Development of an in vivo approach using GFP for P(athogenesis)-R(elated) protein localization and promoter analysis after Agrobacterium-mediated transient expression.
Fabian BARRACATO 1, Maxime Eugène 2, Jacques DOMMES 2 and Patrick MOTTE 1
1Laboratoire de Biologie Cellulaire Végétale, Département des Sciences de la Vie, Bât. B22, Université de Liège
2Laboratoire de Biologie Moléculaire et Hormonologie, Département des Sciences de la Vie, Université de Liège
Plants respond to abiotic and biotic stresses by activating a broad array of molecular, cellular and biochemical responses. Among those, production of reactive oxygen and nitrogen species, accumulation of phenolic compounds, de novo synthesis of proteins and localized cell death are used by plants to inhibit growth and multiplication of pathogens. Pathogenesis-Related (PR) proteins are synthesized to high levels. They are defined as proteins coded for by the host plant, but induced specifically in pathological and related situation. PR proteins have been identified in many plant species and are classified into 17 families. Different acidic and basic isoforms have been characterized. By cell fractionation and immunocytochemistry, it has been suggested that acidic and basic PR proteins are targeted to precise cellular compartments. The goal of our study was to evaluate an Agrobacterium-mediated transient expression to monitor the trafficking of GFP-tagged PR proteins using confocal laser microscopy. We developed several constructs encoding translationally fused PR-GFP proteins under the control of the constitutive CaMV35S promoter. Consistent with previous reports using cell fractionation and immunolocalization, detection of GFP fluorescence clearly showed a precise and defined sub-cellular accumulation of different tomato and tobacco PR proteins, validating our approach. Moreover, to facilitate in vivo analysis of promoters of PR proteins, we have also developed an efficient and rapid transient expression assay based on GFP fluorescence and confocal microscopy. Tobacco cells were transiently transformed with a fusion between PR1a promoter and GFP. By quantification of GFP fluorescence, we demonstrated an induction of this promoter by treatments with salicylic acid and BTH. Kinetic analysis of GFP expression was also achieved, showing an increase of GFP fluorescence with treatment duration.
Analysis of pathogen (a)virulence factors in the Brassica oleracea – Peronospora parasitica interaction transcriptome
Sandra Casimiro1,2, Zé-Zé, L.1, Rogério Tenreiro1 and António A. Monteiro2
1 Universidade de Lisboa, Faculdade de Ciências, Centro de Genética e Biologia Molecular and Instituto de Ciência Aplicada e Tecnologia, Edifício ICAT, Campus da FCUL, Campo Grande, 1749-016 Lisboa, Portugal, 2 Departamento de Produção Agrícola e Animal, Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017, Lisboa Portugal.
Crucifer downy mildew is a worldwide spread and economically important disease of brassica crops. The identification and study of pathogenesis-related virulence factors and avirulence genes is crucial for a successful breeding for resistance. In compatible interactions between Brassica oleracea hosts and the biotrophic oomycete Peronospora parasitica the lack of the R gene or the corresponding Avr gene leads to the growth of fungal mycelium and sporulation, whereas in incompatible interactions the presence of both R and Avr genes activate several defence mechanisms, as the hypersensitive response, preventing pathogen development. The analysis of B. oleracea–P. parasitica interaction transcriptomes was performed by Differential-display PCR. Based on the theoretical estimation of the number of arbitrary primers necessary to cover most of the genes expressed in a cell, about 80% of the interaction transcriptome was analyzed, leading to the identification of 743 specific cDNAs, showing differential expression in Brassica oleracea seedlings infected with Peronospora parasitica. All cDNA clones were tested by Reverse Northern Blot, revealing a small rate of false positives. Sequencing analysis and/or specific PCR of positive clones gave information about the nature and origin of mRNAs being expressed during infection. So far, at least 7 P. parasitica cDNA genes have been identified. RT-PCR assays showed that the level of expression of these genes is related to the interaction type. 5’-RACE allowed cloning of full-length cDNA genes corresponding to some of these DD-PCR fragments.
A proteomic approach to identify proteins differentially regulated during Peronospora viciae infection in susceptible and resistant pea cultivars.
Richard Amey, Laura Taylor, Tanja Schleicher, Heather Macdonald, Steve Neill & Peter Spencer-Phillips.
Centre for Research in Plant Science and Bristol Genomics Research Institute, University of the West of England, Bristol, Coldharbour Lane, Bristol, BS16 1QY.(email: )
Pea downy mildew caused by the oomycete pathogen Peronospora viciae reduces yield by up to 55% where plant resistance and fungicide treatment are ineffective. Therefore, the development of resistant pea varieties is essential for effective control of the pathogen, and for reducing fungicide applications in order to prolong their effectiveness and reducing pesticide use. This BSPP-funded research uses two-dimensional electrophoresis and mass spectrometry to examine differences in protein expression during P. viciae infection between the resistant cultivar Early Onward, and the susceptible cultivar Leviolleta. Data relating to those proteins identified as being up- or down-regulated during the plant pathogen interaction in the two cultivars will be presented.