Reactive oxygen species dosage in Arabidopsis chloroplasts can improve resistance towards Colletotrichum higginsianum by the induction of WRKY33.

Arabidopsis plants overexpressing glycolate oxidase in chloroplasts (GO5) and loss-of-function mutants of the major peroxisomal catalase isoform, cat2-2, produce increased hydrogen peroxide (H2 O2 ) amounts from the respective organelles when subjected to photorespiratory conditions like increased light intensity. Here, we have investigated if and how the signaling processes triggered by H2 O2 production in response to shifts in environmental conditions and the concomitant induction of indole phytoalexin biosynthesis in GO5 affect susceptibility towards the hemibiotrophic fungus Colletotrichum higginsianum. Combining histological, biochemical, and molecular assays, we found that the accumulation of the phytoalexin camalexin was comparable between GO genotypes and cat2-2 in the absence of pathogen. Compared with wild-type, GO5 showed improved resistance after light-shift-mediated production of H2 O2 , whereas cat2-2 became more susceptible and allowed significantly more pathogen entry. Unlike GO5, cat2-2 suffered from severe oxidative stress after light shifts, as indicated by glutathione pool size and oxidation state. We discuss a connection between elevated oxidative stress and dampened induction of salicylic acid mediated defense in cat2-2. Genetic analyses demonstrated that induced resistance of GO5 is dependent on WRKY33, but not on camalexin production. We propose that indole carbonyl nitriles might play a role in defense against C. higginsianum.

Galactose metabolism and toxicity in Ustilago maydis.

In most organisms, galactose is metabolized via the Leloir pathway, which is conserved from bacteria to mammals. Utilization of galactose requires a close interplay of the metabolic enzymes, as misregulation or malfunction of individual components can lead to the accumulation of toxic intermediate compounds. For the phytopathogenic basidiomycete Ustilago maydis, galactose is toxic for wildtype strains, i.e. leads to growth repression despite the presence of favorable carbon sources as sucrose. The galactose sensitivity can be relieved by two independent modifications: (1) by disruption of Hxt1, which we identify as the major transporter for galactose, and (2) by a point mutation in the gene encoding the galactokinase Gal1, the first enzyme of the Leloir pathway. The mutation in gal1(Y67F) leads to reduced enzymatic activity of Gal1 and thus may limit the formation of putatively toxic galactose-1-phosphate. However, systematic deletions and double deletions of different genes involved in galactose metabolism point to a minor role of galactose-1-phosphate in galactose toxicity. Our results show that molecular triggers for galactose toxicity in U. maydis differ from yeast and mammals.

Cell wall composition and penetration resistance against the fungal pathogen Colletotrichum higginsianum are affected by impaired starch turnover in Arabidopsis mutants.

Penetration resistance represents the first level of plant defense against phytopathogenic fungi. Here, we report that the starch-deficient Arabidopsis thaliana phosphoglucomutase (pgm) mutant has impaired penetration resistance against the hemibiotrophic fungus Colletotrichum higginsianum. We could not determine any changes in leaf cutin and epicuticular wax composition or indolic glucosinolate levels, but detected complex alterations in the cell wall monosaccharide composition of pgm. Notably, other mutants deficient in starch biosynthesis (adg1) or mobilization (sex1) had similarly affected cell wall composition and penetration resistance. Glycome profiling analysis showed that both overall cell wall polysaccharide extractability and relative extractability of specific pectin and xylan epitopes were affected in pgm, suggesting extensive structural changes in pgm cell walls. Screening of mutants with alterations in content or modification of specific cell wall monosaccharides indicated an important function of pectic polymers for penetration resistance and hyphal growth of C. higginsianum during the biotrophic interaction phase. While mutants with affected pectic rhamnogalacturonan-I (mur8) were hypersusceptible, penetration frequency and morphology of fungal hyphae were impaired on pmr5 pmr6 mutants with increased pectin levels. Our results reveal a strong impact of starch metabolism on cell wall composition and suggest a link between carbohydrate availability, cell wall pectin and penetration resistance.

The receptor kinase IMPAIRED OOMYCETE SUSCEPTIBILITY1 attenuates abscisic acid responses in Arabidopsis.

In plants, membrane-bound receptor kinases are essential for developmental processes, immune responses to pathogens and the establishment of symbiosis. We previously identified the Arabidopsis (Arabidopsis thaliana) receptor kinase IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as required for successful infection with the downy mildew pathogen Hyaloperonospora arabidopsidis. We report here that IOS1 is also required for full susceptibility of Arabidopsis to unrelated (hemi)biotrophic filamentous oomycete and fungal pathogens. Impaired susceptibility in the absence of IOS1 appeared to be independent of plant defense mechanism. Instead, we found that ios1-1 plants were hypersensitive to the plant hormone abscisic acid (ABA), displaying enhanced ABA-mediated inhibition of seed germination, root elongation, and stomatal opening. These findings suggest that IOS1 negatively regulates ABA signaling in Arabidopsis. The expression of ABA-sensitive COLD REGULATED and RESISTANCE TO DESICCATION genes was diminished in Arabidopsis during infection. This effect on ABA signaling was alleviated in the ios1-1 mutant background. Accordingly, ABA-insensitive and ABA-hypersensitive mutants were more susceptible and resistant to oomycete infection, respectively, showing that the intensity of ABA signaling affects the outcome of downy mildew disease. Taken together, our findings suggest that filamentous (hemi)biotrophs attenuate ABA signaling in Arabidopsis during the infection process and that IOS1 participates in this pathogen-mediated reprogramming of the host.

Tocopherol deficiency reduces sucrose export from salt-stressed potato leaves independently of oxidative stress and symplastic obstruction by callose.

Tocopherol cyclase, encoded by the gene SUCROSE EXPORT DEFECTIVE1, catalyses the second step in the synthesis of the antioxidant tocopherol. Depletion of SXD1 activity in maize and potato leaves leads to tocopherol deficiency and a 'sugar export block' phenotype that comprises massive starch accumulation and obstruction of plasmodesmata in paraveinal tissue by callose. We grew two transgenic StSXD1:RNAi potato lines with severe tocopherol deficiency under moderate light conditions and subjected them to salt stress. After three weeks of salt exposure, we observed a strongly reduced sugar exudation rate and a lack of starch mobilization in leaves of salt-stressed transgenic plants, but not in wild-type plants. However, callose accumulation in the vasculature declined upon salt stress in all genotypes, indicating that callose plugging of plasmodesmata was not the sole cause of the sugar export block phenotype in tocopherol-deficient leaves. Based on comprehensive gene expression analyses, we propose that enhanced responsiveness of SnRK1 target genes in mesophyll cells and altered redox regulation of phloem loading by SUT1 contribute to the attenuation of sucrose export from salt-stressed SXD:RNAi source leaves. Furthermore, we could not find any indication that elevated oxidative stress may have served as a trigger for the salt-induced carbohydrate phenotype of SXD1:RNAi transgenic plants. In leaves of the SXD1:RNAi plants, sodium accumulation was diminished, while proline accumulation and pools of soluble antioxidants were increased. As supported by phytohormone contents, these differences seem to increase longevity and prevent senescence of SXD:RNAi leaves under salt stress.

Reduced carbohydrate availability enhances the susceptibility of Arabidopsis toward Colletotrichum higginsianum.

Colletotrichum higginsianum is a hemibiotrophic ascomycete fungus that is adapted to Arabidopsis (Arabidopsis thaliana). After breaching the host surface, the fungus establishes an initial biotrophic phase in the penetrated epidermis cell, before necrotrophic growth is initiated upon further host colonization. We observed that partitioning of major leaf carbohydrates was shifted in favor of sucrose and at the expense of starch during necrotrophic fungal growth. Arabidopsis mutants with impaired starch turnover were more susceptible toward C. higginsianum infection, exhibiting a strong negative correlation between diurnal carbohydrate accumulation and fungal proliferation for the tested genotypes. By altering the length of the light phase and employing additional genotypes impaired in nocturnal carbon mobilization, we revealed that reduced availability of carbon enhances susceptibility in the investigated pathosystem. Systematic starvation experiments resulted in two important findings. First, we showed that carbohydrate supply by the host is dispensable during biotrophic growth of C. higginsianum, while carbon deficiency was most harmful to the host during the necrotrophic colonization phase. Compared with the wild type, the increases in the total salicylic acid pool and camalexin accumulation were reduced in starch-free mutants at late interaction stages, while an increased ratio of free to total salicylic acid did not convey elevated pathogenesis-related gene expression in starch-free mutants. These observations suggest that reduced carbon availability dampens induced defense responses. In contrast, starch-free mutants were more resistant toward the fungal biotroph Erysiphe cruciferarum, indicating that reduced carbohydrate availability influences susceptibility differently in the interaction with the investigated hemibiotrophic and biotrophic fungal pathogens.

Loss of the two major leaf isoforms of sucrose-phosphate synthase in Arabidopsis thaliana limits sucrose synthesis and nocturnal starch degradation but does not alter carbon partitioning during photosynthesis.

Sucrose (Suc)-phosphate synthase (SPS) catalyses one of the rate-limiting steps in the synthesis of Suc in plants. The Arabidopsis genome contains four annotated SPS genes which can be grouped into three different families (SPSA1, SPSA2, SPSB, and SPSC). However, the functional significance of this multiplicity of SPS genes is as yet only poorly understood. All four SPS isoforms show enzymatic activity when expressed in yeast although there is variation in sensitivity towards allosteric effectors. Promoter-reporter gene analyses and quantitative real-time reverse transcription-PCR studies indicate that no two SPS genes have the same expression pattern and that AtSPSA1 and AtSPSC represent the major isoforms expressed in leaves. An spsa1 knock-out mutant showed a 44% decrease in leaf SPS activity and a slight increase in leaf starch content at the end of the light period as well as at the end of the dark period. The spsc null mutant displayed reduced Suc contents towards the end of the photoperiod and a concomitant 25% reduction in SPS activity. In contrast, an spsa1/spsc double mutant was strongly impaired in growth and accumulated high levels of starch. This increase in starch was probably not due to an increased partitioning of carbon into starch, but was rather caused by an impaired starch mobilization during the night. Suc export from excised petioles harvested from spsa1/spsc double mutant plants was significantly reduced under illumination as well as during the dark period. It is concluded that loss of the two major SPS isoforms in leaves limits Suc synthesis without grossly changing carbon partitioning in favour of starch during the light period but limits starch degradation during the dark period.

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform.

Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy time scales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating highly active chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots). We show that these systems work with conventionally used T7 RNA polymerase as well as the endogenous chloroplast polymerases, allowing for detailed characterization and prototyping of regulatory sequences at both transcription and translation levels. To demonstrate the platform for characterization of promoters and 5' and 3' untranslated regions (UTRs) in higher plant chloroplast gene expression, we analyze a collection of 23 5'UTRs, 10 3'UTRs, and 6 chloroplast promoters, assessed their expression in spinach and wheat extracts, and found consistency in expression patterns, suggesting cross-species compatibility. Looking forward, our chloroplast CFE systems open new avenues for plant synthetic biology, offering prototyping tools for both understanding gene expression and developing engineered plants, which could help meet the demands of a changing global climate.

Two recently duplicated maize NAC transcription factor paralogs are induced in response to Colletotrichum graminicola infection.

BACKGROUND: NAC transcription factors belong to a large family of plant-specific transcription factors with more than 100 family members in monocot and dicot species. To date, the majority of the studied NAC proteins are involved in the response to abiotic stress, to biotic stress and in the regulation of developmental processes. Maize NAC transcription factors involved in the biotic stress response have not yet been identified. RESULTS: We have found that two NAC transcription factors, ZmNAC41 and ZmNAC100, are transcriptionally induced both during the initial biotrophic as well as the ensuing necrotrophic colonization of maize leaves by the hemibiotrophic ascomycete fungus C. graminicola. ZmNAC41 transcripts were also induced upon infection with C. graminicola mutants that are defective in host penetration, while the induction of ZmNAC100 did not occur in such interactions. While ZmNAC41 transcripts accumulated specifically in response to jasmonate (JA), ZmNAC100 transcripts were also induced by the salicylic acid analog 2,6-dichloroisonicotinic acid (INA).To assess the phylogenetic relation of ZmNAC41 and ZmNAC100, we studied the family of maize NAC transcription factors based on the recently annotated B73 genome information. We identified 116 maize NAC transcription factor genes that clustered into 12 clades. ZmNAC41 and ZmNAC100 both belong to clade G and appear to have arisen by a recent gene duplication event. Including four other defence-related NAC transcription factors of maize and functionally characterized Arabidopsis and rice NAC transcription factors, we observed an enrichment of NAC transcription factors involved in host defense regulation in clade G. In silico analyses identified putative binding elements for the defence-induced ERF, Myc2, TGA and WRKY transcription factors in the promoters of four out of the six defence-related maize NAC transcription factors, while one of the analysed maize NAC did not contain any of these potential binding sites. CONCLUSIONS: Our study provides a systematic in silico analysis of maize NAC transcription factors in which we propose a nomenclature for maize genes encoding NAC transcription factors, based on their chromosomal position. We have further identified five pathogen-responsive maize NAC transcription factors that harbour putative binding elements for other defence-associated transcription factors in the proximal promoter region, indicating an involvement of the described NACs in the maize defence network. Our phylogenetic analysis has revealed that the majority of the yet described pathogen responsive NAC proteins from all plant species belong to clade G and suggests that they are phylogenetically related.

Early senescence and cell death in Arabidopsis saul1 mutants involves the PAD4-dependent salicylic acid pathway.

Age-dependent leaf senescence and cell death in Arabidopsis (Arabidopsis thaliana) requires activation of the transcription factor ORESARA1 (ORE1) and is not initiated prior to a leaf age of 28 d. Here, we investigate the conditional execution of events that regulate early senescence and cell death in senescence-associated ubiquitin ligase1 (saul1) mutants, deficient in the PLANT U-BOX-ARMADILLO E3 ubiquitin ligase SAUL1. In saul1 mutants challenged with low light, the switch of age-dependent cell death was turned on prematurely, as indicated by the accumulation of ORE1 transcripts, induction of the senescence marker gene SENESCENCE-ASSOCIATED GENE12, and cell death. However, ORE1 accumulation by itself was not sufficient to cause saul1 phenotypes, as demonstrated by double mutant analysis. Exposure of saul1 mutants to low light for only 24 h did not result in visible symptoms of senescence; however, the senescence-promoting transcription factor genes WRKY53, WRKY6, and NAC-LIKE ACTIVATED BY AP3/PI were up-regulated, indicating that senescence in saul1 seedlings was already initiated. To resolve the time course of gene expression, microarray experiments were performed at narrow intervals. Differential expression of the genes involved in salicylic acid and defense mechanisms were the earliest events detected, suggesting a central role for salicylic acid in saul1 senescence and cell death. The salicylic acid content increased in low-light-treated saul1 mutants, and application of exogenous salicylic acid was indeed sufficient to trigger saul1 senescence in permissive light conditions. Double mutant analyses showed that PHYTOALEXIN DEFICIENT4 (PAD4) but not NONEXPRESSER OF PR GENES1 (NPR1) is essential for saul1 phenotypes. Our results indicate that saul1 senescence depends on the PAD4-dependent salicylic acid pathway but does not require NPR1 signaling.

The Ustilago maydis Nit2 homolog regulates nitrogen utilization and is required for efficient induction of filamentous growth.

Nitrogen catabolite repression (NCR) is a regulatory strategy found in microorganisms that restricts the utilization of complex and unfavored nitrogen sources in the presence of favored nitrogen sources. In fungi, this concept has been best studied in yeasts and filamentous ascomycetes, where the GATA transcription factors Gln3p and Gat1p (in yeasts) and Nit2/AreA (in ascomycetes) constitute the main positive regulators of NCR. The reason why functional Nit2 homologs of some phytopathogenic fungi are required for full virulence in their hosts has remained elusive. We have identified the Nit2 homolog in the basidiomycetous phytopathogen Ustilago maydis and show that it is a major, but not the exclusive, positive regulator of nitrogen utilization. By transcriptome analysis of sporidia grown on artificial media devoid of favored nitrogen sources, we show that only a subset of nitrogen-responsive genes are regulated by Nit2, including the Gal4-like transcription factor Ton1 (a target of Nit2). Ustilagic acid biosynthesis is not under the control of Nit2, while nitrogen starvation-induced filamentous growth is largely dependent on functional Nit2. nit2 deletion mutants show the delayed initiation of filamentous growth on maize leaves and exhibit strongly compromised virulence, demonstrating that Nit2 is required to efficiently initiate the pathogenicity program of U. maydis.

Transcriptome and metabolome profiling of field-grown transgenic barley lack induced differences but show cultivar-specific variances.

The aim of the present study was to assess possible adverse effects of transgene expression in leaves of field-grown barley relative to the influence of genetic background and the effect of plant interaction with arbuscular mycorrhizal fungi. We conducted transcript profiling, metabolome profiling, and metabolic fingerprinting of wild-type accessions and barley transgenics with seed-specific expression of (1,3-1, 4)-beta-glucanase (GluB) in Baronesse (B) as well as of transgenics in Golden Promise (GP) background with ubiquitous expression of codon-optimized Trichoderma harzianum endochitinase (ChGP). We found more than 1,600 differential transcripts between varieties GP and B, with defense genes being strongly overrepresented in B, indicating a divergent response to subclinical pathogen challenge in the field. In contrast, no statistically significant differences between ChGP and GP could be detected based on transcriptome or metabolome analysis, although 22 genes and 4 metabolites were differentially abundant when comparing GluB and B, leading to the distinction of these two genotypes in principle component analysis. The coregulation of most of these genes in GluB and GP, as well as simple sequence repeat-marker analysis, suggests that the distinctive alleles in GluB are inherited from GP. Thus, the effect of the two investigated transgenes on the global transcript profile is substantially lower than the effect of a minor number of alleles that differ as a consequence of crop breeding. Exposing roots to the spores of the mycorrhizal Glomus sp. had little effect on the leaf transcriptome, but central leaf metabolism was consistently altered in all genotypes.

AtHsp70-15-deficient Arabidopsis plants are characterized by reduced growth, a constitutive cytosolic protein response and enhanced resistance to TuMV.

Arabidopsis thaliana contains 18 genes encoding Hsp70s. This heat shock protein superfamily is divided into two sub-families: DnaK and Hsp110/SSE. In order to functionally characterize members of the Hsp70 superfamily, loss-of-function mutants with reduced cytosolic Hsp70 expression were studied. AtHsp70-1 and AtHsp70-2 are constitutively expressed and represent the major cytosolic Hsp70 isoforms under ambient conditions. Analysis of single and double mutants did not reveal any difference compared to wild-type controls. In yeast, SSE protein has been shown to act as a nucleotide exchange factor, essential for Hsp70 function. To test whether members of the Hsp110/SSE sub-family serve essential functions in plants, two members of the sub-family, AtHsp70-14 and AtHsp70-15, were analysed. Both genes are highly homologous and constitutively expressed. Deficiency of AtHsp70-15 but not of AtHsp70-14 led to severe growth retardation. AtHsp70-15-deficient plants were smaller than wild-type and exhibited a slightly different leaf shape. Stomatal closure under ambient conditions and in response to ABA was impaired in the AtHsp70-15 transgenic plants, but ABA-dependent inhibition of germination was not affected. Heat treatment of AtHsp70-15-deficient plants resulted in drastically increased mortality, indicating that AtHsp70-15 plays an essential role during normal growth and in the heat response of Arabidopsis plants. AtHsp70-15-deficient plants are more tolerant to infection by turnip mosaic virus. Comparative transcriptome analysis revealed that AtHsp70-15-deficient plants display a constitutive stress response similar to the cytosolic protein response. Based on these results, AtHsp70-15 is likely to be a key factor in proper folding of cytosolic proteins, and may function as nucleotide exchange factor as proposed for yeast.

Indole derivative production by the root endophyte Piriformospora indica is not required for growth promotion but for biotrophic colonization of barley roots.

Beneficial effects elicited by the root endophyte Piriformospora indica are widely known, but the mechanism by which these are achieved is still unclear. It is proposed that phytohormones produced by the fungal symbiont play a crucial role in the interaction with the plant roots. Biochemical analyses of the underlying biosynthetic pathways for auxin production have shown that, on tryptophan feeding, P. indica can produce the phytohormones indole-3-acetic acid (IAA) and indole-3-lactate (ILA) through the intermediate indole-3-pyruvic acid (IPA). Time course transcriptional analyses after exposure to tryptophan designated the piTam1 gene as a key player. A green fluorescence protein (GFP) reporter study and transcriptional analysis of colonized barley roots showed that piTam1 is induced during the biotrophic phase. Piriformospora indica strains in which the piTam1 gene was silenced via an RNA interference (RNAi) approach were compromised in IAA and ILA production and displayed reduced colonization of barley (Hordeum vulgare) roots in the biotrophic phase, but the elicitation of growth promotion was not affected compared with the wild-type situation. Our results suggest that IAA is involved in the establishment of biotrophy in P. indica-barley symbiosis and might represent a compatibility factor in this system.

Loss of cytosolic NADP-malic enzyme 2 in Arabidopsis thaliana is associated with enhanced susceptibility to Colletotrichum higginsianum.

• While photosynthetic NADP-malic enzyme (NADP-ME) has a prominent role in the C(4) cycle, the biological function of nonphotosynthetic isoforms remains elusive. Here, we analysed the link between Arabidopsis thaliana cytosolic NADP-ME2 and the plant defence response. • Arabidopsis thaliana plants with wild-type and modified NADP-ME2 expression levels were analysed after elicitation with pathogen-associated molecular patterns (PAMPs) and during the interaction with the hemibiotrophic fungal pathogen Colletotrichum higginsianum. • Under normal growth conditions, the lack or gain of NADP-ME2 activity produced large changes in plant metabolite pool sizes without any effect on morphology or development. Total NADP-ME activity and NADP-ME2 transcript level were enhanced after PAMP treatment and pathogen infection. During infection with C. higginsianum, loss-of-function mutants of NADP-ME2 (nadp-me2) showed enhanced susceptibility. Transient apoplastic reactive oxygen species (ROS) production after elicitation and callose papilla formation after infection were dampened in nadp-me2. Late salicylic acid (SA)-dependent and SA-independent defence responses were not affected. • Taken together, our results indicate that NADP-ME2 is an important player in plant basal defence, where it appears to be involved in the generation of ROS. Moreover, NADP-ME2 was found to be dispensable for later defence responses.

Barley leaf transcriptome and metabolite analysis reveals new aspects of compatibility and Piriformospora indica-mediated systemic induced resistance to powdery mildew.

Colonization of barley roots with the basidiomycete fungus Piriformospora indica (Sebacinales) induces systemic resistance against the biotrophic leaf pathogen Blumeria graminis f. sp. hordei (B. graminis). To identify genes involved in this mycorrhiza-induced systemic resistance, we compared the leaf transcriptome of P. indica-colonized and noncolonized barley plants 12, 24, and 96 h after challenge with a virulent race of B. graminis. The leaf pathogen induced specific gene sets (e.g., LRR receptor kinases and WRKY transcription factors) at 12 h postinoculation (hpi) (prepenetration phase) and vesicle-localized gene products 24 hpi (haustorium establishment). Metabolic analysis revealed a progressing shift of steady state contents of the intermediates glucose-1-phosphate, uridinediphosphate-glucose, and phosphoenolpyruvate 24 and 96 hpi, indicating that B. graminis shifts central carbohydrate metabolism in favor of sucrose biosynthesis. Both B. graminis and P. indica increased glutamine and alanine contents, whereas substrates for starch and nitrogen assimilation (adenosinediphosphate- glucose and oxoglutarate) decreased. In plants that were more B. graminis resistant due to P. indica root colonization, 22 transcripts, including those of pathogenesis-related genes and genes encoding heat-shock proteins, were differentially expressed ?twofold in leaves after B. graminis inoculation compared with non-mycorrhized plants. Detailed expression analysis revealed a faster induction after B. graminis inoculation between 8 and 16 hpi, suggesting that priming of these genes is an important mechanism of P. indica-induced systemic disease resistance.

Stimulation of nonselective amino acid export by glutamine dumper proteins.

Phloem and xylem transport of amino acids involves two steps: export from one cell type to the apoplasm, and subsequent import into adjacent cells. High-affinity import is mediated by proton/amino acid cotransporters, while the mechanism of export remains unclear. Enhanced expression of the plant-specific type I membrane protein Glutamine Dumper1 (GDU1) has previously been shown to induce the secretion of glutamine from hydathodes and increased amino acid content in leaf apoplasm and xylem sap. In this work, tolerance to low concentrations of amino acids and transport analyses using radiolabeled amino acids demonstrate that net amino acid uptake is reduced in the glutamine-secreting GDU1 overexpressor gdu1-1D. The net uptake rate of phenylalanine decreased over time, and amino acid net efflux was increased in gdu1-1D compared with the wild type, indicating increased amino acid export from cells. Independence of the export from proton gradients and ATP suggests that overexpression of GDU1 affects a passive export system. Each of the seven Arabidopsis (Arabidopsis thaliana) GDU genes led to similar phenotypes, including increased efflux of a wide spectrum of amino acids. Differences in expression profiles and functional properties suggested that the GDU genes fulfill different roles in roots, vasculature, and reproductive organs. Taken together, the GDUs appear to stimulate amino acid export by activating nonselective amino acid facilitators.

Ustilago maydis infection strongly alters organic nitrogen allocation in maize and stimulates productivity of systemic source leaves.

The basidiomycete Ustilago maydis is the causal agent of corn smut disease and induces tumor formation during biotrophic growth in its host maize (Zea mays). We have conducted a combined metabolome and transcriptome survey of infected leaves between 1 d post infection (dpi) and 8 dpi, representing infected leaf primordia and fully developed tumors, respectively. At 4 and 8 dpi, we observed a substantial increase in contents of the nitrogen-rich amino acids glutamine and asparagine, while the activities of enzymes involved in primary nitrogen assimilation and the content of ammonia and nitrate were reduced by 50% in tumors compared with mock controls. Employing stable isotope labeling, we could demonstrate that U. maydis-induced tumors show a reduced assimilation of soil-derived (15)NO(3)(-) and represent strong sinks for nitrogen. Specific labeling of the free amino acid pool of systemic source leaves with [(15)N]urea revealed an increased import of organic nitrogen from systemic leaves to tumor tissue, indicating that organic nitrogen provision supports the formation of U. maydis-induced tumors. In turn, amino acid export from systemic source leaves was doubled in infected plants. The analysis of the phloem amino acid pool revealed that glutamine and asparagine are not transported to the tumor tissue, although these two amino acids were found to accumulate within the tumor. Photosynthesis was increased and senescence was delayed in systemic source leaves upon tumor development on infected plants, indicating that the elevated sink demand for nitrogen could determine photosynthetic rates in source leaves.

Manipulation of plant innate immunity and gibberellin as factor of compatibility in the mutualistic association of barley roots with Piriformospora indica.

Fungi of the order Sebacinales (Basidiomycota) are involved in a wide spectrum of mutualistic symbioses with various plants, thereby exhibiting unique potential for biocontrol strategies. Piriformospora indica, a model organism of this fungal order, is able to increase the biomass and grain yield of crop plants, and induces local and systemic resistance to fungal diseases and tolerance to abiotic stress. To elucidate the molecular basis for root colonization, we characterized the interaction of P. indica with barley roots by combining global gene expression profiling, metabolic profiling, and genetic studies. At the metabolic level, we show that fungal colonization reduces the availability of free sugars and amino acids to the root tip. At the transcriptional level, consecutive interaction stages covering pre-penetration-associated events and progressing through to root colonization showed differential regulation of signal perception and transduction components, secondary metabolism, and genes associated with membrane transport. Moreover, we observed stage-specific up-regulation of genes involved in phytohormone metabolism, mainly encompassing gibberellin, auxin and abscisic acid, but salicylic acid-associated gene expression was suppressed. The changes in hormone homoeostasis were accompanied with a general suppression of the plant innate immune system. Further genetic studies showed reduced fungal colonization in mutants that are impaired in gibberellin synthesis as well as perception, and implicate gibberellin as a modulator of the root's basal defence. Our data further reveal the complexity of compatibility mechanisms in host-microbe interactions, and identify gibberellin signaling as potential target for successful fungi.

Reprogramming a maize plant: transcriptional and metabolic changes induced by the fungal biotroph Ustilago maydis.

The fungal pathogen Ustilago maydis establishes a biotrophic relationship with its host plant maize (Zea mays). Hallmarks of the disease are large plant tumours in which fungal proliferation occurs. Previous studies suggested that classical defence pathways are not activated. Confocal microscopy, global expression profiling and metabolic profiling now shows that U. maydis is recognized early and triggers defence responses. Many of these early response genes are downregulated at later time points, whereas several genes associated with suppression of cell death are induced. The interplay between fungus and host involves changes in hormone signalling, induction of antioxidant and secondary metabolism, as well as the prevention of source leaf establishment. Our data provide novel insights into the complexity of a biotrophic interaction.