Biomedical subjects

M I Roncero

Publications and source records attributed to M I Roncero.

17 recordsLinked to original sources

PubMed2001

Molecular characterization of a subtilase from the vascular wilt fungus Fusarium oxysporum.

The gene prt1 was isolated from the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici, whose predicted amino acid sequence shows significant homology with subtilisin-like fungal proteinases. Prt1 is a single-copy gene, and its structure is highly conserved among different formae speciales of F. oxysporum. Prt1 is expressed constitutively at low levels during growth on different carbon and nitrogen sources and strongly induced in medium containing collagen and glucose. As shown by reverse transcription-polymerase chain reaction and fluorescence microscopy of F. oxysporum strains carrying a prt1-promoter-green fluorescent protein fusion, prt1 is expressed at low levels during the entire cycle of infection on tomato plants. F. oxysporum strains transformed with an expression vector containing the prt1 coding region fused to the inducible endopolygalacturonase pg1 gene promoter and grown under promoter-inducing conditions secreted high levels of extracellular subtilase activity that resolved into a single peak of pI 4.0 upon isoelectric focusing. The active fraction produced two clearing bands of 29 and 32 kDa in sodium dodecyl sulfate gels containing gelatin. Targeted inactivation of prt1 in F. oxysporum f. sp. lycopersici had no detectable effect on mycelial growth, sporulation, and pathogenicity on tomato plants.

Amino Acid Sequence↗

PubMed2001

Molecular characterization of an endopolygalacturonase from Fusarium oxysporum expressed during early stages of infection.

The tomato vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici produces an array of pectinolytic enzymes that may contribute to penetration and colonization of the host plant. Here we report the isolation of pg5, encoding a novel extracellular endopolygalacturonase (endoPG) that is highly conserved among different formae speciales of F. oxysporum. The putative mature pg5 product has a calculated molecular mass of 35 kDa and a pI of 8.3 and is more closely related to endoPGs from other fungal plant pathogens than to PG1, the major endoPG of F. oxysporum. Overexpression of pg5 in a bacterial heterologous system produced a 35-kDa protein with endoPG activity. Accumulation of pg5 transcript is induced by citrus pectin and D-galacturonic acid and repressed by glucose. As shown by reverse transcription-PCR, pg5 is expressed by F. oxysporum in tomato roots during the initial stages of infection. Targeted inactivation of pg5 has no detectable effect on virulence toward tomato plants.

Amino Acid Sequence↗

PubMed2001

A MAP kinase of the vascular wilt fungus Fusarium oxysporum is essential for root penetration and pathogenesis.

The soil-borne vascular wilt fungus Fusarium oxysporum infects a wide variety of plant species by directly penetrating roots, invading the cortex and colonizing the vascular tissue. We have identified fmk1, encoding a mitogen-activated protein kinase (MAPK) of F. oxysporum that belongs to the yeast and fungal extracellular signal-regulated kinase (YERK1) subfamily. Targeted mutants of F. oxysporum f. sp. lycopersici carrying an inactivated copy of fmk1 have lost pathogenicity on tomato plants but show normal vegetative growth and conidiation in culture. Colonies of the fmk1 mutants are easily wettable, and hyphae are impaired in breaching the liquid-air interface, suggesting defects in surface hydrophobicity. Fmk1 mutants also show reduced invasive growth on tomato fruit tissue and drastically reduced transcript levels of pl1 encoding the cell wall-degrading enzyme pectate lyase. Conidia of the mutants germinating in the tomato rhizosphere fail to differentiate penetration hyphae, resulting in greatly impaired root attachment. The orthologous MAPK gene Pmk1 from the rice leaf pathogen Magnaporthe grisea complements invasive growth and partially restores surface hydrophobicity, root attachment and pathogenicity in an fmk1 mutant. These results demonstrate that FMK1 controls several key steps in the pathogenesis of F. oxysporum and suggest a fundamentally conserved role for the corresponding MAPK pathway in soil-borne and foliar plant pathogens.

Amino Acid Sequence↗

PubMed2000

Cloning and disruption of pgx4 encoding an in planta expressed exopolygalacturonase from Fusarium oxysporum.

Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato vascular wilt, produces an array of pectinolytic enzymes, including at least two exo-alpha1,4-polygalacturonases (exoPGs). A gene encoding an exoPG, pgx4, was isolated with degenerate polymerase chain reaction primers derived from amino acid sequences conserved in two fungal exoPGs. pgx4 encodes a 454 amino acid polypeptide with nine potential N-glycosylation sites and a putative 21 amino acid N-terminal signal peptide. The deduced mature protein has a calculated molecular mass of 47.9 kDa, a pI of 8.0, and 51 and 49% identity with the exoPGs of Cochliobolus carbonum and Aspergillus tubingensis, respectively. The gene is present in a single copy in different formae speciales of F. oxysporum. Expression of pgx4 was detected during in vitro growth on pectin, polygalacturonic acid, and tomato vascular tissue and in roots and stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Two mutants of F. oxysporum f. sp. lycopersici with a copy of pgx4 inactivated by gene replacement were as virulent on tomato plants as the wild-type strain.

Amino Acid Sequence↗

PubMed2000

Role of cell wall-degrading enzymes in pathogenicity of Fusarium oxysporum.

Fusarium oxysporum invades its host plants through the roots and colonizes the vascular system. It produces a great variety of cell-wall degrading enzymes (CWDE), such as cellulases, xylanases, pectinases and proteases. Our group has purified and characterized an endopolygalacturonase (PG1), two exopolygalacturonases (PG2 and PG3), an endoxylanase (XYL1) and an endo pectatelyase (PL1). We have isolated the following CWDE-encoding genes: pg1, pgx4, pg5, xyl2, xyl3, prt1 and pl1. Gene expression in different culture conditions has been determined by Northern analysis. The occurrence of these genes in different formae speciales has been analyzed by Southern analysis and PCR. All these genes are expressed during different stages of the interaction with the host plant indicating a possible role in pathogenesis. At present, targeted gene disruption is being carried out, in order to determine the role of each gene in the pathogenicity process.

Journal Article↗

PubMed1999

Folyt1, a new member of the hAT family, is active in the genome of the plant pathogen Fusarium oxysporum.

An active transposable element, Folyt1, has been isolated from the tomato pathogen Fusarium oxysporum f. sp. lycopersici as an insertion sequence within the coding region of the nitrate reductase gene (nit 1) in two independent mutants (CO66 and CO108). Folyt1 was 2615 bp in length and contained 9-bp imperfect inverted terminal repeats (ITRs) and 8 bp duplicated at the target site upon insertion. The element contained a long open reading frame interrupted by a single putative intron. The predicted amino acid sequence showed similarity to conserved domains of transposases from hobo, Ac, and Tam3 elements, which belong to the hAT family. The excision frequency of Folyt1 was determined to be less than 10(-5) in both mutants. These events restored the nit 1 wild-type allele without leaving footprints in all the revertants of strain CO66. Nevertheless, some revertants of strain CO108 showed a point mutation footprint at the target sequence. Expression of the Folyt1 transposase was detected by Northern analysis as a 2.1-kb transcript. The element exists in about 10 copies per genome in F. oxysporum f. sp. lycopersici and appears to be widely distributed among different formae speciales of F. oxysporum.

Amino Acid Sequence↗

PubMed1999

Cloning and characterization of pl1 encoding an in planta-secreted pectate lyase of Fusarium oxysporum.

A pectate lyase (PL1) from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici was previously characterized, and evidence was obtained for its production in planta. The gene encoding PL1 was isolated from a genomic library of F. oxysporum f. sp. lycopersici. Pl1 encodes a 240 amino-acid polypeptide with one putative N-glycosylation site and a 15 amino-acid N-terminal signal peptide. PL1 showed 89%, 67%, 55% and 56% identity with the products of the Fusarium solani f.sp. pisi pelA, pelB, pelC and pelD genes, respectively. A single copy of the gene was detected in different formae speciales of F. oxysporum. The pl1 transcript was observed during growth on polygalacturonic acid sodium salt and tomato vascular tissue, but not on pectin or glucose. RT-PCR showed pl1 expression in roots and stems of tomato plants infected by F. oxysporum f.sp. lycopersici.

Amino Acid Sequence↗

PubMed1999

Two xylanase genes of the vascular wilt pathogen Fusarium oxysporum are differentially expressed during infection of tomato plants.

Two genes encoding putative family F xylanases from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici have been cloned and sequenced. The two genes, designated xyl2 and xyl3, encode proteins with calculated molecular masses of 33 and 39.3 kDa and isoelectric points of 8.9 and 6.7, respectively. The predicted amino acid sequences show significant homology to other family F xylanases. XYL3 contains a cellulose-binding domain in its N-terminal region. Southern analysis suggested that xyl2 and xyl3 homologs are also present in other formae speciales of F. oxysporum. Both genes were expressed during growth on oat spelt xylan and tomato vascular tissue in vitro. RT-PCR revealed that xyl3 is expressed in roots and in the lower stems of tomato plants infected by F. oxysporum f.sp. lycopersici throughout the whole disease cycle, whereas xyl2 is only expressed during the final stages of disease.

Amino Acid Sequence↗

PubMed1998

Cloning, expression, and role in pathogenicity of pg1 encoding the major extracellular endopolygalacturonase of the vascular wilt pathogen Fusarium oxysporum.

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 three-fold. Transformants exhibited the same degree of virulence toward susceptible muskmelon cultivars as the wild-type isolate and were avirulent on a resistant cultivar.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of an exo-polygalacturonase from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici.

An exo-polygalacturonase (EC 3.2.1.15) was purified to apparent homogeneity from cultures of Fusarium oxysporum f.sp. lycopersici on synthetic medium supplemented with citrus pectin, using preparative isoelectric focusing. The enzyme, denominated PG2, had an apparent M(r) of 74000 Da upon SDS-PAGE. The pI of the main PG2 isoform was 4.5, and pH and temperature optima were 5.0 and 55 degrees C, respectively. PG2 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by anaysis of degradation products. The enzyme was N-glycosylated. The N-terminal amino acid sequence, L-A-F-N-V-P-S-K-P-P, has no identify to other known polygalacturonases.

Electrophoresis, Polyacrylamide Gel↗

PubMed1996

Stress-induced rearrangement of Fusarium retrotransposon sequences.

Rearrangement of fusarium oxysporum retrotransposon skippy was induced by growth in the presence of potassium chlorate. Three fungal strains, one sensitive to chlorate (Co60) and two resistant to chlorate and deficient for nitrate reductase (Co65 and Co94), were studied by Southern analysis of their genomic DNA. Polymorphism was detected in their hybridization banding pattern, relative to the wild type grown in the absence of chlorate, using various enzymes with or without restriction sites within the retrotransposon. Results were consistent with the assumption that three different events had occurred in strain Co60: genomic amplification of skippy yielding tandem arrays of the element, generation of new skippy sequences, and deletion of skippy sequences. Amplification of Co60 genomic DNA using the polymerase chain reaction and divergent primers derived from the retrotransposon generated a new band, corresponding to one long terminal repeat plus flanking sequences, that was not present in the wild-type strain. Molecular analysis of nitrate reductase-deficient mutants showed that generation and deletion of skippy sequences, but not genomic amplification in tandem repeats, had occurred in their genomes.

Blotting, Southern↗

PubMed1996

A homologous and self-replicating system for efficient transformation of Fusarium oxysporum.

A highly efficient transformation system has been developed for Fusarium oxysporum f. sp. lycopersici based on the complementation of a nitrate-reductase mutant with the homologous nit1 gene and on the presence of ARS and telomeric sequences in the vector. Preliminary transformation experiments with the niaD gene from Aspergillus niger generated self-replicating plasmids within the transformed entity that contained extra-fungal DNA. A fragment of the extra DNA was inserted into pUC19 together with the F. oxysporum nit1 gene, resulting in plasmid pFNit-Lam. This allowed the isolation of a new linear plasmid within self-replicative F. oxysporum transformants (pFNit-Lam-TLam, linear). The circular form of this vector yielded 5600 fungal transformants per microgram of DNA. All of the transformants contained autonomous linear plasmids harboring direct repeats of fungal DNA at both ends. The sequence of the 1.2-kb fragment from F. oxysporum responsible for autonomous replication, and maintenance as linear plasmid molecules, has been determined. Comparison analysis with the ARS from different organisms has shown that this fragment contained the commonly identified ARS consensus sequence, 5'A/TTTTATA/GTTTA/T3' and, in addition to this core, ten copies of the ARS-box, 5'TNTA/GAA3'. Adjacent to this presumed ARS, the telomeric hexanucleotide sequence (TTAGGG)n was present in six tandem copies followed by 18 copies of its complementary sequence.

Base Sequence↗

PubMed1995

Skippy, a retrotransposon from the fungal plant pathogen Fusarium oxysporum.

A retrotransposon from the fungal plant pathogen Fusarium oxysporum f. sp. lycopersici has been isolated and characterized. The element, designated skippy (skp) is 7846 bp in length, flanked by identical long terminal repeats (LTR) of 429 bp showing structural features characteristic of retroviral and retrotransposon LTRs. Target-site duplications of 5 bp were found. Two long overlapping open reading frames (ORF) were identified. The first ORF, 2562 bp in length, shows homology to retroviral gag genes. The second ORF, 3888 bp in length, has homology to the protease, reverse transcriptase. RNase H and integrase domains of retroelement pol genes in that order. Sequence comparisons and the order of the predicted proteins from skippy indicate that the element is closely related to the gypsy family of LTR-retrotransposons. The element is present in similar copy numbers in the two races investigated, although RFLP analysis showed differences in banding patterns. The number of LTR sequences present in the genome is higher than the number of copies of complete elements, indicating excision by homologous recombination between LTR sequences.

Amino Acid Sequence↗

PubMed1991

Transformation of a methionine auxotrophic mutant of Mucor circinelloides by direct cloning of the corresponding wild type gene.

A transformation system has been developed for Mucor circinelloides, by direct cloning of a wild-type methionine gene that complements the auxotrophic mutation. The marker gene isolated was associated with an autonomous replication sequence (ARS) functional in this zygomycete. Southern hybridisation analyses of transformants showed sequence homology both with vector DNA and with Mucor wild-type DNA. The transformation frequency (up to 6000 per microgram DNA) and the mitotic instability of the transformed cells were studied. The hybridisation pattern of undigested DNA from the transformants suggests that the inserts contain a novel autonomous replication element for this filamentous fungus.

Blotting, Southern↗

PubMed1989

Characterization of a leuA gene and an ARS element from Mucor circinelloides.

A 4.4-kb PstI restriction endonuclease fragment of Mucor circinelloides DNA has previously been shown to both complement a leuA- mutation, and to enable the autonomous replication of plasmids within this organism. The complete nucleotide (nt) sequence of this fragment has been determined and an open reading frame of 1935 bp with no introns has been identified, which exhibits significant similarity (75% at the nt level) with 114 bp of the 5' coding region of the Saccharomyces cerevisiae LEU1 gene. Based on this and on the fact that the fragment weakly complements a leu1 auxotroph of S. cerevisiae, we concluded that the Mucor leu gene encodes alpha-isopropylmalate (alpha-IPM) isomerase and designated it leuA+ accordingly. Primer extension analysis of leuA mRNA and Northern-blot hybridization, indicated the leuA transcript to be approx. 2.3 kb in size, with 5'- and 3'-untranslated regions of 16-20 nt and approx. 450 nt, respectively. Specific Mucor ARS sequence(s) were not identified, although the general location of ARS was indicated by subcloning experiments. Nucleotide sequences are present within this region, which show some similarity with the core consensus of the S. cerevisiae ARS; however, any functional homology is doubtful, since insertion of the 4.4-kb PstI fragment into YIp5 did not increase the transformation frequency of S. cerevisiae with such a vector.

Amino Acid Sequence↗

PubMed1984

Mutagenesis in multinucleate cells: the effects of N-methyl-N'-nitro-N-nitrosoguanidine on Phycomyces spores.

Multinucleate cells, such as the spores of the fungus Phycomyces, are unsuitable for the isolation of recessive mutants. Nuclear killing by N-methyl-N'-nitro-N-nitrosoguanidine (henceforth nitrosoguanidine) eliminates all but one of the nuclei in some of the cells and allows the expression of recessive mutations. Even in the best conditions, only about 35% of the survivors have a single functional nucleus. Functionally uninucleate cells can be positively selected. This involves the exposure to nitrosoguanidine of the spores of a heterokaryon and selection for a recessive marker present in a small fraction of its nuclei. The optimal conditions for nitrosoguanidine mutagenesis in Phycomyces differ from those for bacteria and yeast. Buffer composition and pH are less important than in other organisms. Survival is an exponential function and mutation induction a linear function of the dose of the mutagen (concentration X time). Spore germination leads to an immediate increase in the number of gene copies per cell, thus further hindering the expression of recessive mutations; dominant mutations are then nearly always isolated in heterokaryotic form.

Cell Nucleus↗

PubMed1983

Genes controlling xylan utilization by Bacillus subtilis.

Eight mutants of Bacillus subtilis deficient in xylan utilization were isolated and characterized genetically and biochemically. Each mutant was obtained independently after nitrosoguanidine mutagenesis. All of the analyzed mutations were shown to be linked. Reciprocal transformation crosses revealed the existence of two genes controlling xylan utilization which have been designated xynA and xynB. Available data have indicated that these two genes code for two xylan-degrading enzymes existing in the wild-type strains, an extracellular beta-xylanase (xynA) and a cell-associated beta-xylosidase (xynB).

Bacillus subtilis↗