Regulación del ciclo celular durante el proceso infectivo de "Ustilago maydis"

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 17-12-200

Programmed cell cycle arrest is required for infection of corn plants by the fungus Ustilago maydis

Ustilago maydis is a plant pathogen that requires a specific structure called infective filament to penetrate the plant tissue. Although able to grow, this filament is cell cycle arrested on the plant surface. This cell cycle arrest is released once the filament penetrates the plant tissue.The reasons and mechanisms for this cell cycle arrest are unknown. Here, we have tried to address these questions. We reached three conclusions from our studies. First, the observed cell cycle arrest is the result of the cooperation of at least two distinct mechanisms: one involving the activation of the DNA damage response (DDR) cascade; and the other relying on the transcriptional downregulation of Hsl1, a kinase that modulates the G2/M transition. Second, a sustained cell cycle arrest during the infective filament step is necessary for thevirulence in U. maydis, as a strain unable to arrest the cell cycle was severely impaired in its ability to infect corn plants. Third, production of the appressorium, a structure required for plant penetration, is incompatible with an active cell cycle. The inability to infect plants bystrains defective in cell cycle arrest seems to be caused by their failure to induce the appressorium formation process. In summary, our findings uncover genetic circuits to arrest the cell cycle during the growth of this fungus on the plant surface, thus allowing the penetration into plant tissue.Fil: Castanheira, Sónia. Consejo Superior de Investigaciones Científicas. Instituto de Biología Funcional y Genómica; EspañaFil: Mielnichuk, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. Consejo Superior de Investigaciones Científicas. Instituto de Biología Funcional y Genómica; EspañaFil: Pérez Martín, José. Consejo Superior de Investigaciones Científicas. Instituto de Biología Funcional y Genómica; Españ

Xanthan pyruvilation is essential for the virulence of Xanthomonas campestris pv. campestris

Xanthan, the main exopolysaccharide (EPS) synthesized by Xanthomonas spp., contributes to bacterial stress tolerance and enhances attachment to plant surfaces by helping in biofilm formation. Therefore, xanthan is essential for successful colonization and growth in planta and has also been proposed to be involved in the promotion of pathogenesis by calcium ion chelation and, hence, in the suppression of the plant defense responses in which this cation acts as a signal. The aim of this work was to study the relationship between xanthan structure and its role as a virulence factor. We analyzed four Xanthomonas campestris pv. campestris mutants that synthesize structural variants of xanthan. We found that the lack of acetyl groups that decorate the internal mannose residues, ketal-pyruvate groups, and external mannose residues affects bacterial adhesion and biofilm architecture. In addition, the mutants that synthesized EPS without pyruvilation or without the external mannose residues did not develop disease symptoms in Arabidopsis thaliana. We also observed that the presence of the external mannose residues and, hence, pyruvilation is required for xanthan to suppress callose deposition as well as to interfere with stomatal defense. In conclusion, pyruvilation of xanthan seems to be essential for Xanthomonas campestris pv. campestris virulence.Fil: Bianco, María Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología ; ArgentinaFil: Toum, Laila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología ; ArgentinaFil: Yaryura, Pablo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; ArgentinaFil: Mielnichuk, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología ; ArgentinaFil: Gudesblat, Gustavo Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Roeschlin, Roxana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Marano, María Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ielpi, Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Vojnov, Adrián Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología ; Argentin

14-3-3 regulates the G2/M transition in the basidiomycete Ustilago maydis

14-3-3 proteins are a family of highly conserved polypeptides that function as small adaptors that facilitate a diverse array of cellular processes by binding phosphorylated target proteins. One of these processes is the regulation of the cell cycle. Here we characterized the role of Bmh1, a 14-3-3 protein, in the cell cycle regulation of the fungus Ustilago maydis. We found that this protein is essential in U. maydis and that it has roles during the G2/M transition in this organism. The function of 14-3-3 in U. maydis seems to mirror the proposed role for this protein during Schizosaccharomyces pombe cell cycle regulation. We provided evidence that in U. maydis 14-3-3 protein binds to the mitotic regulator Cdc25. Comparison of the roles of 14-3-3 during cell cycle regulation in other fungal system let us to discuss the connections between morphogenesis, cell cycle regulation and the evolutionary role of 14-3-3 proteins in fungi.This work was supported by grants from Spanish government (BIO2005-02998) and EU (MRTN-CT-2005-019277). NM was a recipient of I3P contract from CSIC.Peer reviewe

A role for the DNA-damage checkpoint kinase Chk1 in the virulence program of the fungus Ustilago maydis

During induction of the virulence program in the phytopathogenic fungus Ustilago maydis, the cell cycle is arrested on the plant surface and it is not resumed until the fungus enters the plant. The mechanism of this cell cycle arrest is unknown, but it is thought that it is necessary for the correct implementation of the virulence program. Here, we show that this arrest takes place in the G2 phase, as a result of an increase in the inhibitory phosphorylation of the catalytic subunit of the mitotic cyclin-dependent kinase Cdk1. Sequestration in the cytoplasm of the Cdc25 phosphatase seems to be one of the reasons for the increase in inhibitory phosphorylation. Strikingly, we also report the DNA-damage checkpoint kinase Chk1 appears to be involved in this process. Our results support the emerging idea that checkpoint kinases have roles other than in the DNA-damage response, by virtue of their ability to interact with the cell cycle machinery.N.M. was supported by JAE. This work was supported by a Grant from the Spanish Government (BIO2008-04054)

Biocontrol of tomato bacterial spot by novel Bacillus and Pseudomonas strains

Bacterial spot is a disease that affects tomato worldwide reducing its yield and quality. It is caused by different Xanthomonas spp., among which is Xanthomonas vesicatoria. Copper-based bactericides are generally used to control this disease, although nowadays sustainable strategies are being searched to efficiently replace their use. Our aim was to select native bacteria from tomato rhizosphere with biocontrol properties against X. vesicatoria. We selected, characterized, and identified three novel strains, two closely related to Bacillus velezensis (VMA11p and VM05) and one closely related to Pseudomonas soli (VMAP1), that in vitro antagonized X. vesicatoria. We evaluated the efficacy of the three rhizobacteria and their cell-free supernatants to control bacterial spot using the model tomato-X. vesicatoria in plants grown in pots, in greenhouse conditions. Bacterial suspensions of VMA11p and VMAP1, applied to the soil by irrigation, significantly (P < 0.05) reduced bacterial spot severity by 53.9% and 44.2%, respectively. Nevertheless, the most effective strategy to control bacterial spot was achieved using the cell-free supernatant produced by VMA11p, VM05 or VMAP1 applied as foliar spray, which significantly (P < 0.05) reduced the severity of the disease by 98.5%, 94.2% and 75.2%, respectively. None of the treatments reduced the growth of tomato plants. Our results suggest that the use of these novel strains of Bacillus and Pseudomonas and/or their metabolic products could be used for the development of biocontrol strategies for the management of bacterial spot in tomato.Fil: Felipe, Verónica. Universidad Nacional de Villa María. Instituto Académico Pedagógico de Ciencias Básicas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; ArgentinaFil: Bianco, María Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Terrestre, M.. Universidad Nacional de Villa María. Instituto Académico Pedagógico de Ciencias Básicas y Aplicadas; ArgentinaFil: Mielnichuk, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Romero, A. M.. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Vegetal. Cátedra de Fitopatología; ArgentinaFil: Yaryura, Pablo Marcelo. Universidad Nacional de Villa María. Instituto Académico Pedagógico de Ciencias Básicas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentin

Virulence factors analysis of native isolates of Xanthomonas albilineans and Xanthomonas sacchari from Tucumán, Argentina, reveals differences in pathogenic strategies

Xanthomonas albilineans (Xa) and X. sacchari (Xs) are both sugarcane pathogens. Xa is the causal agent of leaf scald disease, but there is limited information about the pathogenicity of Xs. The aim of this work was to study virulence factors of native strains of Xa (Xa32, Xa33, and XaM6) and Xs (Xs14 and Xs15) previously isolated from sugarcane with leaf scald symptoms, to gain insight into the biology of each microorganism. We analysed epiphytic survival, sensitivity to oxidative stress, extracellular degradative enzymes, cell motilities, exopolysaccharide (EPS) characteristics, cell adhesion, biofilm development, and control of stomatal regulation of the five strains. We observed that each species presented similar phenotypes for every factor analysed. Xa strains appeared to be more sensitive to oxidative stress and presented lower epiphytic survival than Xs. All strains presented endoglucanase activity; however, we could only detect protease and amylase activities in Xs strains. Swimming and sliding were higher in Xs, but twitching was variable among species. We also observed that only Xs strains produced a xanthan-like EPS, presented a strong cell adhesion, and structured biofilm. We detected some intraspecific variations showing that higher amounts of EPS produced by Xs14 correlated with its higher sliding motility and its homogenous and more adhesive biofilm. In addition, EPSs of Xs14 and Xs15 presented differences in strand height and acetyl percentage. Finally, we found that strains of both species were able to interfere with stomatal aperture mechanism. All these differences could influence the colonization strategies and/or disease progression in each species.Fil: Mielnichuk, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Bianco, María Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Yaryura, Pablo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; ArgentinaFil: Bertani, Romina Priscila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Toum, Laila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Daglio, Yasmin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Colonnella, Maria Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Lizarraga, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Castagnaro, Atilio Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Vojnov, Adrián Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentin

Molecular variability and genetic relationship among Brazilian strains of the sugarcane smut fungus

Sporisorium scitamineum is the fungus that causes sugarcane smut disease. Despite of the importance of sugarcane for Brazilian agribusiness and the persistence of the pathogen in most cropping areas, genetic variation studies are still missing for Brazilian isolates. In this study, sets of isolates were analyzed using two molecular markers (AFLP and telRFLP) and ITS sequencing. Twenty-two whips were collected from symptomatic plants in cultivated sugarcane fields of Brazil. A total of 41 haploid strains of compatible mating types were selected from individual teliospores and used for molecular genetic analyses. telRFLP and ITS analyses were expanded to six Argentine isolates, where the sugarcane smut was first recorded in America. Genetic relationship among strains suggests the human-mediated dispersal of S. scitamineum within the Brazilian territory and between the two neighboring countries. Two genetically distinct groups were defined by the combined analysis of AFLP and telRFLP. The opposite mating-type strains derived from single teliospores were clustered together into these main groups, but had not always identical haplotypes. telRFLP markers analyzed over two generations of selfing and controlled outcrossing confirmed the potential for emergence of new variants and occurrence of recombination, which are relevant events for evolution of virulence and environmental adaptation.Fil: Benevenuto, Juliana. Universidade de Sao Paulo; BrasilFil: Longatto, Daniel P.. Universidade de Sao Paulo; BrasilFil: Reis, Gislaine V.. Universidade de Sao Paulo; BrasilFil: Mielnichuk, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Palhares, Alessandra C.. Universidade de Sao Paulo; BrasilFil: Carvalho, Giselle. Universidade de Sao Paulo; BrasilFil: Saito, Suzane. Universidade de Sao Paulo; BrasilFil: Quecine, Maria C.. Universidade de Sao Paulo; BrasilFil: Sanguino, Alvaro. Agencia Paulista de Tecnologia Dos Agronegocios; BrasilFil: Vieira, Maria Lucia C.. Universidade de Sao Paulo; BrasilFil: Camargo, Luis Eduardo A.. Universidade de Sao Paulo; BrasilFil: Creste, Silvana. Agencia Paulista de Tecnologia Dos Agronegocios; BrasilFil: Monteiro-Vitorello, Claudia B.. Universidade de Sao Paulo; Brasi