Transcriptional profiling of Piriformospora indica colonizing living and dead barley roots
ABSTRACT: Recent sequencing projects have provided deep insight into fungal lifestyle-associated genomic adaptations. Here we report on the 25 Mb genome of the mutualistic root symbiont Piriformospora indica (Sebacinales, Basidiomycota) and provide a global characterization of fungal transcriptional responses associated with the colonization of living and dead roots. Extensive comparative analysis of the P. indica genome with other Basidiomycota and Ascomycota fungi that have diverse lifestyles strategies identified features typically associated with both, biotrophism and saprotrophism. The tightly controlled expression of the lifestyle-associated gene sets during the onset of the symbiosis, revealed by microarrays analysis, argues for a biphasic root colonization strategy of P. indica. Our finding provides a significant advance in understanding development of biotrophic plant symbionts and suggests a series of incremental shifts along the continuum from saprotrophy towards biotrophy in the evolution of mycorrhizal association from decomposer fungi. P. indica (DSM 11827, DSMZ) was cultivated on complex medium agar plates or liquid medium as described before (Zuccaro et al., 2009). Barley seeds (Hordeum vulgare L. cv. Golden Promise) were surface sterilized with 3 % sodium hypochlorite, rinsed in water and pregerminated for 3 days. To address the experimental design four different treatments were done (P. indica on barley roots on 1/10 PNM medium, P. indica on autoclaved barley roots on 1/10 PNM medium, P. indica on 1/10 PNM medium and P. indica on CM medium), each in three independent biological replications. Root and fungal material was harvested in liquid nitrogen after 24, 36, 48, 72, 120 and 168 hpi. For each time point roots from 15 to 20 living plants or 21 to 36 autoclaved plants were pooled.
Project description:Broad-host root endophytes establish long-term interactions with a large variety of plants, thereby playing a significant role in natural and managed ecosystems and in evolution of land plants. To exploit plants as living substrates and to establish a compatible interaction with morphologically and biochemically extremely different hosts, endophytes must respond and adapt to different plant signals and host metabolic states. Here we identified host-adapted colonization strategies and host-specific effector candidates of the mutualistic root endophyte Piriformospora indica by a global investigation of fungal transcriptional responses to barley and Arabidopsis at different symbiotic stages. Additionally we examined the role played by nitrogen in these two diverse associations. Cytological studies and colonization analyses of a barley mutant and fungal RNAi strains show that distinct physiological and metabolic signals regulate host-specific lifestyle in P. indica. This is the foundation for exploring how distinct fungal and host symbiosis determinants modulate biotrophy in one host and saprotrophy in another host and, ultimately, gives hints into the mechanisms underlying host adaptation in root symbioses. Arabidopsis and barley roots were inoculated with Piriformospora indica and grown for 14 days. Additionally P. indica was grown on 1/10 PNM medium alone. Samples were taken 3 and 14 dpi (Arabidopsis), 14 dpi (barley) and 3dpi (1/10 PNM). Each experiment was performed in three independent biological repetitions. Piriformospora indica gene expression examined only.
Project description:The fungal mutualist Piriformospora indica is colonising barley roots thereby mediating various beneficial effects to its host. The interaction is characterised by an initial biotrophic interaction stage which is followed by a cell death-dependent colonisation phase. We used microarrays to identify the global programme of gene expression during the colonisation process of barley roots by P. indica and to obtain informations into plant defense and metabolic reprogramming. In three independent experiments plants were inoculated with Piriformospora indica. Samples from inoculated roots were taken at 1, 3, and 7 days after inoculation. Samples from uninfected control plants were taken at the same time points.
Project description:Colonization of barley roots with the basidiomycete fungus Piriformospora indica enhances resistance against the leaf pathogen Blumeria graminis f.sp. hordei (Bgh). To identify genes involved in this mycorrhiza-induced systemic resistance, we used the Affymetrix Barley1 22K gene chip for leaf transcriptome analysis of P. indica-colonized and non-colonized barley plants 12, 24 and 96 hours post inoculation (hpi) with a compatible Bgh strain.
Project description:Piriformospora indica, an endophytic fungus of Sebacinales, colonizes the roots of many plant species including Arabidopsis thaliana. The symbiotic interaction promotes plant per-formance, growth and resistance/tolerance against abiotic and biotic stress. We demonstrate that exudated compounds from the fungus activate stress and defense responses in the Arabidopsis roots and shoots before the two partners are in physical contact. They induce stomata closure, stimulate reactive oxygen species (ROS) production, stress-related phytohormone accumulation and activate defense and stress genes in the roots and/or shoots. Once a physical contact is established, the stomata re-open, ROS and phytohormone levels decline, and the gene expression pattern indicates a shift from defense to mutualistic interaction. We propose that exudated compounds from P. indica induce stress and defense responses in the host. Root colonization results in the downregulation of defense responses and the activation of genes involved in promoting plant growth, metabolism and performance. Twelve day-old (48 h cold treatment and 10 days of illumination) Arabidopsis seedlings of equal sizes were selected for co-cultivation experiments. They were transferred to PNM plates with a nylone membrane on the top (Johnson et al. 2011) and exposed to a fungal plug 5 mm in diameter or a KM plug of the same size without fungal hyphae (control). The plugs were placed 3 cm away from the closest root part . The light intensity (80 ± 5 μmol m-2 sec-1) was checked every third day to ensure that both P. indica- and mock-treated seedlings receive equal amounts of light.
Project description:The fungal mutualist Piriformospora indica is colonising barley roots thereby mediating various beneficial effects to its host. The interaction is characterised by an initial biotrophic interaction stage which is followed by a cell death-dependent colonisation phase. We used microarrays to identify the global programme of gene expression during the colonisation process of barley roots by P. indica and to obtain informations into plant defense and metabolic reprogramming. Overall design: In three independent experiments plants were inoculated with Piriformospora indica. Samples from inoculated roots were taken at 1, 3, and 7 days after inoculation. Samples from uninfected control plants were taken at the same time points.
Project description:The recent release of a large number of genomes from ectomycorrhizal, orchid mycorrhizal and root endophytic fungi have provided deep insight into fungal lifestyle-associated genomic adaptation. Comparative analyses of symbiotic fungal taxa showed that similar outcomes of interactions in distant related root symbioses are examples of convergent evolution. The order Sebacinales represents a sister group to the Agaricomycetes (Basidiomycota) that is comprised of ectomycorrhizal, ericoid-, orchid- mycorrhizal, root endophytic fungi and saprotrophs (Oberwinkler et al., 2013). Sebacinoid taxa are widely distributed from arctic to temperate to tropical ecosystems and are among the most common and species-rich groups of ECM, OM and endophytic fungi (Tedersoo et al., 2012, Tedersoo et al., 2010, Oberwinkler et al., 2013). The root endophyte Piriformospora indica and the orchid mycorrhizal fungus S. vermifera (MAFF 305830) are non-obligate root symbionts which were shown to be able to interact with many different experimental hosts, including the non-mycorrhizal plant Arabidopsis thaliana. These two fungi display similar colonization strategies in barley and in Arabidopsis and the ability to establish beneficial interactions with different hosts (Deshmukh et al., 2006). Colonization of the roots by P. indica and S. vermifera results in enhanced seed germination and biomass production as well as increased resistance against biotic and abiotic stresses in its experimental hosts, including various members of the Brassicaceae family, barley, Nicotiana attenuata and switchgrass (Ghimire, 2011, Ghimire et al., 2009, Ghimire et al., 2011, Waller et al., 2008, Barazani et al., 2007, Deshmukh et al., 2006). Microarray experiments were performed to identify and characterize conserved sebacinoid genes as key determinants in the Sebacinales symbioses. Overall design: Arabidopsis roots were inoculated with either Sebacina vermifera or Piriformospora indica and grown for up to 14 days. Additionally S. vermifera and A. thaliana were grown on 1/10 PNM medium alone. Samples were taken 3, 7 and 14 dpi (Arabidopsis), 3dpi (1/10 PNM, S. vermifera) and 7dpi (1/10 PNM, A. thaliana). Each experiment was performed in three independent biological repetitions. The experiments are follow-ups of previous experiments deposited under GSE47775.
Project description:Identification of transcripts that change expression in roots of Nicotiana attenuata plants with reduced expression of HSPRO and in association with Piriformospora indica. Gene expression in roots of Nicotiana attenuata wild type and ir-hspro seedlings was measured at 14 days after inoculation with Piriformospora indica. Three independent experiments were performed with wild type plants and three independent experiments were performed with ir-hspro plants.
Project description:The endophytic fungus Piriformospora indica is a member of Sebacinales. It colonizes the roots of many plant species including the model plant Arabidospis thaliana (Peskan-Berghofer et al. 2004). P. indica promotes plant growth (Peskan-Berghofer et al. 2004), seed production (Shahollari et al. 2007) and confers resistance to biotic (Camehl et al. 2011) and abiotic stresses (Sherameti et al., 2008, Matsuo et al., 2015). Vadassery et al. (2009) have shown that a cell wall extract from P. indica induces cytoplasmic calcium elevation in Arabidopsis Col-0 roots. Johnson et al. (2017) isolated an ethylmethane-induced mutant cycam (cytoplasmic calcium elevation mutant) which fails to induce cytoplasmic calcium elevation in response to the cell wall preparation. The active compound was identified as cellotriose (CT) (Johnson et al., 2017). The cycam mutant shows a wild-type response to treatment with chitin. The expression profiles compare the response of WT and cycam roots to treatments with CT and chitin. References: Camehl I, Drzewiecki C, Vadassery J, Shahollari B, Sherameti I, Forzani C, Munnik T, Hirt H, Oelmüller R. (2011) The OXI1 kinase pathway mediates Piriformospora indica-induced growth promotion in Arabidopsis. PLoS Pathog. 7(5):e1002051. Johnson JM, Thürich J, Petutschnig EK, Altschmied L, Meichsner D, Sherameti I, Dindas J, Mrozinska A, Paetz C, Scholz S, Furch A, Lipka V, Hedrich R, Schneider B, Svatoš S, Oelmüller R. (2017) Cellotriose-induced cytoplasmic calcium elevation is controlled by a poly(A) ribonuclease in Arabidopsis root. (submitted to elife). Matsuo M, Johnson JM, Hieno A, Tokizawa M, Nomoto M, Tada Y, Godfrey R, Obokata J, Sherameti I, Yamamoto YY, Böhmer FD, Oelmüller R. (2015) High REDOX RESPONSIVE TRANSCRIPTION FACTOR1 Levels Result in Accumulation of Reactive Oxygen Species in Arabidopsis thaliana Shoots and Roots. Mol Plant. 8(8):1253-73. Peškan-Berghöfer T, Shahollari B, Giang PH, Hehl S, Markert C, Blanke V, Varma AK, Oelmüller R. (2004) Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant–microbe interactions and involves early plant protein modifications in the endoplasmatic reticulum and at the plasma membrane. Physiol. Plant. 122: 465–477. Shahollari B, Vadassery J, Varma A, Oelmüller R. (2007) A leucine-rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thaliana. Plant J. 50(1):1-13. Sherameti, I., Tripathi, S., Varma, A., and Oelmüller, R. (2008). The root-colonizing endophyte Pirifomospora indica confers drought tolerance in Arabidopsis by stimulating the expression of drought stress-related genes in leaves. Mol. Plant Microbe Interact. 21: 799-807. Vadassery, J., Ranf, S., Drzewiecki, C., Mithöfer, A., Mazars, C., Scheel, D., Lee, J., and Oelmüller, R. (2009) A cell wall extract from the endophytic fungus Piriformospora indica promotes growth of Arabidopsis seedlings and induces intracellular calcium elevation in roots. Plant J. 59: 193-206. Overall design: 13 day-old Arabidopsis seedlings, Col-0 and the cycam mutant, were grown under long day conditions and light (60-70 µM m-2 sec-1, 16 h light/8 h dark) at 22°C. For CT and chitin (chitohexaose) treatments (10 µM), the roots of 13-day old seedlings were soaked in a solution containing either CT or chitin; autoclaved H2O (control) was used as control. The seedlings were then transferred to long day conditions before the roots were harvested at 0 h, 4 h and 8 h after the treatments. The roots were then frozen in liquid nitrogen before RNA extraction. Data are based on 3 independent biological replicates. For additional experimental information, cf. Johnson et al. (2017).