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The downy mildew oomyceteHyaloperonospora arabidopsidis, an obligate filamentous pathogen, infects Arabidopsis by forming feeding structures called haustoria inside host cells. Previous transcriptome analyses revealed host genes are specifically induced during infection; however, RNA profiling from infected tissues may fail to capture key transcriptional events occurring exclusively in haustoriated host cells where the pathogen injects virulence effectors to modulate host immunity. To determine interactions between Arabidopsis andH. arabidopsidisat the cellular level, we devised a new translating ribosome affinity purification system applicable to inducible, including pathogen-responsive, promoters thus enabling haustoriated cell-specific RNA profiling. Among the host genes specifically expressed inH. arabidopsidis-haustoriated cells, we found genes that promote either susceptibility or resistance to the pathogen, providing new insights into the Arabidopsis/downy mildew interaction. We propose that our novel protocol for profiling cell-specific transcripts will be applicable to several stimulus-specific contexts and other plant-pathogen interactions.

The soilborne filamentous fungus Fusarium oxysporum causes devastating diseases of many cultivated plant species. F. oxysporum f. sp. raphani and f. sp. rapae are two of four formae speciales that are pathogenic to Brassicaceae plants. Here, we present high-quality genome sequences of F. oxysporum f. sp. raphani strain Tf1262 and F. oxysporum f. sp. rapae strain Tf1208 that were isolated from radish (Raphanus sativus) and turnip (Brassica rapa var. rapa), respectively. These genome resources should facilitate in-depth investigation of interactions between F. oxysporum and Brassicaceae plants, and enable comparative genomics of the F. oxysporum species complex to uncover how pathogenicity evolved within F. oxysporum. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Transposable elements (TEs) are accumulated in both intergenic and intragenic regions in plant genomes. Intragenic TEs often act as regulatory elements of associated genes and are also co-transcribed with genes, generating chimeric TE-gene transcripts. Despite the potential impact on mRNA regulation and gene function, the prevalence and transcriptional regulation of TE-gene transcripts are poorly understood. By long-read direct RNA sequencing and a dedicated bioinformatics pipeline, “ParasiTE”, we investigated the transcription and RNA processing of TE-gene transcripts in Arabidopsis thaliana. We identified a global production of TE-gene transcripts in thousands of A. thaliana gene loci, with TE sequences often being associated with alternative transcription start sites or transcription termination sites. The epigenetic state of intragenic TEs affects RNAPII elongation and usage of alternative poly(A) signals within TE sequences, regulating alternative TE-gene isoform production. Co-transcription and inclusion of TE-derived sequences into gene transcripts impact regulation of RNA stability and environmental responses of some loci. Our study provides novel insights into TE-gene interactions that contributes to mRNA regulation, transcriptome diversity, and environmental responses in plants.

In plants, many invading microbial pathogens are recognized by cell-surface pattern recognition receptors (PRRs), inducing defense responses; yet how PRRs perceive pathogen sphingolipids remains unclear. Here, we show that the ceramide Pi-Cer D from a plant pathogenic oomycetePhytophthora infestanstriggers defense responses in Arabidopsis. Pi-Cer D is cleaved by an Arabidopsis apoplastic ceramidase, NCER2, and the resulting 9-methyl-branched sphingoid base is recognized by a plasma membrane lectin receptor-like kinase, RDA2. Importantly, 9-methyl-branched sphingoid base, which is unique to microbes, induces plant immune responses by interacting with RDA2. Loss ofRDA2orNCER2function compromised Arabidopsis resistance against an oomycete pathogen, indicating that these are crucial for defense. We provide new insights that help elucidate the recognition mechanisms of pathogen-derived lipid molecules in plants.One Sentence SummaryOomycete-derived ceramide is cleaved into sphingoid base by ceramidase and recognized by an Arabidopsis receptor kinase.

Pseudomonas amygdali pv. tabaci strain 6605 is the bacterial pathogen causing tobacco wildfire disease that has been used as a model for elucidating virulence mechanisms. Here, we present the complete genome sequence of P. amygdali pv. tabaci 6605 as a circular chromosome from reads using a PacBio sequencer.

Many plant pathogenic fungi contain conditionally dispensable (CD) chromosomes that are associated with virulence, but not growth in vitro. Virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes that may contribute significantly to determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. Among a large number of host-specific forms, F. oxysporum f. sp. conglutinans (Focn) can infect Brassicaceae plants including Arabidopsis and cabbage. Here we show that Focn has multiple CD chromosomes involved in virulence but also in vegetative growth, which is an atypical feature of CD chromosomes. We identified specific CD chromosomes that are required for virulence on Arabidopsis, cabbage, or both, and describe a pair of effectors encoded on one of the CD chromosomes that is required for suppression of Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis, but not of other plants. This study provides insight into how host specificity of F. oxysporum may be determined by a pair of effector genes on a transmissible CD chromosome.

Fusarium oxysporum f. sp. cubense is the causal agent of banana Fusarium wilt, also known as Panama disease. Here, we present a high-quality genome sequence of F. oxysporum f. sp. cubense strain 160527. The genome assembly is composed of 12 contigs with a total assembly length of 51,139,495 bp (N50 contig length, 4,884,632 bp).

The original version of this article contained an error in the author affiliations. Oliver J. Furzer was incorrectly associated with Department of Plant Sciences, College of Life Sciences, Wuhan University, 430072, Wuhan, China.This has now been corrected in the HTML version of the article. The PDF version of the article was correct at the time of publication.Furthermore, the original version of this article stated that correspondence and requests for materials should be addressed to Heidelberg.Center.for.Personalized.Oncology, DKFZ-HIPO, DKFZ, Heidelberg 69120Germany S.A. (email: shuta.asai@riken.jp) or to J.D.G.J. (email: jonathan.jones@tsl.ac.uk). The words “Heidelberg.Center.for.Personalized.Oncology, DKFZ-HIPO, DKFZ, Heidelberg 69120Germany” were introduced inadvertently.This has now been corrected in the PDF version of the article. The HTML version of the article was correct at the time of publication.

Pathogen co-evolution with plants involves selection for evasion of host surveillance systems. The oomycete Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis, and race-specific interactions between Arabidopsis accessions and Hpa isolates fit the gene-for-gene model in which host resistance or susceptibility are determined by matching pairs of plant Resistance (R) genes and pathogen Avirulence (AVR) genes. Arabidopsis Col-0 carries R gene RPP4 that confers resistance to Hpa isolates Emoy2 and Emwa1, but its cognate recognized effector(s) were unknown. We report here the identification of the Emoy2 AVR effector gene recognized by RPP4 and show resistance-breaking isolates of Hpa on RPP4-containing Arabidopsis carry the alleles that either are not expressed, or show cytoplasmic instead of nuclear subcellular localization., Plant pathogens have evolved to evade detection by their hosts. Here, Asai et al. show that virulent isolates of the oomycete Hyaloperonospora arabidopsidis can break resistance conferred by the Arabidopsis RPP4 resistance gene via variation in effector expression or subcellular localization.

Plant immune responses are attributable to hierarchical immunities, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). The plant NADPH oxidase, RBOH, is a major player in the pathogen-responsive reactive oxygen species (ROS) burst. Nicotiana benthamiana RBOHB is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Our understanding of the activation mechanisms controlling RBOH during PTI and ETI has increased in recent years. MAPKs are involved in the ETI ROS burst by transactivation of NbRBOHB , but not in the PTI ROS burst. We also introduce mechanisms of RBOH regulation at the post-transcriptional level by diverse protein kinases.

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.

The oomycete pathogenHyaloperonospora arabidopsidis(Hpa) causes downy mildew disease onArabidopsis. During infection,Hpalike other biotrophic pathogens, suppresses activation of plant innate immunity by translocating effector proteins into host cells. Some of these effectors localize to the host cell nucleus where they may manipulate transcriptional reprogramming of plant defense genes. Here we report that the nuclear-localizedHpaeffector HaRxL106, when expressed inArabidopsis, induces shade avoidance and attenuates the transcriptional response to the defense signaling molecule salicylic acid. HaRxL106 interacts with RADICAL-INDUCED CELL DEATH1 (RCD1) and loss of RCD1 function rendersArabidopsisresilient against HaRxL106-mediated suppression of immunity. To further characterize the molecular functions of RCD1 we solved a crystal structure of RCD1’s Poly-(ADP-ribose)-Polymerase (PARP) domain and, based on non-conservation of amino acids constituting the active site of canonical PARPs, conclude that RCD1 has no PARP activity. We report that RCD1-type proteins are phosphorylated and identified histone-modifying Mut9-like kinases (MLKs) as RCD1-interacting proteins. Amlk1,3,4triple mutant exhibits stronger SA-induced defense marker gene expression compared to wild-type plants. Our data suggest that HaRxL106 suppressesArabidopsisinnate immunity by manipulating the function(s) of RCD1 in the host cell nucleus and point towards a role of RCD1 as a transcriptional co-regulator that integrates signals from light and pathogen sensors.

Background Plants are exposed to diverse pathogens and pests, yet most plants are resistant to most plant pathogens. Non-host resistance describes the ability of all members of a plant species to successfully prevent colonization by any given member of a pathogen species. White blister rust caused by Albugo species can overcome non-host resistance and enable secondary infection and reproduction of usually non-virulent pathogens, including the potato late blight pathogen Phytophthora infestans on Arabidopsis thaliana. However, the molecular basis of host defense suppression in this complex plant–microbe interaction is unclear. Here, we investigate specific defense mechanisms in Arabidopsis that are suppressed by Albugo infection. Results Gene expression profiling revealed that two species of Albugo upregulate genes associated with tryptophan-derived antimicrobial metabolites in Arabidopsis. Albugo laibachii-infected tissue has altered levels of these metabolites, with lower indol-3-yl methylglucosinolate and higher camalexin accumulation than uninfected tissue. We investigated the contribution of these Albugo-imposed phenotypes to suppression of non-host resistance to P. infestans. Absence of tryptophan-derived antimicrobial compounds enables P. infestans colonization of Arabidopsis, although to a lesser extent than Albugo-infected tissue. A. laibachii also suppresses a subset of genes regulated by salicylic acid; however, salicylic acid plays only a minor role in non-host resistance to P. infestans. Conclusions Albugo sp. alter tryptophan-derived metabolites and suppress elements of the responses to salicylic acid in Arabidopsis. Albugo sp. imposed alterations in tryptophan-derived metabolites may play a role in Arabidopsis non-host resistance to P. infestans. Understanding the basis of non-host resistance to pathogens such as P. infestans could assist in development of strategies to elevate food security. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0360-z) contains supplementary material, which is available to authorized users.

Two-way ANOVA results from qRT-PCR of tryptophan-derived secondary metabolite genes at 48 hours post P. infestans inoculation. ANOVA table. (DOCX 13 kb)

Primers used in the study. Details of the primers used to conduct qRT-PCR in the study. (DOCX 25 kb)

AlNc14 suppresses benzo-(1,2,3)-thiadiazole-7-carbothioic acid (BTH) and salicylic acid (SA) induction of PR1. To visualize reporter gene induction and pathogen growth in the same leaf, leaves were collected and stained with magenta-GUS to reveal GUS activity, followed by trypan blue to reveal pathogen growth. Leaves of Col-0 pro(PR1)::GUS were previously inoculated with water or AlNc14 (13 dpi) and infiltrated with DMSO (mock), BTH (200 μM) or SA (200 μM) for 8 hours, then stained and examined with a microscope. The percentage of each leaf stained with GUS was determined using ImageJ. (A) Open circles represent mean ± SE of the raw data (percentage of leaf stained) of three independent biological replicates with between two and seven technical replicates per biological replicate (bars left to right n = 10, 12, 13, 15, 14 and 16). (B) Open circles represent mean ± SE of the transformed data (arcsine square root transformation followed by log10 transformation) of three independent biological replicates with between two and seven technical replicates per biological replicate (bars left to right n = 10, 12, 13, 15, 14 and 16). Different letters indicate significant differences P

Two-way ANOVA results from qRT-PCR of tryptophan-derived secondary metabolite genes at 6 hours post P. infestans inoculation. ANOVA table. (DOCX 13 kb)

B. cinerea detects less available camalexin in Albugo-infected tissue. Leaves underwent protein extraction and GUS enzyme activity was determined using a fluorescence-based assay. Results were normalized to B. cinerea weight proportion of each sample using qRT-PCR on Botrytis and Arabidopsis genomic DNA. Open circles and bar dots represent the mean ± SE of three independent biological replicates with three or four technical replicates per biological replicate. Closed, black circles denote the individual data points. Asterisk indicates significant differences measured at P

Calcium-dependent protein kinases (CDPKs) are Ca(2+) sensors that regulate diverse biological processes in plants and apicomplexans. However, how CDPKs discriminate specific substrates in vivo is still largely unknown. Previously, we found that a potato StCDPK5 is dominantly localized to the plasma membrane and activates the plasma membrane NADPH oxidase (RBOH; for respiratory burst oxidase homolog) StRBOHB by direct phosphorylation of the N-terminal region. Here, we report the contribution of the StCDPK5 N-terminal variable (V) domain to activation of StRBOHB in vivo using heterologous expression system in Nicotiana benthamiana. Mutations of N-terminal myristoylation and palmitoylation sites in the V domain eliminated the predominantly plasma membrane localization and the capacity of StCDPK5 to activate StRBOHB in vivo. A tomato SlCDPK2, which also contains myristoylation and palmitoylation sites in its N terminus, phosphorylated StRBOHB in vitro but not in vivo. Functional domains responsible for activation and phosphorylation of StRBOHB were identified by swapping regions for each domain between StCDPK5 and SlCDPK2. The substitution of the V domain of StCDPK5 with that of SlCDPK2 abolished the activation and phosphorylation abilities of StRBOHB in vivo and relocalized the chimeric CDPK to the trans-Golgi network, as observed for SlCDPK2. Conversely, SlCDPK2 substituted with the V domain of StCDPK5 localized to the plasma membrane and activated StRBOHB. These results suggest that the V domains confer substrate specificity in vivo by dictating proper subcellular localization of CDPKs.

Sequential recognition of invading microbes and rapid induction of plant immune responses comprise at least two recognition systems. Early basal defenses are initiated by pathogen-associated molecular patterns and pattern recognition receptors (PRR) in the plasma membrane. Pathogens produce effectors to suppress defense but plants, in turn, can sense such effectors by dominant plant resistance (R) gene products. Plant PRR and R proteins modulate signaling networks for defense responses that rely on rapid production of reactive nitrogen species (RNS) and reactive oxygen species (ROS). Recent research has shown that nitric oxide (NO) mainly mediates biological function through chemical reactions between locally controlled accumulation of RNS and proteins leading to potential alteration of protein function. Many proteins specifically regulated by NO and participating in signaling during plant defense response have been identified, highlighting the physiological relevance of these modifications in plant immunity. ROS function independently or in cooperation with NO during defense, modulating the RNS signaling functions through the entire process. This review provides an overview of current knowledge about regulatory mechanisms for NO burst and signaling, and crosstalk with ROS in response to pathogen attack.

Plant immunity protects plants from numerous potentially pathogenic microbes. The biological network that controls plant inducible immunity must function effectively even when network components are targeted and disabled by pathogen effectors. Network buffering could confer this resilience by allowing different parts of the network to compensate for loss of one another's functions. Networks rich in buffering rely on interactions within the network, but these mechanisms are difficult to study by simple genetic means. Through a network reconstitution strategy, in which we disassemble and stepwise reassemble the plant immune network that mediates Pattern-Triggered-Immunity, we have resolved systems-level regulatory mechanisms underlying the Arabidopsis transcriptome response to the immune stimulant flagellin-22 (flg22). These mechanisms show widespread evidence of interactions among major sub-networks-we call these sectors-in the flg22-responsive transcriptome. Many of these interactions result in network buffering. Resolved regulatory mechanisms show unexpected patterns for how the jasmonate (JA), ethylene (ET), phytoalexin-deficient 4 (PAD4), and salicylate (SA) signaling sectors control the transcriptional response to flg22. We demonstrate that many of the regulatory mechanisms we resolved are not detectable by the traditional genetic approach of single-gene null-mutant analysis. Similar to potential pathogenic perturbations, null-mutant effects on immune signaling can be buffered by the network.

• Potato (Solanum tuberosum) calcium-dependent protein kinase (StCDPK5) has been shown to phosphorylate the N-terminal region of plasma membrane RBOH (respiratory burst oxidase homolog) proteins, and participate in StRBOHB-mediated reactive oxygen species (ROS) burst. The constitutively active form, StCDPK5VK, provides a useful tool for gain-of-function analysis of RBOH in defense responses. • StCDPK5- and StCDPK5VK-green fluorescent protein fusion proteins were predominantly targeted to the plasma membrane, and conditional expression of StCDPK5VK activated StRBOHA-D. The interaction was confirmed by bimolecular fluorescence complementation assay. We generated transgenic potato plants containing StCDPK5VK under the control of a pathogen-inducible promoter to investigate the role of ROS burst on defense responses to blight pathogens. • Virulent isolates of the late blight pathogen Phytophthora infestans and the early blight pathogen Alternaria solani induced hypersensitive response-like cell death accompanied by ROS production at the infection sites of transgenic plants. Transgenic plants showed resistance to the near-obligate hemibiotrophic pathogen P. infestans and, by contrast, increased susceptibility to the necrotrophic pathogen A. solani. • These results indicate that RBOH-dependent ROS contribute to basal defense against near-obligate pathogens, but have a negative role in resistance or have a positive role in expansion of disease lesions caused by necrotrophic pathogens.

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.

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in diverse physiological processes, such as programmed cell death, development, cell elongation and hormonal signaling, in plants. Much attention has been paid to the regulation of plant innate immunity by these signal molecules. Recent studies provide evidence that an NADPH oxidase, respiratory burst oxidase homolog, is responsible for pathogen-responsive ROS burst. However, we still do not know about NO-producing enzymes, except for nitrate reductase, although many studies suggest the existence of NO synthase-like activity responsible for NO burst in plants. Here, we introduce regulatory mechanisms of NO and ROS bursts by mitogen-activated protein kinase cascades, calcium-dependent protein kinase or riboflavin and its derivatives, flavin mononucleotide and flavin adenine dinucleotide, and we discuss the roles of the bursts in defense responses against plant pathogens.

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in the supply of reduced nicotine amide cofactors for resistance to biotic and abiotic stresses. Here, we show participation of the plastidic P2 isoform of G6PDH in plant immunity. A cytosolic isoform (NbG6PDH-Cyto) and two plastidic isoforms (NbG6PDH-P1 and NbG6PDH-P2) cloned from Nicotiana benthamiana were localized in cytosol and chloroplasts, respectively. Hypersensitive response (HR) cell death and NADPH oxidase (RBOH; respiratory burst oxidase homolog)-dependent reactive oxygen species (ROS) production after recognition of INF1 elicitin, secreted by oomycete Phytophthora infestans, decreased in NbG6PDH-P2-silenced plants, but not in NbG6PDH-Cyto- and NbG6PDH-P1-silenced plants. Silencing of the cytosolic NAD kinase NbNADK1, which phosphorylates NADH to form NADPH, compromised HR cell death and ROS production, and concomitant silencing with NbG6PDH-P2 reduced HR cell death and ROS to levels near those in NbG6PDH-P2-silenced plants. Similarly, silencing NbG6PDH-P2 and NbNADK1 resulted in high susceptibility to P. infestans. These results suggest that NADPH produced by the P2 isoform of G6PDH in chloroplasts is responsible for HR cell death and ROS production mediated by RBOH and that NbNADK1 is involved in this pathway.

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. A potato calcium-dependent protein kinase (StCDPK5) activates an NADPH oxidase StRBOHA to D by direct phosphorylation of N-terminal regions, and heterologous expression of StCDPK5 and StRBOHs in Nicotiana benthamiana results in oxidative burst. The transgenic potato plants that carry a constitutively active StCDPK5 driven by a pathogen-inducible promoter of the potato showed high resistance to late blight pathogen Phytophthora infestans accompanied by HR-like cell death and H(2)O(2) accumulation in the attacked cells. In contrast, these plants showed high susceptibility to early blight necrotrophic pathogen Alternaria solani, suggesting that oxidative burst confers high resistance to biotrophic pathogen, but high susceptibility to necrotrophic pathogen. NO and ROS synergistically function in defense responses. Two MAPK cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NbRBOHB gene in N. benthamiana. On the other hand, NO burst is regulated by the MEK2-SIPK cascade. Conditional activation of SIPK in potato plants induces oxidative and NO bursts, and confers resistance to both biotrophic and necrotrophic pathogens, indicating the plants may have obtained during evolution the signaling pathway which regulates both NO and ROS production to adapt to wide-spectrum pathogens.

During defense responses, plant cells produce nitric oxide (NO), which may control many physiological processes. In a previous study, we reported that nitrate reductase (NR) is responsible in part for INF1 elicitor-induced NO production in Nicotiana benthamiana, but the possibility remains that other NO-generating system(s) contribute to NO production. In mammalian cells, NO production is catalyzed by NO synthase (NOS). However, NOS-like enzyme(s) have never been identified in plants, and only the gene for Arabidopsis thaliana nitric oxide-associated 1 (AtNOA1) has been identified as a putative regulator of NOS activity in plants. In this study, we cloned NbNOA1, a homolog of AtNOA1, from N. benthamiana and investigated its involvement in NO production induced by INF1. The NbNOA1 gene was silenced by a virus-induced gene-silencing (VIGS) technique. NbNOA1-silenced plants had yellowish leaves. Silencing NbNOA1 partially decreased INF1-induced NO production, while overexpressing NbNOA1 did not affect NO production. Silencing NbNOA1 suppressed INF1-induced PR1a gene expression and increased susceptibility to Colletotrichum lagenarium. These results suggest that NbNOA1 is involved in INF1-mediated NO production and is required for defense responses.

Non-host resistance is the most general form of disease resistance in plants because it is effective against most phytopathogens. The importance of hypersensitive responses (HRs) in non-host resistance of Nicotiana species to the oomycete Phytophthora is clear. INF1 elicitin, an elicitor obtained from the late-blight pathogen Phytophthora infestans, is sufficient to induce a typical HR in Nicotiana species. The molecular mechanisms that underlie the non-host resistance component of plant defence responses have been investigated using differential-display polymerase chain reaction (PCR) in a model HR system between INF1 elicitin and tobacco BY-2 cells. Differential-display PCR has revealed that Cdc27B is down-regulated in tobacco BY- 2 cells after treatment with INF1 elicitin. Cdc27B is one of 13 essential components of the anaphase- promoting complex or cyclosome ( APC/ C)-type E3 ubiquitin ligase complex in yeast. This APC/C-type E3 ubiquitin ligase complex regulates G2-to-M phase transition of the cell cycle by proteolytic degradation. In this study, we investigated the roles of this gene, NbCdc27B, in plant defence responses using virus-induced gene silencing. Suppression of NbCdc27B in Nicotiana benthamiana plants induced defence responses and a gain of resistance to Colletotrichum lagenarium fungus. Elicitin-induced hypersensitive cell death (HCD) was inhibited mildly in plants silenced with tobacco rattle virus:: Cdc27B. Cdc27B could manage the signalling pathways of plant defence responses as a negative regulator without HCD.

Pathogen-secreted effector proteins enable pathogens to manipulate plant immunity for successful infection. To penetrate host apoplastic space, pathogens reopen the stomata. Once the invasion into the apoplast occurs, pathogens deceive the host detection system by deploying apoplastic effectors. Pathogens also deliver an arsenal of cytosolic effectors into the host cells, which undermine host immunity such as salicylic acid (SA)-dependent immunity. Here we summarize recent findings that highlight the functions of the effectors from fungal, oomycete and bacterial pathogens in the key steps of infection at the stomata, in the apoplast, and inside the cell. We also discuss cell type-specific responses in the host during infection and the necessity of further investigation of plant-pathogen interactions at spatial and temporal resolution.

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. Fil: Wirthmueller, Lennart. John Innes Institute; Reino Unido. The Sainsbury Laboratory; Reino Unido Fil: Roth, Charlotte. Georg‐August‐University; Alemania Fil: Fabro, Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Caillaud, Marie-Cécile. The Sainsbury Laboratory; Reino Unido Fil: Rallapalli, Ghanasyam. The Sainsbury Laboratory; Reino Unido Fil: Asai, Shuta. The Sainsbury Laboratory; Reino Unido Fil: Sklenar, Jan. John Innes Institute; Reino Unido Fil: Jones, Alexandra M. E.. John Innes Institute; Reino Unido Fil: Wiermer, Marcel. Georg‐August‐University; Alemania Fil: Jones, Jonathan D. G.. The Sainsbury Laboratory; Reino Unido Fil: Banfield, Mark J.. Georg‐August‐University; Alemania

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., Author Summary A comprehensive understanding of host-pathogen interactions requires knowledge of the dynamics of gene expression changes in both the host and the pathogen during a time course of infection. However, expression profiling has often focused on either the host or the pathogen due to limitations of methods that involve microarrays. We report here gene expression changes in both Arabidopsis and its parasite Hyaloperonospora arabidopsidis (Hpa) simultaneously during infection using a high-throughput RNA sequencing method. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of cognate recognized effector ATR1. We also found that Hpa suppresses responsiveness to salicylic acid (SA) in haustoriated cells into which host-translocated effectors are delivered. An Hpa effector HaRxL62, previously shown to enhance host susceptibility, was highly expressed in this assay, and we found it suppresses responsiveness to SA. Expression profiling of both pathogen effector genes and host genes involved in immunity allows us to suggest distinct mechanisms of effector-mediated susceptibility and reveals interesting Hpa effectors for detailed mechanistic investigation in future experiments.

Nitric oxide (NO) and reactive oxygen species (ROS) are important signaling molecules in plant immunity. However, roles of NO and ROS in disease resistance to necrotrophic pathogens are not fully understood. We have recently demonstrated that NO plays a pivotal role in basal defense against Botrytis cinerea and the expression of the salicylic acid (SA) -responsive gene PR-1in Nicotiana benthamiana. By contrast, ROS function negatively in resistance or positively in expansion of disease lesions during B. cinerea-N. benthamiana interaction. Here, analysis in NahG-transgenic N. benthamiana showed that SA signaling is not involved in resistance to B. cinerea in N. benthamiana. We discuss how NO and ROS participate in disease resistance to necrotrophic pathogens on the basis of recent reports.

HaRxL44, a secreted effector from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis, enhances disease susceptibility by interacting with and degrading Mediator subunit MED19a, thereby perturbing plant defense gene transcription., Plants are continually exposed to pathogen attack but usually remain healthy because they can activate defences upon perception of microbes. However, pathogens have evolved to overcome plant immunity by delivering effectors into the plant cell to attenuate defence, resulting in disease. Recent studies suggest that some effectors may manipulate host transcription, but the specific mechanisms by which such effectors promote susceptibility remain unclear. We study the oomycete downy mildew pathogen of Arabidopsis, Hyaloperonospora arabidopsidis (Hpa), and show here that the nuclear-localized effector HaRxL44 interacts with Mediator subunit 19a (MED19a), resulting in the degradation of MED19a in a proteasome-dependent manner. The Mediator complex of ∼25 proteins is broadly conserved in eukaryotes and mediates the interaction between transcriptional regulators and RNA polymerase II. We found MED19a to be a positive regulator of immunity against Hpa. Expression profiling experiments reveal transcriptional changes resembling jasmonic acid/ethylene (JA/ET) signalling in the presence of HaRxL44, and also 3 d after infection with Hpa. Elevated JA/ET signalling is associated with a decrease in salicylic acid (SA)–triggered immunity (SATI) in Arabidopsis plants expressing HaRxL44 and in med19a loss-of-function mutants, whereas SATI is elevated in plants overexpressing MED19a. Using a PR1::GUS reporter, we discovered that Hpa suppresses PR1 expression specifically in cells containing haustoria, into which RxLR effectors are delivered, but not in nonhaustoriated adjacent cells, which show high PR1::GUS expression levels. Thus, HaRxL44 interferes with Mediator function by degrading MED19, shifting the balance of defence transcription from SA-responsive defence to JA/ET-signalling, and enhancing susceptibility to biotrophs by attenuating SA-dependent gene expression., Author Summary The highly conserved Mediator complex plays an essential role in transcriptional regulation by providing a molecular bridge between transcription factors and RNA polymerase II. Recent studies in Arabidopsis have revealed that it also performs an essential role in plant defence. However, it remains unknown how pathogens manipulate Mediator function in order to increase a plant's susceptibility to infection. In this article, we show that a secreted effector, HaRxL44, from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa), interacts with and degrades the Mediator subunit MED19a, resulting in the alteration of plant defence gene transcription. This effector-mediated interference with host transcriptional regulation perturbs the balance between jasmonic acid/ethylene (JA/ET) and salicylic acid (SA)–dependent defence. HaRxL44 interaction with MED19a results in reduced SA-regulated gene expression, indicating that this pathogen effector modulates host transcription to promote virulence. The resulting alteration in defence transcription patterns compromises the plant's ability to defend itself against pathogens, such as Hpa, that establish long-term parasitic interactions with living host cells via haustoria (a pathogen structure that creates an expanded host/parasite interface to extract nutrients) but not against necrotrophic pathogens that kill host cells. HaRxL44 is unlikely to be the sole effector that accomplishes this shift in hormonal balance, and other nuclear HaRxL proteins were reported by other researchers to interact with Mediator components, as well as with other regulators of the JA/ET signalling pathway. Functional analyses of these effectors should facilitate the discovery of new components of the plant immune system. These data show that pathogens can target fundamental mechanisms of host regulation in order to tip the balance of signalling pathways to suppress defence and favour parasitism.