PLANT INNATE IMMUNITY AND RESISTANCE TO NECROTROPHIC FUNGI
- Díaz González, Sandra - PhD Student
- Escudero Welsch, Viviana - Postdoctoral Fellow
- Fernández Calvo, Patricia - Postdoctoral Fellow
- Hierro García, Irene del - PhD Student
- Jordá Miró, Lucía - Assistant Professor
- López García, Gemma - Technician
- Martín Antoranza, Rubén - Technician
- Mélida Martínez, Hugo - Postdoctoral Fellow
- Muñoz Barrios, Antonio - PhD Student
- Prado, Palmira del - Technician
- Rebaque Morán, Diego - PhD Student
- Sacristán Benayas, María Soledad - Associate Professor
- Téllez de Pablos, Julio - PhD Student
- Torres Lacruz, Miguel Ángel - Assistant Professor
The group interest is the characterization of the molecular and genetic bases of plant resistance to necrotrophic fungi, a group of pathogens causing devastating diseases in crops. We use as model patho-system the interaction between Arabidopsis thaliana and the ascomycete fungus Plectosphaerella cucumerina, a pathogen that colonizes Arabidopsis plants in their natural habitats. The main goal of our group is to understanding how plants sense this type of pathogens and activate immune responses to confer enhanced disease resistance, which involves several defensive signaling pathways. In parallel, the group is trying to decipher the molecular mechanisms determining P. cucumerina virulence and pathogenicity. Several P. cucumerina isolates with different lifestyles on Arabidopsis plants (e.g. pathogenic, non-pathogenic and endophytic) have been characterized and their genomes sequenced and annotated. Using comparative and evolutionary genomic studies we have identified fungal determinants explaining the differential interactions of these isolates with Arabidopsis plants. The group also studies how plants shape their fungal endophytic microbiota in natural habitats and whether this type of fungi positively regulates plant physiology and fitness (Ecological and molecular factors involved in fungal endophytism and pathogenesis).
The main research lines of the group are:
1) Arabidopsis innate immune responses and resistance to necrotrophic fungi: fungal recognition and immune response regulation.
Arabidopsis resistance to necrotrophic fungi is complex and depends on the interplay of different signaling pathways (Figure 1), such as those mediated by the defensive hormones ethylene, jasmonic acid and salicylic acid, but also abscisic acid and auxins (Llorente et al., 2008; Hernández-Blanco et al., 2007; Berrocal-Lobo et al., 2008; Berrocal-Lobo et al., 2002 Sanchez-Vallet et al., 2012; Denance et al., 2013). We have also demonstrated that non-host resistance, secondary metabolites derived from tryptophan metabolism and antimicrobial peptides (APs) play a key role in plant resistance to necrotrophs (Lipka et al., 2005; Stein et al., 2006; Bednarek et al., 2009; Sanchez-Vallet et al., 2010; Frerigmann et al., 2016; Harris et al., 2014; Denancé et al., 2013; Harris et al., 2014; Yeung et al., 2016).
Figure 1.Signalling pathways and molecular mechanisms regulating immune response and resistance to necrotrophic fungi.
The group is specially focused in deciphering how plants recognize P. cucumerina and activate immune responses. Plants possess a cell autonomous monitoring system that comprises a collection of Pattern-Recognition Receptors (PRRs), such as Receptor-Like Kinases (RLKs) and Receptor Like Proteins (RLPs), that .upon pathogen perception, activate early immune responses, such as Ca+2 influx, ROS protein kinase phosphorylation and gene expression. Our group has identified several genetic components controlling these immune responses, such as the RLK ERECTA (ER), or the heterotrimeric G protein (Llorente et al., 2005). ER, in addition to control the resistance to P. cucumerina and other pathogens, regulates different developmental processes, such as stomata development, lateral organ shape and plant architecture (Llorente et al. 2005; Sánchez-Rodriguez et al., 2009). ER regulates these developmental processes by interacting with the ER family (ERf) paralogs, ER-like 1 (ERL1) and ERL2, the RLP TOO MANY MOUTHS (TMM) and SERKs RLKs (including BAK1 protein). Our recent results indicate that this multiproteic receptorome formed by ERf, TMM and BAK1 also modulates Arabidopsis thaliana resistance to PcBMM (Figure 2) Remarkably, the secreted Epidermal Pattern Factor peptides (EPF1 and EPF2), which are perceived by ERf members, do not regulates ER-mediated immune response further suggesting that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering (Jordá et al., 2016).
Figure 2. Model of ERFs/TMM and BAK1 function in the regulation of immunityand development processes. The ERFs/TMM/BAK1 putative complex (orange box) regulates immunity and developmental processes through the recognition of unknown DAMPs/MAPMPs and EPFs peptides, respectively. Ligan binding activates MAPK cascades probably involving the YDA-MKK4/5-MPK3/6 module, and downstream effectors will lead to different cellular responses. Additional RLPs and RLKs might be requiered for the activity of the complex in immuniy and development processes. The dotted lines indicated uncharacterised genetic or biochemical interactions
We have identified specific genetic elements of ER-mediated immunity, such as an Arabidopsis thaliana MAP3K functioning downstream ER. This MAP3K is a key regulator of a novel, non-canonical immune pathway whose constitutive activation (CA-MAP3K) results in broad-spectrum disease resistance to different type of pathogens, including fungi, oomycetes and bacteria with different life styles (Sopeña et al., submitted). CA-MAP3K plants constitutively express defense-associated genes that are induced upon plant infection with different types of pathogens and show alterations in their cell wall composition/structure (Sopeña-Torres et al., submitted).
Figure 3. Response of pD::LUC (promoter of RbohD , pD) and pF::LUC (promoter of RbohF, pF) transgenic lines to inoculation with pathogenic (PcBMM) or non-pathogenic (non-adaptaded, Pc2127) isolates of P. cucumerina fungus. The hours postinoculation (hpi) are indicated.
Heterotrimeric G protein, particularly the β-subunit (AGB1) and the two g-subunits (AGG1 and AGG2), has been recognized as important mediator of Arabidopsis thaliana immunity to different pathogens including P. cucumerina (Delgado-Cerezo et al., 2012; Jiang et al., 2012; Trusov et al., 2010). These regulatory protein subunits interact with additional genetic components that could modulate different plant responses, such as cell wall integrity/structure (Klopffleisch et al., 2011; Jiang et al., 2012; Delgado et al., 2013) or ROS production (Torres et al., 2013; Morales et al., 2016; Figure 3). We have identified novel components (sgb9-sgb13) of AGB1-mediated signaling pathway in a suppressors screening of agb1 susceptibility to P. cucumerina. The functional characterization of these sgb mutants indicates that AGB1 regulates a complex regulatory network and that novel, uncharacterized immune responses are activated in the sgb plants.
2) SignWALLing: cell wall integrity and cell wall-derived signals regulating plant immune responses.
Plant cell wall is a complex structure constantly subjected to dynamic remodelling in response to internal cues and external constraints. Wall adaptation to these developmental and environmental signals is regulated by a dedicated plant cell wall integrity (CWI) monitoring system that initiate compensatory responses to restore wall integrity. This CWI system consists of a set of wall sensor/receptors that specifically bind wall-derived ligands, so-called Damage-Associated Molecular Patterns (DAMPs) that are released upon alteration of wall integrity. This plant monitoring system also functions during pathogen infection, since microbes modify wall composition to favour colonization by means of secreted cell wall degrading enzymes. The perception of wall DAMPs and microbial molecules (so-called Pathogen-Associated Molecular Patterns PAMPs) by specific plant Pattern Recognition Receptors (PRRs) triggers immune responses (Miedes et al., 2013;Sánchez-Rodríguez et al., 2010).
We have identified several Arabidopsis cell wall mutants (e.g. ern1/irx1/lew2 impaired in AtCESA8 required for secondary cell wall cellulose synthesis) displaying broad-spectrum resistance to pathogens and drought. This resistance to stresses relies on novel mechanisms involving ABA signaling and the accumulation of secondary metabolites and APs (Hernandez-Blanco et al, 2007; Sanchez-Vallet et al., 2010). In line with this relevant function of plant cell wall in immunity, we found that er mutant displays alterations in the cell wall compared with wild-type plants, which were partially restored to wild-type phenotype by the suppressor of er (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). Similarly, map3kmutant shows cell wall alterations quite similar to those of er plants, which are restored to wild-type phenotypes by MAP3K-overexpression (Sopeña et al., submitted). Moreover, Arabidopsis heterotrimeric G protein has been implicated in CWI regulation (Delgado-Cerezo et al., 2012; Torres et al., 2013).
To explore this regulatory effect of the cell wall, we have performed a detailed analysis of the resistance to different pathogens of a collection of Arabidopsis primary and secondary cell wall mutants. In this biased screening, a significant high number of cell wall mutants (cwm) showed altered susceptibility/resistance to one or more pathogens compared with wild-type plants (Miedes et al., in preparation). Resistance and developmental phenotypes from the cwm collection and their biochemical cell wall compositions have been mathematically integrated to built a predictive model correlating specific changes in cell wall epitopes with resistance/growth phenotypes. This model suggests that modulation of CWI might be an efficient strategy to improve plant resistance to environmental threats and to obtain crop varieties with improved resistance to biotic and abiotic stresses (Miedes et al., 2014; Miedes et al., in preparation).
3) Functional genomics of the necrotrophic fungus Plectosphaerella cucumerina
In order to fully understand how plants defend themselves against the attack of pathogens it is crucial to understand the colonization mechanisms used by the pathogens. The group has established the patho-system P. cucumerina-Arabidopsis as the model to study the genetic and molecular bases of necrotrophic fungi pathogenicity. Several molecular tools have been established to study fungal colonization (Ramos et al., 2013, 2015). Three P. cucumerina isolates that differ in the life style and interaction with Arabidopsis have been characterized (Figure 4): PcBMM, is a fully pathogenic isolate that colonizes Arabidopsis causing extensive necrosis; Pc2127, does not cause necrosis nor colonizes wild-type Arabidopsis plants, but infects immune deficient Arabidopsis mutants (cyp79b2cyp79b3); and Pc0831, is a endophytic isolate identified in wild Arabidopsis populations that causes not harm to the plant (Sanchez-Vallet et al, 2010; Ramos et al., 2013;García et al., 2013;Muñoz et al., in preparation). The genomes of these three isolates have been sequenced and annotated, and comparative and evolutionary genomic analyses are underway to identify fungal determinants explaining the different interactions of these isolates with Arabidopsis plants (Muñoz et al., in preparation). Similar studies have been performed with the interaction between the fungal endophyte Colletotrichum tofieldiae and Arabidopsis, revealing specific genomic and transcriptomic features related to the beneficial responses of this mutualistic fungal isolate (Hacquard et al., 2016;Hiruma et al., 2016). Both Colletrichum and Plectosphaerella endophytic isolates have been obtained by our group from natural populations of Arabidopsis thaliana, within the research line “Ecological and molecular factors involved in fungal endophytism and pathogenesis” (Ecological and molecular factors involved in fungal endophytism and pathogenesis). In this research line, we study how plants shape their fungal endophytic microbiota in natural habitats and whether this type of fungi positively regulates plant physiology and fitness.
Figure 4. A. Disease symptoms in wild-type plants (Col-o) and the immune deficient mutant cyp79b2b3 at 7 days postinoculation with either a pathogenic (PcBMM), a non-pathogenic (Pc2127) or and edophytic (Pc0831) isolate of P. cucumerina fungus. The control plants (Mock) were treated with water. B. Confocal microscopy images if wild-type plants (Col-0) and the immune deficient mutant agb1-1 at 24 hours after inoculation with PcBMM-GFP and Pc2127-GFP transformants constitutively expressing GFP.
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