown to enhance virulence in a murine skin infection model. Deletion mutants and complemented clones will be evaluated in vivo to identify culprit genes and determine the effect of lineage-specific genetic variation on the phenotype. Complementary studies include a comprehensive characterization of phage and bacterial genes expressed during lysogeny in vitro using RNA sequencing (RNA-Seq), and in vivo using a targeted approach focusing on known bacterial virulence and phage lytic pathways as well as candidate genes identified by in vitro studies. RESULTS/ANTICIPATED RESULTS: Comparison of otherwise isogenic lab strains showed that the mosaic block of phage genes present in USA300-BKV enhance skin abscess size in mice, confirming previous results. As this region of the phage, named mΦ11, does not contain known toxin genes, we hypothesize that mΦ11 modulates expression of bacterial host genes to enhance virulence. Thus, transcriptional profiles of CA-MRSA containing mΦ11 and selected deletion mutants are expected to reveal changes in known or novel virulence factors compared to controls. Candidate regulators specific to the mosaic block include an adenine methyltransferase linked to changes in global gene expression of other bacterial species. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results will broaden scientific understanding of phage-bacterial interactions and determine the mechanisms by which phage impact virulence independent from toxin gene carriage. Identification of phage-encoded gene(s) enhancing CA-MRSA contagion will inform surveillance efforts and identify novel therapeutic targets.

Deletion mutants of protective antigen that inhibit anthrax toxin both in vitro and in vivo

2003 ◽  
Vol 307 (3) ◽  
pp. 446-450 ◽  
Author(s):  
Nidhi Ahuja ◽  
Praveen Kumar ◽  
Sheeba Alam ◽  
Megha Gupta ◽  
Rakesh Bhatnagar
Keyword(s):  
Protective Antigen ◽  
Anthrax Toxin ◽  
Deletion Mutants

scholarly journals Invading the Yeast Nucleus: a Nuclear Localization Signal at the C Terminus of Ty1 Integrase Is Required for Transposition In Vivo

1998 ◽  
Vol 18 (2) ◽  
pp. 1115-1124 ◽  
Author(s):  
Margaret A. Kenna ◽  
Carrie Baker Brachmann ◽  
Scott E. Devine ◽  
Jef D. Boeke
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Reverse Transcriptase Activity ◽  
Deletion Mutants ◽  
Amino Acid Residues ◽  
Wild Type ◽  
C Terminus ◽  
Localization Signal

ABSTRACT Retrotransposon Ty1 faces a formidable cell barrier during transposition—the yeast nuclear membrane which remains intact throughout the cell cycle. We investigated the mechanism by which transposition intermediates are transported from the cytoplasm (the presumed site of Ty1 DNA synthesis) to the nucleus, where they are integrated into the genome. Ty1 integrase has a nuclear localization signal (NLS) at its C terminus. Both full-length integrase and a C-terminal fragment localize to the nucleus. C-terminal deletion mutants in Ty1 integrase were used to map the putative NLS to the last 74 amino acid residues of integrase. Mutations in basic segments within this region decreased retrotransposition at least 50-fold in vivo. Furthermore, these mutant integrase proteins failed to localize to the nucleus. Production of virus-like particles, reverse transcriptase activity, and complete in vitro Ty1 integration resembled wild-type levels, consistent with failure of the mutant integrases to enter the nucleus.

scholarly journals DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter.

1982 ◽  
Vol 2 (7) ◽  
pp. 737-751 ◽  
Author(s):  
P Jat ◽  
U Novak ◽  
A Cowie ◽  
C Tyndall ◽  
R Kamen
Keyword(s):  
Dna Sequences ◽  
In Vitro Transcription ◽  
Polyoma Virus ◽  
Tata Box ◽  
Deletion Mutants ◽  
Base Pairs ◽  
Transcription Analysis ◽  
In Vivo Expression

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

scholarly journals Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene.

1984 ◽  
Vol 4 (4) ◽  
pp. 657-665 ◽  
Author(s):  
K J Shaw ◽  
M V Olson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Deletion Mutants ◽  
Flanking Sequence ◽  
Base Pairs ◽  
In Vivo Expression ◽  
Flanking Sequences

Deletion mutations ending in the 5'-flanking sequences of the Saccharomyces cerevisiae SUP4-o gene have been analyzed for their effects on gene expression. This ochre-suppressing tRNATyr gene was cloned into a S. cerevisiae centromeric plasmid, and its level of in vivo expression was monitored by observing the suppressor phenotype of the gene after transformation into S. cerevisiae. A deletion mutant that retains only four base pairs of the 5'-flanking sequence is profoundly deficient in expression; deletion mutants extending to positions -18, -17, -16, or -15 are moderately deficient; deletion mutants extending to positions -36 or -27 are slightly defective; and mutants retaining more than 60 base pairs of the original 5'-flanking DNA are expressed normally. In some cases, the cloning procedure led to the introduction of multiple BamHI linkers at the SUP4-o-vector fusion site, and in one instance, the resulting structure dramatically affects gene function: the presence of three linkers abutting a -18 deletion completely inhibits the in vivo expression of SUP4-o. In contrast, three linkers that abut a -77 deletion have no effect on in vivo expression. The template properties of these plasmids in a homologous in vitro transcription system parallel the levels of in vivo expression, suggesting that the mutations predominantly affect transcription. The data demonstrate that there are significant functional constraints on the 5'-flanking sequences of this RNA polymerase III-transcribed gene. The dramatic effects of the multiple linker insertion at position -18 suggest that there may be extensive melting of the DNA in this region during normal transcription initiation.

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