The group is focused in the characterization of the molecular and genetic bases of plant resistance to necrotrophic and vascular fungi, using as model pathosystem the interaction between Arabidopsis thaliana and the ascomycete fungus Plectosphaerella cucumerina, a natural pathogen of Arabidopsis plants. The main research lines of the group are:
1) Identification and characterization of genes and regulatory pathways controlling Arabidopsis thaliana resistance to necrotrophic fungi.
Arabidopsis resistance to this type of fungi is complex and depends on the interplay between different signalling pathways, such as those mediated by the hormones ethylene (ET), jasmonic acid (JA) and salicylic acid (SA), but also these regulated by abscisic acid (ABA) and auxins (Llorente et al., 2008; Hernández-Blanco et al., 2007; Berrocal-Lobo et al., 2008; Berrocal-Lobo et al., 2002). Some additional key regulatory processes, such as the preteaosome activity, have been found to control resistance to these fungi (Llorente et al., 2008). Also, it has been demonstrated that non-host resistance, and in particular the metabolites derived from PEN2 and PEN3 activities, are relevant for plant resistance to necrotrophs (Lipka et al., 2005; Stein et al., 2006; Bednarek et al., 2009).
|To gain more knowledge on the mechanisms controlling plant resistance to necrotrophs, we performed a screening to isolate and to characterize Arabidopsis ern (enhanced resistance to necrotrophs) and esn (enhanced susceptibility to necrotrophs) mutants. In these analyses were identified the ern1/irx1/lew2 mutant, impaired in AtCESA8 protein required for the synthesis of cellulose from secondary cell wall (Hernandez-Blanco et al, 2007) and the esn1/er/qrp1 mutant, that corresponds to the ERECTA protein (Llorente et al., 2005).||
The irx1/ern1 mutant shows a broad spectrum resistance to pathogens and drought, that relies on novel mechanisms regulated by ABA signalling and that involves the accumulation of secondary metabolites and antimicrobial peptides (APs). The contribution of these metabolites and APs on plants resistance to adapted and nonadapted necrotrophs has been demonstrated recently (Sanchez-Vallet et al., 2010).
2) Perception of nectrotrophic fungi by Arabidopsis and triggering of innate immunity responses.
The group have identify several genetic components controlling these processes such as ERECTA (ER), ELK2 or the heterotrimeric G protein (Llorente et al., 2005). ER is a Leucine Rich Repeat (LRR)-Receptor Like Kinase (RLK), that controls different developmental processes, but also is required for Arabidopsis resistance to several pathogens, including P. cucumerina (Llorente et al. 2005). In contrast to the well-characterized ER-mediated developmental signalling pathway, relatively few genetic elements of ER-mediated immunity have been identified.
We have demonstrated that genes required for ER-regulated developmental processes (e.g ERL1, ERL2 and TMM) do not regulate Arabidopsis resistance to P. cucumerina. In a suppressor screening of er susceptibility, we have identified specific genetic elements (SER1 and SER2) of ER-mediated immunity. Interestingly, a comparative analysis of the cell wall structure/composition and defence responses in er, ser1, ser2 and wild-type plants revealed that composition of the host cell wall may be a determinant of fungal infection success. Thus, er mutant show cell wall structure/composition alterations compared with wild-type plants, which were partially restored to wild-type phenotype by the ser mutations. These results suggest that ER plays a role in regulating cell wall-mediated resistance to pathogens that is distinct from its role in plant development (Sanchez-Rodriguez et al., 2009).
The ELK2 gene was recently cloned and found to encode a MAP3K, which has been previously found to regulate developmental processes. Our data indicate a genetic interaction between ELK2, ER and additional RLKs recently identified in the lab, suggesting that all these proteins may form part of the same functional complex required for fungal recognition and signalling activation. To identify novel components of AGB1-mediated signalling pathway, we selected suppressors of the agb1 (sgb9-sgb13) susceptibility to P. cucumerina. The functional characterization of these sgb mutants indicates that AGB1 regulates a complex regulatory network and that novel, uncharacterized immune response may be activated in these mutants.
3) Function of cell wall integrity in Arabidopsis resistance to pathogens and innate immunity triggering
The characterization of irx1, er and ser mutants mutant suggested that cell wall integrity regulates immune responses. To explore this regulatory effect of the cell wall, we have performed a detailed analysis of the resistance to different pathogens of primary or secondary Arabidopsis cell wall mutants. In this biased screening a significant number of mutants showed altered susceptibility/resistance to one or more pathogens compared with wild-type plants. Phenotypic clustering allowed the classification of these mutants in different functional immune response categories (Riviere et al., in preparation). Also, some potential cell wall derived signals have been identified, which are able to activate immune response in Arabidopsis. The biochemical nature of this cell wall-derived “elicitors” and their potential use in crop protection are under study.
4) Functional genomics of P. cucumerina
The group has established the pathosystem P.cucumerina-Arabidopsis to study the genetic and molecular bases of necrotrophic fungi pathogenicity. The Agrobacterium-mediated transformation of this fungus has been established in the lab and P.cucumerina transformants overexpressin GFP have been obtained nd used to study, by confocal microscopy, fungal colonization of plants. A collection of T-DNA insertional mutants have been generated, their virulence tested and some genes required for pathogenicity identified.
Fig. 3: Confocal microscope analisys of the colonization by GFP-P cucumerina of leaves from different Arabidopsis genotypes. Differents hours post inoculation (hpi) are showed. Chloroplast in alive cells are seen in red colour.
Arabidopsis resistance to pathogens and osmotic stress
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