Obligate Biotrophic Protist Research Articles

Comparative Metagenomic Analysis Reveals Rhizosphere Microbiome Assembly and Functional Adaptation Changes Caused by Clubroot Disease in Chinese Cabbage.

Clubroot is a major disease and severe threat to Chinese cabbage, and it is caused by the pathogen Plasmodiophora brassicae Woron. This pathogen is an obligate biotrophic protist and can persist in soil in the form of resting spores for more than 18 years, which can easily be transmitted through a number of agents, resulting in significant economic losses to global Chinese cabbage production. Rhizosphere microbiomes play fundamental roles in the occurrence and development of plant diseases. The changes in the rhizosphere microorganisms could reveal the severity of plant diseases and provide the basis for their control. Here, we studied the rhizosphere microbiota after clubroot disease infections with different severities by employing metagenomic sequencing, with the aim of exploring the relationships between plant health, rhizosphere microbial communities, and soil environments; then, we identified potential biomarker microbes of clubroot disease. The results showed that clubroot disease severity significantly affected the microbial community composition and structure of the rhizosphere soil, and microbial functions were also dramatically influenced by it. Four different microbes that had great potential in the biocontrol of clubroot disease were identified from the obtained results; they were the genera Pseudomonas, Gemmatimonas, Sphingomonas, and Nocardioides. Soil pH, organic matter contents, total nitrogen, and cation exchange capacity were the major environmental factors modulating plant microbiome assembly. In addition, microbial environmental information processing was extremely strengthened when the plant was subjected to pathogen invasion, but weakened when the disease became serious. In particular, oxidative phosphorylation and glycerol-1-phosphatase might have critical functions in enhancing Chinese cabbage's resistance to clubroot disease. This work revealed the interactions and potential mechanisms among Chinese cabbage, soil environmental factors, clubroot disease, and microbial community structure and functions, which may provide a novel foundation for further studies using microbiological or metabolic methods to develop disease-resistant cultivation technologies.

Multiple transcription factors involved in the response of Chinese cabbage against Plasmodiophora brassicae.

Clubroot disease, which is caused by the obligate biotrophic protist Plasmodiophora brassicae, leads to the formation of galls, commonly known as pathogen-induced tumors, on the roots of infected plants. The identification of crucial regulators of host tumor formation is essential to unravel the mechanisms underlying the proliferation and differentiation of P. brassicae within plant cells. To gain insight into this process, transcriptomic analysis was conducted to identify key genes associated with both primary and secondary infection of P. brassicae in Chinese cabbage. Our results demonstrate that the k-means clustering of subclass 1, which exhibited specific trends, was closely linked to the infection process of P. brassicae. Of the 1610 differentially expressed genes (DEGs) annotated in subclass 1, 782 were identified as transcription factors belonging to 49 transcription factor families, including bHLH, B3, NAC, MYB_related, WRKY, bZIP, C2H2, and ERF. In the primary infection, several genes, including the predicted Brassica rapa probable pectate lyase, RPM1-interacting protein 4-like, L-type lectin-domain-containing receptor kinase, G-type lectin S-receptor-like serine, B. rapa photosystem II 22 kDa protein, and MLP-like protein, showed significant upregulation. In the secondary infection stage, 45 of 50 overlapping DEGs were upregulated. These upregulated DEGs included the predicted B. rapa endoglucanase, long-chain acyl-CoA synthetase, WRKY transcription factor, NAC domain-containing protein, cell division control protein, auxin-induced protein, and protein variation in compound-triggered root growth response-like and xyloglucan glycosyltransferases. In both the primary and secondary infection stages, the DEGs were predicted to be Brassica rapa putative disease resistance proteins, L-type lectin domain-containing receptor kinases, ferredoxin-NADP reductases, 1-aminocyclopropane-1-carboxylate synthases, histone deacetylases, UDP-glycosyltransferases, putative glycerol-3-phosphate transporters, and chlorophyll a-binding proteins, which are closely associated with plant defense responses, biosynthetic processes, carbohydrate transport, and photosynthesis. This study revealed the pivotal role of transcription factors in the initiation of infection and establishment of intracellular parasitic relationships during the primary infection stage, as well as the proliferation and differentiation of the pathogen within the host cell during the secondary infection stage.

The GH3 Protein from the Clubroot Pathogen Plasmodiophora brassicae Causes Hormone-Related Phenotypes in Arabidopsis thaliana

In the genome of the obligate biotrophic protist Plasmodiophora brassicae, which causes clubroot disease in the Brassicaceae family, a single gene was identified with homology to the group of GH3 proteins synthesizing amino acid conjugates with carboxylic acids. In comparison to plant members the P. brassicae GH3 protein seems to be very promiscuous with respect to the substrates converted. In vitro assays showed conversion of three different auxins (indole-3-acetic, IAA; indole-propionic, IPA; and indole-3-butyric acids, IBA), jasmonic acid (JA) and also 12-oxo-phytodienoic acid (OPDA). Auxin and Methyl-JA responsive elements were found in the promoter sequence of PbGH3. For further analysis Arabidopsis thaliana was transformed with PbGH3. Seedlings with confirmed constitutive PbGH3 protein production were grown to adult plants that were shown to produce fewer inflorescences per plant. Growth on 10 µM IAA or JA levels also altered the phenotype of the seedlings, but not so much between wild types and transgenic lines. The hormone profiles in seedlings and adult plants were examined for IAA, JA, OPDA, and respective conjugates. Transgenic seedlings displayed changes that could be partially attributed to the overexpression of a GH3 gene like lower levels of free IAA and the JA precursor cis-OPDA under control conditions, and higher levels of some auxin conjugates accumulated after IAA treatment compared to the corresponding wild types. However, the hormone patterns in adult plants were more complex and varied based on different tissue types analyzed. After inoculation with P. brassicae the transgenic plants did not show any changes in infection rate and disease index, but had higher shoot and lower root weight in infected plants compared to controls. Based on the above findings we discuss a role for PbGH3 during pathogenesis.

Early-stage responses to Plasmodiophora brassicae at the transcriptome and metabolome levels in clubroot resistant and susceptible oilseed Brassica napus.

Clubroot, a devastating soil-borne root disease, in Brassicaceae is caused by Plasmodiophora brassicae Woronin (P. brassicae W.), an obligate biotrophic protist. Plant growth and development, as well as seed yield of Brassica crops, are severely affected due to this disease. Several reports described the molecular responses of B. napus to P. brassicae; however, information on the early stages of pathogenesis is limited. In this study, we have used transcriptomics and metabolomics to characterize P. brassicae pathogenesis at 1-, 4-, and 7-days post-inoculation (DPI) in clubroot resistant (CR) and susceptible (CS) doubled-haploid (DH) canola lines. When we compared between inoculated and uninoculated groups, a total of 214 and 324 putative genes exhibited differential expression (q-value < 0.05) at one or more time-points in the CR and CS genotypes, respectively. When the inoculated CR and inoculated CS genotypes were compared, 4765 DEGs were differentially expressed (q-value < 0.05) at one or more time-points. Several metabolites related to organic acids (e.g., citrate, pyruvate), amino acids (e.g., proline, aspartate), sugars, and mannitol, were differentially accumulated in roots in response to pathogen infection when the CR and CS genotypes were compared. Several DEGs also corresponded to differentially accumulated metabolites, including pyrroline-5-carboxylate reductase (BnaC04g11450D), citrate synthase (BnaC02g39080D), and pyruvate kinase (BnaC04g23180D) as detected by transcriptome analysis. Our results suggest important roles for these genes in mediating resistance to clubroot disease. To our knowledge, this is the first report of an integrated transcriptome and metabolome analysis aimed at characterizing the molecular basis of resistance to clubroot in canola.

ITRAQ-based quantitative analysis reveals proteomic changes in Chinese cabbage (Brassica rapa L.) in response to Plasmodiophora brassicae infection

Clubroot disease is one of the major diseases affecting Brassica crops, especially Chinese cabbage (Brassica rapa L. ssp. pekinensis), which is known to be highly susceptible to the disease. In this study, the obligate biotrophic protist Plasmodiophora brassicae Woronin was used to infect the roots of Chinese cabbage seedlings. The disease symptoms were noticeable at 28 and 35 days after inoculation (DAI) in the susceptible (CM) line. Using isobaric tags for relative and absolute quantitation (iTRAQ) analysis, a total of 5,003 proteins of differential abundance were identified in the resistant/susceptible lines, which could be quantitated by dipeptide or polypeptide segments. Gene ontology (GO) analysis indicated that the differentially expressed proteins (DEPs) between the susceptible (CM) and resistant (CCR) lines were associated with the glutathione transferase activity pathway, which could catalyze the combination of glutathione and other electrophilic compounds to protect plants from disease. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEPs may be significantly enriched cytokinin signaling or arginine biosynthesis pathways, both of which are responses to stimuli and are plant defense reactions. The cytokinins may facilitate cell division in the shoot, resulting in the hypertrophy and formation of galls and the presentation of typical clubroot symptoms. In this study, the proteomic results provide a new perspective for creating germplasm resistance to P. brassicae, as well as a genetic basis for breeding to improve Chinese cabbage.

Role of Cytokinins in Clubroot Disease Development

Clubroot, caused by the obligate biotrophic protist Plasmodiophora brassicae is a devastating disease of crucifers that causes substantial economic loss worldwide. The disease is characterized by the formation of galls in the root and hypocotyl of infected plants which restricts host vascular cambium development inhibiting efficient water and nutrient uptake by the plant. The pathogen- driven interference of hormonal homeostasis, particularly of cytokinin, in the root tissue is intricately linked with induction of hypertrophy and cell divisions leading to formation of galls. Levels of cytokinins and cell division generally increase at the onset of the disease which declines at the later stages of gall formation. The genes involved cytokinin biosynthesis such as cytokinin oxidase/ dehydrogenases and isopentenyl transferases shows differential expressions during clubroot infection and gall expansion in root tissues. Wider understanding of the roles of cytokinins and associated genes along the development of the disease will be helpful in unravelling plants defense mechanism against clubroot disease.

Jasmonic Acid-Mediated Aliphatic Glucosinolate Metabolism Is Involved in Clubroot Disease Development in Brassica napus L.

Glucosinolate (GSL) is associated with clubroot disease, which is caused by the obligate biotrophic protist Plasmodiophora brassicae. Due to the complicated composition of GSLs, their exact role in clubroot disease development remains unclear. By investigating clubroot disease resistance in cruciferous plants and characterizing the GSL content in seeds, we can determine if clubroot disease development is related to the components of GSLs. The difference in the infection process between Matthiola incana L. (resistant) and Brassica napus L. (susceptible) was determined. Root hair infection was definitely observed in both resistant and susceptible hosts, but no infection was observed during the cortical infection stage in resistant roots; this finding was verified by molecular detection of P. brassicae via PCR amplification at various times after inoculation. Based on the time course detection of the contents and compositions of GSLs after P. brassicae inoculation, susceptible roots exhibited increased accumulation of aliphatic, indolic, and aromatic GSLs in B. napus, but only aromatic GSLs were significantly increased in M. incana. Gluconapin, which was the main aliphatic GSL in B. napus and present only in B. napus, was significantly increased during the secondary infection stage. Quantification of the internal jasmonic acid (JA) concentration showed that both resistant and susceptible plants exhibited an enhanced level of JA, particularly in susceptible roots. The exogenous JA treatment induced aliphatic GSLs in B. napus and aromatic GSLs in M. incana. JA-induced aromatic GSLs may be involved in the defense against P. brassicae, whereas aliphatic GSLs induced by JA in B. napus likely play a role during the secondary infection stage. Three candidate MYB28 genes regulate the content of aliphatic GSLs identified in B. napus; one such gene was BnMYB28.1, which was significantly increased following both the treatment with exogenous JA and P. brassicae inoculation. In summary, the increased content of JA during the secondary infection stage may induce the expression of BnMYB28.1, which caused the accumulation of aliphatic GSLs in clubroot disease development.

Genomic evidence for genes encoding leucine-rich repeat receptors linked to resistance against the eukaryotic extra- and intracellular Brassica napus pathogens Leptosphaeria maculans and Plasmodiophora brassicae.

Genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) control resistance against intracellular (cell-penetrating) pathogens. However, evidence for a role of genes coding for proteins with LRR domains in resistance against extracellular (apoplastic) fungal pathogens is limited. Here, the distribution of genes coding for proteins with eLRR domains but lacking kinase domains was determined for the Brassica napus genome. Predictions of signal peptide and transmembrane regions divided these genes into 184 coding for receptor-like proteins (RLPs) and 121 coding for secreted proteins (SPs). Together with previously annotated NLRs, a total of 720 LRR genes were found. Leptosphaeria maculans-induced expression during a compatible interaction with cultivar Topas differed between RLP, SP and NLR gene families; NLR genes were induced relatively late, during the necrotrophic phase of pathogen colonization. Seven RLP, one SP and two NLR genes were found in Rlm1 and Rlm3/Rlm4/Rlm7/Rlm9 loci for resistance against L. maculans on chromosome A07 of B. napus. One NLR gene at the Rlm9 locus was positively selected, as was the RLP gene on chromosome A10 with LepR3 and Rlm2 alleles conferring resistance against L. maculans races with corresponding effectors AvrLm1 and AvrLm2, respectively. Known loci for resistance against L. maculans (extracellular hemi-biotrophic fungus), Sclerotinia sclerotiorum (necrotrophic fungus) and Plasmodiophora brassicae (intracellular, obligate biotrophic protist) were examined for presence of RLPs, SPs and NLRs in these regions. Whereas loci for resistance against P. brassicae were enriched for NLRs, no such signature was observed for the other pathogens. These findings demonstrate involvement of (i) NLR genes in resistance against the intracellular pathogen P. brassicae and a putative NLR gene in Rlm9-mediated resistance against the extracellular pathogen L. maculans.

Manipulation of Auxin and Cytokinin Balance During the Plasmodiophora brassicae-Arabidopsis thaliana Interaction.

The symptoms of the clubroot disease on Brassica species caused by the obligate biotrophic protist Plasmodiophora brassicae relies, among other factors, on the modulation of plant hormones. Signaling, transport as well as biosynthesis and metabolism are key features how the levels of auxins and cytokinins are controlled. We here describe (a) how to inoculate the model plant Arabidopsis thaliana with P. brassicae, (b) qualitative and quantitative methods to evaluate disease severity in auxin and cytokinin mutants,

Arabidopsis Mutant bik1 Exhibits Strong Resistance to Plasmodiophora brassicae.

Botrytis-induced kinase1 (BIK1), a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1, and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2, and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40–50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS) at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA) treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2) and npr1-1 [non-expresser of PR genes that regulate systemic acquired resistance (SAR)] mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms.

PBT1, a novel antimicrobial protein from the biocontrol agent Bacillus subtilis XF-1 against Plasmodiophora brassicae

Plasmodiophora brassicae is a Plasmodiophorid and obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. It causes clubroot disease of cruciferous crops by producing resting spores that can survive in the soil for more than ten years. Although up to date there is no effective chemical control but now a strain of the bacterium Bacillus subtilis XF-1, isolated from China, has been found to inhibit P. brassicae and the mycelial growth of other plant-pathogenic fungi, such as Magnaporthe oryzae, Curvularia lunata,Fusarium oxysporum and so on. The antimicrobial activity of this biocontrol bacterium lies in its water-soluble protein (PBT1) which was isolated and purified by means of molecular sieve and affinity chromatography. The single band of the purified protein has a molecular mass of 25 kDa, estimated by a sodium dodecyl sulfate- polyacrylamide gel electrophoresis and it was heat resistant. The result of the sequencing proved PBT1 to be a new protein. Recombinant plasmid pQE81L-PBT1 was constructed using the coding gene of Pbt1 and plasmid PQE81-L, and expressed in Escherichia coli BL21. The fusion protein of BL21 exhibited significant detrimental effects on P. brassicae spores. In addition, pQE81L-PBT1 was successfully inserted into Pseudomonas fluorescens strain 2P24 which developed antagonistic effect to the clubroot pathogen that was not suppressed by 2P24 alone. The results showed that PBT1 protein might play a key role in the biocontrol of clubroot disease of crucifers.

Histological methods to detect the clubroot pathogen Plasmodiophora brassicae during its complex life cycle

The clubroot pathogen Plasmodiophora brassicae is an obligate biotrophic protist that lives in close relationship with its host cell. The roots of the host plants are colonized and the plant growth is altered upon infection. While shoots can be stunted and show wilt symptoms after longer infection periods, the root system is converted to a tumorous root tissue, called ‘clubroot’, by alterations of the plant growth promoting hormones auxin, cytokinin and brassinosteroid. Because the life cycle occurs largely within the host cells, this leads to dramatic changes in host root morphology and anatomy. Thus, the identification of the respective protist structures in the host tissue by microscopy is challenging. Different staining methods as well as fluorescence and electron microscopy of thin sections can reveal specific life stages of P. brassicae and can yield additional information on the changes in the host tissues concerning, for example, cell wall properties. In addition, promoter–reporter fusions, immunostaining methods and in situ hybridization techniques can be used to gain additional information on the changes in the host roots.

Putative role of IAA during the early response of Brassica napus L. to Plasmodiophora brassicae

This study examined the role of indole-3-acetic acid (IAA) during the early stage of infection of rapeseed (Brassica napus L.) by the obligate biotrophic protist Plasmodiophora brassicae Woron., which causes clubroot disease. Treatment of infected B. napus seedlings with exogenous IAA promoted the development of clubroot disease, including an increase in the number and size of galls, whereas treatment with N-1-naphthylphthalamic acid (NPA) attenuated the disease and altered the location and size of the root nodules, resulting in a reduced disease index. An increased level of free IAA was detected in the roots at 3 and 7 days after inoculation (DAI) compared with the control, suggesting the activation of IAA signaling in the early stage of P. brassicae infection. Moreover, IAA treatment caused no significant change in the germination rate of the resting spores but resulted in enhanced root hair infection. RT–PCR and quantitative real-time PCR (qPCR) were used to analyze the expression of IAA homeostasis-related genes (3, 7, and 10 DAI) to identify gene expression patterns in the roots and leaves of B. napus after inoculation. Five IAA biosynthesis-related genes examined in this study were induced in the roots after inoculation with P. brassicae but not in the leaves. The rapid induction of BnAAO4 expression at 3 DAI may be responsible for the overproduction of IAA during the early stages of infection. This study suggests that IAA acts as a signaling molecule that putatively stimulates root hair infection in the early response of B. napus to P. brassicae infection.

Response of Arabidopsis thaliana Roots with Altered Lipid Transfer Protein (LTP) Gene Expression to the Clubroot Disease and Salt Stress.

The clubroot disease of Brassicaceae is caused by the obligate biotrophic protist Plasmodiophora brassicae. The disease is characterized by abnormal tumorous swellings of infected roots that result in reduced drought resistance and insufficient distribution of nutrients, leading to reduced crop yield. It is one of the most damaging diseases among cruciferous crops worldwide. The acquisition of nutrients by the protist is not well understood. Gene expression profiles in Arabidopsis thaliana clubroots indicate that lipid transfer proteins (LTPs) could be involved in disease development or at least in adaptation to the disease symptoms. Therefore, the aim of the study was to examine the role of some, of the still enigmatic LTPs during clubroot development. For a functional approach, we have generated transgenic plants that overexpress LTP genes in a root specific manner or show reduced LTP gene expression. Our results showed that overexpression of some of the LTP genes resulted in reduced disease severity whereas the lipid content in clubs of LTP mutants seems to be unaffected. Additional studies indicate a role for some LTPs during salt stress conditions in roots of A. thaliana.

New kid on the block – the clubroot pathogen genome moves the plasmodiophorids into the genomic era

Plasmodiophora brassicae causes clubroot on cruciferous plants and causes worldwide huge economical losses on important Brassica crops. P. brassicae infection produces large root galls, the clubroots, which can also affect the upper plant parts by reduced water and nutrient uptake and redirection of assimilates from leaves to roots. P. brassicae is an obligate biotrophic protist in the plasmodiophorids within the eukaryote supergroup of Rhizaria and is unrelated to other known plant pathogens. Plasmodiophorids can be parasites of plants and oomycetes. The recently released genome of P. brassicae is only the third in the poorly studied Rhizaria and the first plant pathogenic genome of this eukaryotic group. The P. brassicae genome was estimated to be 25.5 Mb in size and predicted to contain 9730 gene models. A transcriptome of P. brassicae and Spongospora subterranea, the potato scab pathogen was also presented. Consequently, for the first time large scale data for a eukaryotic plant pathogen group outside the fungi and oomycetes are now available. This review highlights selected characteristics of the P. brassicae genome including molecular events shown or predicted to take place in each phase of its life-cycle, such as manipulation of: 1) host primary metabolism, 2) plant hormone homeostasis, and 3) plant defense. Further, future directions and challenges in the P. brassicae and plasmodiophorid genomic research are discussed.

The Plasmodiophora brassicae genome reveals insights in its life cycle and ancestry of chitin synthases

Plasmodiophora brassicae causes clubroot, a major disease of Brassica oil and vegetable crops worldwide. P. brassicae is a Plasmodiophorid, obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. Here we present the 25.5 Mb genome draft of P. brassicae, developmental stage-specific transcriptomes and a transcriptome of Spongospora subterranea, the Plasmodiophorid causing powdery scab on potato. Like other biotrophic pathogens both Plasmodiophorids are reduced in metabolic pathways. Phytohormones contribute to the gall phenotypes of infected roots. We report a protein (PbGH3) that can modify auxin and jasmonic acid. Plasmodiophorids contain chitin in cell walls of the resilient resting spores. If recognized, chitin can trigger defense responses in plants. Interestingly, chitin-related enzymes of Plasmodiophorids built specific families and the carbohydrate/chitin binding (CBM18) domain is enriched in the Plasmodiophorid secretome. Plasmodiophorids chitin synthases belong to two families, which were present before the split of the eukaryotic Stramenopiles/Alveolates/Rhizaria/Plantae and Metazoa/Fungi/Amoebozoa megagroups, suggesting chitin synthesis to be an ancient feature of eukaryotes. This exemplifies the importance of genomic data from unexplored eukaryotic groups, such as the Plasmodiophorids, to decipher evolutionary relationships and gene diversification of early eukaryotes.

배추 뿌리혹병균 Plasmodiophora brassicae의 종 특이적 프라이머 개발

Clubroot caused by the obligate biotrophic protist Plasmodiophora brassicae Woronin is one of the most damaging diseases of Brassicaceae family. In this study, we developed species-specific primer sets for rapid and accurate detection of P. brassicae. The primer sets developed amplified a specific fragment only from P. brassicae DNA while they did not amplify a band from 10 other soilborne pathogens or from Kimchi cabbage. In sensitivity test, the species-specific primer set ITS1-1/ITS1-2 could work for approximately 10 spores/ml of genomic DNA showing more sensitivity and accuracy than previous methods. With quantitative real-time PCR test, the primer set detected less spores of P. brassicae than before, confirming that the species-specific primer set could be useful for rapid and accurate detection of P. brassicae.

Cross-kingdom host shifts of phytomyxid parasites

BackgroundPhytomyxids (plasmodiophorids and phagomyxids) are cosmopolitan, obligate biotrophic protist parasites of plants, diatoms, oomycetes and brown algae. Plasmodiophorids are best known as pathogens or vectors for viruses of arable crops (e.g. clubroot in brassicas, powdery potato scab, and rhizomania in sugar beet). Some phytomyxid parasites are of considerable economic and ecologic importance globally, and their hosts include important species in marine and terrestrial environments. However most phytomyxid diversity remains uncharacterised and knowledge of their relationships with host taxa is very fragmentary.ResultsOur molecular and morphological analyses of phytomyxid isolates–including for the first time oomycete and sea-grass parasites–demonstrate two cross-kingdom host shifts between closely related parasite species: between angiosperms and oomycetes, and from diatoms/brown algae to angiosperms. Switching between such phylogenetically distant hosts is generally unknown in host-dependent eukaryote parasites. We reveal novel plasmodiophorid lineages in soils, suggesting a much higher diversity than previously known, and also present the most comprehensive phytomyxid phylogeny to date.ConclusionSuch large-scale host shifts between closely related obligate biotrophic eukaryote parasites is to our knowledge unique to phytomyxids. Phytomyxids may readily adapt to a wide diversity of new hosts because they have retained the ability to covertly infect alternative hosts. A high cryptic diversity and ubiquitous distribution in agricultural and natural habitats implies that in a changing environment phytomyxids could threaten the productivity of key species in marine and terrestrial environments alike via host shift speciation.

Laser Microdissection Coupled to Transcriptional Profiling of Arabidopsis Roots Inoculated by Plasmodiophora brassicae Indicates a Role for Brassinosteroids in Clubroot Formation

The clubroot disease caused by the obligate biotrophic protist Plasmodiophora brassicae on host plants of the Brassicaceae family is characterized by enhanced cell division and cell expansion. Since a typical root section of an infected plant always includes different stages of the pathogen as well as uninfected cells, we were interested in investigating specific developmental stages of the pathogen and their effect on host transcriptional changes. We extended previous microarray studies on whole roots by using laser microdissection and pressure catapulting (LMPC) to isolate individual cells harboring defined developmental stages of the pathogen. In addition, we compared the central cylinder of infected plants with that of control plants. We were especially interested in elucidating the stage-specific hormonal network. The up-regulation of genes involved in auxin and cytokinin metabolism and signaling was confirmed. In addition, we found evidence that brassinosteroid (BR) synthesis and signal perception genes were in many cases up-regulated in enlarged cells and the central cylinder. This was confirmed by quantitative PCR. Treatment of wild-type plants with the BR biosynthesis inhibitor propiconazole reduced gall formation, and the analysis of the BR receptor mutant bri1-6 revealed less severe gall formation than in the respective wild type. Our results identify novel hormone pathways involved in clubroot development. Using LMPC to generate pools of homogeneous cell type populations combined with transcriptome analysis has been very useful to elucidate the regulation of gall growth by this obligate biotropic pathogen in a cell- and stage-specific manner.

The Clubroot Pathogen (Plasmodiophora brassicae) Influences Auxin Signaling to Regulate Auxin Homeostasis in Arabidopsis.

The clubroot disease, caused by the obligate biotrophic protist Plasmodiophora brassicae, affects cruciferous crops worldwide. It is characterized by root swellings as symptoms, which are dependent on the alteration of auxin and cytokinin metabolism. Here, we describe that two different classes of auxin receptors, the TIR family and the auxin binding protein 1 (ABP1) in Arabidopsis thaliana are transcriptionally upregulated upon gall formation. Mutations in the TIR family resulted in more susceptible reactions to the root pathogen. As target genes for the different pathways we have investigated the transcriptional regulation of selected transcriptional repressors (Aux/IAA) and transcription factors (ARF). As the TIR pathway controls auxin homeostasis via the upregulation of some auxin conjugate synthetases (GH3), the expression of selected GH3 genes was also investigated, showing in most cases upregulation. A double gh3 mutant showed also slightly higher susceptibility to P. brassicae infection, while all tested single mutants did not show any alteration in the clubroot phenotype. As targets for the ABP1-induced cell elongation the effect of potassium channel blockers on clubroot formation was investigated. Treatment with tetraethylammonium (TEA) resulted in less severe clubroot symptoms. This research provides evidence for the involvement of two auxin signaling pathways in Arabidopsis needed for the establishment of the root galls by P. brassicae.