Oomycete Pathogen Hyaloperonospora Arabidopsidis Research Articles

Pathogen challenge in Arabidopsis cotyledons induces enhanced disease resistance at newly formed rosette leaves via sustained upregulation of WRKY70.

Pathogenic microorganisms often target seedlings shortly after germination. If plants exhibit resistance or resilience to pathogens, those exposed to pathogen challenge may grow further and form new unchallenged leaves. The purpose of this study was to examine disease resistance in the newly formed leaves of plants subjected to pathogen challenge. We used Arabidopsis thaliana and the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) as the model pathosystem. We found that Arabidopsis seedlings primarily challenged with the avirulent isolate Hpa exhibited enhanced disease resistance against the virulent isolate Hpa in newly formed rosette leaves (NFRLs). Our observations indicated that the transcript levels of the transcription factor gene WRKY70, which is essential for full resistance to the virulent isolate HpaNoco2, were elevated and maintained at high levels in the NFRLs. In contrast, the transcript levels of the salicylic acid marker gene PR1 and systemic acquired resistance-related genes did not exhibit sustained elevation. The maintenance of increased transcript levels of WRKY70 operated independently of non-expressor of pathogenesis-related gene 1. These findings suggest that prolonged upregulation of WRKY70 represents a defensive state synchronized with plant development to ensure survival against subsequent infections.

A fungal RNA-dependent RNA polymerase is a novel player in plant infection and cross-kingdom RNA interference.

Small RNAs act as fungal pathogen effectors that silence host target genes to promote infection, a virulence mechanism termed cross-kingdom RNA interference (RNAi). The essential pathogen factors of cross-kingdom small RNA production are largely unknown. We here characterized the RNA-dependent RNA polymerase (RDR)1 in the fungal plant pathogen Botrytis cinerea that is required for pathogenicity and cross-kingdom RNAi. B. cinerea bcrdr1 knockout (ko) mutants exhibited reduced pathogenicity and loss of cross-kingdom small RNAs. We developed a "switch-on" GFP reporter to study cross-kingdom RNAi in real-time within the living plant tissue which highlighted that bcrdr1 ko mutants were compromised in cross-kingdom RNAi. Moreover, blocking seven pathogen cross-kingdom small RNAs by expressing a short-tandem target mimic RNA in transgenic Arabidopsis thaliana led to reduced infection levels of the fungal pathogen B. cinerea and the oomycete pathogen Hyaloperonospora arabidopsidis. These results demonstrate that cross-kingdom RNAi is significant to promote host infection and making pathogen small RNAs an effective target for crop protection.

GIGANTEA regulates PAD4 transcription to promote pathogen defense against Hyaloperonospora arabidopsidis in Arabidopsis thaliana

ABSTRACT Plants have evolved a network of complex signaling pathways that allow them to cope with the fluctuations of internal and external environmental cues. GIGANTEA (GI), a well-known, highly conserved plant nuclear protein, has been shown to regulate multiple biological functions in plants such as circadian rhythm, light signaling, cold tolerance, hormone signaling, and photoperiodic flowering. Recently, the role of GI in disease tolerance against different pathogens has come to light; however, a detailed mechanism to understand the role of GI in pathogen defense remains largely unexplained. Here, we report that GIGANTEA is upregulated upon infection with a virulent oomycete pathogen, Hyaloperonospora arabidopsidis (Hpa), in Arabidopsis thaliana accession Col-0. To investigate the role of GI in Arabidopsis defense, we examined the pathogen infection phenotype of gi mutant plants and found that gi-100 mutant was highly susceptible to Hpa Noco2 infection. Notably, the quantitative real-time PCR showed that PHYTOALEXIN DEFICIENT4 (PAD4) and several PAD4-regulated downstream genes were downregulated upon Noco2 infection in gi-100 mutant as compared to Col-0 plants. Furthermore, the chromatin immunoprecipitation results show that GI can directly bind to the intronic region of the PAD4 gene, which might explain the mechanism of GI function in regulating disease resistance in plants. Taken together, our results suggest that GI expression is induced upon Hpa pathogen infection and GI can regulate the expression of PAD4 to promote resistance against the oomycete pathogen Hyaloperonospora arabidopsidis in Arabidopsis thaliana.

Insect eggs trigger systemic acquired resistance against a fungal and an oomycete pathogen.

Summary Plants are able to detect insect eggs deposited on leaves. In Arabidopsis, eggs of the butterfly species Pieris brassicae (common name large white) induce plant defenses and activate the salicylic acid (SA) pathway. We previously discovered that oviposition triggers a systemic acquired resistance (SAR) against the bacterial hemibiotroph pathogen Pseudomonas syringae.Here, we show that insect eggs or treatment with egg extract (EE) induce SAR against the fungal necrotroph Botrytis cinerea BMM and the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. This response is abolished in ics1, ald1 and fmo1, indicating that the SA pathway and the N‐hydroxypipecolic acid (NHP) pathway are involved.Establishment of EE‐induced SAR in distal leaves potentially involves tryptophan‐derived metabolites, including camalexin. Indeed, SAR is abolished in the biosynthesis mutants cyp79B2 cyp79B3, cyp71a12 cyp71a13 and pad3‐1, and camalexin is toxic to B. cinerea in vitro.This study reveals an interesting mechanism by which lepidopteran eggs interfere with plant–pathogen interactions.

CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.

Sweet basil (Ocimum basilicum) is an economically important allotetraploid (2n = 4x = 48) herb whose global production is threatened by downy mildew disease caused by the obligate biotrophic oomycete, Peronospora belbahrii. Generation of disease resistant cultivars by mutagenesis of susceptibility (S) genes via CRISPR/Cas9 is currently one of the most promising strategies to maintain favored traits while improving disease resistance. Previous studies have identified Arabidopsis DMR6 (Downy Mildew Resistance 6) as an S gene required for pathogenesis of the downy mildew-causing oomycete pathogen Hyaloperonospora arabidopsidis. In this study, a sweet basil homolog of DMR6, designated ObDMR6, was identified in the popular sweet basil cultivar Genoveser and found to exist with a high copy number in the genome with polymorphisms among the variants. Two CRISPR/Cas9 constructs expressing one or two single guide RNAs (sgRNAs) targeting the conserved regions of ObDMR6 variants were generated and used to transform Genoveser via Agrobacterium-mediated transformation. 56 T0 lines were generated, and mutations of ObDMR6 were detected by analyzing the Sanger sequencing chromatograms of an ObDMR6 fragment using the Interference of CRISPR Edits (ICE) software. Among 54 lines containing mutations in the targeted sites, 13 had an indel percentage greater than 96% suggesting a near-complete knockout (KO) of ObDMR6. Three representative transgene-free lines with near-complete KO of ObDMR6 determined by ICE were identified in the T1 segregating populations derived from three independent T0 lines. The mutations were further confirmed using amplicon deep sequencing. Disease assays conducted on T2 seedlings of the above T1 lines showed a reduction in production of sporangia by 61–68% compared to the wild-type plants and 69–93% reduction in relative pathogen biomass determined by quantitative PCR (qPCR). This study not only has generated transgene-free sweet basil varieties with improved downy mildew resistance, but also contributed to our understanding of the molecular interactions of sweet basil-P. belbahrii.

A family of pathogen-induced cysteine-rich transmembrane proteins is involved in plant disease resistance

Main conclusionOverexpression of pathogen-induced cysteine-rich transmembrane proteins (PCMs) in Arabidopsis thaliana enhances resistance against biotrophic pathogens and stimulates hypocotyl growth, suggesting a potential role for PCMs in connecting both biological processes.Plants possess a sophisticated immune system to protect themselves against pathogen attack. The defense hormone salicylic acid (SA) is an important player in the plant immune gene regulatory network. Using RNA-seq time series data of Arabidopsis thaliana leaves treated with SA, we identified a largely uncharacterized SA-responsive gene family of eight members that are all activated in response to various pathogens or their immune elicitors and encode small proteins with cysteine-rich transmembrane domains. Based on their nucleotide similarity and chromosomal position, the designated Pathogen-induced Cysteine-rich transMembrane protein (PCM) genes were subdivided into three subgroups consisting of PCM1-3 (subgroup I), PCM4-6 (subgroup II), and PCM7-8 (subgroup III). Of the PCM genes, only PCM4 (also known as PCC1) has previously been implicated in plant immunity. Transient expression assays in Nicotiana benthamiana indicated that most PCM proteins localize to the plasma membrane. Ectopic overexpression of the PCMs in Arabidopsis thaliana resulted in all eight cases in enhanced resistance against the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis Noco2. Additionally, overexpression of PCM subgroup I genes conferred enhanced resistance to the hemi-biotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000. The PCM-overexpression lines were found to be also affected in the expression of genes related to light signaling and development, and accordingly, PCM-overexpressing seedlings displayed elongated hypocotyl growth. These results point to a function of PCMs in both disease resistance and photomorphogenesis, connecting both biological processes, possibly via effects on membrane structure or activity of interacting proteins at the plasma membrane.

Arabidopsis downy mildew effector HaRxLL470 suppresses plant immunity by attenuating the DNA-binding activity of bZIP transcription factor HY5.

The oomycete pathogen Hyaloperonospora arabidopsidis delivers diverse effector proteins into host plant cells to suppress the plant's innate immunity. In this study, we investigate the mechanism of action of a conserved RxLR effector, HaRxLL470, in suppressing plant immunity. Genomic, molecular and biochemical analyses were performed to investigate the function of HaRxLL470 and the mechanism of the interaction between HaRxLL470 and the target host protein during H. arabidopsidis infection. We report that HaRxLL470 enhances plant susceptibility to H. arabidopsidis isolate Noco2 by interacting with the host photomorphogenesis regulator protein HY5. Our results demonstrate that HY5 is not only an important component in the regulation of light signalling, but also positively regulates host plant immunity against H. arabidopsidis by transcriptional activation of defense-related genes. We show that the interaction between HaRxLL470 and HY5 compromises the function of HY5 as a transcription factor by attenuating its DNA-binding activity. The present study demonstrates that HY5 positively regulates host plant defense against H. arabidopsidis whereas HaRxLL470, a conserved RxLR effector across oomycete pathogens, enhances pathogenicity by interacting with HY5 and suppressing transcriptional activation of defense-related genes.

Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis.

Over the last decade, it has been noticed that microbial pathogens and pests deliver small RNA (sRNA) effectors into their host plants to manipulate plant physiology and immunity for infection, known as cross kingdom RNA interference. In this process, fungal and oomycete parasite sRNAs hijack the plant ARGONAUTE (AGO)/RNA-induced silencing complex to post-transcriptionally silence host target genes. We hereby describe the methodological details of how we recovered cross kingdom sRNA effectors of the oomycete pathogen Hyaloperonospora arabidopsidis during infection of its host plant Arabidopsis thaliana. This Bio-protocol contains two parts: first, a detailed description on the procedure of plant AGO/sRNA co-immunopurification and sRNA recovery for Illumina high throughput sequencing analysis. Second, we explain how to perform bioinformatics analysis of sRNA sequence reads using a Galaxy server. In principle, this protocol is suitable to investigate AGO-bound sRNAs from diverse host plants and plant-interacting (micro)organisms.

A rust fungal effector binds plant DNA and modulates transcription

The basidiomycete Melampsora larici-populina causes poplar rust disease by invading leaf tissues and secreting effector proteins through specialized infection structures known as haustoria. The mechanisms by which rust effectors promote pathogen virulence are poorly understood. The present study characterized Mlp124478, a candidate effector of M. larici-populina. We used the models Arabidopsis thaliana and Nicotiana benthamiana to investigate the function of Mlp124478 in plant cells. We established that Mlp124478 accumulates in the nucleus and nucleolus, however its nucleolar accumulation is not required to promote growth of the oomycete pathogen Hyaloperonospora arabidopsidis. Stable constitutive expression of Mlp124478 in A. thaliana repressed the expression of genes involved in immune responses, and also altered leaf morphology by increasing the waviness of rosette leaves. Chip-PCR experiments showed that Mlp124478 associats'e with the TGA1a-binding DNA sequence. Our results suggest that Mlp124478 exerts a virulence activity and binds the TGA1a promoter to suppress genes induced in response to pathogen infection.

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

Summary 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.

STOREKEEPER RELATED1/G-Element Binding Protein (STKR1) Interacts with Protein Kinase SnRK1.

Sucrose nonfermenting related kinase1 (SnRK1) is a conserved energy sensor kinase that regulates cellular adaptation to energy deficit in plants. Activation of SnRK1 leads to the down-regulation of ATP-consuming biosynthetic processes and the stimulation of energy-generating catabolic reactions by transcriptional reprogramming and posttranslational modifications. Although considerable progress has been made during the last years in understanding the SnRK1 signaling pathway, many of its components remain unidentified. Here, we show that the catalytic α-subunits KIN10 and KIN11 of the Arabidopsis (Arabidopsis thaliana) SnRK1 complex interact with the STOREKEEPER RELATED1/G-Element Binding Protein (STKR1) inside the plant cell nucleus. Overexpression of STKR1 in transgenic Arabidopsis plants led to reduced growth, a delay in flowering, and strongly attenuated senescence. Metabolite profiling revealed that the transgenic lines exhausted their carbohydrates during the dark period to a greater extent than the wild type and accumulated a range of amino acids. At the global transcriptome level, genes affected by STKR1 overexpression were broadly associated with systemic acquired resistance, and transgenic plants showed enhanced resistance toward a virulent strain of the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis Noco2. We discuss a possible connection of STKR1 function, SnRK1 signaling, and plant immunity.

A Role of the FUZZY ONIONS LIKE Gene in Regulating Cell Death and Defense in Arabidopsis.

Programmed cell death (PCD) is critical for development and responses to environmental stimuli in many organisms. FUZZY ONIONS (FZO) proteins in yeast, flies, and mammals are known to affect mitochondrial fusion and function. Arabidopsis FZO-LIKE (FZL) was shown as a chloroplast protein that regulates chloroplast morphology and cell death. We cloned the FZL gene based on the lesion mimic phenotype conferred by an fzl mutation. Here we provide evidence to support that FZL has evolved new function different from its homologs from other organisms. We found that fzl mutants showed enhanced disease resistance to the bacterial pathogen Pseudomonas syringae and the oomycete pathogen Hyaloperonospora arabidopsidis. Besides altered chloroplast morphology and cell death, fzl showed the activation of reactive oxygen species (ROS) and autophagy pathways. FZL and the defense signaling molecule salicylic acid form a negative feedback loop in defense and cell death control. FZL did not complement the yeast strain lacking the FZO1 gene. Together these data suggest that the Arabidopsis FZL gene is a negative regulator of cell death and disease resistance, possibly through regulating ROS and autophagy pathways in the chloroplast.

Identification of Methylosome Components as Negative Regulators of Plant Immunity Using Chemical Genetics

Nucleotide-binding leucine-rich repeat (NLR) proteins serve as immune receptors in both plants and animals. To identify components required for NLR-mediated immunity, we designed and carried out a chemical genetics screen to search for small molecules that can alter immune responses in Arabidopsis thaliana. From 13 600 compounds, we identified Ro 8-4304 that was able to specifically suppress the severe autoimmune phenotypes of chs3-2D (chilling sensitive 3, 2D), including the arrested growth morphology and heightened PR (Pathogenesis Related) gene expression. Further, six Ro 8-4304 insensitive mutants were uncovered from the Ro 8-4304-insensitive mutant (rim) screen using a mutagenized chs3-2D population. Positional cloning revealed that rim1 encodes an allele of AtICln (I, currents; Cl, chloride; n, nucleotide). Genetic and biochemical analysis demonstrated that AtICln is in the same protein complex with the methylosome components small nuclear ribonucleoprotein D3b (SmD3b) and protein arginine methyltransferase 5 (PRMT5), which are required for the biogenesis of small nuclear ribonucleoproteins (snRNPs) involved in mRNA splicing. Double mutant analysis revealed that SmD3b is also involved in the sensitivity to Ro 8-4304, and the prmt5-1 chs3-2D double mutant is lethal. Loss of AtICln, SmD3b, or PRMT5 function results in enhanced disease resistance against the virulent oomycete pathogen Hyaloperonospora arabidopsidis Noco2, suggesting that mRNA splicing plays a previously unknown negative role in plant immunity. The successful implementation of a high-throughput chemical genetic screen and the identification of a small-molecule compound affecting plant immunity indicate that chemical genetics is a powerful tool to study whole-organism plant defense pathways.

AtCDC48A is involved in the turnover of an NLR immune receptor.

Plants rely on different immune receptors to recognize pathogens and defend against pathogen attacks. Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play a major role as intracellular immune receptors. Their homeostasis must be maintained at optimal levels in order to effectively recognize pathogens without causing autoimmunity. Previous studies have shown that the activity of the ubiquitin-proteasome system is essential to prevent excessive accumulation of NLR proteins such as Suppressor of NPR1, Constitutive 1 (SNC1). Attenuation of the ubiquitin E3 ligase SCFCPR1 (Constitutive expressor of Pathogenesis Related genes 1) or the E4 protein MUSE3 (Mutant, SNC1-Enhancing 3) leads to NLR accumulation and autoimmunity. In the current study, we report the identification of AtCDC48A as a negative regulator of NLR-mediated immunity. Plants carrying Atcdc48A-4, a partial loss-of-function allele of AtCDC48A, exhibit dwarf morphology and enhanced disease resistance to the oomycete pathogen Hyaloperonospora arabidopsidis (H.a.) Noco2. The SNC1 level is increased in Atcdc48A-4 plants and AtCDC48A interacts with MUSE3 in co-immunoprecipitation experiments, supporting a role for AtCDC48A in NLR turnover. While Arabidopsis contains four other paralogs of AtCDC48A, knockout mutants of these genes do not show obvious immunity-related phenotypes, suggesting functional divergence within this family. As an AAA-ATPase, AtCDC48A likely serves to process the poly-ubiquitinated NLR substrate for final protein degradation by the 26S proteasome.

Spatial dissection of the Arabidopsis thaliana transcriptional response to downy mildew using Fluorescence Activated Cell Sorting.

Changes in gene expression form a crucial part of the plant response to infection. In the last decade, whole-leaf expression profiling has played a valuable role in identifying genes and processes that contribute to the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, with some pathogens such as downy mildew caused by the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response encompassing responses of non-infected as well as infected cells within the leaf. Highly localized expression changes that occur in infected cells may be diluted by the comparative abundance of non-infected cells. Furthermore, local and systemic Hpa responses of a differing nature may become conflated. To address this we applied the technique of Fluorescence Activated Cell Sorting (FACS), typically used for analyzing plant abiotic responses, to the study of plant-pathogen interactions. We isolated haustoriated (Hpa-proximal) and non-haustoriated (Hpa-distal) cells from infected seedling samples using FACS, and measured global gene expression. When compared with an uninfected control, 278 transcripts were identified as significantly differentially expressed, the vast majority of which were differentially expressed specifically in Hpa-proximal cells. By comparing our data to previous, whole organ studies, we discovered many highly locally regulated genes that can be implicated as novel in the Hpa response, and that were uncovered for the first time using our sensitive FACS technique.

The Chromatin Remodeler SPLAYED Negatively Regulates SNC1-Mediated Immunity.

SNC1 (SUPPRESSOR OF NPR1, CONSTITUTIVE 1) is one of a suite of intracellular Arabidopsis NOD-like receptor (NLR) proteins which, upon activation, result in the induction of defense responses. However, the molecular mechanisms underlying NLR activation and the subsequent provocation of immune responses are only partially characterized. To identify negative regulators of NLR-mediated immunity, a forward genetic screen was undertaken to search for enhancers of the dwarf, autoimmune gain-of-function snc1 mutant. To avoid lethality resulting from severe dwarfism, the screen was conducted using mos4 (modifier of snc1, 4) snc1 plants, which display wild-type-like morphology and resistance. M2 progeny were screened for mutant, snc1-enhancing (muse) mutants displaying a reversion to snc1-like phenotypes. The muse9 mos4 snc1 triple mutant was found to exhibit dwarf morphology, elevated expression of the pPR2-GUS defense marker reporter gene and enhanced resistance to the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. Via map-based cloning and Illumina sequencing, it was determined that the muse9 mutation is in the gene encoding the SWI/SNF chromatin remodeler SYD (SPLAYED), and was thus renamed syd-10. The syd-10 single mutant has no observable alteration from wild-type-like resistance, although the syd-4 T-DNA insertion allele displays enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Transcription of SNC1 is increased in both syd-4 and syd-10. These data suggest that SYD plays a subtle, specific role in the regulation of SNC1 expression and SNC1-mediated immunity. SYD may work with other proteins at the chromatin level to repress SNC1 transcription; such regulation is important for fine-tuning the expression of NLR-encoding genes to prevent unpropitious autoimmunity.

Recognition and activation domains contribute to allele-specific responses of an Arabidopsis NLR receptor to an oomycete effector protein.

In plants, specific recognition of pathogen effector proteins by nucleotide-binding leucine-rich repeat (NLR) receptors leads to activation of immune responses. RPP1, an NLR from Arabidopsis thaliana, recognizes the effector ATR1, from the oomycete pathogen Hyaloperonospora arabidopsidis, by direct association via C-terminal leucine-rich repeats (LRRs). Two RPP1 alleles, RPP1-NdA and RPP1-WsB, have narrow and broad recognition spectra, respectively, with RPP1-NdA recognizing a subset of the ATR1 variants recognized by RPP1-WsB. In this work, we further characterized direct effector recognition through random mutagenesis of an unrecognized ATR1 allele, ATR1-Cala2, screening for gain-of-recognition phenotypes in a tobacco hypersensitive response assay. We identified ATR1 mutants that a) confirm surface-exposed residues contribute to recognition by RPP1, and b) are recognized by and activate the narrow-spectrum allele RPP1-NdA, but not RPP1-WsB, in co-immunoprecipitation and bacterial growth inhibition assays. Thus, RPP1 alleles have distinct recognition specificities, rather than simply different sensitivity to activation. Using chimeric RPP1 constructs, we showed that RPP1-NdA LRRs were sufficient for allele-specific recognition (association with ATR1), but insufficient for receptor activation in the form of HR. Additional inclusion of the RPP1-NdA ARC2 subdomain, from the central NB-ARC domain, was required for a full range of activation specificity. Thus, cooperation between recognition and activation domains seems to be essential for NLR function.

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.

Convergent targeting of a common host protein-network by pathogen effectors from three kingdoms of life.

While conceptual principles governing plant immunity are becoming clear, its systems-level organization and the evolutionary dynamic of the host-pathogeninterface are still obscure. We generated a systematic protein-protein interaction network of virulence effectors from the ascomycete pathogen Golovinomyces orontii and Arabidopsis thaliana host proteins. We combined this data set with corresponding data for the eubacterial pathogen Pseudomonas syringae and the oomycete pathogen Hyaloperonospora arabidopsidis. The resulting network identifies host proteins onto which intraspecies and interspecies pathogen effectors converge. Phenotyping of 124 Arabidopsis effector-interactor mutants revealed a correlation between intraspecies and interspecies convergence and several altered immune response phenotypes. Several effectors and the most heavily targeted host protein colocalized in subnuclear foci. Products of adaptively selected Arabidopsis genes are enriched for interactions with effector targets. Our data suggest the existenceof a molecular host-pathogen interface that isconserved across Arabidopsis accessions, while evolutionary adaptation occurs in the immediate network neighborhood of effector targets.

Arabidopsis GOLDEN2‐LIKE (GLK) transcription factors activate jasmonic acid (JA)‐dependent disease susceptibility to the biotrophic pathogen Hyaloperonospora arabidopsidis, as well as JA‐independent plant immunity against the necrotrophic pathogen Botrytis cinerea

Arabidopsis thaliana GOLDEN2-LIKE (GLK1 and 2) transcription factors regulate chloroplast development in a redundant manner. Overexpression of AtGLK1 (35S:AtGLK1) in Arabidopsis also confers resistance to the cereal pathogen Fusarium graminearum. To further elucidate the role of GLK transcription factors in plant defence, the Arabidopsis glk1 glk2 double-mutant and 35S:AtGLK1 plants were challenged with the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) Noco2. Compared with Col-0, glk1 glk2 plants were highly resistant to Hpa Noco2, whereas 35S:AtGLK1 plants showed enhanced susceptibility to this pathogen. Genetic studies suggested that AtGLK-mediated plant defence to Hpa Noco2 was partially dependent on salicylic acid (SA) accumulation, but independent of the SA signalling protein NONEXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1). Pretreatment with jasmonic acid (JA) dramatically reversed Hpa Noco2 resistance in the glk1 glk2 double mutant, but only marginally affected the 35S:AtGLK1 plants. In addition, overexpression of AtGLK1 in the JA signalling mutant coi1-16 did not increase susceptibility to Hpa Noco2. Together, our GLK gain-of-function and loss-of-function experiments suggest that GLK acts upstream of JA signalling in disease susceptibility to Hpa Noco2. In contrast, glk1 glk2 plants were more susceptible to the necrotrophic fungal pathogen Botrytis cinerea, whereas 35S:AtGLK1 plants exhibited heightened resistance which could be maintained in the absence of JA signalling. Together, the data reveal that AtGLK1 is involved in JA-dependent susceptibility to the biotrophic pathogen Hpa Noco2 and in JA-independent resistance to the necrotrophic pathogen B. cinerea.