Molecular Genetics and Functional Genomics of Plant-Microorganism Interactions

Summary

Plants are constantly exposed to stress factors. Biotic stress is produced by living organisms such as pathogens while abiotic stress by unfavourable environmental conditions. A wide crosstalk has been observed between both stress pathways. The biotic stress can be generated by plants, insects or pathogenic microorganisms as fungi, bacteria or viruses. In our group plant-pathogen interactions that lead to plant disease or defence responses are studied. For that purpose we use bacteria and plants with agronomic relevance as models. These interactions are mainly studied using plant functional genomics and bacterial molecular genetics techniques. The knowledge generated in these studies will make possible to design new strategies which could be used in molecular breeding for the genetic improvement of plant stress resistance.

Research Lines

Influence of environmental factors in the regulation of genes involved in the pathogenesis of Xanthomonas citri subsp. citri in citrus canker.

Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker, a disease that causes great economic losses in the citrus growing areas. The sequencing of Xcc genome has allowed the in silico identification of genes encoding blue light (two BLUF protein and one LOV protein) and red light (one bacterio-phytochrome) photoreceptors. The presence of these genes allows us to hypothesize a light regulation of the virulence of this plant pathogen. This project focuses on the study of these novel bacterial photoreceptors, both, its structure and photochemical activity, as its role during the interaction of Xcc with host plants (compatible interaction leading to disease) and non-host plants (incompatible interaction). To achieve the desired objectives we propose the use of biophysical methods such as steady state absorption and fluorescence spectroscopy for the detection of photo-induced species and time resolved methods such as flash photolysis and laser-induced optoacoustic spectroscopy for the identification of transient excited states, as well as enzymatic methods for the characterization of the activity of the different photoreceptors. Moreover, the generation of Xcc strains mutated in genes encoding such proteins will be used to evaluate the role of the photoreceptors in bacterial physiology and in disease development during infection of different types of plants. The effect of light on the physiology of heterotrophic bacteria is a new paradigm that has attracted interest worldwide. Knowledge of the role of light in the virulence process of Xcc will contribute to the study of the mechanisms involved in plant-pathogen interactions. Moreover, the study of the structure and mechanisms of photochemical activation of Xcc photoreceptors will contribute to the knowledge of this new group of bacterial light-sensing proteins.

Transcriptome analysis of Rutaceous plants during the response to bacterial pathogens.

Citrus is one of the most important crops in the world. Significant losses of these crops are produced worldwide as a result of pathogen attack. In this project, we are studying the transcriptional changes in Rutaceous plants challenged with phytopathogenic bacteria of the Xanthomonas genus, with the aim of detecting the molecular mechanisms involved during the plant disease or defense responses. The different transcriptomes are analyzed in order to find new candidates genes for use in molecular breeding strategies. The knowledge of the defense responses used by perennial plants like citrus during biotic stress will make possible, in the long term, to improve citrus stress resistance to pathogens. Furthermore, the production of transgenic citrus plants tolerant or resistant to pathogens could be an effective and not expensive method to control diseases.

Study of the molecular mechanisms of post-translational modification in Ralstonia solanacearum and its relevance for virulence.

Protein glycosylation is a common posttranslational modification in bacteria. Glycosylated proteins are often exposed on the cell surface and therefore they could play a role in bacterial virulence. In many animal pathogenic bacteria the en bloc transfer of an oligosaccharide to protein acceptors by an oligosaccharyltransferase (OTase) constitutes a general glycosylation system for many structural and functional diverse membrane proteins.
Ralstonia solanacearum is a Gram-negative soil-borne bacterium that causes disease on more than 200 plant species, including many economically important crops. We have recently identified an open reading frame in the R. solanaceraum genome with high homology to OTases involved in protein glycosylation. The aim of the present project is to evaluate the global function of this enzyme and to identify its putative protein targets on the cell surface. We also propose to evaluate the regulation of this gene by major transcriptional regulators that control the expression of pathogenicity determinants in R. solanacearum. These studies will allow us to determine whether the OTase is part of the virulence machinery turned on in R. solanacearum during the interaction with host plants.

Selected Publications

  • Daurelio L.D., Romero M.S., Petrocelli S., Merelo P., Cortadi A.A., Talón M., Tadeo F.R., Orellano E.G. (2013) Characterization of Citrus sinensis transcription factors closely associated with the non-host response to Xanthomonas campestris pv. vesicatoria. J Plant Physiol. 170(10):934-42. doi: 10.1016/j.jplph.2013.01.011.
  • Tondo M.L., Hurtado-Guerrero R., Ceccarelli E.A., Medina M., Orellano E.G., Martínez-Júlvez M. (2013) Crystal structure of the FAD-containing ferredoxin-NADP+ reductase from the plant pathogen Xanthomonas axonopodis pv. citri. Biomed Res Int. 2013:906572. doi: 10.1155/2013/906572.
  • Kraiselburd I., Daurelio L.D., Tondo M.L., Merelo P., Cortadi A.A., Talón M., Tadeo F.R., Orellano E.G. (2013) The LOV protein of Xanthomonas citri subsp. citri plays a significant role in the counteraction of plant immune responses during citrus canker. PLoS One 8(11):e80930. doi: 10.1371/journal.pone.0080930.
  • Petrocelli S., Tondo M.L., Daurelio L.D., Orellano E.G. (2012) Modifications of Xanthomonas axonopodis pv. citri lipopolysaccharide affect the basal response and the virulence process during citrus canker. PLoS One 7(7):e40051. doi: 10.1371/journal.pone.0040051.
  • Kraiselburd I., Alet A.I., Tondo M.L., Petrocelli S., Daurelio L.D., Monzón J., Ruiz O.A., Losi A., Orellano E.G. (2012) A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization. PLoS One 7(6):e38226. doi: 10.1371/journal.pone.0038226.
  • Casabuono A.*, Petrocelli S.*, Ottado J., Orellano E.G.#, Couto A.S.#(2011) Structural analysis and involvement in plant innate immunity of Xanthomonas axonopodis pv. citri lipopolysaccharide. J Biol Chem. 286(29):25628-43. doi: 10.1074/jbc.M110.186049. *These authors contributed equally to this work; #Corresponding authors.
  • Daurelio L.D., Petrocelli S., Blanco F., Holuigue L., Ottado J., Orellano E.G. (2011) Transcriptome analysis reveals novel genes involved in nonhost response to bacterial infection in tobacco. J Plant Physiol. 168(4):382-91. doi: 10.1016/j.jplph.2010.07.014.
  • Tondo M.L., Musumeci M.A., Delprato M.L., Ceccarelli E.A., Orellano E.G. (2011) Structural-functional characterization and physiological significance of ferredoxin-NADP reductase from Xanthomonas axonopodis pv. citri. PLoS One 6(11):e27124. doi: 10.1371/journal.pone.0027124.
  • Tondo M.L., Petrocelli S., Ottado J., Orellano E.G. (2010) The monofunctional catalase KatE of Xanthomonas axonopodis pv. citri is required for full virulence in citrus plants. PLoS One 5(5):e10803. doi: 10.1371/journal.pone.0010803.
  • Daurelio L.D., Checa S.K., Barrio J.M., Ottado J., Orellano E.G. (2009) Characterization of Citrus sinensis type 1 mitochondrial alternative oxidase and expression analysis in biotic stress. Biosci Rep. 30(1):59-71. doi: 10.1042/BSR20080180.

Collaborators

Dr. Francisco Tadeo (Centre de Genómica, IVIA, España)
Dr. Manuel Talón (Centre de Genómica, IVIA, España)
Dr. Stephan Genin (LIPM, INRA-CNRS, Francia)
Dra. Milagros Medina (Universidad de Zaragoza, BIFI, España)
Dra. María Julia Pianzzola (Universidad de la República, Uruguay)
Dra. María Inés Siri (Universidad de la República, Uruguay)
Dra. María Eugenia Farías (PROIMI, CCT-Tucumán, Argentina)
Dra. Alicia Couto (Centro de Investigaciones en Hidratos de Carbono, Universidad de Buenos Aires, Argentina)
Dr. Leandro Moreira (DECBI, NUPEB, Universidad Federal de Ouro Preto, Brasil)
Dra. Aba Losi (Departamento de Física, Universidad de Parma, Italia)
Dr. Wolfgang Gärtner (Max Planck Institute für chemische Energiekonversion, Mülheim un der Ruhr, Alemania)
Dr. Chuck Farah (Instituto de Química, Universidad de San Pablo, Brasil)

Grants

• PICT 2010-1762, Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT): “Regulación por luz de la patogenicidad de Xanthomonas en plantas”.
• PIP 0873, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET): “Regulación por luz de la patogenicidad de Xanthomonas en plantas”.
• BIO 1BIO269, Universidad Nacional de Rosario (UNR): “Influencia de factores ambientales en la regulación de genes implicados en la patogénesis y/o virulencia de Xanthomonas axonopodis pv. citri durante la cancrosis de los cítricos”.
• PIP 0394, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET): “Análisis de los cambios transcripcionales en plantas Rutáceas en respuesta a patógenos bacterianos. Búsqueda de genes y regiones promotoras en cítricos que promuevan su resistencia a la cancrosis”.

Director

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Orellano, Elena G.
Core Faculty
Email: orellano@ibr-conicet.gov.ar
Phone: +54 341 4350596/4350661
Office Extension: 106
Laboratory Extension: 106

Researchers

Lucas D. Daurelio
María Laura Tondo

Doctoral fellows

  • Laura Moyano
  • Analía Carrau
  • Ma. Josefina Tano
  • Vandecaveye Agustina
  • Nannini Julian

Imágenes

Infección de hojas de cítricos con Xanthomonas citri subsp. citri salvaje (WT) y mutantes en el lipopolisacárido (Xacwzt y Xacrfb303) y curvas de crecimiento bacteriano in planta.