Obligate Biotrophic Oomycete Research Articles

Subcellular targeting of an evolutionarily conserved plant defensin MtDef4.2 determines the outcome of plant–pathogen interaction in transgenic Arabidopsis

The Medicago truncatula gene encoding an evolutionarily conserved antifungal defensin MtDef4.2 was cloned and characterized. In silico expression analysis indicated that MtDef4.2 is expressed in many tissues during the normal growth and development of M. truncatula. MtDef4.2 exhibits potent broad-spectrum antifungal activity against various Fusarium spp. Transgenic Arabidopsis thaliana lines in which MtDef4.2 was targeted to three different subcellular compartments were generated. These lines were tested for resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 and the hemibiotrophic fungal pathogen Fusarium graminearum PH-1. MtDef4.2 directed to the extracellular space, but not to the vacuole or retained in the endoplasmic reticulum, conferred robust resistance to H. arabidopsidis. Siliques of transgenic Arabidopsis lines expressing either extracellularly or intracellularly targeted MtDef4.2 displayed low levels of resistance to F. graminearum, but accumulated substantially reduced levels of the mycotoxin deoxynivalenol. The data presented here suggest that extracellularly targeted MtDef4.2 is sufficient to provide strong resistance to the biotrophic oomycete, consistent with the extracellular lifestyle of this pathogen. However, the co-expression of extracellular and intracellular MtDef4.2 is probably required to achieve strong resistance to the hemibiotrophic pathogen F. graminearum which grows extracellularly and intracellularly.

Overexpression of Xanthomonas campestris pv. vesicatoria effector AvrBsT in Arabidopsis triggers plant cell death, disease and defense responses

Recognition of bacterial effector proteins by plant cells is crucial for plant disease and defense response signaling. The Xanthomonas campestris pv. vesicatoria (Xcv) type III effector protein, AvrBsT, is secreted into plant cells from Xcv strain Bv5-4a. Here, we demonstrate that dexamethasone (DEX): avrBsT overexpression triggers cell death signaling in healthy transgenic Arabidopsis plants. AvrBsT overexpression in Arabidopsis also reduced susceptibility to infection with the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Overexpression of avrBsT significantly induced some defense-related genes in Arabidopsis leaves. A high-throughput in planta proteomics screen identified TCP-1 chaperonin, SEC7-like guanine nucleotide exchange protein and calmodulin-like protein, which were differentially expressed in DEX:avrBsT-overexpression (OX) Arabidopsis plants during Hp. arabidopsidis infection. Treatment with purified GST-tagged AvrBsT proteins distinctly inhibited the growth and sporulation of Hp. arabidopsidis on Arabdiopsis cotyledons. In contrast, DEX:avrBsT-OX plants exhibited enhanced susceptibility to Pseudomonas syringae pv. tomato (Pst) DC3000 infection. Notably, susceptible cell death and enhanced electrolyte leakage were significantly induced in the Pst-infected leaves of DEX:avrBsT-OX plants. Together, these results suggest that Xcv effector AvrBsT overexpression triggers plant cell death, disease and defense signaling leading to both disease and defense responses to microbial pathogens of different lifestyles.

Nontoxic Nep1-Like Proteins of the Downy Mildew Pathogen Hyaloperonospora arabidopsidis: Repression of Necrosis-Inducing Activity by a Surface-Exposed Region

The genome of the downy mildew pathogen Hyaloperonospora arabidopsidis encodes necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP). Although NLP are widely distributed in eukaryotic and prokaryotic plant pathogens, it was surprising to find these proteins in the obligate biotrophic oomycete H. arabidopsidis. Therefore, we analyzed the H. arabidopsidis NLP (HaNLP) family and identified 12 HaNLP genes and 15 pseudogenes. Most of the 27 genes form an H. arabidopsidis-specific cluster when compared with other oomycete NLP genes, suggesting this class of effectors has recently expanded in H. arabidopsidis. HaNLP transcripts were mainly detected during early infection stages. Agrobacterium tumefaciens-mediated transient expression and infiltration of recombinant NLP into tobacco and Arabidopsis leaves revealed that all HaNLP tested are noncytotoxic proteins. Even HaNLP3, which is most similar to necrosis-inducing NLP proteins of other oomycetes and which contains all amino acids that are critical for necrosis-inducing activity, did not induce necrosis. Chimeras constructed between HaNLP3 and the necrosis-inducing PsojNIP protein demonstrated that most of the HaNLP3 protein is functionally equivalent to PsojNIP, except for an exposed domain that prevents necrosis induction. The early expression and species-specific expansion of the HaNLP genes is suggestive of an alternative function of noncytolytic NLP proteins during biotrophic infection of plants.

Computational Prediction and Molecular Characterization of an Oomycete Effector and the Cognate Arabidopsis Resistance Gene

Hyaloperonospora arabidopsidis (Hpa) is an obligate biotroph oomycete pathogen of the model plant Arabidopsis thaliana and contains a large set of effector proteins that are translocated to the host to exert virulence functions or trigger immune responses. These effectors are characterized by conserved amino-terminal translocation sequences and highly divergent carboxyl-terminal functional domains. The availability of the Hpa genome sequence allowed the computational prediction of effectors and the development of effector delivery systems enabled validation of the predicted effectors in Arabidopsis. In this study, we identified a novel effector ATR39-1 by computational methods, which was found to trigger a resistance response in the Arabidopsis ecotype Weiningen (Wei-0). The allelic variant of this effector, ATR39-2, is not recognized, and two amino acid residues were identified and shown to be critical for this loss of recognition. The resistance protein responsible for recognition of the ATR39-1 effector in Arabidopsis is RPP39 and was identified by map-based cloning. RPP39 is a member of the CC-NBS-LRR family of resistance proteins and requires the signaling gene NDR1 for full activity. Recognition of ATR39-1 in Wei-0 does not inhibit growth of Hpa strains expressing the effector, suggesting complex mechanisms of pathogen evasion of recognition, and is similar to what has been shown in several other cases of plant-oomycete interactions. Identification of this resistance gene/effector pair adds to our knowledge of plant resistance mechanisms and provides the basis for further functional analyses.

De novo sequence assembly of Albugo candida reveals a small genome relative to other biotrophic oomycetes

BackgroundAlbugo candida is a biotrophic oomycete that parasitizes various species of Brassicaceae, causing a disease (white blister rust) with remarkable convergence in behaviour to unrelated rusts of basidiomycete fungi.ResultsA recent genome analysis of the oomycete Hyaloperonospora arabidopsidis suggests that a reduction in the number of genes encoding secreted pathogenicity proteins, enzymes for assimilation of inorganic nitrogen and sulphur represent a genomic signature for the evolution of obligate biotrophy. Here, we report a draft reference genome of a major crop pathogen Albugo candida (another obligate biotrophic oomycete) with an estimated genome of 45.3 Mb. This is very similar to the genome size of a necrotrophic oomycete Pythium ultimum (43 Mb) but less than half that of H. arabidopsidis (99 Mb). Sequencing of A. candida transcripts from infected host tissue and zoosporangia combined with genome-wide annotation revealed 15,824 predicted genes. Most of the predicted genes lack significant similarity with sequences from other oomycetes. Most intriguingly, A. candida appears to have a much smaller repertoire of pathogenicity-related proteins than H. arabidopsidis including genes that encode RXLR effector proteins, CRINKLER-like genes, and elicitins. Necrosis and Ethylene inducing Peptides were not detected in the genome of A. candida. Putative orthologs of tat-C, a component of the twin arginine translocase system, were identified from multiple oomycete genera along with proteins containing putative tat-secretion signal peptides.ConclusionAlbugo candida has a comparatively small genome amongst oomycetes, retains motility of sporangial inoculum, and harbours a much smaller repertoire of candidate effectors than was recently reported for H. arabidopsidis. This minimal gene repertoire could indicate a lack of expansion, rather than a reduction, in the number of genes that signify the evolution of biotrophy in oomycetes.

A molecular insight into algal-oomycete warfare: cDNA analysis of Ectocarpus siliculosus infected with the basal oomycete Eurychasma dicksonii.

Brown algae are the predominant primary producers in coastal habitats, and like land plants are subject to disease and parasitism. Eurychasma dicksonii is an abundant, and probably cosmopolitan, obligate biotrophic oomycete pathogen of marine brown algae. Oomycetes (or water moulds) are pathogenic or saprophytic non-photosynthetic Stramenopiles, mostly known for causing devastating agricultural and aquacultural diseases. Whilst molecular knowledge is restricted to crop pathogens, pathogenic oomycetes actually infect hosts from most eukaryotic lineages. Molecular evidence indicates that Eu. dicksonii belongs to the most early-branching oomycete clade known so far. Therefore Eu. dicksonii is of considerable interest due to its presumed environmental impact and phylogenetic position. Here we report the first large scale functional molecular data acquired on the most basal oomycete to date. 9873 unigenes, totalling over 3.5Mb of sequence data, were produced from Sanger-sequenced and pyrosequenced EST libraries of infected Ectocarpus siliculosus. 6787 unigenes (70%) were of algal origin, and 3086 (30%) oomycete origin. 57% of Eu. dicksonii sequences had no similarity to published sequence data, indicating that this dataset is largely unique. We were unable to positively identify sequences belonging to the RXLR and CRN groups of oomycete effectors identified in higher oomycetes, however we uncovered other unique pathogenicity factors. These included putative algal cell wall degrading enzymes, cell surface proteins, and cyclophilin-like proteins. A first look at the host response to infection has also revealed movement of the host nucleus to the site of infection as well as expression of genes responsible for strengthening the cell wall, and secretion of proteins such as protease inhibitors. We also found evidence of transcriptional reprogramming of E. siliculosus transposable elements and of a viral gene inserted in the host genome.

Why Biotrophs Can't Live Alone

Why Biotrophs Can't Live Alone

Gene Gain and Loss during Evolution of Obligate Parasitism in the White Rust Pathogen of Arabidopsis thaliana

Biotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells. Evolution to biotrophy occurred independently in fungal rusts and powdery mildews, and in oomycete white rusts and downy mildews. Biotroph evolution and molecular mechanisms of biotrophy are poorly understood. It has been proposed, but not shown, that obligate biotrophy results from (i) reduced selection for maintenance of biosynthetic pathways and (ii) gain of mechanisms to evade host recognition or suppress host defence. Here we use Illumina sequencing to define the genome, transcriptome, and gene models for the obligate biotroph oomycete and Arabidopsis parasite, Albugo laibachii. A. laibachii is a member of the Chromalveolata, which incorporates Heterokonts (containing the oomycetes), Apicomplexa (which includes human parasites like Plasmodium falciparum and Toxoplasma gondii), and four other taxa. From comparisons with other oomycete plant pathogens and other chromalveolates, we reveal independent loss of molybdenum-cofactor-requiring enzymes in downy mildews, white rusts, and the malaria parasite P. falciparum. Biotrophy also requires “effectors” to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes, and also discover and verify a novel class of effectors, the “CHXCs”, by showing effector delivery and effector functionality. Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways, and particularly reduced selection for retention of molybdopterin-requiring biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata.

Interconnected Metabolism of Host Plants and Obligate Biotrophic Pathogens

Downy mildew disease, caused by obligate biotrophic oomycetes in the family Peronsporaceae, affects a wide range of flowering plants, including numerous monocot and dicot crops. Species from the Hyaloperonospora genus are causal agents of downy mildew in Arabidopsis thaliana and many other Cruciferae species, including crops such as broccoli, cabbage, and canola, and the Arabidopsis/ Hyaloperonospora arabidopsidis pathosystem is an established model for downy mildews (Coates and Beynon, 2010). Obligate biotrophic parasites depend on living host cells for their growth and reproduction (reviewed in O’Connell and Panstruga, 2006). This means that, following successful infection, the parasite must establish connections within plant cells (e.g., haustoria) and obtain nourishment from the host without causing immediate host cell death. Therefore, changes in the metabolic state of the host might be expected to influence the growth of obligate biotrophic pathogens and the development of disease. In new work, Stuttmann et al. (pages 2788–2803) describe two distinct mutants of Arabidopsis ,b oth of which show increased resistance to H. arabidopsidis (Hpa) resulting from a disruption in amino acid metabolism causing overaccumulation of the amino acid Thr. Although the mutant plants overaccumulated all three Asp-derived amino acids, Thr, Ile, and Met, enhanced resistance was found to be related specifically to Thr accumulation. Mutant plants that overaccumulate only Met were not hyperresistant to Hpa, and pretreatment of plants with Thr, but not Ile, led to reduced Hpa sporulation (see figure). In addition, the mutations did not cause constitutive plant defense activation, and enhanced resistance did not appear to be linked to plant immunity. Rather, the effect appeared to be due to a loss of susceptibility (i.e., Thr overaccumulation somehow rendered the plant unsuitable for Hpa infection). Interestingly, this effect was specific for Hpa susceptibility, since pretreatment of plants with Thr did not affect sporulation of another obligate biotroph, the powdery mildew ascomycete Golovinomyces orontii (Go). The authors addressed the question of how overaccumulation of Thr might affect Hpa but not Go. Excess Thr is known to be toxic to Arabidopsis, and, indeed, the mutants described in this study were smaller than wild-type plants. An assessment of genes and biochemical pathway components present in the different phyla provided support for the notion that oomycetes and plants might use similar pathways of amino acid biosynthesis and metabolism in a distinct manner from the more distantly related ascomycete. Thus, the toxic effect of high Thr accumulation might be shared among plants and oomycetes, but not by the fungal ascomycete Go. These data provide solid examples of how plant host metabolic state can influence growth and pathogenicity of adapted biotrophic pathogens.

Perturbation of Arabidopsis Amino Acid Metabolism Causes Incompatibility with the Adapted Biotrophic Pathogen Hyaloperonospora arabidopsidis

Reliance of biotrophic pathogens on living plant tissues to propagate implies strong interdependence between host metabolism and nutrient uptake by the pathogen. However, factors determining host suitability and establishment of infection are largely unknown. We describe a loss-of-inhibition allele of ASPARTATE KINASE2 and a loss-of-function allele of DIHYDRODIPICOLINATE SYNTHASE2 identified in a screen for Arabidopsis thaliana mutants with increased resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa). Through different molecular mechanisms, these mutations perturb amino acid homeostasis leading to overaccumulation of the Asp-derived amino acids Met, Thr, and Ile. Although detrimental for the plant, the mutations do not cause defense activation, and both mutants retain full susceptibility to the adapted obligate biotrophic fungus Golovinomyces orontii (Go). Chemical treatments mimicking the mutants' metabolic state identified Thr as the amino acid suppressing Hpa but not Go colonization. We conclude that perturbations in amino acid homeostasis render the mutant plants unsuitable as an infection substrate for Hpa. This may be explained by deployment of the same amino acid biosynthetic pathways by oomycetes and plants. Our data show that the plant host metabolic state can, in specific ways, influence the ability of adapted biotrophic strains to cause disease.

The Arabidopsis Leucine-Rich Repeat Receptor–Like Kinases BAK1/SERK3 and BKK1/SERK4 Are Required for Innate Immunity to Hemibiotrophic and Biotrophic Pathogens

Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) constitutes an important layer of innate immunity in plants. The leucine-rich repeat (LRR) receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN SENSING2 (FLS2) are the PRRs for the peptide PAMPs elf18 and flg22, which are derived from bacterial EF-Tu and flagellin, respectively. Using coimmunoprecipitation and mass spectrometry analyses, we demonstrated that EFR and FLS2 undergo ligand-induced heteromerization in planta with several LRR receptor-like kinases that belong to the SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1/SERK3 (BAK1/SERK3) and BAK1-LIKE1/SERK4 (BKK1/SERK4). Using a novel bak1 allele that does not exhibit pleiotropic defects in brassinosteroid and cell death responses, we determined that BAK1 and BKK1 cooperate genetically to achieve full signaling capability in response to elf18 and flg22 and to the damage-associated molecular pattern AtPep1. Furthermore, we demonstrated that BAK1 and BKK1 contribute to disease resistance against the hemibiotrophic bacterium Pseudomonas syringae and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Our work reveals that the establishment of PAMP-triggered immunity (PTI) relies on the rapid ligand-induced recruitment of multiple SERKs within PRR complexes and provides insight into the early PTI signaling events underlying this important layer of plant innate immunity.

Optimisation and comparison of transient expression methods to express the green fluorescent protein in the obligate biotrophic oomycete Plasmopara viticola

Grape downy mildew is caused by Plasmopara viticola , an obligate biotrophic oomycete and a major pathogen of grapevine. Studying obligate biotrophic pathogens is difficult as they cannot grow without their host. We therefore attempted to develop a method where the pathogen could be visualized and quantified in planta without killing the host plant. To this end P. viticola was transformed with the marker gene gfp coding for the green fluorescent protein. Various transformation methods, namely electroporation, particle bombardment and transformation with Agrobacterium tumefaciens were applied. Although some methods yielded positive transformation events, no stable strain of P. viticola expressing gfp could be generated. Using the electroporation method, we obtained transient P. viticola transformants expressing gfp over 4 generations. In contrast, particle bombardment failed in transforming P. viticola . Transformation with A. tumefaciens had a low efficiency, only some structures were fluorescent and fluorescence was never observed in the subsequent generations.

Are there alternatives to copper for controlling grapevine downy mildew in organic viticulture?

Downy mildew caused by the obligate biotrophic oomycete Plasmopara viticola is one of the most important and devastating diseases of grapevine. Copper-based fungicides are used to control the disease in organic vineyards. However, since 2006, copper use has been limited by an European Commission regulation. In this study conducted from 2004 to 2007, we tested materials that might replace copper in organic viticulture, under controlled greenhouse and field conditions at two locations in Europe (northern Italy and Switzerland). We tested 112 different treatments, including biocontrol agents, materials of animal origin, homeopathic preparations, inorganic materials, microbial extracts, natural derivatives, plant extracts, physical methods and synthetic materials. Good control results were observed for two new copper formulations, but one of them induced phytotoxic effects. Two of the plant extracts provided good control on both leaves and clusters, and six of them controlled the infection on clusters only. One biocontrol agent reduced symptoms on leaves and bunches. Three of the other treatments provided good control on leaves, and four of them provided good control on bunches. Clay was as effective as copper on both leaves and bunches. Currently, even if there is no treatment that is as effective as copper for controlling grapevine downy mildew in organic vineyards, Yucca schidigera and Salvia officinalis extracts and Trichoderma harzianum, could be considered as promising candidates to be developed. This study may help the development of an integrated management program in which a less effective product is applied in combination with copper, to reduce the total quantities of copper applied.

Identification of genes expressed during the compatible interaction of grapevine with Plasmopara viticola through suppression subtractive hybridization (SSH)

Grapevine (Vitis vinifera) is the most widely cultivated and economically important fruit crop, but is susceptible to a large number of diseases. Downy mildew, caused by the obligate biotrophic oomycete pathogen Plasmopara viticola, is a common disease present in all regions where vines are cultivated. We used suppression subtractive hybridization (SSH) to generate two cDNA libraries enriched for transcripts induced and repressed, respectively, in the susceptible grapevine cultivar Chasselas 24 h after inoculation with P. viticola. Differential screening on glass slide microarrays yielded over 800 putative genes that were up-regulated in response to P. viticola infection and over 200 that were down-regulated. One hundred and ninety four of these, were sequenced, identified and functionally categorised. Transcript abundance of twelve genes over a 48 h time course was examined by reverse transcriptase quantitative real-time PCR (RT-qPCR). Ten of these genes were induced/enhanced by P. viticola challenge, confirming the results of the SSH. The vast majority of the genes identified are related to defence. Interestingly, many genes involved in photosynthesis were down-regulated.

Evolution of diversity in Albugo is driven by high host specificity and multiple speciation events on closely related Brassicaceae

The Albuginaceae, responsible for white blister rust disease on various angiosperms, are obligate biotrophic oomycetes that are only distantly related to downy mildews (Peronosporaceae). Their diversity has been much underestimated during the past decades, mainly because of the paucity of morphological characters for species delimitation, which led to the application of a broad species concept. Recent phylogenetic analyses have revealed three new species within Albugo parasitic to Brassicaceae, but the overall evolution of these plant pathogens remains poorly understood. Especially the diversity of Albugo in various plant genera is almost completely unknown. Based on ITS and cox2 sequence data of 72 Albugo specimens, predominantly from herbarium archives, and focusing on the widespread genus Cardamine, a high degree of phylogenetic diversity was revealed in Albugo. In particular, the hypothesis that one host genus can be colonised by more than one white blister rust species is confirmed. In addition, it is revealed that there are hitherto overlooked lineages with close relationships to the generalist species Albugo candida. Evidence for at least three different species of Albugo infecting Cardamine is presented in this study. Based on molecular phylogenetic and morphological data three new white blister rust species are described, Albugo hohenheimia, Albugo hesleri, and Albugo leimonios infecting Cardamine hirsuta, Cardamine diphylla and Cardamine pratensis, respectively. The fact that these species each have different ecological niches, suggests that environmental factors may have played a role in the speciation process in Albugo. Our findings suggest that other larger genera of the Brassicaceae may harbour unrecognized white blister rust species and that only a small fraction of the true biodiversity of white blister rusts is known at present.

Repression of the Auxin Response Pathway Increases Arabidopsis Susceptibility to Necrotrophic Fungi

In plants, resistance to necrotrophic pathogens depends on the interplay between different hormone systems, such as those regulated by salicylic acid (SA), jasmonic acid (JA), ethylene, and abscisic acid. Repression of auxin signaling by the SA pathway was recently shown to contribute to antibacterial resistance. Here, we demonstrate that Arabidopsis auxin signaling mutants axr1, axr2, and axr6 that have defects in the auxin-stimulated SCF (Skp1-Cullin-F-box) ubiquitination pathway exhibit increased susceptibility to the necrotrophic fungi Plectosphaerella cucumerina and Botrytis cinerea. Also, stabilization of the auxin transcriptional repressor AXR3 that is normally targeted for removal by the SCF-ubiquitin/proteasome machinery occurs upon P. cucumerina infection. Pharmacological inhibition of auxin transport or proteasome function each compromise necrotroph resistance of wild-type plants to a similar extent as in non-treated auxin response mutants. These results suggest that auxin signaling is important for resistance to the necrotrophic fungi P. cucumerina and B. cinerea. SGT1b (one of two Arabidopsis SGT1 genes encoding HSP90/HSC70 co-chaperones) promotes the functions of SCF E3-ubiquitin ligase complexes in auxin and JA responses and resistance conditioned by certain Resistance (R) genes to biotrophic pathogens. We find that sgt1b mutants are as resistant to P. cucumerina as wild-type plants. Conversely, auxin/SCF signaling mutants are uncompromised in RPP4-triggered resistance to the obligate biotrophic oomycete, Hyaloperonospora parasitica. Thus, the predominant action of SGT1b in R gene-conditioned resistance to oomycetes appears to be at a site other than assisting SCF E3-ubiquitin ligases. However, genetic additivity of sgt1b axr1 double mutants in susceptibility to H. parasitica suggests that SCF-mediated ubiquitination contributes to limiting biotrophic pathogen colonization once plant-pathogen compatibility is established.

Two simplified fluorescent staining techniques to observe infection structures of the oomycete Plasmopara viticola in grapevine leaf tissues

Plasmopara viticola, the causal agent of grapevine downy mildew, is an obligate biotrophic oomycete that grows in the intercellular spaces of host tissues and develops haustoria in the cells. Histological observations are the most effective methods to visualize and quantify the development of the infection structures. We chose two staining techniques leading to high resolution and contrast between parasite structures and host-plant tissues with a minimum of sample preparation: Blankophor and KOH-aniline blue fluorescent stainings. Blankophor (50 ppm in water or 15% KOH) staining was used to study the zoospore encystement on the leaf surface after release from sporangia. The aniline blue dye (0.05% in 0.067 M K 2HPO 4, pH 9–9.5, after hot KOH whitening) was used to observe the invasive structures inside host tissues that lead to the production of sporangiophores and infectious sporangia. We tested modifications of some parameters of the procedures to determine the most appropriate for high throughput analyses adapted to our pathosystem and equipment facilities.

Age-related resistance to Pseudomonas syringae pv. tomato is associated with the transition to flowering in Arabidopsis and is effective against Peronospora parasitica

As plants mature it has been observed that some become more resistant to normally virulent pathogens. The ability to manifest the Age-Related Resistance (ARR) response in Arabidopsis to Pseudomonas syringae pathovars tomato ( Pst) coincided with the transition to flowering in plants both delayed and accelerated in the transition to flowering. ARR was also associated with a change in PR-1 gene expression, such that young plants expressed PR-1 abundantly at 3 days post inoculation (dpi) while mature plants expressed much less. The Arabidopsis ARR response requires SA accumulation via isochorismate synthase ( ICS1) [24]. ICS1 was expressed one dpi with virulent and avirulent Pst in both young and mature plants. The ARR response was also effective versus avirulent Pst providing an additional 4-fold limitation in bacterial growth. Arabidopsis ARR was found to be ineffective against two necrotrophs, Erwinia carotovora subspecies carotovora (bacterium) and Botrytis cinerea (fungus) and one obligate biotroph, Erysiphe cichoracearum (fungus). However, mature wild type, SA-deficient sid2 and NahG plants supported little growth of the obligate biotrophic oomycete, Peronospora parasitica. Therefore ARR to P. parasitica appears to be SA-independent, however the level of ARR resistance was somewhat reduced in these mutants in some experiments. Thus, there may be numerous defence pathways that contribute to adult plant resistance in Arabidopsis.

Use of suppression subtractive hybridization to identify downy mildew genes expressed during infection of Arabidopsis thaliana

SUMMARY Peronospora parasitica is an obligate biotrophic oomycete that causes downy mildew in Arabidopsis thaliana and Brassica species. Our goal is to identify P. parasitica (At) genes that are involved in pathogenicity. We used suppression subtractive hybridization (SSH) to generate cDNA libraries enriched for in planta-expressed parasite genes and up-regulated host genes. A total of 1345 clones were sequenced representing cDNA fragments from 25 putative P. parasitica (At) genes (Ppat 1-25) and 618 Arabidopsis genes. Analyses of expression patterns showed that 15 Ppats were expressed only in planta. Eleven Ppats encoded peptides with homology (BlastP values < 1e-05) to proteins with roles in membrane or cell wall biosynthesis, amino acid metabolism, osmoregulation, cation transport, phosphorylation or protein secretion. The other 14 represent potentially novel oomycete genes with none having homologues in an extensive Phytophthora species EST database. A full-length sequence was obtained for four Ppats and each encoded small cysteine-rich proteins with amino-terminal signal peptide sequences. These results demonstrate the utility of SSH in obtaining novel in planta-expressed genes from P. parasitica (At) that complements other gene discovery approaches such as EST sequencing.