The Viscum album Gene Space database.

The hemiparasitic flowering plant Viscum album (European mistletoe) is known for its very special life cycle, extraordinary biochemical properties, and extremely large genome. The size of its genome is estimated to be 30 times larger than the human genome and 600 times larger than the genome of the model plant Arabidopsis thaliana. To achieve insights into the Gene Space of the genome, which is defined as the space including and surrounding protein-coding regions, a transcriptome project based on PacBio sequencing has recently been conducted. A database resulting from this project contains sequences of 39,092 different open reading frames encoding 32,064 distinct proteins. Based on 'Benchmarking Universal Single-Copy Orthologs' (BUSCO) analysis, the completeness of the database was estimated to be in the range of 78%. To further develop this database, we performed a transcriptome project of V. album organs harvested in summer and winter based on Illumina sequencing. Data from both sequencing strategies were combined. The new V. album Gene Space database II (VaGs II) contains 90,039 sequences and has a completeness of 93% as revealed by BUSCO analysis. Sequences from other organisms, particularly fungi, which are known to colonize mistletoe leaves, have been removed. To evaluate the quality of the new database, proteome data of a mitochondrial fraction of V. album were re-analyzed. Compared to the original evaluation published five years ago, nearly 1000 additional proteins could be identified in the mitochondrial fraction, providing new insights into the Oxidative Phosphorylation System of V. album. The VaGs II database is available at https://viscumalbum.pflanzenproteomik.de/. Furthermore, all V. album sequences have been uploaded at the European Nucleotide Archive (ENA).

The gene space of European mistletoe (Viscum album).

European mistletoe (Viscum album) is a hemiparasitic flowering plant that is known for its very special life cycle and extraordinary biochemical properties. Particularly, V. album has an unusual mode of cellular respiration that takes place in the absence of mitochondrial complex I. However, insights into the molecular biology of V. album so far are very limited. Since the genome of V. album is extremely large (estimated 600 times larger than the genome of the model plant Arabidopsis thaliana) it has not been sequenced up to now. We here report sequencing of the V. album gene space (defined as the space including and surrounding genic regions, encompassing coding as well as 5' and 3' non-coding regions). mRNA fractions were isolated from different V. album organs harvested in summer or winter and were analyzed via single-molecule real-time sequencing. We determined sequences of 39 092 distinct open reading frames encoding 32 064 V. album proteins (designated V. album protein space). Our data give new insights into the metabolism and molecular biology of V. album, including the biosynthesis of lectins and viscotoxins. The benefits of the V. album gene space information are demonstrated by re-evaluating mass spectrometry-based data of the V. album mitochondrial proteome, which previously had been evaluated using the A. thaliana genome sequence. Our re-examination allowed the additional identification of nearly 200 mitochondrial proteins, including four proteins related to complex I, which all have a secondary function not related to respiratory electron transport. The V. album gene space sequences are available at the NCBI.

Insights into the complex role of GRAS transcription factors in the arbuscular mycorrhiza symbiosis.

To improve access to limiting nutrients, the vast majority of land plants forms arbuscular mycorrhizal (AM) symbioses with Glomeromycota fungi. We show here that AM-related GRAS transcription factors from different subgroups are upregulated during a time course of mycorrhization. Based on expression studies in mutants defective in arbuscule branching (ram1-1, with a deleted MtRam1 GRAS transcription factor gene) or in the formation of functional arbuscules (pt4-2, mutated in the phosphate transporter gene MtPt4), we demonstrate that the five AM-related GRAS transcription factor genes MtGras1, MtGras4, MtGras6, MtGras7, and MtRad1 can be differentiated by their dependency on MtRAM1 and MtPT4, indicating that the network of AM-related GRAS transcription factors consists of at least two regulatory modules. One module involves the MtRAM1- and MtPT4-independent transcription factor MtGRAS4 that activates MtGras7. Another module is controlled by the MtRAM1- and MtPT4-dependent transcription factor MtGRAS1. Genome-wide expression profiles of mycorrhized MtGras1 knockdown and ram1-1 roots differ substantially, indicating different targets. Although an MtGras1 knockdown reduces transcription of AM-related GRAS transcription factor genes including MtRam1 and MtGras7, MtGras1 overexpression alone is not sufficient to activate MtGras genes. MtGras1 knockdown roots display normal fungal colonization, with a trend towards the formation of smaller arbuscules.

Molecular Background of Pi Deficiency-Induced Root Hair Growth in Brassica carinata - A Fasciclin-Like Arabinogalactan Protein Is Involved.

Formation of longer root hairs under limiting phosphate (P) conditions can increase the inorganic P (Pi) uptake. Here, regulatory candidate genes for Pi deficiency-induced root hair growth were identified by comparison of massive analysis of cDNA ends (MACE) provided expression profiles of two Brassica carinata cultivars (cv.) differing in their root hair response to Pi deficiency: cv. Bale develops longer root hairs under Pi deficiency, but not cv. Bacho. A split-root experiment was conducted for the differentiation between locally and systemically regulated genes. Furthermore, plants were exposed to nitrogen and potassium deficiency to identify P-specific reacting genes. The latter were knocked out by CRISPR/Cas9 and the effect on the root hair length was determined. About 500 genes were differentially expressed under Pi deficiency in cv. Bale, while these genes did not respond to the low P supply in cv. Bacho. Thirty-three candidate genes with a potential regulatory role were selected and the transcriptional regulation of 30 genes was confirmed by quantitative PCR. Only five candidate genes seemed to be either exclusively regulated locally (two) or systemically (three), whereas 25 genes seemed to be involved in both local and systemic signaling pathways. Potassium deficiency affected neither the root hair length nor the expression of the 30 candidate genes. By contrast, both P and nitrogen deficiency increased the root hair length, and both affected the transcript levels in 26 cases. However, four genes reacted specifically to Pi starvation. These genes and, additionally, INORGANIC PHOSPHATE TRANSPORTER 1 (BcPHT1) were targeted by CRISPR/Cas9. However, even if the transcript levels of five of these genes were clearly decreased, FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 1 (BcFLA1) was the only gene whose downregulation reduced the root hair length in transgenic hairy roots under Pi-deficient conditions. To the best of our knowledge, this is the first study describing a fasciclin-like arabinogalactan protein with a predicted role in the Pi deficiency-induced root hair elongation.

The mycorrhiza-dependent defensin MtDefMd1 of Medicago truncatula acts during the late restructuring stages of arbuscule-containing cells.

Different symbiotic and pathogenic plant-microbe interactions involve the production of cysteine-rich antimicrobial defensins. In Medicago truncatula, the expression of four MtDefMd genes, encoding arbuscular mycorrhiza-dependent defensins containing an N-terminal signal peptide and exhibiting some differences to non-symbiotic defensins, raised over the time of fungal colonization. Whereas the MtDefMd1 and MtDefMd2 promoters were inactive in cells containing young arbuscules, cells with fully developed arbuscules displayed different levels of promoter activities, indicating an up-regulation towards later stages of arbuscule formation. MtDefMd1 and MtDefMd2 expression was absent or strongly down-regulated in mycorrhized ram1-1 and pt4-2 mutants, known for defects in arbuscule branching or premature arbuscule degeneration, respectively. A ~97% knock-down of MtDefMd1/MtDefMd2 expression did not significantly affect arbuscule size. Although overexpression of MtDefMd1 in arbuscule-containing cells led to an up-regulation of MtRam1, encoding a key transcriptional regulator of arbuscule formation, no morphological changes were evident. Co-localization of an MtDefMd1-mGFP6 fusion with additional, subcellular markers revealed that this defensin is associated with arbuscules in later stages of their life-cycle. MtDefMd1-mGFP6 was detected in cells with older arbuscules about to collapse, and ultimately in vacuolar compartments. Comparisons with mycorrhized roots expressing a tonoplast marker indicated that MtDefMd1 acts during late restructuring processes of arbuscule-containing cells, upon their transition into a post-symbiotic state.

Pre-announcement of symbiotic guests: transcriptional reprogramming by mycorrhizal lipochitooligosaccharides shows a strict co-dependency on the GRAS transcription factors NSP1 and RAM1.

BACKGROUND: More than 80 % of all terrestrial plant species establish an arbuscular mycorrhiza (AM) symbiosis with Glomeromycota fungi. This plant-microbe interaction primarily improves phosphate uptake, but also supports nitrogen, mineral, and water aquisition. During the pre-contact stage, the AM symbiosis is controled by an exchange of diffusible factors from either partner. Amongst others, fungal signals were identified as a mix of sulfated and non-sulfated lipochitooligosaccharides (LCOs), being structurally related to rhizobial nodulation (Nod)-factor LCOs that in legumes induce the formation of nitrogen-fixing root nodules. LCO signals are transduced via a common symbiotic signaling pathway (CSSP) that activates a group of GRAS transcription factors (TFs). Using complex gene expression fingerprints as molecular phenotypes, this study primarily intended to shed light on the importance of the GRAS TFs NSP1 and RAM1 for LCO-activated gene expression during pre-symbiotic signaling. RESULTS: We investigated the genome-wide transcriptional responses in 5 days old primary roots of the Medicago truncatula wild type and four symbiotic mutants to a 6 h challenge with LCO signals supplied at 10(-7/-8) M. We were able to show that during the pre-symbiotic stage, sulfated Myc-, non-sulfated Myc-, and Nod-LCO-activated gene expression almost exclusively depends on the LysM receptor kinase NFP and is largely controled by the CSSP, although responses independent of this pathway exist. Our results show that downstream of the CSSP, gene expression activation by Myc-LCOs supplied at 10(-7/-8) M strictly required both the GRAS transcription factors RAM1 and NSP1, whereas those genes either co- or specifically activated by Nod-LCOs displayed a preferential NSP1-dependency. RAM1, a central regulator of root colonization by AM fungi, controled genes activated by non-sulfated Myc-LCOs during the pre-symbiotic stage that are also up-regulated in areas with early physical contact, e.g. hyphopodia and infecting hyphae; linking responses to externally applied LCOs with early root colonization. CONCLUSIONS: Since both RAM1 and NSP1 were essential for the pre-symbiotic transcriptional reprogramming by Myc-LCOs, we propose that downstream of the CSSP, these GRAS transcription factors act synergistically in the transduction of those diffusible signals that pre-announce the presence of symbiotic fungi.

Spatial gene expression analysis in tomato hypocotyls suggests cysteine as key precursor of vascular sulfur accumulation implicated in Verticillium dahliae defense.

Verticillium dahliae is a prominent generator of plant vascular wilting disease and sulfur (S)-enhanced defense (SED) mechanisms contribute to its in-planta elimination. The accumulation of S-containing defense compounds (SDCs) including elemental S (S(0) ) has been described based on the comparison of two near-isogenic tomato (Solanum lycopersicum) lines differing in fungal susceptibility. To better understand the effect of S nutrition on V. dahliae resistance both lines were supplied with low, optimal or supraoptimal sulfate-S. An absolute quantification demonstrated a most effective fungal elimination due to luxury plant S nutrition. High-pressure liquid chromatography (HPLC) showed a strong regulation of Cys levels and an S-responsive GSH pool rise in the bulk hypocotyl. High-frequency S peak accumulations were detected in vascular bundles of resistant tomato plants after fungal colonization by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Global transcriptomic analysis suggested that early steps of the primary S metabolism did not promote the SDCs synthesis in the whole hypocotyl as gene expression was downregulated after infection. Enhanced S fertilization mostly alleviated the repressive fungal effect but did not reverse it. Upregulation of glutathione (GSH)-associated genes in bulk hypocotyls but not in vascular bundles indicated a global antioxidative role of GSH. To finally assign the contribution of S metabolism-associated genes to high S(0) accumulations exclusively found in the resistant tomato line, a spatial gene expression approach was applied. Laser microdissection of infected vascular bundles revealed a switch toward transcription of genes connected with cysteine (Cys) synthesis. The upregulation of LeOASTLp1 suggests a role for Cys as key precursor for local S accumulations (possibly S(0) ) in the vascular bundles of the V. dahliae-resistant tomato line.

Through the doors of perception to function in arbuscular mycorrhizal symbioses.

The formation of an arbuscular mycorrhizal (AM) symbiosis is initiated by the bidirectional exchange of diffusible molecules. While strigolactone hormones, secreted from plant roots,stimulate hyphal branching and fungal metabolism, fungal short-chain chitin oligomers as well assulfated and nonsulfated lipochitooligosaccharides (s/nsMyc-LCOs) elicit pre-symbiosis responses in the host. Fungal LCO signals are structurally related to rhizobial Nod-factor LCOs. Genome-wide expression studies demonstrated that defined sets of genes were induced by Nod-, sMyc- and nsMyc-LCOs, indicating LCO-specific perception in the pre-symbiosis phase. During hyphopodium formation and the subsequent root colonization, cross-talk between plant roots and AM fungi also involves phytohormones. Notably, gibberellins control arbuscule formation via DELLA proteins, which themselves serve as positive regulators of arbuscule formation. The establishment of arbuscules is accompanied by a substantial transcriptional and post-transcriptional reprogramming of host roots, ultimately defining the unique protein composition of arbuscule-containing cells. Based on cellular expression profiles, key check points of AM development as well as candidate genes encoding transcriptional regulators and regulatory microRNAs were identified. Detailed functional analyses of promoters specified short motifs sufficient for cell-autonomous gene regulation in cells harboring arbuscules, and suggested simultaneous, multi-level regulation of the mycorrhizal phosphate uptake pathway by integrating AM symbiosis and phosphate starvation response signaling.

Transcriptome-wide mapping of pea seed ageing reveals a pivotal role for genes related to oxidative stress and programmed cell death.

Understanding of seed ageing, which leads to viability loss during storage, is vital for ex situ plant conservation and agriculture alike. Yet the potential for regulation at the transcriptional level has not been fully investigated. Here, we studied the relationship between seed viability, gene expression and glutathione redox status during artificial ageing of pea (Pisum sativum) seeds. Transcriptome-wide analysis using microarrays was complemented with qRT-PCR analysis of selected genes and a multilevel analysis of the antioxidant glutathione. Partial degradation of DNA and RNA occurred from the onset of artificial ageing at 60% RH and 50°C, and transcriptome profiling showed that the expression of genes associated with programmed cell death, oxidative stress and protein ubiquitination were altered prior to any sign of viability loss. After 25 days of ageing viability started to decline in conjunction with progressively oxidising cellular conditions, as indicated by a shift of the glutathione redox state towards more positive values (>-190 mV). The unravelling of the molecular basis of seed ageing revealed that transcriptome reprogramming is a key component of the ageing process, which influences the progression of programmed cell death and decline in antioxidant capacity that ultimately lead to seed viability loss.

cell- and tissue-specific transcriptome analyses of Medicago truncatula root nodules.

Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital "in situ". This digital "in situ" offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies.

A roadmap of cell-type specific gene expression during sequential stages of the arbuscular mycorrhiza symbiosis.

BACKGROUND: About 80% of today's land plants are able to establish an arbuscular mycorrhizal (AM) symbiosis with Glomeromycota fungi to improve their access to nutrients and water in the soil. On the molecular level, the development of AM symbioses is only partly understood, due to the asynchronous development of the microsymbionts in the host roots. Although many genes specifically activated during fungal colonization have been identified, genome-wide information on the exact place and time point of their activation remains limited. RESULTS: In this study, we relied on a combination of laser-microdissection and the use of Medicago GeneChips to perform a genome-wide analysis of transcription patterns in defined cell-types of Medicago truncatula roots mycorrhized with Glomus intraradices. To cover major stages of AM development, we harvested cells at 5-6 and at 21 days post inoculation (dpi). Early developmental stages of the AM symbiosis were analysed by monitoring gene expression in appressorial and non-appressorial areas from roots harbouring infection units at 5-6 dpi. Here, the use of laser-microdissection for the first time enabled the targeted harvest of those sites, where fungal hyphae first penetrate the root. Circumventing contamination with developing arbuscules, we were able to specifically detect gene expression related to early infection events. To cover the late stages of AM formation, we studied arbusculated cells, cortical cells colonized by intraradical hyphae, and epidermal cells from mature mycorrhizal roots at 21 dpi. Taken together, the cell-specific expression patterns of 18014 genes were revealed, including 1392 genes whose transcription was influenced by mycorrhizal colonization at different stages, namely the pre-contact phase, the infection of roots via fungal appressoria, the subsequent colonization of the cortex by fungal hyphae, and finally the formation of arbuscules. Our cellular expression patterns identified distinct groups of AM-activated genes governing the sequential reprogramming of host roots towards an accommodation of microsymbionts, including 42 AM-activated transcription factor genes. CONCLUSIONS: Our genome-wide analysis provides novel information on the cell-specific activity of AM-activated genes during both early and late stages of AM development, together revealing the road map of fine-tuned adjustments of transcript accumulation within root tissues during AM fungal colonization.

Involvement of bacterial TonB-dependent signaling in the generation of an oligogalacturonide damage-associated molecular pattern from plant cell walls exposed to Xanthomonas campestris pv. campestris pectate lyases.

BACKGROUND: Efficient perception of attacking pathogens is essential for plants. Plant defense is evoked by molecules termed elicitors. Endogenous elicitors or damage-associated molecular patterns (DAMPs) originate from plant materials upon injury or pathogen activity. While there are comparably well-characterized examples for DAMPs, often oligogalacturonides (OGAs), generated by the activity of fungal pathogens, endogenous elicitors evoked by bacterial pathogens have been rarely described. In particular, the signal perception and transduction processes involved in DAMP generation are poorly characterized. RESULTS: A mutant strain of the phytopathogenic bacterium Xanthomonas campestris pv. campestris deficient in exbD2, which encodes a component of its unusual elaborate TonB system, had impaired pectate lyase activity and caused no visible symptoms for defense on the non-host plant pepper (Capsicum annuum). A co-incubation of X. campestris pv. campestris with isolated cell wall material from C. annuum led to the release of compounds which induced an oxidative burst in cell suspension cultures of the non-host plant. Lipopolysaccharides and proteins were ruled out as elicitors by polymyxin B and heat treatment, respectively. After hydrolysis with trifluoroacetic acid and subsequent HPAE chromatography, the elicitor preparation contained galacturonic acid, the monosaccharide constituent of pectate. OGAs were isolated from this crude elicitor preparation by HPAEC and tested for their biological activity. While small OGAs were unable to induce an oxidative burst, the elicitor activity in cell suspension cultures of the non-host plants tobacco and pepper increased with the degree of polymerization (DP). Maximal elicitor activity was observed for DPs exceeding 8. In contrast to the X. campestris pv. campestris wild type B100, the exbD2 mutant was unable to generate elicitor activity from plant cell wall material or from pectin. CONCLUSIONS: To our knowledge, this is the second report on a DAMP generated by bacterial features. The generation of the OGA elicitor is embedded in a complex exchange of signals within the framework of the plant-microbe interaction of C. annuum and X. campestris pv. campestris. The bacterial TonB-system is essential for the substrate-induced generation of extracellular pectate lyase activity. This is the first demonstration that a TonB-system is involved in bacterial trans-envelope signaling in the context of a pathogenic interaction with a plant.

Transcriptional responses toward diffusible signals from symbiotic microbes reveal MtNFP- and MtDMI3-dependent reprogramming of host gene expression by arbuscular mycorrhizal fungal lipochitooligosaccharides.

The formation of root nodules and arbuscular mycorrhizal (AM) roots is controlled by a common signaling pathway including the calcium/calmodulin-dependent kinase Doesn't Make Infection3 (DMI3). While nodule initiation by lipochitooligosaccharide (LCO) Nod factors is well characterized, diffusible AM fungal signals were only recently identified as sulfated and nonsulfated LCOs. Irrespective of different outcomes, the perception of symbiotic LCOs in Medicago truncatula is mediated by the LysM receptor kinase M. truncatula Nod factor perception (MtNFP). To shed light on transcriptional responses toward symbiotic LCOs and their dependence on MtNFP and Ca(2+) signaling, we performed genome-wide expression studies of wild-type, Nod-factor-perception mutant1, and dmi3 mutant roots challenged with Myc- and Nod-LCOs. We show that Myc-LCOs lead to transient, quick responses in the wild type, whereas Nod-LCOs require prolonged incubation for maximal expression activation. While Nod-LCOs are most efficient for an induction of persistent transcriptional changes, sulfated Myc-LCOs are less active, and nonsulfated Myc-LCOs display the lowest capacity to activate and sustain expression. Although all symbiotic LCOs up-regulated a common set of genes, discrete subsets were induced by individual LCOs, suggesting common and specific functions for these in presymbiotic signaling. Surprisingly, even sulfated fungal Myc-LCOs and Sinorhizobium meliloti Nod-LCOs, having very similar structures, each elicited discrete subsets of genes, while a mixture of both Myc-LCOs activated responses deviating from those induced by single treatments. Focusing on the precontact phase, we identified signaling-related and transcription factor genes specifically up-regulated by Myc-LCOs. Comparative gene expression studies in symbiotic mutants demonstrated that transcriptional reprogramming by AM fungal LCOs strictly depends on MtNFP and largely requires MtDMI3.

Phoma medicaginis stimulates the induction of the octadecanoid and phenylpropanoid pathways in Medicago truncatula.

Gene expression changes and metabolite abundances were measured during the interaction of Medicago truncatula with the fungal necrotrophic pathogen Phoma medicaginis in leaf tissue of susceptible and resistant accessions. Over 330 genes were differentially expressed in plants infected with P. medicaginis relative to mock-inoculated plants at 12 h post-inoculation. Of these, 191 were induced in either the resistant or the susceptible accession, with 143 genes repressed. Expression changes were observed in genes involved in the oxidative burst, cell wall strengthening and lipid metabolism, as well as several transcription factors. Genes related to salicylic acid, jasmonate and ethylene responses were up-regulated, as well as genes leading to the production of jasmonic acid. Significant induction of genes in the phenylpropanoid pathway leading to lignin and isoflavonoid biosynthesis occurred. High-pressure liquid chromatography with UV detection (HPLC-UV) identified several phenolic compounds induced by P. medicaginis, as well as constitutively higher levels of phenolic compounds, in the resistant M. truncatula accession. Differentially regulated genes induced in both the resistant and susceptible accessions, but with different kinetics, and constitutively more highly expressed and induced phenolic compounds provide candidates for functional analysis. Taken together, these results highlight the importance of the octadecanoid and phenylpropanoid pathways in defence against this necrotrophic pathogen.

Laser microdissection unravels cell-type-specific transcription in arbuscular mycorrhizal roots, including CAAT-box transcription factor gene expression correlating with fungal contact and spread.

Arbuscular mycorrhizae (AM) are the most widespread symbioses on Earth, promoting nutrient supply of most terrestrial plant species. To unravel gene expression in defined stages of Medicago truncatula root colonization by AM fungi, we here combined genome-wide transcriptome profiling based on whole mycorrhizal roots with real-time reverse transcription-PCR experiments that relied on characteristic cell types obtained via laser microdissection. Our genome-wide approach delivered a core set of 512 genes significantly activated by the two mycorrhizal fungi Glomus intraradices and Glomus mossae. Focusing on 62 of these genes being related to membrane transport, signaling, and transcriptional regulation, we distinguished whether they are activated in arbuscule-containing or the neighboring cortical cells harboring fungal hyphae. In addition, cortical cells from nonmycorrhizal roots served as a reference for gene expression under noncolonized conditions. Our analysis identified 25 novel arbuscule-specific genes and 37 genes expressed both in the arbuscule-containing and the adjacent cortical cells colonized by fungal hyphae. Among the AM-induced genes specifying transcriptional regulators were two members encoding CAAT-box binding transcription factors (CBFs), designated MtCbf1 and MtCbf2. Promoter analyses demonstrated that both genes were already activated by the first physical contact between the symbionts. Subsequently, and corresponding to our cell-type expression patterns, they were progressively up-regulated in those cortical areas colonized by fungal hyphae, including the arbuscule-containing cells. The encoded CBFs thus represent excellent candidates for regulators that mediate a sequential reprogramming of root tissues during the establishment of an AM symbiosis.

Identification of genes differentially expressed in a resistant reaction to Mycosphaerella pinodes in pea using microarray technology.

BACKGROUND: Ascochyta blight, caused by Mycosphaerella pinodes is one of the most important pea pathogens. However, little is known about the genes and mechanisms of resistance acting against M. pinodes in pea. Resistance identified so far to this pathogen is incomplete, polygenic and scarce in pea, being most common in Pisum relatives. The identification of the genes underlying resistance would increase our knowledge about M. pinodes-pea interaction and would facilitate the introgression of resistance into pea varieties. In the present study differentially expressed genes in the resistant P. sativum ssp. syriacum accession P665 comparing to the susceptible pea cv. Messire after inoculation with M. pinodes have been identified using a M. truncatula microarray. RESULTS: Of the 16,470 sequences analysed, 346 were differentially regulated. Differentially regulated genes belonged to almost all functional categories and included genes involved in defense such as genes involved in cell wall reinforcement, phenylpropanoid and phytoalexins metabolism, pathogenesis- related (PR) proteins and detoxification processes. Genes associated with jasmonic acid (JA) and ethylene signal transduction pathways were induced suggesting that the response to M. pinodes in pea is regulated via JA and ET pathways. Expression levels of ten differentially regulated genes were validated in inoculated and control plants using qRT-PCR showing that the P665 accession shows constitutively an increased expression of the defense related genes as peroxidases, disease resistance response protein 39 (DRR230-b), glutathione S-transferase (GST) and 6a-hydroxymaackiain methyltransferase. CONCLUSIONS: Through this study a global view of genes expressed during resistance to M. pinodes has been obtained, giving relevant information about the mechanisms and pathways conferring resistance to this important disease. In addition, the M. truncatula microarray represents an efficient tool to identify candidate genes controlling resistance to M. pinodes in pea.

Abscisic acid deficiency of developing pea embryos achieved by immunomodulation attenuates developmental phase transition and storage metabolism.

The transition of pea embryos from pre-storage to maturation is partially controlled by abscisic acid (ABA). Immunomodulation in pea embryos specifically reduces free ABA levels during transition stages. Such seeds are, therefore, suitable models for studying ABA deficiency by global transcript and metabolite analysis. Compared with the wild type, anti-ABA seeds are smaller, contain fewer globulins and show lower dry matter accumulation and delayed differentiation. Free sugars are decreased, indicating lower uptake and/or elevated mobilisation. Lower levels of trans-zeatins suggest that ABA reduction influences rates of cytokinin synthesis and/or its level of accumulation. Abscisic acid deficiency leads to a general downregulation of gene expression related to transcription and translation. At the transcriptional level, anti-ABA embryos reveal a wide-range repression of carbohydrate oxidation, downregulated sucrose mobilisation, glycolysis and the tricarboxylic acid cycle/Krebs cycle (TCA cycle). Genes related to starch, amino acid and storage protein biosynthesis are downregulated, indicating a general decrease in metabolic fluxes. We conclude that during embryo differentiation ABA triggers broad upregulation of gene activity and genetic reprogramming, involving regulated protein degradation via the ubiquitin/proteasome system. Abscisic acid deficiency affects gene expression associated with transport processes and stimulation of membrane energisation. Our study identified mediators and downstream signalling elements of ABA during embryo differentiation, such as the transcription factor FUSCA3, SnRK1 kinase and Ca(2+) signalling processes. This suggests that ABA interacts with SnRK1 complexes, thus connecting SnRK1, sugar and stress signalling with ABA. Certain protein kinases/phosphatases known to negatively respond to ABA are upregulated in the modulated line, whilst those which respond positively are downregulated, pointing to a highly coordinated response of the gene network to ABA levels.

MediPlEx - a tool to combine in silico & experimental gene expression profiles of the model legume Medicago truncatula.

BACKGROUND: Expressed Sequence Tags (ESTs) are in general used to gain a first insight into gene activities from a species of interest. Subsequently, and typically based on a combination of EST and genome sequences, microarray-based expression analyses are performed for a variety of conditions. In some cases, a multitude of EST and microarray experiments are conducted for one species, covering different tissues, cell states, and cell types. Under these circumstances, the challenge arises to combine results derived from the different expression profiling strategies, with the goal to uncover novel information on the basis of the integrated datasets. FINDINGS: Using our new analysis tool, MediPlEx (MEDIcago truncatula multiPLe EXpression analysis), expression data from EST experiments, oligonucleotide microarrays and Affymetrix GeneChips® can be combined and analyzed, leading to a novel approach to integrated transcriptome analysis. We have validated our tool via the identification of a set of well-characterized AM-specific and AM-induced marker genes, identified by MediPlEx on the basis of in silico and experimental gene expression profiles from roots colonized with AM fungi. CONCLUSIONS: MediPlEx offers an integrated analysis pipeline for different sets of expression data generated for the model legume Medicago truncatula. As expected, in silico and experimental gene expression data that cover the same biological condition correlate well. The collection of differentially expressed genes identified via MediPlEx provides a starting point for functional studies in plant mutants. MediPlEx can freely be used at http://www.cebitec.uni-bielefeld.de/mediplex.