scholarly journals Cloning, Expression, and Role in Pathogenicity of pg1 Encoding the Major Extracellular Endopolygalacturonase of the Vascular Wilt Pathogen Fusarium oxysporum

1998 ◽  
Vol 11 (2) ◽  
pp. 91-98 ◽  
Author(s):  
Antonio Di Pietro ◽  
M. Isabel G. Roncero
Keyword(s):  
Molecular Mass ◽  
Fusarium Oxysporum ◽  
Galacturonic Acid ◽  
Vascular Wilt ◽  
Wild Type ◽  
Citrus Pectin ◽  
Calculated Molecular Mass ◽  
Type Isolate ◽  
Cloned Gene

pg1 encoding the major in vitro extracellular endopolygalacturonase of the tomato vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici was cloned and sequenced. The deduced mature protein had a calculated molecular mass of 35.5 kDa and a pI of 6.2, and showed significant similarity with other fungal endoPGs. pg1 mRNA was induced in vitro by citrus pectin, tomato vascular tissue, 0.1% D-galacturonic acid, and polygalacturonic acid, and repressed by 1% D-galacturonic acid and 1% glucose. Reverse transcription-polymerase chain reaction revealed pg1 expression in roots and lower stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Three naturally occurring F. oxysporum f. sp. melonis isolates deficient in PG1 were transformed with the cloned gene. The PG1 enzyme secreted by the transformants had the same molecular mass, pI, and glycosylation pattern as those of the donor isolate. Polygalacturonase activity in cultures of transformants grown in vitro on citrus pectin and on melon plants, but not on glucose, increased 10- to 20-fold, compared with the PG1-deficient wild-type isolate, whereas mycelial dry weight increased two- to threefold. Transformants exhibited the same degree of virulence toward susceptible muskmelon cultivars as the wild-type isolate and were avirulent on a resistant cultivar.

scholarly journals Recovery of Fusarium oxysporum Fo47 Mutants Affected in Their Biocontrol Activity After Transposition of the Fot1 Element

2002 ◽  
Vol 92 (9) ◽  
pp. 936-945 ◽  
Author(s):  
Sophie Trouvelot ◽  
Chantal Olivain ◽  
Ghislaine Recorbet ◽  
Quirico Migheli ◽  
Claude Alabouvette
Keyword(s):  
Fusarium Oxysporum ◽  
Insertional Mutagenesis ◽  
Wild Type Strain ◽  
Growth Habit ◽  
Antagonistic Activity ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Biocontrol Activity

To investigate the biocontrol mechanisms by which the antagonistic Fusarium oxysporum strain Fo47 is active against Fusarium wilt, a Fot1 transposon-mediated insertional mutagenesis approach was adopted to generate mutants affected in their antagonistic activity. Ninety strains in which an active Fot1 copy had transposed were identified with a phenotypic assay for excision and tested for their biocontrol activity against F. oxysporum f. sp. lini on flax in greenhouse experiments. Sixteen strains were affected in their capacity to protect flax plants, either positively (more antagonistic than Fo47) or negatively (less antagonistic). The molecular characterization of these mutants confirms the excision of Fot1 and its reinsertion in most of the cases. Moreover, we demonstrate that other transposable elements such as Fot2, impala, and Hop have no transposition activity in the mutant genomes. The phenotypic characterization of these mutants shows that they are affected neither in their in vitro growth habit nor in their competitiveness in soil compared with wild-type strain Fo47. These results show that mutants are not impaired in their saprophytic phase and suggest that the altered biocontrol phenotype should likely be expressed during the interaction with the host plant.

scholarly journals Molecular Characterization of Saccharomyces cerevisiae TFIID

2002 ◽  
Vol 22 (16) ◽  
pp. 6000-6013 ◽  
Author(s):  
Steven L. Sanders ◽  
Krassimira A. Garbett ◽  
P. Anthony Weil
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Molecular Mass ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Calculated Molecular Mass ◽  
General Transcription Factor

ABSTRACT We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.

scholarly journals FoSTUA, Encoding a Basic Helix-Loop-Helix Protein, Differentially Regulates Development of Three Kinds of Asexual Spores, Macroconidia, Microconidia, and Chlamydospores, in the Fungal Plant Pathogen Fusarium oxysporum

2004 ◽  
Vol 3 (6) ◽  
pp. 1412-1422 ◽  
Author(s):  
Toshiaki Ohara ◽  
Takashi Tsuge
Keyword(s):  
Fusarium Oxysporum ◽  
Fluorescent Protein ◽  
Negative Regulator ◽  
Vascular Wilt ◽  
Wild Type ◽  
Low Frequencies ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Chlamydospore Formation ◽  
Green Fluorescent

ABSTRACT The soil-borne fungus Fusarium oxysporum causes vascular wilt of a wide variety of plant species. F. oxysporum produces three kinds of asexual spores, macroconidia, microconidia, and chlamydospores. Falcate macroconidia are formed generally from terminal phialides on conidiophores and rarely from intercalary phialides on hyphae. Ellipsoidal microconidia are formed from intercalary phialides on hyphae. Globose chlamydospores with thick walls are developed by the modification of hyphal and conidial cells. Here we describe FoSTUA of F. oxysporum, which differentially regulates the development of macroconidia, microconidia, and chlamydospores. FoSTUA encodes a basic helix-loop-helix protein with similarity to Aspergillus nidulans StuA, which has been identified as a transcriptional regulator controlling conidiation. Nuclear localization of FoStuA was verified by using strains expressing FoStuA-green fluorescent protein fusions. The FoSTUA-targeted mutants exhibited normal microconidium formation in cultures. However, the mutants lacked conidiophores and produced macroconidia at low frequencies only from intercalary phialides. Thus, FoSTUA appears to be necessary to induce conidiophore differentiation. In contrast, chlamydospore formation was dramatically promoted in the mutants. These data demonstrate that FoStuA is a positive regulator and a negative regulator for the development of macroconidia and chlamydospores, respectively, and is dispensable for microconidium formation in cultures. The disease-causing ability of F. oxysporum was not affected by mutations in FoSTUA. However, the mutants produced markedly fewer macroconidia and microconidia in infected plants than the wild type. These results suggest that FoSTUA also has an important role for microconidium formation specifically in infected plants.

scholarly journals Role of SraP, a Serine-Rich Surface Protein of Staphylococcus aureus, in Binding to Human Platelets

2005 ◽  
Vol 73 (4) ◽  
pp. 2273-2280 ◽  
Author(s):  
Ian R. Siboo ◽  
Henry F. Chambers ◽  
Paul M. Sullam
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Molecular Mass ◽  
Signal Sequence ◽  
Rabbit Model ◽  
Human Platelets ◽  
Ligand Interaction ◽  
Calculated Molecular Mass ◽  
Platelet Binding

ABSTRACT The binding of bacteria to platelets is a postulated central event in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus gordonii is mediated in large part by GspB, a high-molecular-mass cell wall glycoprotein. Although Staphylococcus aureus has a GspB homolog (SraP), little is known about its function. SraP has a calculated molecular mass of 227 kDa and, like GspB, is predicted to contain an atypical N-terminal signal sequence, two serine-rich repeat regions (srr1 and srr2) separated by a nonrepeat region, and a C-terminal cell wall anchoring motif (LPDTG). To assess whether SraP contributes to platelet binding, we compared the binding to human platelets of S. aureus strain ISP479C and of an isogenic variant (strain PS767) in which sraP had been disrupted by allelic replacement. Platelet binding in vitro by PS767 was 47% ± 17% (mean ± standard deviation) lower than that of ISP479C (P < 0.001). In addition, a recombinant fragment of SraP containing srr1 and the nonrepeat region was found to bind platelets directly. Binding was saturable, suggesting a receptor-ligand interaction. When tested in a rabbit model of endocarditis, in which each animal was simultaneously infected with ISP479C and PS767 at a ratio of approximately 1:1, the titers of the mutant strain within vegetations were significantly lower than those of the parent strain at 1 and 24 h postinfection. These results indicate that SraP can mediate the direct binding of S. aureus to platelets and that the platelet-binding domain of this glycoprotein is located within its N-terminal region. Moreover, the expression of SraP appears to be a virulence determinant in endovascular infection.

scholarly journals Isolation of Nonpathogenic Mutants of Fusarium oxysporum f. sp. melonis for Biological Control of Fusarium Wilt in Cucurbits

2002 ◽  
Vol 92 (2) ◽  
pp. 164-168 ◽  
Author(s):  
Stanley Freeman ◽  
Aida Zveibil ◽  
Haim Vintal ◽  
Marcel Maymon
Keyword(s):  
Biological Control ◽  
Fusarium Oxysporum ◽  
Fungal Pathogens ◽  
Conidial Suspension ◽  
Wild Type ◽  
Seedling Mortality ◽  
Type Isolate ◽  
Novel Approach ◽  
Mutant Strains ◽  
Race 2

Two nonpathogenic mutant strains 4/4 and 15/15 of Fusarium oxysporum f. sp. melonis (race 1,2) were isolated by a continuous dipinoculation technique following UV mutagenesis of the virulent wild-type isolate FOM1.2. No disease symptoms or detrimental effects were observed following inoculation of muskmelon seedlings by strain 4/4. In contrast, strain 15/15 caused mortality of susceptible cultivars although to a lesser extent than the wild-type isolate. Strain 4/4 colonized a variety of muskmelon and watermelon cultivars. In muskmelon cv. Ein Dor, seedlings were dipped in a conidial suspension of strain 4/4 and planted in medium amended with the mutant to achieve 100% colonization of roots and between 30 to 70% of the lower stem tissues 7 days after planting. Similar percent colonization of watermelon seedlings by strain 4/4 was recorded. In cross-protection experiments with muskmelon cultivars, significant reduction in seedling mortality was observed between 4/4-colonized FOM1.2. challenged plants compared with that of wild-type challenged plants alone. Similarly, strain 4/4 was able to significantly reduce mortality of watermelon seedlings caused by F. oxysporum f. sp. niveum race 2. This novel approach of generating nonpathogenic mutants for biological control in Fusarium spp. and other fungal pathogens from virulent wild-type isolates may be beneficial for control, because the mutant strains, lacking only in pathogenicity, may compete more efficiently than other biocontrol organisms against the pathogen of origin.

An endophytic chitinase-producing isolate of Actinoplanes missouriensis, with potential for biological control of root rot of lupin caused by Plectosporium tabacinum

2003 ◽  
Vol 51 (3) ◽  
pp. 257 ◽  
Author(s):  
Khaled A. El-Tarabily
Keyword(s):  
Biological Control ◽  
Cell Wall ◽  
Root Rot ◽  
Germ Tube ◽  
First Record ◽  
Plant Roots ◽  
Wild Type ◽  
Type Isolate ◽  
Cell Wall Lysis

Twenty-one streptomycete and 15 non-streptomycete actinomycetes were isolated from surface-disinfested lupin roots and evaluated for their potential to produce chitinase and to inhibit the growth of Plectosporium tabacinum, the causal agent of lupin root rot in Egypt. The most inhibitory isolate was identified as Actinoplanes missouriensis which produced relatively high levels of chitinase and degraded the hyphae of P.�tabacinum in vitro, causing extensive plasmolysis and cell-wall lysis. A crude culture filtrate of A. missouriensis exhibited antifungal activity and significantly (P < 0.05) reduced spore germination and germ-tube growth of the pathogen. The antagonist was recovered from inside the root at all samplings up to 8 weeks after inoculation, indicating that the roots of healthy lupin may be a habitat for the endophyte. A. missouriensis significantly (P < 0.05) reduced the severity of root rot under glasshouse conditions. An endophytic isolate of Actinoplanes italicus incapable of producing chitinase and a mutant strain of A. missouriensis that did not produce detectable levels of chitinase, did not lyse hyphae of P. tabacinum or reduce root rot in the glasshouse experiments, although colonisation of the lupin root by both these isolates was similar to that of the chitinase-producing wild-type isolate of A. missouriensis. This study is the first record of control of a soil-borne plant pathogen by a chitinolytic actinomycete, endophytic in plant roots.

scholarly journals Bombyx mori Nucleopolyhedrovirus Encodes a DNA-Binding Protein Capable of Destabilizing Duplex DNA

1998 ◽  
Vol 72 (4) ◽  
pp. 3107-3116 ◽  
Author(s):  
Victor S. Mikhailov ◽  
Alla L. Mikhailova ◽  
Masashi Iwanaga ◽  
Sumiko Gomi ◽  
Susumu Maeda
Keyword(s):  
Dna Binding ◽  
Molecular Mass ◽  
Bombyx Mori ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Amino Acid Sequences ◽  
Single Stranded Dna ◽  
Calculated Molecular Mass ◽  
Reading Frame

ABSTRACT A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived fromBombyx mori) infected with the B. morinucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no. L33180 ). This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3′→5′ exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.

Effects of light- and altered-cercosporin phenotypes on gene expression in Cercospora kikuchii

1993 ◽  
Vol 39 (1) ◽  
pp. 118-124 ◽  
Author(s):  
J. A. Rollins ◽  
M. Ehrenshaft ◽  
R. G. Upchurch
Keyword(s):  
In Vitro Translation ◽  
Complete Medium ◽  
Wild Type ◽  
In Planta ◽  
Logarithmic Growth Phase ◽  
Two Dimensional Gel Electrophoresis ◽  
Cercospora Kikuchii ◽  
Potato Dextrose Broth ◽  
Type Isolate

Cercospora kikuchii is a fungal pathogen of soybean that produces a photosensitizing and phytotoxic polyketide, cercosporin, in culture and in planta. We have studied the influence of growth stage, light, and growth medium on cercosporin accumulation in a wild-type isolate and three mutant strains with altered toxin phenotypes. After an initial logarithmic growth phase, the wild-type isolate accumulated high levels of cercosporin on either complete medium or potato dextrose broth, but only when cultured in the light. Dark-grown cultures of the wild-type and light-grown cultures of two uv-induced mutant derivatives accumulated 100-fold lower cercosporin levels. A third mutant strain accumulated wild-type cercosporin levels, but only when cultured in the light in potato dextrose broth. Two-dimensional gel electrophoresis of both extracted proteins and in vitro translation products from wild-type cultures revealed the presence of polypeptides and poly A + RNAs whose accumulation was positively regulated by light. Comparison of translated polypeptide patterns from wild-type and mutant cultures also demonstrated differential accumulation of translatable poly A + RNAs in cercosporin-producing and nonproducing cultures.Key words: nonspecific toxin, photo induction, in vitro translation.

STUDIES ON THE WILT OF PEAS CAUSED BY FUSARIUM OXYSPORUM SCHL. F. PISI (LINF.) S. & H., RACE

1963 ◽  
Vol 41 (11) ◽  
pp. 1569-1584 ◽  
Author(s):  
George Fleischmann
Keyword(s):  
Fusarium Oxysporum ◽  
Ultraviolet Irradiation ◽  
Limiting Factor ◽  
Wild Type ◽  
Experimental Conditions ◽  
Parasexual Cycle ◽  
Race 1 ◽  
Substrate Medium ◽  
Physiological Characters

Fusarium oxysporum Schl. f. pisi (Linf.) S. & H., race 1, was found associated with pea wilt in two localities on the Niagara Peninsula. Under experimental conditions, low soil temperature was found to be the primary limiting factor to the development of this disease. Secondary factors governing disease severity included the nature and concentration of pathogenic inoculum, the microbial population of the soil, and the particular host variety infected. Two pathogenic isolates of race 1 could not be distinguished in vitro with respect to morphological and physiological characters examined.A stable cultural variant isolated from a sectored region of a wild-type colony was found to differ from the parental strain in colonial characters, pigmentation, rate of growth and sporulation, pH drifts of the substrate medium incident to the growth of the fungus, sensitivity to the antifungal antibiotic Acti-dione, and inactivation by ultraviolet irradiation. The variant strain was also less virulent on host varieties susceptible to the wild-type race 1 isolate.Auxotrophic mutants were induced by ultraviolet irradiation and were used in the synthesis of heterocaryons. A diploid strain was screened from one such induced heterocaryon. Positive evidence for the heterocaryotic and diploid stages of the parasexual cycle in this pathogen is presented.

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