Arabidopsis thaliana expressing PbBSMT, a gene encoding a SABATH-type methyltransferase from the plant pathogenic protist Plasmodiophora brassicae, show leaf chlorosis and altered host susceptibility.

The plant pathogenic protist Plasmodiophora brassicae causes clubroot disease of Brassicaceae. This biotrophic organism can down-regulate plant defence responses. The previously characterised P. brassicae PbBSMT methyltransferase has substrate specificity for salicylic, benzoic and anthranilic acids. We therefore propose a role for the methylation of SA in attenuating plant defence response in infected roots as a novel strategy for intracellular parasitism. We overexpressed PbBSMT under the control of an inducible promoter in Arabidopsis thaliana and performed physiological, molecular and phytopathological analyses with the transgenic plants under control and induced conditions in comparison to the wild type. Upon induction, transcription of PbBSMT was associated with: (1) strong leaf phenotypes from anthocyanin accumulation and chlorosis followed by browning; (2) increased plant susceptibility after infection with P. brassicae that was manifested as more yellow leaves and reduced growth of upper plant parts; and (3) induced transgenic plants were not able to support large galls and had a brownish appearance of some clubs. Microarray data indicated that chlorophyll loss was accompanied by reduced transcription of genes involved in photosynthesis, while genes encoding glucose metabolism, mitochondrial functions and cell wall synthesis were up-regulated. Our results indicate a role for PbBSMT in attenuation of host defence responses in the roots by metabolising a plant defence signal.

Biochemical and epigenetic changes in phytoplasma-recovered periwinkle after indole-3-butyric acid treatment.

AIM: To elucidate the possible mechanism of phytoplasma elimination from periwinkle shoots caused by indole-3-butyric acid (IBA) treatment. METHODS AND RESULTS: It has been shown that a transfer of in vitro-grown phytoplasma-infected Catharanthus roseus (periwinkle) plantlets from medium supplemented with 6-benzylaminopurine (BA) to one supplemented with IBA can induce remission of symptoms and even permanent elimination of 'Candidatus Phytoplasma asteris' reference strain HYDB. Endogenous auxin levels and general methylation levels in noninfected periwinkles, periwinkles infected with two 'Candidatus Phytoplasma' species and phytoplasma-recovered periwinkles were measured and compared. After the transfer from cytokinin- to auxin-containing media, healthy shoots maintained their phenotype, methylation levels and hormone concentrations. Phytoplasma infection caused a change in the endogenous indole-3-acetic acid to IBA ratio in periwinkle shoots infected with two 'Candidatus Phytoplasma' species, but general methylation was significantly changed only in shoots infected with 'Ca. P. asteris', which resulted in the only phytoplasma species eliminated from shoots after transfer to IBA-containing medium. Both phytoplasma infection and treatment with plant growth regulators influenced callose deposition in phloem tissue, concentrations of photosynthetic pigments and soluble proteins, H(2) O(2) levels and activities of catalase (CAT) and ascorbate peroxidase (APX). CONCLUSION: Lower level of host genome methylation in 'Ca. P. asteris'-infected periwinkles on medium supplemented with BA was significantly elevated after IBA treatment, while IBA treatment had no effect on cytosine methylation in periwinkles infected with 'Candidatus Phytoplasma ulmi' strain EY-C. SIGNIFICANCE AND IMPACT OF THE STUDY: Hormone-dependent recovery is a distinct phenomenon from natural recovery. As opposed to spontaneously recovered plants in which elevated peroxide levels and differential expression of peroxide-related enzymes were observed, in hormone-dependent recovery changes in global host genome, methylation coincide with the presence/absence of phytoplasma.

Indole-3-acetic acid protein conjugates: novel players in auxin homeostasis.

Indole-3-acetic acid (IAA) is found in plants in both free and conjugated forms. Within the group of conjugated IAA there is a unique class of proteins and peptides where IAA is attached directly to the polypeptide structure as a prosthetic group. The first gene, IAP1, encoding for a protein with IAA as a prosthetic group, was cloned from bean (Phaseolus vulgaris). It was shown that the expression of IAP1 as a major IAA modified protein in bean seed (PvIAP1) was correlated to a developmental period of rapid growth during seed development. Moreover, this protein underwent rapid degradation during germination. Since further molecular analysis was difficult in bean, the IAP1 gene was transformed into Arabidopsis thaliana and Medicago truncatula. Expression of the bean IAP1 gene in both plant species under the control of its native promoter targeted protein expression to the seeds. In Arabidopsis no IAA was found to be attached to PvIAP1. These results show that there is specificity to protein modification by IAA and suggests that protein conjugation may be catalyzed by species specific enzymes. Furthermore, subcellular localization showed that in Arabidopsis PvIAP1 was predominantly associated with the microsomal fraction. In addition, a related protein and several smaller peptides that are conjugated to IAA were identified in Arabidopsis. Further research on this novel class of proteins from Arabidopsis will both advance our knowledge of IAA proteins and explore aspects of auxin homeostasis that were not fully revealed by studies of free IAA and lower molecular weight conjugates.

A family of auxin conjugate hydrolases from Brassica rapa: characterization and expression during clubroot disease.

During the obligate biotrophic interaction of Plasmodiophora brassicae with members of the Brassicaceae, the host roots show hypertrophy and galls are established. An increased auxin pool appears to correlate with cell expansion and cell division, but the origin of the free auxin is not yet clear. As previous results point to increased IAA-hydrolytic activity in infected roots of Brassica rapa at later time points of infection, we isolated IAA-amidohydrolase-like genes from various tissues. We cloned full-length cDNAs of two genes with high homology to the Arabidopsis IAR3 (Br-IAR3) as well as full-length clones corresponding to the Arabidopsis ILL2/ILL1 (Br-ILL2) and ILL6 (Br-ILL6) hydrolase genes. Using heterologous expression in Escherichia coli, we showed that Br-IAR3 24 and Br-ILL2 possess hydrolytic activity in vitro. Real-time reverse transcription (RT)-PCR revealed that only Br-IAR3 25 and Br-ILL6 are expressed differentially during clubroot disease, but showed a decreased expression at later time point of infection. These findings are discussed with regard to a negative regulation in IAA homeostasis during clubroot disease.

The tu8 mutation of Arabidopsis thaliana encoding a heterochromatin protein 1 homolog causes defects in the induction of secondary metabolite biosynthesis.

The tu8 mutant of Arabidopsis thaliana (L.) Heynh. was previously described as deficient in pathogen-induced auxin and glucosinolate (GSL) accumulation, as well as in heat-induced accumulation of cytosolic Hsp90, the latter feature was accom"panied by reduced thermotolerance at higher temperatures. The mutated gene was shown to be a novel allele of TERMINAL FLOWER2, encoding the only Arabidopsis homolog for heterochromatin protein 1 (Kim et al., 2004). In this report, we investigated the influence of heat stress on auxin and GSL content, as well as the accumulation of several secondary metabolites derived from the phenylpropanoid pathway, including anthocyanins and sinapine derivatives, in the mutant tu8. tu8 had less sinapine and sinapoyl esters compared to the wild type. In addition, the induction of sinapine by heat shock in Columbia was not found in tu8. Anthocyanins were also induced by heat stress in wild type plants, whereas tu8 showed only slight induction of these compounds and only at higher temperatures. GSLs were induced at higher temperatures in the wild type, but induction was absent in tu8. Transcript levels known to be involved in IAA/glucosinolate synthesis and metabolism (nitrilase and myrosinase) were examined and both showed developmental regulation, while only nitrilase mRNA levels differed between wild type and mutant seedlings. Treatment of Columbia and tu8 with jasmonic acid (JA), a known inducer of glucosinolates, showed differences between wild type and tu8 with respect to induction of individual GSLs and anthocyanins. However, the transcript level of the TU8/TFL2 gene after heat shock and jasmonate treatment did not change. Loss of function or altered function in the heterochromatin protein most likely lead to the pleiotropic phenotype observed for the tu8 mutant.

A glucosinolate mutant of Arabidopsis is thermosensitive and defective in cytosolic Hsp90 expression after heat stress.

The TU8 mutant of Arabidopsis previously described to be deficient in glucosinolate metabolism and pathogen-induced auxin accumulation was found to be remarkably less tolerant upon exposure to elevated temperatures than wild-type plants. Although moderately increased temperature only affected shoot growth, exposure to severe heat stress led to a dramatic decay of mutant plants. By contrast, wild-type seedlings showed little or no damage under the same conditions. Analysis of different heat stress proteins (Hsps) in TU8 seedlings revealed that only expression of cytoplasmic Hsp90 was affected in these plants. Although Hsp90 was present under control conditions, its level declined in mutant plants at elevated temperatures. Northern-blot analysis indicated that the decrease in Hsp90 protein was accompanied with a reduction of hsp90 transcript levels. Transient expression of Hsp90 in mutant protoplasts increased their survival rate at higher temperatures to near equivalent that of wild-type protoplasts. These data suggest that the reduced level of Hsp90 in TU8 mutants may be the primary cause for the observed reduction in thermostability.

[Kohlhernie - neue Ansätze zur Bekämpfung der gefürchteten Kohlerkrankung]

Durch Pflanzenkrankheiten entstehen weltweit hohe Ernteverluste. Eine der häufigsten Infektionen in der Familie der Kreuzb&luml;tengewächse wird durch den Pilz Plasmodiophora brassicae verursacht. Die Krankheit äußert sich in Form von Wurzeltumoren, den "Kohlherniegallen". Da es auf verseuchten Böden zu hohen Ernteausfällen kommt, besteht ein enormes Interesse an der Aufklärung der Vorgänge, die zu diesem Krankheitsbild führen.

Expression and localization of nitrilase during symptom development of the clubroot disease in Arabidopsis.

The expression of nitrilase in Arabidopsis during the development of the clubroot disease caused by the obligate biotroph Plasmodiophora brassicae was investigated. A time course study showed that only during the exponential growth phase of the clubs was nitrilase prominently enhanced in infected roots compared with controls. NIT1 and NIT2 are the nitrilase isoforms predominantly expressed in clubroot tissue, as shown by investigating promoter-beta-glucuronidase fusions of each. Two peaks of beta-glucuronidase activity were visible: an earlier peak (21 d post inoculation) consisting only of the expression of NIT1, and a second peak at about 32 d post inoculation, which predominantly consisted of NIT2 expression. Using a polyclonal antibody against nitrilase, it was shown that the protein was mainly found in infected cells containing sporulating plasmodia, whereas in cells of healthy roots and in uninfected cells of inoculated roots only a few immunosignals were detected. To determine which effect a missing nitrilase isoform might have on symptom development, the P. brassicae infection in a nitrilase mutant (nit1-3) of Arabidopsis was investigated. As a comparison, transgenic plants overexpressing NIT2 under the control of the cauliflower mosaic virus 35S promoter were studied. Root galls were smaller in nit1-3 plants compared with the wild type. The phenotype of smaller clubs in the mutant was correlated with a lower free indole-3-acetic acid content in the clubs compared with the wild type. Overexpression of nitrilase did not result in larger clubs compared with the wild type. The putative role of nitrilase and auxins during symptom development is discussed.

Differences in glucosinolate patterns and arbuscular mycorrhizal status of glucosinolate-containing plant species.

Under defined laboratory conditions it was shown that two glucosinolate-containing plant species, Tropaeolum majus and Carica papaya, were colonized by arbuscular mycorrhizal (AM) fungi, whereas it was not possible to detect AM fungal structures in other glucosinolate-containing plants (including several Brassicaceae). Benzylglucosinolate was present in all of the T. majus cultivars and in C. papaya it was the major glucosinolate. 2-Phenylethylglucosinolate was found in most of the non-host plants tested. Its absence in the AM host plants indicates a possible role for the isothiocyanate produced from its myrosinase-catalysed hydrolysis as a general AM inhibitory factor in non-host plants. The results suggest that some of the indole glucosinolates might also be involved in preventing AM formation in some of the species. In all plants tested, both AM hosts and non-hosts, the glucosinolate pattern was altered after inoculation with one of three different AM fungi (Glomus mosseae, Glomus intraradices and Gigaspora rosea), indicating signals between AM fungi and plants even before root colonization. The glucosinolate induction was not specifically dependent on the AM fungus. A time-course study in T. majus showed that glucosinolate induction was present during all stages of mycorrhizal colonization.

Indole glucosinolate and auxin biosynthesis in Arabidopsis thaliana (L.) Heynh. glucosinolate mutants and the development of clubroot disease.

Mutants and wild type plants of Arabidopsis thaliana were analysed for differences in glucosinolate accumulation patterns, indole-3-acetic acid (IAA) biosynthesis and phenotype. A previously identified series of mutants, termed TU, with altered glucosinolate patterns was used in this study. Only the line TU8 was affected in shoot phenotype (shorter stems, altered branching pattern). Synthesis of IAA and metabolism were not much affected in the TU8 mutant during seedling development, although the content of free IAA peaked earlier in TU8 during plant development than in the wild type. Indole glucosinolates and IAA may, however, be involved in the development of clubroot disease caused by the obligate biotrophic fungus Plasmodiophora brassicae since the TU3 line had a lower infection rate than the wild type, and lines TU3 and TU8 showed decreased symptom development. The decline in clubroot formation was accompanied by a reduced number of fungal structures within the root cortex and slower development of the fungus. Indole glucosinolates were lower in infected roots of TU3 and TU8 than in control roots of these lines, whereas in wild-type plants the differences were not as prominent. Free IAA and indole-3-acetonitrile (IAN) were increased in infected roots of the wild type and mutants with normal clubroot symptoms, whereas they were reduced in infected roots of mutants TU3 and TU8. These results indicate a role for indole glucosinolates and IAN/IAA in relation to symptom development in clubroot disease.

High-level expression of a viscotoxin in Arabidopsis thaliana gives enhanced resistance against Plasmodiophora brassicae.

Viscotoxins are a group of toxic thionins found in several mistletoe species. The constitutive CaMV-omega promoter was used to drive the expression of the viscotoxin A3 cDNA from Viscum album in transgenic Arabidopsis thaliana C24. Lines with high viscotoxin A3 levels in all parts of the plant were selected and tested for resistance against the clubroot pathogen Plasmodiophora brassicae. The transgenic lines were more resistant to infection by this pathogen than the parental line.

Occurrence and in Vivo Biosynthesis of Indole-3-Butyric Acid in Corn (Zea mays L.).

Indole-3-butyric acid (IBA) was identified as an endogenous compound in leaves and roots of maize (Zea mays L.) var Inrakorn by thin layer chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry. Its presence was also confirmed in the variety Hazera 224. Indole-3-acetic acid (IAA) was metabolized to IBA in vivo by seedlings of the two maize varieties. The reaction product was identified by thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry after incubating the corn seedlings with [(14)C]IAA and [(13)C(6)]IAA. The in vivo conversion of IAA to IBA and the characteristics of IBA formation in two different maize varieties of Zea mays L. (Hazera 224 and Inrakorn) were investigated. IBA-forming activity was examined in the roots, leaves, and coleoptiles of both maize varieties. Whereas in the variety Hazera 224, IBA was formed mostly in the leaves, in the variety Inrakorn, IBA synthesis was detected in the roots as well as in the leaves. A time course study of IBA formation showed that maximum activity was reached in Inrakorn after 1 hour and in Hazera after 2 hours. The pH optimum for the uptake of IAA was 6.0, and that for IBA formation was 7.0. The K(m) value for IBA formation was 17 micromolar for Inrakorn and 25 micromolar for Hazera 224. The results are discussed with respect to the possible functions of IBA in the plant.