RESUMEN
Previous studies have demonstrated the essential role of morphogenetic regulation in Fusarium oxysporum pathogenesis, including processes such as cell-wall biogenesis, cell division, and differentiation of infection-like structures. We identified three F. oxysporum genes encoding predicted transcription factors showing significant identities to Magnaporthe oryzae Con7p, Con7-1, plus two identical copies of Con7-2. Targeted deletion of con7-1 produced nonpathogenic mutants with altered morphogenesis, including defects in cell wall structure, polar growth, hyphal branching, and conidiation. By contrast, simultaneous inactivation of both con7-2 copies caused no detectable defects in the resulting mutants. Comparative microarray-based gene expression analysis indicated that Con7-1 modulates the expression of a large number of genes involved in different biological functions, including host-pathogen interactions, morphogenesis and development, signal perception and transduction, transcriptional regulation, and primary and secondary metabolism. Taken together, our results point to Con7-1 as general regulator of morphogenesis and virulence in F. oxysporum.
Asunto(s)
Fusarium/genética , Regulación Fúngica de la Expresión Génica/genética , Enfermedades de las Plantas/microbiología , Solanum lycopersicum/microbiología , Factores de Transcripción/genética , Empalme Alternativo , Animales , Pared Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/crecimiento & desarrollo , Fusarium/patogenicidad , Fusarium/ultraestructura , Perfilación de la Expresión Génica , Glucosa/metabolismo , Interacciones Huésped-Patógeno , Hifa , Larva , Magnaporthe/genética , Mariposas Nocturnas , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Raíces de Plantas/microbiología , Eliminación de Secuencia , Esporas Fúngicas , Factores de Transcripción/metabolismo , VirulenciaRESUMEN
Ca(2+) signaling is an early and necessary event in plant immunity. The tomato (Solanum lycopersicum) kinase Pto triggers localized programmed cell death (PCD) upon recognition of Pseudomonas syringae effectors AvrPto or AvrPtoB. In a virus-induced gene silencing screen in Nicotiana benthamiana, we independently identified two components of a Ca(2+)-signaling system, Cbl10 (for calcineurin B-like protein) and Cipk6 (for calcineurin B-like interacting protein kinase), as their silencing inhibited Pto/AvrPto-elicited PCD. N. benthamiana Cbl10 and Cipk6 are also required for PCD triggered by other plant resistance genes and virus, oomycete, and nematode effectors and for host susceptibility to two P. syringae pathogens. Tomato Cipk6 interacts with Cbl10 and its in vitro kinase activity is enhanced in the presence of Cbl10 and Ca(2+), suggesting that tomato Cbl10 and Cipk6 constitute a Ca(2+)-regulated signaling module. Overexpression of tomato Cipk6 in N. benthamiana leaves causes accumulation of reactive oxygen species (ROS), which requires the respiratory burst homolog RbohB. Tomato Cbl10 and Cipk6 interact with RbohB at the plasma membrane. Finally, Cbl10 and Cipk6 contribute to ROS generated during effector-triggered immunity in the interaction of P. syringae pv tomato DC3000 and N. benthamiana. We identify a role for the Cbl/Cipk signaling module in PCD, establishing a mechanistic link between Ca(2+) and ROS signaling in plant immunity.
Asunto(s)
Inmunidad de la Planta/fisiología , Proteínas de Plantas/metabolismo , Transducción de Señal/fisiología , Solanum lycopersicum/metabolismo , Secuencia de Aminoácidos , Apoptosis/genética , Apoptosis/fisiología , Calcio/metabolismo , Calcio/farmacología , Membrana Celular/metabolismo , Técnicas de Silenciamiento del Gen , Interacciones Huésped-Patógeno , Immunoblotting , Solanum lycopersicum/genética , Solanum lycopersicum/microbiología , Microscopía Confocal , Datos de Secuencia Molecular , Filogenia , Inmunidad de la Planta/efectos de los fármacos , Inmunidad de la Planta/genética , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Unión Proteica/efectos de los fármacos , Pseudomonas syringae/fisiología , Especies Reactivas de Oxígeno/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologíaRESUMEN
With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor ß-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity.
Asunto(s)
Pared Celular/enzimología , Fusarium/enzimología , Fusarium/patogenicidad , Genes Fúngicos/genética , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Azul Alcián , Adhesión Celular/fisiología , Fraccionamiento Celular , Pared Celular/ultraestructura , Clonación Molecular , Biología Computacional , Matriz Extracelular/ultraestructura , Citometría de Flujo , Glicosilación , Funciones de Verosimilitud , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Modelos Genéticos , Mutación/genética , Oligonucleótidos/genética , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Ultracentrifugación , VirulenciaRESUMEN
A mutant of the root pathogen Fusarium oxysporum f. sp. lycopersici, deficient in class V chitin synthase, has been shown previously to be nonvirulent. In this study, we tested the hypothesis that the cause of its avirulence could be the elicitation of the induced plant defence response, leading to the restriction of fungal infection. Co-inoculation of tomato plants with the wild-type strain and the DeltachsV mutant resulted in a significant reduction in symptom development, supporting a protective mechanism exerted by the mutant. The ability of the mutant to penetrate and colonize plant tissues was determined by scanning and transmission electron microscopy, as well as fluorescence microscopy using green fluorescent protein- or cherry fluorescent protein-labelled fungal strains. The extent of wild-type strain colonization in co-inoculated plants decreased steadily throughout the infection process, as shown by the quantification of fungal biomass using real-time polymerase chain reaction. The hypothesis that defence responses are activated by the DeltachsV mutant was confirmed by the analysis of plant pathogenesis-related genes using real-time reverse transcriptase-polymerase chain reaction. Tomato plants inoculated with the DeltachsV mutant showed a three fold increase in endochitinase activity in comparison with wild-type inoculated plants. Taken together, these results suggest that the perturbation of fungal cell wall biosynthesis results in elicitation of the plant defence response during the infection process.
Asunto(s)
Quitina Sintasa/genética , Fusarium/enzimología , Fusarium/genética , Solanum lycopersicum/microbiología , Quitina Sintasa/deficiencia , Quitinasas/genética , Quitinasas/metabolismo , Fusarium/inmunología , Solanum lycopersicum/inmunología , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiologíaRESUMEN
Saponin detoxification enzymes from pathogenic fungi are involved in the infection process of their host plants. Fusarium oxysporum f. sp lycopersici, a tomato pathogen, produces the tomatinase enzyme Tom1, which degrades alpha-tomatine to less toxic derivates. To study the role of the tom1 gene in the virulence of F. oxysporum, we performed targeted disruption and overexpression of the gene. The infection process of tomato plants inoculated with transformants constitutively producing Tom1 resulted in an increase of symptom development. By contrast, tomato plants infected with the knockout mutants showed a delay in the disease process, indicating that Tom1, although not essential for pathogenicity, is required for the full virulence of F. oxysporum. Total tomatinase activity in the disrupted strains was reduced only 25%, leading to beta(2)-tomatine as the main hydrolysis product of the saponin in vitro. In silico analysis of the F. oxysporum genome revealed the existence of four additional putative tomatinase genes with identities to tomatinases from family 3 of glycosyl hydrolases. These might be responsible for the remaining tomatinase activity in the Deltatom1 mutants. Our results indicate that detoxification of alpha-tomatine in F. oxysporum is carried out by several tomatinase activities, suggesting the importance of these enzymes during the infection process.