Introduction to a Virtual Special Issue on cell biology at the plant-microbe interface.

Abstract

Plants can be inhabited by a broad range of microbes, including prokaryotic bacteria, eukaryotic fungi and oomycetes. These taxonomically very diverse microbes are able to colonize all types of plant organs/tissues, including roots, stems, leaves, flowers and fruits. Plant–microbe interactions can be mutually beneficial, neutral or parasitic. While the former give rise to so-called symbiotic relationships, the latter typically results in disease. Seemingly neutral microbial plant residents (‘endophytes’) have been neglected for a long time but recently gained increasing attention with the advent of metagenomic studies that are driven by new sequencing technologies. Historically, plant–microbe interactions have been often studied at the level of entire plants or tissues. With the advancement of molecular, biochemical and particularly cell biological tools and techniques, individual cells have increasingly moved to the forefront of interest (Lipka & Panstruga, 2005; Hückelhoven & Panstruga, 2011; Gutjahr & Parniske, 2013; Dörmann et al., 2014). It became clear that each cell provides the interface for the molecular dialogue between plant and microbe. Amongst other factors, the delivery of microbial effectors to plant cells is a key factor that shapes this molecular conversation (Kuhn & Panstruga, 2014). Both compatible and incompatible interactions are accompanied by a plethora of cellular changes and adaptations on the plant and microbial site. The combination of advanced imaging devices and genetically encoded fluorophores now enables unprecedented insights into the microcosm of cellular events that occur at the plant–microbe interface. In this Virtual Special Issue, devoted to the forthcoming 36th New Phytologist Symposium ‘Cell biology at the plant–microbe interface’ (http://www.newphytologist.org/symposiums/view/38), we bring together a group of papers published recently in New Phytologist that illustrate various aspects of cell biology in different types of plant–microbe interactions. The nine cell biological themes highlighted in this Virtual Special Issue are also depicted in Fig. 1. The distinction between beneficial or detrimental microbes is one of the major tasks plants have to fulfil in order to initiate the proper response(s) to potential invaders or appreciated interactors. The first steps towards a correct discrimination of friend or foe relies on the recognition of molecular clues and microbe-associated molecular patterns (MAMPs) by receptors localized to the plant plasma membrane. As part of a special feature on ‘Molecular plant–microbe interactions’ published in New Phytologist in December 2014, Antolín-Llovera and colleagues provide an excellent overview of leucine-rich repeat (LRR) and lysine motif (LysM) receptor-like kinases (RLKs) and receptor-like proteins (RLPs). These protein classes represent the major types of pattern recognition receptors (PRRs) that mediate signalling leading to the initiation of symbiosis and defence (Antolín-Llovera et al., 2014). After recognition of MAMPs the first line of plant defence is initiated, conferring pattern-triggered immunity (PTI). In order to counteract this resistance pathogens evolved to secrete effectors that interfere with critical components of the plant defence machinery. In the case of recognition of pathogenic effectors by dedicated cytoplasmic receptors (plant resistance (R) proteins), a second layer of host defence is activated that is termed effector-triggered immunity (ETI) (Jones & Dangl, 2006). LysM motif-containing PRRs perceive chitin oligomers, which represent the best-studied fungal MAMP. However, LysM motifs are additionally present in symbiotic receptors involved in the perception of lipo-chitooligosaccharides (LCOs) such as rhizobial Nod factors (NFs) or LCOs secreted by arbuscular mycorrhizal fungi (Myc-LCOs). Legumes, like Medicago truncatula, are able to perceive and differentiate beneficial microsymbionts from antagonistic pathogens. In M. truncatula, LysM-RLK NFP (Nod factor perception) and LYK3 (LysM domain-containing receptor-like kinase 3) are required for nodulation, while only NFP is critical for NF perception and furthermore involved in Myc-LCO perception and signalling. More recently, data supporting an additional involvement of NFP in recognition of the root-colonizing oomycete Aphanomyces euteiches points to a more general role of the receptor in the perception of N-acetylglucosamine-based molecules (Rey et al., 2013). Corresponding to fungal chitin, the bacterial protein flagellin (FliC) contributes to the recognition of bacteria by plant cells. Whereas the perception of the flagellar peptide flg22 by the Arabidopsis LRR-RLK FLS2 (FLAGELLIN SENSING 2) is well documented, the epitope flgll-28, which is sufficient to trigger immunity in tomato, has been less studied. Recognition and elicitation of immune responses by flgll-28 is restricted to solanaceous plant species. However, mutations in the epitope in context of the entire FliC protein have FLS2-dependent effects on virulence in Arabidopsis without interfering with bacterial motility. As tomato FLS2 is not involved in recognition of flgll-28, the epitope seems to be perceived by a yet unidentified receptor in the Solanaceae, despite having an FLS2-dependent virulence effect in Arabidopsis. Clarke and colleagues suggest that a putative FLS3 protein of Arabidopsis might require a larger region of FliC to interact with flgII-28 and postulate that FLS2 acts as a co-receptor for FLS3 (Clarke et al., 2013). Calcium (Ca2+) ions are secondary messenger molecules that modulate a plethora of intracellular activities. Ca2+ is usually kept at low cytoplasmic concentrations, but its levels dramatically increase by influx from extracellular (apoplast) and intracellular stores upon particular stimuli, including biotic stress (Brownlee & Hetherington, 2011). The time-resolved pattern of changes in Ca2+ concentrations (the so-called Ca2+ signature) is subject to the coordinated action of Ca2+ influx and efflux pathways and varies considerably in response to different cues (McAinsh & Pittman, 2009). Although the details of the cellular ‘Ca2+ code’ remain largely mysterious, some first studies have begun to shed light on the underlying molecular mechanisms. It seems that different types of Ca2+ signatures are linked to particular downstream gene expression patterns (Whalley & Knight, 2013; Liu et al., 2015). It has been known for a long time that the establishment of mutualistic (symbiotic) interactions of plants with either rhizobia or arbuscular mycorrhizal (AM) fungi involves oscillatory Ca2+ spiking in the nuclei of plant root cells. The capability to mount such Ca2+ oscillations in response to symbiotic bacteria appears to be a common feature of nodulating species within the clade of nitrogen-fixing plants (Granqvist et al., 2015). In the case of rhizobia, NFs have been identified as the primary triggers for this response (Ehrhardt et al., 1996). Similar molecules, designated as Myc-LCOs, represent the signalling counterparts of AM fungi (Maillet et al., 2011). Recently, short-chain chitin oligomers were shown to contribute also to mycorrhiza-related Ca2+ spiking (Genre et al., 2013). Besides nuclear Ca2+ oscillations, NFs induce apoplastic Ca2+ influx at the root hair tip. By exploiting Sinorhizobium meliloti mutants that produce chemically altered NFs Allan Downie's group showed that the two events can be differentially activated and play separate roles in the establishment of rhizobial symbiosis (Morieri et al., 2013). Traditionally, most of the work regarding Ca2+ signatures focused on the plant side of symbiotic interactions. Like other AM fungi, Gigaspora margarita is recalcitrant to genetic manipulations such as transformation. In an attempt to explore possibilities to deliver recombinant proteins via a protein translocator to the fungal symbionts, the delivery of a fusion protein with the Ca2+ sensor (apo-) aequorin was successfully achieved. Subsequent aequorin-based measurements revealed rapid increases in cytoplasmic Ca2+ concentration following a range of diverse stress stimuli in G. margarita (Moscatiello et al., 2014). While an oscillatory pattern is well known for the Ca2+ signatures occurring in symbiotic interactions, the details of the Ca2+ signatures that occur upon the perception of pathogenic signals such as MAMPs are less understood. This lack of knowledge is largely due to the fact that most measurements using MAMP-triggers have been performed at the level of whole seedlings using the Ca2+ sensor aequorin. Single cell recordings in Arabidopsis guard cells using the fluorescent Ca2+ sensor Yellow Cameleon 3.6 recently revealed oscillatory Ca2+ patterns upon cellular stimulation with the bacterial MAMP flg22. Although essentially similar to Ca2+ spiking in symbiotic interactions, the flg22-induced Ca2+ oscillations differ in a number of features from their symbiotic counterparts (Downie, 2014; Thor & Peiter, 2014). Cellular responses to interacting microorganisms are tightly linked to an active trafficking of components between the different membranous compartments of plant cells. Maintenance of a signalling-competent plasma membrane (PM)-resident pool of PRRs such as FLS2 depends on delivery, recycling of nonactivated and depletion of activated receptors to and from the PM (Beck et al., 2012). This task is mediated by secretory and endosomal pathways connecting endomembranous compartments such as the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), multi-vesicular bodies (MVBs) and lytic vacuole to the PM. The plant response to pathogens involves targeting of anti-microbial and cell wall-strengthening compounds by the host secretory system to the site of attack (Yun et al., 2008). Secretion requires delivery, tethering and fusion of secretory vesicles with the target membrane that could either be the PM or microbe-enclosing membranes such as the extrahaustorial membrane (EHM) that encompasses intracellular fungal/oomycete feeding structures. Assembly of the exocyst complex is pivotal to the tethering of secretory vesicles to the PM during the last step of exocytosis. In contrast to other exocyst subunits conserved in plants, the Exo70 gene family exhibits dynamic expression patterns as well as remarkable expansion in land plants. Multiple Exo70 genes are present in a variety of land plants (e.g. 23 in Arabidopsis). Interestingly, transcript abundance of several members of the AtEXO70H subclade is increased during pathogen infections and upon elicitor treatments. Localization patterns of the Arabidopsis exocyst subunits are similar to those found in yeast and animal cells, in line with a conserved role of the plant exocyst complex (Chong et al., 2010). Furthermore, an observed co-localization with endosomal markers is consistent with a role of the exocyst in vesicular trafficking from the recycling endosomal compartment proposed to reside at the TGN/early endosome (Žárský et al., 2009). Secretory vesicle fusion with the PM is finally mediated by SNARE (soluble N-ethylmaleimide-sensitive-factor attachment receptor) complexes. This process is critical for defence against a variety of plant pathogens in monocotyledonous and dicotyledonous plants. A SNARE complex with a known role in pathogen defence in Arabidopsis consists of the two t-SNARE proteins AtPEN1 (PENETRATION 1)/AtSYP121 (SYNTAXIN OF PLANTS 121) and AtSNAP33 (SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE FACTOR ADAPTOR PROTEIN 33) as well as one of the two v-SNAREs (AtVAMP721/AtVAMP722) (Kwon et al., 2008). Silencing of both t-SNARE homologues in potato, the PEN1 relative StSYR1 (SYNTAXIN-RELATED 1) and StSNAP33, results in altered infection phenotypes upon challenge with hemibiotrophic and necrotrophic pathogens. This correlates with constitutive accumulation of the phytohormone salicylic acid (SA) as well as with aberrant callose deposition, pointing to a role of syntaxins in the formation of localized cell wall appositions (papillae) in potato (Eschen-Lippold et al., 2012). The formation of papillae that are thought to hinder penetration attempts is one of the earliest responses after perception of fungal pathogens. A recent detailed histo-immunochemical analysis of barley papillae formed in response to the powdery mildew pathogen Blumeria graminis f.sp. hordei (Bgh) revealed a layered structure with callose concentrated in a central core. In addition to the (1,3)-β-glucan, significant amounts of arabinoxylan are distributed throughout the papillae, and crystalline cellulose accumulates in their outer layer (Chowdhury et al., 2014). Membrane restructuring and rearrangements play a pivotal role during pathogen-induced alterations in plant cell architecture and contribute to the intracellular accommodation of microbes (Wang & Dong, 2011). This includes the formation of new apoplastic and/or perimicrobial compartments surrounded by host-derived membranes, which requires massive re-organization of plant membranous systems and of the host cytoskeleton. Such cell-autonomous responses to pathogenic and symbiotic microbes with emphasis on membrane organization and trafficking were recently portrayed in a detailed up-to-date review by Dörmann et al. (2014). Membrane dynamics and vesicle trafficking are also pivotal processes in the control of stomatal closure in response to pathogens. Stomatal immunity, which prevents pathogen entry into the leaf, correlates with the immense surface area changes of guard cells caused by exchange of membrane material between the PM and internal compartments. Stomatal control mechanisms are targeted by pathogen effectors that either induce opening or intervene with the closing of stomata in order to promote disease (reviewed by McLachlan et al., 2014). The contribution of the plant vesicle trafficking machinery to many aspects of an efficient defence response makes it an appropriate target, prone to intervention by microbial effectors. The bacterial type III effector XopB of Xanthomonas campestris pv. vesicatoria, which is involved in suppression of both intracellular and cell surface-mediated defence, localizes to Golgi vesicles and the plant cytoplasm. XopB interferes with secretory vesicle trafficking, a function that is independent of its role in ETI suppression. This functional separation is indicated by a point mutation of XopB that affects its restriction of ETI- but not PTI-related responses and still maintains its ability to impede vesicle trafficking (Schulze et al., 2012). In contrast to XopB, the Pseudomonas syringae pv. tomato (Pto) DC 3000 effector HopD1 impacts ETI- but not PTI-mediated immunity of Arabidopsis. At the ER, HopD1 interacts with the intracellular membrane-tethered Arabidopsis transcription factor NTL9, which is a positive regulator of plant immunity involved in the induction of defence-related genes during ETI and PTI. Its activity is suppressed by HopD1 by a yet unknown mechanism only in response to ETI, leading to enhanced virulence and pathogen proliferation (Block et al., 2014). Besides exocytosis also endocytosis is crucial for plant–microbe interactions. The role of endocytic pathways during resistance is highlighted by the fact that a mutation of SPL28, a clathrin-associated adaptor protein-1 μ-adaptin 1 (AP1M1), leads to enhanced resistance of rice against the fungal rice blast and bacterial blight disease. SPL28 was identified due to its hypersensitive response (HR)-like lesion mimic phenotype, which is linked to high levels of reactive oxygen species (ROS) and the accumulation of callose, phytoalexins and phenolic compounds. AP1M1 is involved in the regulation of post-Golgi trafficking pathways by mediating vesicular protein sorting between the TGN and endosomes (Qiao et al., 2010). Endocytosis potentially results in the fusion of late endosomes (LEs)/MVBs with lytic vacuoles. Here the endocytic pathway converges with the autophagic one, resulting in the digestion of cargo from autophagosomes and LEs/MVBs by hydrolytic enzymes. Autophagy is a highly conserved cellular process of eukaryotes that is implicated in the reaction to environmental stresses including defence against pathogens. In plants, autophagy plays a critical role during the HR in response to pathogens. In antiviral defence, autophagy is involved in targeting the infecting viral particles to autophagic lysosomes, resulting in degradation of the invaders. However, hijacking of the autophagic machinery of the oceanic alga Emiliania huxleyi by the giant double-stranded DNA coccolithovirus EhV contributes to viral propagation and production of infective viral particles (Schatz et al., 2014). Besides protein biosynthesis, protein degradation is a key regulatory step in plant physiology. In particular the ubiquitin-proteasome system plays a pivotal role in different aspects of plant signalling (Sadanandom et al., 2012). In contrast to other eukaryotic organisms, plant genomes are characterized by a vast abundance of genes coding for ubiquitin-mobilizing enzymes, further highlighting the importance of this pathway in plant life. The pathogen-responsive transcription factor gene SlNAC1 encodes a stress-related tomato NAC transcription factor. Results from pharmacological inhibitor experiments suggest that SlNAC1 is subject to constitutive polyubiquitination and proteasome-dependent turnover. Deletion analyses identified a particular region (degron) within the protein that is essential for degradation of SlNAC1. Proteasome-dependent turnover of transcriptional regulators thus seems to be essential to fine-tune plant responses upon biotic stress cues (Huang et al., 2013). Similar conclusions were drawn by Yu and co-workers who found that the E3 ubiquitin ligase EIRP1 of the Chinese wild grapevine Vitis pseudoreticula positively regulates plant immunity by promoting proteasome-based degradation of the negatively acting transcriptional regulator VpWRKY11 (Yu et al., 2013). Proteolytic activities also seem to play a role in pathogen-triggered plant cell death. HR-associated host cell death is an essential component in many types of R gene-mediated pathogen resistance (ETI). Activity-based profiling of hydrolase activities revealed that changes in the activities of papain-like cysteine proteases and serine hydrolases precede such Avr4/Cf-4-triggered hypersensitive tissue collapse in tomato seedlings (Sueldo et al., 2014). Genetic evidence relying on virus-based gene silencing in Nicotiana benthamiana suggests that several components of the SKP1/Cullin/F-box E3 ubiquitin ligase complex are involved in Agrobacterium tumefaciens-mediated plant transformation. These factors probably target T-complex-associated proteins for polyubiquitination and subsequent degradation by the 26S proteasome (Anand et al., 2012). However, it seems although proteolysis is important for successful plant transformation, the putative Agrobacterium transcriptional activator-like virulence protein VirD5 prevents coat proteins of the T-complex from being (too) quickly degraded by the host ubiquitin-proteasome system (Wang et al., 2014). Proteolytic activities are not only crucial in plant–microbe interactions, but also in root symbioses engaging rhizobacteria or mycorrhizal fungi. For example, the ectomycorrhizal basidiomycete Paxillus involutus secretes a number of peptidases and shows transcriptional upregulation of the respective genes during symbiotic interactions. The peptidase genes are co-regulated with genes encoding transporters and other enzymes involved in the assimilation and metabolism of amino acids/peptides, suggesting that the mobilization of organic nitrogen is a key event in ectomycorrhizal symbiosis (Shah et al., 2013). Host protease activities are also involved in the senescence process of root nodules in M. truncatula. Cysteine protease genes MtCP6 and MtVPE exhibit increased transcript levels during nodule senescence. Furthermore, modulation of the expression of proteinase genes impacts the senescence process (Pierre et al., 2014). Vesicle trafficking during secretion and endocytosis, as well as spatial distribution of organelles and cellular rearrangements in response to pathogen attack or enabling the accommodation of microbes, rely on the host cytoskeleton (Schmidt & Panstruga, 2007). The actin cytoskeleton responds rapidly to biotic stresses and supports numerous fundamental cellular processes during plant innate immunity against fungi, oomycetes and phytopathogenic bacteria. However, viruses exploit cytoskeleton functions for their own needs. Several viral proteins critical for infection are known to travel intracellularly along actin microfilaments in host cells. So far, no such ability has been described for viral nucleocapsid (N) proteins, which are presumably involved in the regulation of viral gene transcription and genome replication in plants and animals. The tomato spotted wilt tospovirus (TSWV) N protein has now been shown to form highly motile cytoplasmic inclusions that move along the ER and actin network. Integrity of the actin cytoskeleton contributes to local and systemic TSWV symptoms suggesting a role of N protein trafficking in viral intracellular and intercellular spread (Feng et al., 2013). Protein–protein interactions and the formation of protein complexes are decisive events in all types of signalling pathways, including those activated in the course of plant–microbe interactions (Bogdanove, 2002; Ellis et al., 2002). The analysis of protein–protein interactions and protein complexes by a variety of experimental approaches is thus a central aspect of the detailed molecular dissection of these pathways. Mitogen-activated protein kinase (MAPK) cascades play a pivotal role during the initiation of plant immune responses (Meng & Zhang, 2013). The substrate proteins of MAPKs remain, however, largely unknown. The combination of various experimental approaches led to the identification of a subset of Arabidopsis VQ motif-containing proteins (VQPs) as potential downstream MAPK substrates. Some of these VQPs interact with WRKY transcription factors, thereby possibly modulating defence gene expression upon MAMP signalling (Pecher et al., 2014). Molecular chaperones, including heat shock proteins (HSPs), are believed to be essential for the folding and function of nucleotide-binding leucine-rich repeat (NB-LRR)-type immune receptors (Elmore et al., 2011). The Arabidopsis chilling-sensitive mutant chs2-1 (rpp4-1d) represents a gain-of-function mutant of the resistance gene RPP4 (RECOGNITION OF PERONOSPORA PARASITICA 4) that leads to constitutive activation of defence responses at low temperatures. Mutations in genes encoding HSP90.2 and HSP90.3 suppress the chilling sensitivity of chs2-1, and HSP90.3 interacts with both RPP4 and its mutated version, rpp4. Together these results suggest that HSP90 is required to activate the RPP4 protein (Bao et al., 2014). The plant-specific GRAS transcriptional regulators NSP1 (NODULATION SIGNALING PATHWAY 1) and NSP2 are involved in mediating the colonization by nitrogen-fixing rhizobia and AM fungi. In M. truncatula, the two proteins form a transcription factor complex and bind to the promoters of genes that are activated during symbiotic interactions. The C-terminal GRAS domain of Lotus japonicus NSP2 was found to interact with the coiled-coil domain of a MYB transcription factor that was designated as IPN2 (INTERACTING PROTEIN of NSP2). IPN2 has strong transactivation activity and localizes to nuclei of L. japonicus root cells. RNAi-mediated IPN2 gene silencing compromised rhizobial infection and nodule formation, suggesting that NSP2 and IPN2 may form a transcription factor complex to promote the expression of a set of genes that are essential for nodule development (Kang et al., 2014). Besides mutual interactions between plant proteins, interactions between host and microbe proteins are likewise important. The soybean (Glycine max) homologue of Arabidopsis NONRACE SPECIFIC DISEASE RESISTANCE 1 (GmNDR1) was found to be the target of two effector proteins (AvrB2 and AvrD1) from the bacterial pathogen Pseudomonas syringae pv. glycinea (Psg). Since GmNDR1 is required for the activity of several resistance proteins against Psg, the pathogen might interfere with GmNDR1 function for defence suppression (Selote et al., 2014). The plant nucleus is a major site of action and battleground in plant–microbe interactions. Amongst other activities, it is here that the transcriptional activation of plant defence genes and the reprogramming of host cells for compatibility takes place. It is thus no surprise that many proteins of plant and microbial origin exhibit (re-)localization to the nucleus in the course of these interactions (Wirthmueller et al., 2013). Several plant defence proteins show nucleocytoplasmic shuttling, that is dynamic allocation to the nucleus and the cytoplasm. For example, the tobacco immune receptor N, a protein belonging to the TIR-NB-LRR class of resistance (R) proteins, accumulates in both the cytoplasm and in the nucleus. It seems as if the phosphorylation status of SGT1, a HSP90 co-chaperone, determines the nucleocytoplasmic distribution pattern of N, thereby regulating N activity (Hoser et al., 2013). Similarly, the DNA/RNA-binding cysteine/histidine-rich DC1 domain protein CaDC1, which is a positive regulator of defence and cell death in pepper, exhibits localization to both the cytoplasm and the nucleus (Hwang et al., 2014). Effector proteins from all classes of microbes localize to the plant nucleus to manipulate the host cell. For example, an effector (Mi-EFF1) of the root-knot nematode Meloidogyne incognita that is injected into giant feeding cells through the nematode stylet is targeted to the plant nucleus (Jaouannet et al., 2012). Another instance is the phytoplasma-derived virulence effector SAP11, which is a modular protein with a bipartite nuclear localization signal (NLS). Dissection of domain function revealed that nuclear localization of SAP11 is not required for the destabilization of its Arabidopsis TCP transcription factor targets (Sugio et al., 2014). Effectors with localization in the plant nucleus are also delivered by bacterial pathogens. The bacterial (Xanthomonas campestris) effector XopDXcc8004 targets the plant DELLA protein RGA to delay its degradation. RGA is one out of five Arabidopsis DELLA proteins that repress signalling through the pathway induced by the phytohormone gibberellic acid (Tan et al., 2014). Transcription activator-like effectors (TALEs) are well described effector proteins secreted by Xanthomonas species in order to induce the expression of susceptibility-mediating genes after binding to respective host promoters. Ralstonia solanacearum biovars encode closely related proteins termed RipTALs (R. solanacearum injected effector proteins (Rips)) of which Brg11 (hrpB-regulated 11) is the first identified member. Functional characterization of Brg11 revealed common mechanisms of action to Xanthomonas TALEs with functional interchangeability of DNA-binding and transcriptional activation domains. However differences in response element structure and reduced target sequence diversity are also reported (de Lange et al., 2013). In the course of plant–microbe interactions there is typically a mass transfer of small molecules between the plant and the microbe. While phytopathogens rely on the acquisition of nutrients such as carbohydrates and amino acids from their host plants, plants harbouring root symbionts aspire to take up nitrogen (in the form of ammonium) or inorganic phosphate. PM-localized plant transporter proteins facilitate the efflux and uptake of these solutes. SWEET (Sugars Will Eventually bE Transported) proteins are PM-resident carbohydrate uniporters that primarily serve a role in phloem loading of photoassimilates. However, several types of plant pathogens including bacteria and fungi stimulate elevated expression levels of SWEET genes, suggesting that the respective gene products serve a decisive role in microbial nutrition by exporting carbohydrates to the apoplastic space. Indeed, certain bacterial species (e.g. Xanthomonas oryzae) evolved dedicated TALEs that bind to the promoters of particular SWEET genes to activate their transcription (Streubel et al., 2013; Chen, 2014). Similarly to the SWEET genes, expression of two particular isoforms of genes encoding ammonium transporters (SbAMT3; 1 and SbAMT4) is locally induced in the interaction of Sorghum bicolor with AM fungi (Glomus intraradices (now Rhizophagus irregularis) and G. mosseae (now Funneliformis mosseae)). Transcript levels of the two genes are predominantly elevated in root cells containing arbuscules and in neighbouring cells (Koegel et al., 2013). A similar expression pattern was found for two phosphate transporter genes (AsPT1 and AsPT4) in arbuscule-containing cortical root cells of Astragalus sinicus (Xie et al., 2013). The membrane trafficking events that contribute to shrinkage of the guard cell surface area during stomatal closure (see section 3 ‘4’) are associated with volume and turgor changes (McLachlan et al., 2014). This process is initiated by activation of guard cell outward anion channels leading to membrane depolarization and eventually to reduction of the osmotic potential by a decrease in cytosolic [K+]. While regulation of abscisic acid (ABA)-mediated stomatal behaviour induced by abiotic stress is well described, the contribution of ABA-controlled regulators to microbe-initiated closure is still under discussion. Using noninvasive nanoinfusion and patch clamp techniques Guzel Deger et al. (2015) provided evidence that that both signaling pathways converge at the OST1 (OPEN STOMATA 1) kinase, which induces SLAC1 and SLAH3 anion channels. However, mutation of the upstream phosphatase ABI1, which resulted in insensitivity to ABA due to constitutive inhibition of OST1, was overcome by flg22-induced stomatal closure (Guzel Deger et al., 2015). The study of plant–microbe interactions has always benefitted from the visualization of plant cells’ reaction towards interacting microbes. For several years refined techniques have allowed for improved dynamic in vivo imaging with subcellular resolution, thereby advancing our knowledge of dynamic cellular processes. Due to the availability and further development of existing fluorescent proteins, stains, probes and sensors, along with novel microscopy technologies and quantitative analysis tools, a wide range of plant cell behaviour can now be studied in vivo. Cellular processes that can now be additionally monitored in their three-dimensional (3D) context include protein localization, topology and movement, protein–protein interactions, vesicle trafficking, organelle transport, as well as ion, ROS and redox dynamics. Recently published data further represents advances based on the interdisciplinary interactions of plant biologists with chemists, physicists, mathematicians and computer scientists. In particular the collaborations with bioinformaticians will in future enable and promote high-throughput approaches that become increasingly important in plant cell biology (Zhou et al., 2012; Fitzgibbon et al., 2013; Beck et al., 2014). Simultaneous in vivo quantification of ROS accumulation and mitochondrial membrane potential, as well as glutathione abundance and its redox potential, monitored the occurrence of these features during the development of Magnaporthe oryzae infection structures. It was previously known that besides ROS production, fungal redox regulation additionally depends on the redox potential of glutathione as well as mitochondrial activity. Multi parameter live-cell confocal imaging of various fluorescent reporters revealed high levels of mitochondrial activity and ROS in the growing fungal germ tube and appressorium, but a strong reduction and tight regulation of glutathione during pathogenic development. A study by Samalova and colleagues shows that M. oryzae has sufficient anti-oxidative capacity to cope with a strong oxidative host response, with the consequence that ROS toxicity alone is not sufficient to kill the pathogen (Donofrio & Wilson, 2014; Samalova et al., 2014). The permeation of plant cell walls by M. oryzae appressoria largely relies on the establishment of high pressure directed towards the site of penetration. The resurgence of Mach–Zehnder double-beam interferometry now contradicts the paradigm that force-based penetration of plant cells is restricted to melanized appressoria. By measuring the turgor pressure of Asian soybean rust (Phakopsora pachyrhizi) appressoria in vivo, Loehrer and colleagues show that these nonmelanized penetration structures exhibit a pressure close to that of M. oryzae, while being able to penetrate non-biodegradable membranes even more efficiently (Loehrer et al., 2014). The regulation of cytoskeletal dynamics, membrane trafficking and stress responses, which are all critical for plant immunity, involves the membrane phospholipid phosphatidic acid (PA). Although it is known that PA undergoes rapid turnover, a detailed spatio-temporal monitoring of its abundance has not been possible so far for plant cells. Adaption of a genetically encoded fluorescent PA biosensor, previously shown to be functional in yeast and mammalian cells, now enables live-cell imaging of PA dynamics in plant cells. Fusion of the PA binding domain of a yeast SNARE protein to fluorescent reporters visualizes the compound in pollen tubes of tobacco (Nicotiana tabacum) (Potocký et al., 2014). Parallel imaging of conventionally expressed fluorescent proteins in combination with click-chemistry-mediated detection of DNA replication has long been hampered by quenching of the fluorophores by the copper-based click reaction. By restoring both statuses of a photoswitchable fluorophore, Bourge and colleagues have paved the way for quantification of protein biosynthesis via cell cycle analysis in plant cells (Bourge et al., 2015). Secondary ion mass spectrometry (SIMS), a method to identify isotope ratios, has evolved from micro-scale applications to nano-resolution isotope microscopy. In biological systems this technique has only been used for a couple of years. Now, the employment of ultra-high spatial resolution SIMS, combined with stable isotope tracer experiments, has identified both young and senescent fungal pelotons as functional cellular nutrient transfer sites in the orchid mycorrhizal symbiosis (Kuga et al., 2014). The manifold examples outlined earlier highlight the importance of an array of diverse cellular activities for the establishment of plant–microbe interactions and/or the execution of plant immunity (see also Fig. 1). It remains a future challenge to decipher the cellular details of these encounters at even greater detail. Novel approaches and tools, for example advanced imaging techniques such as super-resolution microscopy, promise to provide new and exciting insights into the molecular dialogue between plant and microbe. With this in mind, there is much to look forward to in terms of future research and the 36th New Phytologist Symposium ‘Cell biology at the plant–microbe interface’ (http://www.newphytologist.org/symposiums/view/38), which will be held from 29 November to 1 December 2015 in Munich (Germany) and which will highlight cutting-edge research in this area.