Moss (Physcomitrella patens) GH3 proteins act in auxin homeostasis.

Auxins are hormones involved in many cellular, physiological and developmental processes in seed plants and in mosses such as Physcomitrella patens. Control of auxin levels is achieved in higher plants via synthesis of auxin conjugates by members of the GH3 family. The role of the two GH3-like proteins from P. patens for growth and auxin homeostasis was therefore analysed. The in vivo-function of the two P. patens GH3 genes was investigated using single and double knockout mutants. The two P. patens GH3 proteins were also heterologously expressed to determine their enzymatic activity. Both P. patens GH3 enzymes accepted the auxin indole acetic acid (IAA) as substrate, but with different preferences for the amino acid to which it is attached. Cytoplasmic localization was shown for PpGH3-1 tagged with green fluorescent protein (GFP). Targeted knock-out of either gene exhibited an increased sensitivity to auxin, resulting in growth inhibition. On plain mineral media mutants had higher levels of free IAA and less conjugated IAA than the wild type, and this effect was enhanced when auxin was supplied. The DeltaPpGH3-1/DeltaPpGH3-2 double knockout had almost no IAA amide conjugates but still synthesized ester conjugates. Taken together, these data suggest a developmentally controlled involvement of P. patens GH3 proteins in auxin homeostasis by conjugating excess of physiologically active free auxin to inactive IAA-amide conjugates.

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,

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.

A novel methyltransferase from the intracellular pathogen Plasmodiophora brassicae methylates salicylic acid.

The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana, which is characterized by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. Plasmodiophora brassicae seems to suppress plant defence reactions, but information on how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease. The accumulation of SA and the emission of methylated SA (methyl salicylate, MeSA) were observed in P. brassicae-infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the mutant Atbsmt1, deficient in the methylation of SA, indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. We found that P. brassicae possesses a methyltransferase (PbBSMT) with homology to plant methyltransferases. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses, we showed that PbBSMT can methylate SA, benzoic and anthranilic acids.

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.