Poly(ADP-ribose)-binding protein RCD1 is a plant PARylation reader regulated by Photoregulatory Protein Kinases.

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs.

Functional requirement of the Arabidopsis importin-α nuclear transport receptor family in autoimmunity mediated by the NLR protein SNC1.

IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is one of nine importin-α isoforms in Arabidopsis that recruit nuclear localization signal-containing cargo proteins to the nuclear import machinery. IMP-α3/MOS6 is required genetically for full autoimmunity of the nucleotide-binding leucine-rich repeat immune receptor mutant snc1 (suppressor of npr1-1, constitutive 1) and MOS6 also contributes to basal disease resistance. Here, we investigated the contribution of the other importin-α genes to both types of immune responses, and we analyzed potential interactions of all importin-α isoforms with SNC1. By using reverse-genetic analyses in Arabidopsis and protein-protein interaction assays in Nicotiana benthamiana, we provide evidence that among the nine α-importins in Arabidopsis, IMP-α3/MOS6 is the main nuclear transport receptor of SNC1, and that IMP-α3/MOS6 is required selectively for autoimmunity of snc1 and basal resistance to mildly virulent Pseudomonas syringae in Arabidopsis.

Nuclear Import of Arabidopsis Poly(ADP-Ribose) Polymerase 2 Is Mediated by Importin-α and a Nuclear Localization Sequence Located Between the Predicted SAP Domains.

Proteins of the Poly(ADP-Ribose) Polymerase (PARP) family modify target proteins by covalent attachment of ADP-ribose moieties onto amino acid side chains. In Arabidopsis, PARP proteins contribute to repair of DNA lesions and modulate plant responses to various abiotic and biotic stressors. Arabidopsis PARP1 and PARP2 are nuclear proteins and given that their molecular weights exceed the diffusion limit of nuclear pore complexes, an active import mechanism into the nucleus is likely. Here we use confocal microscopy of fluorescent protein-tagged Arabidopsis PARP2 and PARP2 deletion constructs in combination with site-directed mutagenesis to identify a nuclear localization sequence in PARP2 that is required for nuclear import. We report that in co-immunoprecipitation assays PARP2 interacts with several isoforms of the importin-α group of nuclear transport adapters and that PARP2 binding to IMPORTIN-α2 is mediated by the identified nuclear localization sequence. Our results demonstrate that PARP2 is a cargo protein of the canonical importin-α/ß nuclear import pathway.

Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to RADICAL-INDUCED CELL DEATH1.

The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL-INDUCED CELL DEATH1 (RCD1). We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes. We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)-induced defense genes and alters plant growth responses to light. HaRxL106-mediated suppression of immunity is abolished in RCD1 loss-of-function mutants. We report that RCD1-type proteins are phosphorylated, and we identified Mut9-like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1-interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression compared with wild-type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling. Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity.

Nucleoporin-Regulated MAP Kinase Signaling in Immunity to a Necrotrophic Fungal Pathogen.

Pathogen-responsive mitogen-activated protein kinase (MAPK or MPK) cascades relay signals from activated immune receptors across the nuclear envelope to intranuclear targets. However, in plants, little is known about the spatial control of MAPK signaling. Here, we report that the Arabidopsis (Arabidopsis thaliana) nuclear pore complex protein Nup88/MOS7 is essential for immunity to the necrotrophic fungus Botrytis cinerea The mos7-1 mutation, causing a four-amino acid deletion, compromises B. cinerea-induced activation of the key immunoregulatory MAPKs MPK3/MPK6 and reduces MPK3 protein levels posttranscriptionally. Furthermore, MOS7 contributes to retaining a sufficient MPK3 abundance in the nucleus, which is required for full immunity to B. cinerea Finally, we present a structural model of MOS7 and show that the mos7-1 mutation compromises interactions with Nup98a/b, two phenylalanine-glycine repeat nucleoporins implicated in maintaining the selective nuclear pore complex permeability barrier. Together, our analysis uncovered MOS7 and Nup98 as novel components of plant immunity toward a necrotrophic pathogen and provides mechanistic insights into how these nucleoporins coordinate nucleocytoplasmic transport to mount a robust immune response.

The functional divergence between SPA1 and SPA2 in Arabidopsis photomorphogenesis maps primarily to the respective N-terminal kinase-like domain.

BACKGROUND: Plants have evolved complex mechanisms to adapt growth and development to the light environment. The COP1/SPA complex is a key repressor of photomorphogenesis in dark-grown Arabidopsis plants and acts as an E3 ubiquitin ligase to ubiquitinate transcription factors involved in the light response. In the light, COP1/SPA activity is inhibited by photoreceptors, thereby allowing accumulation of these transcription factors and a subsequent light response. Previous results have shown that the four members of the SPA family exhibit partially divergent functions. In particular, SPA1 and SPA2 strongly differ in their responsiveness to light, while they have indistinguishable activities in darkness. The much higher light-responsiveness of SPA2 is partially explained by the much stronger light-induced degradation of SPA2 when compared to SPA1. Here, we have conducted SPA1/SPA2 domain swap experiments to identify the protein domain(s) responsible for the functional divergence between SPA1 and SPA2. RESULTS: We have individually swapped the three domains between SPA1 and SPA2 - the N-terminal kinase-like domain, the coiled-coil domain and the WD-repeat domain - and expressed them in spa mutant Arabidopsis plants. The phenotypes of transgenic seedlings show that the respective N-terminal kinase-like domain is primarily responsible for the respective light-responsiveness of SPA1 and SPA2. Furthermore, the most divergent part of the N-terminal domain was sufficient to confer a SPA1- or SPA2-like activity to the respective SPA protein. The stronger light-induced degradation of SPA2 when compared to SPA1 was also primarily conferred by the SPA2 N-terminal domain. At last, the different affinities of SPA1 and SPA2 for cryptochrome 2 are defined by the N-terminal domain of the respective SPA protein. In contrast, both SPA1 and SPA2 similarly interacted with COP1 in light-grown seedlings. CONCLUSIONS: Our results show that the distinct activities and protein stabilities of SPA1 and SPA2 in light-grown seedlings are primarily encoded by their N-terminal kinase-like domains. Similarly, the different affinities of SPA1 and SPA2 for cry2 are explained by their respective N-terminal domain. Hence, after a duplication event during evolution, the N-terminal domains of SPA1 and SPA2 underwent subfunctionalization, possibly to allow optimal adaptation of growth and development to a changing light environment.

A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain.

Probing formation of cargo/importin-α transport complexes in plant cells using a pathogen effector.

Importin-αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin-α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin-α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin-α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co-opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin-α paralogs from Arabidopsis thaliana. A crystal structure of the importin-α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin-αs expressed in rosette leaves have an almost identical NLS-binding site. Comparison of the importin-α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin-α, sequence variation at the importin-α NLS-binding sites and tissue-specific expression levels of importin-αs determine formation of cargo/importin-α transport complexes in plant cells.

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.

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.

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

Although a plethora of nuclear envelope (NE) transmembrane proteins (NETs) have been identified in opisthokonts, plant NETs are largely unknown. The only known NET homologues in plants are Sad1/UNC-84 (SUN) proteins, which bind Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. Therefore, de novo identification of plant NETs is necessary. Based on similarities between opisthokont KASH proteins and the only known plant KASH proteins, WPP domain-interacting proteins, we used a computational method to identify the KASH subset of plant NETs. Ten potential plant KASH protein families were identified, and five candidates from four of these families were verified for their NE localization, depending on SUN domain interaction. Of those, Arabidopsis thaliana SINE1 is involved in actin-dependent nuclear positioning in guard cells, whereas its paralogue SINE2 contributes to innate immunity against an oomycete pathogen. This study dramatically expands our knowledge of plant KASH proteins and suggests that plants and opisthokonts have recruited different KASH proteins to perform NE regulatory functions.

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.

Arabidopsis TNL-WRKY domain receptor RRS1 contributes to temperature-conditioned RPS4 auto-immunity.

In plant effector-triggered immunity (ETI), intracellular nucleotide binding-leucine rich repeat (NLR) receptors are activated by specific pathogen effectors. The Arabidopsis TIR (Toll-Interleukin-1 receptor domain)-NLR (denoted TNL) gene pair, RPS4 and RRS1, confers resistance to Pseudomonas syringae pv tomato (Pst) strain DC3000 expressing the Type III-secreted effector, AvrRps4. Nuclear accumulation of AvrRps4, RPS4, and the TNL resistance regulator EDS1 is necessary for ETI. RRS1 possesses a C-terminal "WRKY" transcription factor DNA binding domain suggesting that important RPS4/RRS1 recognition and/or resistance signaling events occur at the nuclear chromatin. In Arabidopsis accession Ws-0, the RPS4(Ws) /RRS1(Ws) allelic pair governs resistance to Pst/AvrRps4 accompanied by host programed cell death (pcd). In accession Col-0, RPS4(Col) /RRS1(Col) effectively limits Pst/AvrRps4 growth without pcd. Constitutive expression of HA-StrepII tagged RPS4(Col) (in a 35S:RPS4-HS line) confers temperature-conditioned EDS1-dependent auto-immunity. Here we show that a high (28°C, non-permissive) to moderate (19°C, permissive) temperature shift of 35S:RPS4-HS plants can be used to follow defense-related transcriptional dynamics without a pathogen effector trigger. By comparing responses of 35S:RPS4-HS with 35S:RPS4-HS rrs1-11 and 35S:RPS4-HS eds1-2 mutants, we establish that RPS4(Col) auto-immunity depends entirely on EDS1 and partially on RRS1(Col) . Examination of gene expression microarray data over 24 h after temperature shift reveals a mainly quantitative RRS1(Col) contribution to up- or down-regulation of a small subset of RPS4(Col) -reprogramed, EDS1-dependent genes. We find significant over-representation of WRKY transcription factor binding W-box cis-elements within the promoters of these genes. Our data show that RRS1(Col) contributes to temperature-conditioned RPS4(Col) auto-immunity and are consistent with activated RPS4(Col) engaging RRS1(Col) for resistance signaling.

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 ß-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.

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.

mADP-RTs: versatile virulence factors from bacterial pathogens of plants and mammals.

Mono ADP-ribosyltransferases (mADP-RTs) are a family of enzymes that cleave NAD(+) and covalently attach the ADP-ribosyl moiety to target proteins. mADP-RTs are well established as important virulence factors of bacteria that infect mammals. Cholera toxin, pertussis toxin, and diphtheria toxin are three of the best-known examples of mADP-RTs. They modify host target proteins in order to promote infection and/or killing of the host cell. Despite low sequence similarity at the primary amino acid level, mADP-RTs share a conserved core catalytic fold and structural biology has made important contributions to elucidating how mADP-RTs modify mammalian host targets. Recently, mADP-RTs were shown to be present in plant pathogenic bacteria, suggesting that mADP-RTs are also important virulence factors of plant pathogens. Crystal structures of plant pathogenic bacterial mADP-RTs are also now available. Here we review the structure/function of mADP-RTs from pathogens of mammals and plants, highlighting both commonalities and differences.

Arabidopsis EDS1 connects pathogen effector recognition to cell compartment-specific immune responses.

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.

Crystallization and preliminary X-ray analysis of the RXLR-type effector RXLR3 from the oomycete pathogen Hyaloperonospora arabidopsidis.

Manipulating defence responses in infected host cells is a prerequisite for filamentous plant pathogens to complete their life cycle on infected host plants. During infection of its host Arabidopsis thaliana, the oomycete pathogen Hyaloperonospora arabidopsidis secretes numerous RXLR-type effector proteins, some of which are translocated into host cells. RXLR-type effectors share conserved N-terminal translocation motifs but show high diversity in their C-terminal `effector domains' that manipulate host defence mechanisms. Therefore, obtaining structural information on the effector domains of RXLR-type effectors will contribute to elucidating their molecular-virulence functions in infected host cells. Here, the expression, purification and crystallization of the effector domain of RXLR3 from H. arabidopsidis isolate Waco9 are reported. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 61.49, b = 27.99, c = 37.59 Å. X-ray data were collected to a resolution of 1.8 Å from a single crystal using synchrotron radiation.