The apoplast as battleground for plant–microbe interactions

Abstract

The plant apoplast is the compartment where the compatibility of plant–microbe interactions is initially determined, and compared to cytoplasmic immune events, the molecular mechanisms occurring within the apoplast are poorly understood. Whether or not a pathogen can accommodate itself in its host tissue is decided during the initial phase of infection. At this stage, the plant immune system recognizes conserved molecular patterns of the invading microbe through surface-localized pattern recognition receptors (PRRs) to initiate pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Additionally, pathogens deploy a broad arsenal of virulence factors that aim to disarm PTI by targeting immune receptors and other signaling components. In turn, these effectors can also be recognized by the plant, inducing a second layer of immunity called effector-triggered immunity (ETI) (Jones & Dangl, 2006). Crucial regulatory processes take place in the apoplast. It comprises intercellular spaces, plant cell walls as well as the host–pathogen interfaces which are formed between the plant cytoplasm and the specialized infection structures of many fungal or oomycete pathogens. While extensive work has been done to understand the signal transduction pathways and gene regulation in plants responding to biotic stresses, the molecular basis of key processes in apoplastic immunity are largely unexplored. Similarly, microbial effectors acting within the host cell, particularly those of pathogenic bacteria, have been studied for several years and significant progress has been achieved in understanding their molecular mechanism (for recent reviews see Macho & Zipfel, 2015; Ökmen & Doehlemann, 2014). By contrast, only relatively few functions of apoplastic effectors have been described so far (Rovenich et al., 2014). With the aim of gathering an overview on the recent advances in the field and to gain novel insights into key mechanisms and components that determine apoplastic immunity, Gunther Doehlemann (University of Cologne, Germany), Bart Thomma (Wageningen University, the Netherlands) and Cyril Zipfel (The Sainsbury Laboratory, Norwich, UK) brought together a group of leading scientists working on different and complementary aspects of apoplastic and intracellular immunity for the 12th New Phytologist Workshop (Fig. 1). This workshop included the most recent advances in studying plant receptors, apoplastic effectors, apoplastic executers, downstream signaling, perception and mechanisms of compatibility. Despite our increasing knowledge about PRRs, only few receptor–ligand pairs have been identified so far. Lipopolysaccharides (LPS) are important structural components in the outer membrane of Gram negative bacteria and recognized as a PAMP in multiple species, including plants (Boller & Felix, 2009). However, the corresponding receptor remained elusive for a long time. Stefanie Ranf (TU Munich, Germany) presented her work on the first described potential LPS receptor in plants, LORE1, a Brassicaceae-specific S locus receptor kinase. Mutants affected in LORE1 are insensitive to LPS and LORE expression in Nicotiana species is sufficient to confer LPS responsiveness (Ranf et al., 2015). An important question remaining is whether LORE1 directly binds to LPS or if additional LPS binding proteins are required (Zipfel, 2015), as exemplified in mammals where LPS is sensed by a complex of TLR4 and MD-2 (Park et al., 2009). Similar to LPS, other structural components of bacterial cell walls can be recognized by plants, such as peptidoglycan (PGN). Arabidopsis perceives PGN by a complex consisting of the LysM-containing receptor kinase (RK) CERK1, and the paralogous LysM-containing receptor-like proteins (RLPs) LYM1 and LYM3 (Willmann et al., 2011; Gust et al., 2012). Andrea Gust (University of Tuebingen, Germany) reported on the progress in understanding how a host hydrolytic enzyme, LYS1, generates soluble PGN ligands accessible for recognition by the presumed CERK1-LYM1/LYM3 complex (Liu et al., 2014). Interestingly, it was found that LYS1 overexpression resulted in enhanced susceptibility, indicating that ‘over-digestion’ of PGN leads to a loss of its immunogenic properties. Future work will show the function and regulation of LYS1. The LysM-RK CERK1 is also important for the perception of the fungal-derived PAMP chitin (Miya et al., 2007; Wan et al., 2008; Macho & Zipfel, 2014; Cook et al., 2015). In a forward genetic screen for Arabidopsis mutants with enhanced responses to powdery mildew infection the laboratory of Volker Lipka (University of Goettingen, Germany) isolated a unique allele of CERK1. The cerk1-4 mutant shows strongly enhanced cell death in response to Blumeria graminis infection which is independent of chitin signaling (Petutschnig et al., 2014). The cerk1-4 phenotype is caused by impaired CERK1 ectodomain shedding (Petutschnig et al., 2014). At the workshop, Volker Lipka presented unpublished data from a cerk1-4 suppressor screen to better understand the shedding mechanism on a molecular level. In addition to the perception of nonself-molecules, plants also possess mechanisms to produce and detect endogenous signals which may act in an amplification loop to boost immune responses (Huffaker & Ryan, 2007; Huffaker et al., 2013). Alisa Huffaker (University of California, San Diego, CA, USA) presented her work on endogenous peptide (Pep) signaling in maize. Interestingly, although Pep elicitor peptides are widely conserved, plants produce specific sets of peptides that are not recognized by other species. Plant genomes contain a great number of potential PRRs with a plethora of genes encoding RKS and RLPs, mostly being uncharacterized to date (Boller & Felix, 2009). Lectin receptor-like kinases (LecRKs) are one of the largest classes of RKs in Arabidopsis thaliana. Klaas Bouwmeester (Wageningen University, the Netherlands) presented data about the impact of LecRKs on Arabidopsis immunity against different pathogens (Bouwmeester et al., 2011, 2014; Wang et al., 2014). He also reported on downstream interactors of LecRK-IX.1 and LecRK-IX.2 (Wang et al., 2015). An interesting question for future research is how this class of receptors mechanistically work and which ligands they might perceive. A key question in understanding apoplastic immunity is how the perception of extracellular molecules by receptor complexes is translated into downstream signaling events and how the amplitude of such responses is regulated. Jian-Min Zhou's laboratory (Chinese Academy of Sciences, Beijing, China) is interested in studying receptor-like cytoplasmic kinases (RLCKs), including BIK1 and related PBS1-like kinases, which are crucial signaling components downstream of multiple PRR complexes (Lu et al., 2010; Zhang et al., 2010; Liu et al., 2013). A recently identified BIK1 substrate is the NADPH oxidase ROBHD (Kadota et al., 2014; Li et al., 2014). Jian-Min Zhou presented novel data identifying further interaction partners of BIK1 and PBL kinases using discovery immunoprecipitation. Further phosphorylation events downstream of receptor complexes include mitogen-activated protein kinases (MAPKs) (Meng & Zhang, 2013). Dierk Scheel (Leibniz Institute of Plant Biochemistry, Halle, Germany) presented recent work from his laboratory on the identification and characterization of MAPK substrates involved in PTI signaling (Maldonado-Bonilla et al., 2014; Pecher et al., 2014). As soon as pathogen challenges cease, negative regulation of plant immunity is crucial to ensure the availability of cellular resources for growth and development. An interesting new mechanism for fine-tuning PTI signaling was proposed by Cyril Zipfel. In a forward genetic screen (Monaghan et al., 2014), his laboratory identified a subtilase that negatively regulates immunity by processing a subset of endogenous peptides (C. Zipfel et al., unpublished). He reported how this negative regulation takes place and proposed a corresponding perception mechanism. The regulation of secreted cysteine proteases plays an important function in apoplastic immunity (Doehlemann & Hemetsberger, 2013). One example was presented by Gunther Doehlemann (University of Cologne, Germany) using the model system maize with its fungal pathogen Ustilago maydis. He reported on apoplastic papain-like cysteine proteases (PLCPs) that are required for the induction of maize immunity (van der Linde et al., 2012a,b). PLCPs can release small peptides that serve as damage-associated molecular patterns (DAMPs) (Boller & Felix, 2009). Analysis of apoplastic fluids revealed the PLCP-dependent release of a small peptide able to upregulate PR gene expression and thus triggering immune responses (G. Doehlemann et al., unpublished). As important components of defense, apoplastic cysteine proteases are targeted by a plethora of pathogen effectors. PLCP activity is inhibited during infection by various pathogens including fungi, oomycetes and nematodes (Jashni et al., 2015). Renier van der Hoorn (University of Oxford, UK) presented his work on different pathogen effectors targeting the most abundant apoplastic cysteine proteases C14, PIP1 and RCR3 in tomato and other plant species (Shabab et al., 2008; Ilyas et al., 2015). His studies remarkably reveal an enzyme-inhibitor arms race during coevolution with host plants (Hörger & Van der Hoorn, 2013). Interestingly, he showed unpublished data about a secreted Pseudomonas syringae effector required for virulence and able to inhibit all three of the earlier mentioned apoplastic cysteine proteases. Another example of effectors targeting apoplastic cysteine proteases was given by Geert Smant (Wageningen University, the Netherlands) who presented recent work on venom allergen-like proteins (VAPs) from root-knot nematodes. Gr-VAP1 from Globodera rostochienses targets tomato Rcr3PIM (Lozano-Torres et al., 2012) and potato C14TUB (Lozano-Torres et al., 2014). A comparison of VAPs from different nematodes revealed that apoplastic cysteine proteases regulate immunity to cyst nematodes in Arabidopsis (Lozano-Torres et al., 2014). A general definition describes effectors as pathogen-derived proteins and small molecules that alter host cell structure and function (Hogenhout et al., 2009). Effectors can act at different levels by for example facilitating infection, triggering defense responses or both. Sophien Kamoun (The Sainsbury Laboratory, Norwich, UK), lit up the history of apoplastic immunity focusing on the work on Phytophthora infestans and the progress achieved in understanding ETI in the course of the last two decades. A hallmark finding was the discovery of the cell death-inducing Phytophthora elicitin INF1 (Kamoun et al., 1993) and the long journey through the very recent discovery of the corresponding receptor ELR isolated from a wild potato species (Du et al., 2015). Bart Thomma presented the work of his laboratory on different apoplastic effectors that modulate plant chitin perception mechanisms. The effector Ecp6 from the fungal tomato pathogen Cladosprium fulvum contains LysM domains able to disturb chitin perception by directly binding to it and thus titrating out chitin perception by plant PRRs (Sánchez-Vallet et al., 2013). Bart Thomma's laboratory currently aims to further dissect the function of different effectors interfering with chitin signaling and analyze their molecular mechanism. Another example of receptors modulating host immunity was presented by Frank Takken (University of Amsterdam, the Netherlands). He reported on the characterization of SIX (secreted in xylem sap) proteins, produced by Fusarium oxysporum during infection of tomato (Houterman et al., 2009; Gawehns et al., 2013). Frank Takken presented data on the function of SIX proteins, which can either increase virulence of the pathogen or induce ETI by recognition through R proteins (Ma et al., 2015). His studies represent an excellent example of the evolutionary arms race between hosts and pathogens. Additionally, Saskia Hogenhout (John Innes Centre, Norwich, UK) reported on understanding the molecular function of effector proteins from the small mycoplasma-like bacterial pathogen phytoplasma, which is transmitted from plant to plant by insects (Sugio et al., 2011b). Those SAP effectors were primarily shown to interfere with plant developmental processes (Sugio et al., 2011a, 2014; MacLean et al., 2014). Remarkably, Saskia Hogenhout's work highlights important molecular functions of effectors besides the suppression of host immune responses. During their interaction with the host plant, pathogens are characterized by distinct lifestyles, following three different strategies: necrotrophy, biotrophy or hemibiotrophy. Biotrophs rely on living host tissue and depend on metabolic processes of the host cells. A well-studied example for a biotrophic fungal pathogen is the maize pathogen U. maydis. Regine Kahmann (Max Planck Institute, Marburg, Germany) presented on U. maydis effectors and how shifts in effector gene expression affect host colonization. Interestingly, expression of the majority of effectors described so far is linked to the initial biotrophic phase (Kamper et al., 2006; Djamei & Kahmann, 2012). A novel aspect highlighted by Regine Kahmann is the finding of a new effector that is mainly up-regulated during late phases of interaction and is important for sporogenesis and karyogamy. She pointed out that stage specific regulation of effectors is crucial for Ustilago virulence. Another model pathogen is the hemibiotroph rice blast fungus Magnaporthe oryzae. George Littlejohn (University of Exeter, UK) presented technical advances in confocal microscopy during M. oryzae infection and the establishment of different subcellular and structural markers. His impressive presentation showed how fluorescently labeled effectors can be used as reporters to analyze the transition from biotrophy to necrotrophy. This enables live imaging to precisely dissect different stages of infection. Similar to fungal pathogens, the apoplastic space is the first compartment bacterial pathogens encounter after entering the leaf. Gail Preston (University of Oxford, UK) reported on changes in the apoplast composition that can affect immunity and disease development in the bacterial pathogen P. syringae. Compatible interactions are highly specific processes and, importantly, also extend to the interaction between plants and beneficial microorganisms. Alga Zuccaro (University of Cologne, Germany) presented an example of the generalist Sebacinales fungus Piriformospora indica in the interaction with Arabidopsis. Transcriptome analysis revealed mainly jasmonic acid (JA) and salycilic acid (SA) pathways being up-regulated in the host during infection. Interestingly, the induction of glucosinolates is important to balance the infection and to prevent over-colonization (Lahrmann et al., 2015). A genomic comparison of P. indica interactions with monocots and dicots identified a carbohydrate binding protein effector that is able to bind glucanases but not chitin (A. Zuccaro et al., unpublished). Arbuscular mycorrhizal (AM) fungi are beneficial microorganisms able to form a symbiotic relationship with plant roots of multiple species. Natalia Requena (Karlsruhe Institute of Technology, Germany) presented work from her group on understanding how AM fungi avoid plant immunity, which is important to establish an extended biotrophic interaction. Natalia Requena's laboratory found that the intrinsically disorganized effector SP7 suppresses PAMP-triggered gene expression in Medicago to establish mycorrization (Kloppholz et al., 2011). Current progress in understanding the molecular mechanism of SP7 and the interference with splicing components to modulate mRNAs was presented. This workshop was an excellent opportunity to discuss recent and unpublished data in apoplastic immunity, contributing to the establishment of highly relevant future research questions and collaborations. The importance of dissecting perception mechanisms, regulation of signal transduction and the understanding of molecular effector functions was discussed with a group of outstanding scientists. Furthermore, this workshop provided excellent opportunities for students, postgraduate students and postdocs to present posters and discuss their results with well-established senior scientists. Both, scientific and social interactions were enjoyable and very productive and contributed to a very successful meeting. We are looking forward to the next advances in plant–pathogen interactions at the apoplastic interface. The authors would like to thank all the participants and speakers for the good and constructive workshop. Special thanks go to the organizers and the people who contributed to the poster session. Funding for the workshop was provided by the New Phytologist Trust and the BASF Plant Science Company GmbH.