Lennart Wirthmueller

Arabidopsis EDS1 Connects Pathogen Effector Recognition to Cell Compartment–Specific Immune Responses

Coordination of different defense pathways across cell compartments produces a fully effective innate immune response. Pathogen effectors are intercepted by plant intracellular nucleotide binding–leucine-rich repeat (NB-LRR) receptors. However, processes linking receptor activation to downstream defenses remain obscure. Nucleo-cytoplasmic basal resistance regulator EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1) is indispensible for immunity mediated by TIR (Toll–interleukin-1 receptor)–NB-LRR receptors. We show that Arabidopsis EDS1 molecularly connects TIR-NB-LRR disease resistance protein RPS4 recognition of bacterial effector AvrRps4 to defense pathways. RPS4-EDS1 and AvrRps4-EDS1 complexes are detected inside nuclei of living tobacco cells after transient coexpression and in Arabidopsis soluble leaf extracts after resistance activation. Forced AvrRps4 localization to the host cytoplasm or nucleus reveals cell compartment–specific RPS4-EDS1 defense branches. Although nuclear processes restrict bacterial growth, programmed cell death and transcriptional resistance reinforcement require nucleo-cytoplasmic coordination. Thus, EDS1 behaves as an effector target and activated TIR-NB-LRR signal transducer for defenses across cell compartments.

Nuclear Pore Complex Component MOS7/Nup88 Is Required for Innate Immunity and Nuclear Accumulation of Defense Regulators in Arabidopsis

Plant immune responses depend on dynamic signaling events across the nuclear envelope through nuclear pores. Nuclear accumulation of certain resistance (R) proteins and downstream signal transducers are critical for their functions, but it is not understood how these processes are controlled. Here, we report the identification, cloning, and analysis of Arabidopsis thaliana modifier of snc1,7 (mos7-1), a partial loss-of-function mutation that suppresses immune responses conditioned by the autoactivated R protein snc1 (for suppressor of npr1-1, constitutive 1). mos7-1 single mutant plants exhibit defects in basal and R protein–mediated immunity and in systemic acquired resistance but do not display obvious pleiotropic defects in development, salt tolerance, or plant hormone responses. MOS7 is homologous to human and Drosophila melanogaster nucleoporin Nup88 and resides at the nuclear envelope. In animals, Nup88 attenuates nuclear export of activated NF-κB transcription factors, resulting in nuclear accumulation of NF-κB. Our analysis shows that nuclear accumulation of snc1 and the defense signaling components Enhanced Disease Susceptibility 1 and Nonexpresser of PR genes 1 is significantly reduced in mos7-1 plants, while nuclear retention of other tested proteins is unaffected. The data suggest that specifically modulating the nuclear concentrations of certain defense proteins regulates defense outputs.

On the front line: structural insights into plant-pathogen interactions

Over the past decade, considerable advances have been made in understanding the molecular mechanisms that underpin the arms race between plant pathogens and their hosts. Alongside genomic, bioinformatic, proteomic, biochemical and cell biological analyses of plant–pathogen interactions, three-dimensional structural studies of virulence proteins deployed by pathogens to promote infection, in some cases complexed with their plant cell targets, have uncovered key insights into the functions of these molecules. Structural information on plant immune receptors, which regulate the response to pathogen attack, is also starting to emerge. Structural studies of bacterial plant pathogen–host systems have been leading the way, but studies of filamentous plant pathogens are gathering pace. In this Review, we summarize the key developments in the structural biology of plant pathogen–host interactions.

The Plasmodesmal Protein PDLP1 Localises to Haustoria-Associated Membranes during Downy Mildew Infection and Regulates Callose Deposition

The downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) is a filamentous oomycete that invades plant cells via sophisticated but poorly understood structures called haustoria. Haustoria are separated from the host cell cytoplasm and surrounded by an extrahaustorial membrane (EHM) of unknown origin. In some interactions, including Hpa-Arabidopsis, haustoria are progressively encased by host-derived, callose-rich materials but the molecular mechanisms by which callose accumulates around haustoria remain unclear. Here, we report that PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1) is expressed at high levels in Hpa infected cells. Unlike other plasma membrane proteins, which are often excluded from the EHM, PDLP1 is located at the EHM in Hpa-infected cells prior to encasement. The transmembrane domain and cytoplasmic tail of PDLP1 are sufficient to convey this localization. PDLP1 also associates with the developing encasement but this association is lost when encasements are fully mature. We found that the pdlp1,2,3 triple mutant is more susceptible to Hpa while overexpression of PDLP1 enhances plant resistance, suggesting that PDLPs enhance basal immunity against Hpa. Haustorial encasements are depleted in callose in pdlp1,2,3 mutant plants whereas PDLP1 over-expression elevates callose deposition around haustoria and across the cell surface. These data indicate that PDLPs contribute to callose encasement of Hpa haustoria and suggests that the deposition of callose at haustoria may involve similar mechanisms to callose deposition at plasmodesmata.

Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants

A new homology search algorithm identifies novel KASH protein family members in Arabidopsis that act at the nuclear envelope in nuclear positioning and innate immunity.

Expression Profiling during Arabidopsis/Downy Mildew Interaction Reveals a Highly-Expressed Effector That Attenuates Responses to Salicylic Acid

Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.

Mapping FLS2 function to structure: LRRs, kinase and its working bits

The plasma membrane-localised FLAGELLIN SENSING 2 (FLS2) receptor is an important component of plant immunity against potentially pathogenic bacteria, acting to recognise the conserved flg22 peptide of flagellin. FLS2 shares the common structure of transmembrane receptor kinases with a receptor-like ectodomain composed of leucine-rich repeats (LRR) and an active intracellular kinase domain. Upon ligand binding, FLS2 dimerises with the regulatory LRR-receptor kinase BRI1-associated kinase 1, which in turn triggers downstream signalling cascades. Although lacking crystal structure data, recent advances have been made in our understanding of flg22 recognition based on structural and functional analyses of FLS2. These studies have revealed critical regions/residues of FLS2 and post-translational modifications that regulate the abundance and activity of this receptor. In this review, we present the current knowledge on the structural mechanism of the FLS2–flg22 interaction and subsequent receptor-mediated signalling.The plasma membrane-localised FLAGELLIN SENSING 2 (FLS2) receptor is an important component of plant immunity against potentially pathogenic bacteria, acting to recognise the conserved flg22 peptide of flagellin. FLS2 shares the common structure of transmembrane receptor kinases with a receptor-like ectodomain composed of leucine-rich repeats (LRR) and an active intracellular kinase domain. Upon ligand binding, FLS2 dimerises with the regulatory LRR-receptor kinase BRI1-associated kinase 1, which in turn triggers downstream signalling cascades. Although lacking crystal structure data, recent advances have been made in our understanding of flg22 recognition based on structural and functional analyses of FLS2. These studies have revealed critical regions/residues of FLS2 and post-translational modifications that regulate the abundance and activity of this receptor. In this review, we present the current knowledge on the structural mechanism of the FLS2–flg22 interaction and subsequent receptor-mediated signalling.

Light exposure of Arabidopsis seedlings causes rapid de‐stabilization as well as selective post‐translational inactivation of the repressor of photomorphogenesis SPA2

The COP1/SPA complex acts as an E3 ubiquitin ligase to repress photomorphogenesis by targeting activators of the light response for degradation. Genetic analysis has shown that the four members of the SPA gene family (SPA1-SPA4) have overlapping but distinct functions. In particular, SPA1 and SPA2 differ in that SPA1 encodes a potent repressor in light- and dark-grown seedlings, but SPA2 fully loses its function when seedlings are exposed to light, indicating that SPA2 function is hyper-inactivated by light. Here, we have used chimeric SPA1/SPA2 constructs to show that the distinct functions of SPA1 and SPA2 genes in light-grown seedlings are due to the SPA protein sequences and independent of the SPA promoter sequences. Biochemical analysis of SPA1 and SPA2 protein levels shows that light exposure leads to rapid proteasomal degradation of SPA2, and, more weakly, of SPA1, but not of COP1. This suggests that light inactivates the COP1/SPA complex partly by reducing SPA protein levels. Although SPA2 was more strongly degraded than SPA1, this was not the sole reason for the lack of SPA2 function in the light. We found that the SPA2 protein is inherently incapable of repressing photomorphogenesis in light-grown seedlings. The data therefore indicate that light inactivates the function of SPA2 through a post-translational mechanism that eliminates the activity of the remaining SPA2 protein in the cell.

Hop-on hop-off: importin-α-guided tours to the nucleus in innate immune signaling

Nuclear translocation of immune regulatory proteins and signal transducers is an essential process in animal and plant defense signaling against pathogenic microbes. Import of proteins containing a nuclear localization signal (NLS) into the nucleus is mediated by nuclear transport receptors termed importins, typically dimers of a cargo-binding α-subunit and a β-subunit that mediates translocation through the nuclear pore complex. Here, we review recent reports of importin-α cargo specificity and mutant phenotypes in plant- and animal–microbe interactions. Using homology modeling of the NLS-binding cleft of nine predicted Arabidopsis α-importins and analyses of their gene expression patterns, we discuss functional redundancy and specialization within this transport receptor family. In addition, we consider how pathogen effector proteins that promote infection by manipulating host cell nuclear processes might compete with endogenous cargo proteins for nuclear uptake.

Mechanisms of Nuclear Suppression of Host Immunity by Effectors from the Arabidopsis Downy Mildew Pathogen Hyaloperonospora arabidopsidis (Hpa)

Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria additional to their more characterized role of suppressing plant defense. Recent studies suggest that effectors may manipulate host transcription or other nuclear regulatory components for the benefit of pathogen development. However, the specific mechanisms by which these effectors promote susceptibility remain unclear. Of two recent screenings, we identified 15 nuclear-localized Hpa effectors (HaRxLs) that interact directly or indirectly with host nuclear components. When stably expressed in planta, nuclear HaRxLs cause diverse developmental phenotypes highlighting that nuclear effectors might interfere with fundamental plant regulatory mechanisms. Here, we report recent advances in understanding how a pathogen can manipulate nuclear processes in order to cause disease.