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Identification, characterization and role of molecular factors associated with the mechanisms of infection of Fagaceae species by Phytophthora cinnamomi


Reference: PTDC/AGR-AAM/67628/2006
Project Type: Nacional / Público
Financial Program: PTDC - Projectos em todos os domínios científicos
Global Funding Value: 145617.00 €; Research Project
Principal Investigator
Start: 2007-01-01
End: 2010-01-01
Description: Phytophthora cinnamomi and P. cambivora are considered by the generality of the authors as the ink disease of Castanea sativa causal agent. Although several epidemiology surveys have shown P. cinnamomi is regularly associated with Quercus suber and Q. ilex sbsp. rotundifolia decline there is non official acknowledge of a decline disease caused by this pathogen. Most Phytophthora species studied secrete large amounts of elicitins, a group of unique highly conserved proteins that are able to induce hypersensitive response (HR) and enhance plant defences SAR responses in a systemic acquired resistance (SAR) manner, against infection by different pathogens. Some other proteins involved in mechanisms of infection by Phytophthora cinnamomi were identified by one partner (ESAB endo-1,3-beta-glucanase (complete cds), exo-glucanase (partial cds) (adhesion, penetration, and colonization of host tissue); glucanase inhibitor protein (GIP) (complete cds) (suppression of host defense responses); necrosis-inducing Phytophthora protein 1 (NPP1) (partial cds), transglutaminase (partial cds) (induction of defence responses and disease-like symptoms). The present proposal aims at the characterization of the mechanisms and the nature (compatible, incompatible) of the interaction between P. cinnamomi and Quercus suber, Q. ilex and Castanea sativa; the action of cinnamomins and of GIP and NPPI on root cell membranes of those hosts will be studied, identifying, comparatively the genes involved in the defence responses and elucidating the function of the various effectors in the infectious process. Hair-root cell lines from cork and holm oak and chestnut will be developed to be used as a simplified model for the study of the action of the pathogen, GIP and NPPI, and beta and alfa cinnamomins as well on the cell membranes. One of the partners (UAlg) has available the recombinant elicitins produced in Pichia pastoris. Heterologous GIP and NPPI will be produced from the respective cDNAs already synthesized. The pathogen will be applied in the form of wild type and transformed isolates in order to better understand the role of the protein effectors through a gene silencing approach. Transformation was achieved for silencing cinnamomin genes by one of the partners (UAlg) and will be performed for GIP and NPPI. The activation of defence genes will be analysed [mRNA induction analysed by cDNA-AFLP]. The sequencing of differentially expressed fragments will allow gene characterization and the study of their expression by RT-PCR in root cells challenged with the pathogen and the effectors. Concomitantly, the transfer of sterols between cinnamomins and root cell walls will be studied. Since the elicitins (including beta cinnamomin) are known to bind sterols with high affinity, we believe that the presence of cinnamomins in the aqueous phase surrounding a plant cell will provoke the efflux of sterols from the plasma membrane of the plant cells. The consequences of such efflux, if not compensated by replenishment from intracellular sterol pools, will be a reduction of the mass fraction of liquid-ordered phase and increase in the mass fraction of the liquid-disordered phases in the cell plasma membranes and will result in a fragilization of these. We propose to test this hypothesis. To our knowledge, this will be the first time that this sort of study will be applied to plant cell membranes. A complementary approach will be carried out comprising in planta studies in Quercus and Castanea sativa seedlings. Invasion of cortical and vascular tissues by cinnamomin silenced transformed P. cinnamomi isolate (produced in UAlg) will be compared with the wild isolate. General injury of the roots tissues will be histological and cytological evaluated measuring the extension of the disorder by light microscopy. In addition the effects of cinnamomins, GIP and NPPI on the ultrastructure of root cells challenged by P. cinnamomi will be studied in order to assess the extent of defence reactions against the pathogen to restrict its invasion. Finally, immunocytochemical localization of cinamomins in tissues infected by P. cinnamomi and their production in function of the growth phase of the pathogen will be assessed by transmission electron microscopy. It is expected to contribute with the present proposal to a better understanding of the mechanisms by which the infectious process of important forest trees by P. cinnamomi develops, fundamental condition for the implementation of control strategies of the decline and ink diseases.