The Pleiades are a cluster of fungal effectors that inhibit host defenses.

Biotrophic plant pathogens secrete effector proteins to manipulate the host physiology. Effectors suppress defenses and induce an environment favorable to disease development. Sequence-based prediction of effector function is impeded by their rapid evolution rate. In the maize pathogen Ustilago maydis, effector-coding genes frequently organize in clusters. Here we describe the functional characterization of the pleiades, a cluster of ten effector genes, by analyzing the micro- and macroscopic phenotype of the cluster deletion and expressing these proteins in planta. Deletion of the pleiades leads to strongly impaired virulence and accumulation of reactive oxygen species (ROS) in infected tissue. Eight of the Pleiades suppress the production of ROS upon perception of pathogen associated molecular patterns (PAMPs). Although functionally redundant, the Pleiades target different host components. The paralogs Taygeta1 and Merope1 suppress ROS production in either the cytoplasm or nucleus, respectively. Merope1 targets and promotes the auto-ubiquitination activity of RFI2, a conserved family of E3 ligases that regulates the production of PAMP-triggered ROS burst in plants.

Ustilago maydis effector Jsi1 interacts with Topless corepressor, hijacking plant jasmonate/ethylene signaling.

Ustilago maydis is the causal agent of maize smut disease. During the colonization process, the fungus secretes effector proteins that suppress immune responses and redirect the host metabolism in favor of the pathogen. As effectors play a critical role during plant colonization, their identification and functional characterization are essential to understanding biotrophy and disease. Using biochemical, molecular, and transcriptomic techniques, we performed a functional characterization of the U. maydis effector Jasmonate/Ethylene signaling inducer 1 (Jsi1). Jsi1 interacts with several members of the plant corepressor family Topless/Topless related (TPL/TPR). Jsi1 expression in Zea mays and Arabidopsis thaliana leads to transcriptional induction of the ethylene response factor (ERF) branch of the jasmonate/ethylene (JA/ET) signaling pathway. In A. thaliana, activation of the ERF branch leads to biotrophic susceptibility. Jsi1 likely activates the ERF branch via an EAR (ET-responsive element binding-factor-associated amphiphilic repression) motif, which resembles EAR motifs from plant ERF transcription factors, that interacts with TPL/TPR proteins. EAR-motif-containing effector candidates were identified from different fungal species, including Magnaporthe oryzae, Sporisorium scitamineum, and Sporisorium reilianum. Interaction between plant TPL proteins and these effector candidates from biotrophic and hemibiotrophic fungi indicates the convergent evolution of effectors modulating the TPL/TPR corepressor hub.

Proline Accumulation Is Regulated by Transcription Factors Associated with Phosphate Starvation.

Pro accumulation in plants is a well-documented physiological response to osmotic stress caused by drought or salinity. In Arabidopsis (Arabidopsis thaliana), the stress and ABA-induced Δ1-PYRROLINE-5-CARBOXYLATE SYNTHETASE1 (P5CS1) gene was previously shown to control Pro biosynthesis in such adverse conditions. To identify regulatory factors that control the transcription of P5CS1, Y1H screens were performed with a genomic fragment of P5CS1, containing 1.2-kB promoter and 0.8-kb transcribed regions. The myeloblastosis (MYB)-type transcription factors PHOSPHATE STARVATION RESPONSE1 (PHR1) and PHR1-LIKE1 (PHL1) were identified to bind to P5CS1 regulatory sequences in the first intron, which carries a conserved PHR1-binding site (P1BS) motif. Binding of PHR1 and PHL1 factors to P1BS was confirmed by Y1H, electrophoretic mobility assay and chromatin immunoprecipitation. Phosphate starvation led to gradual increase in Pro content in wild-type Arabidopsis plants as well as transcriptional activation of P5CS1 and PRO DEHYDROGENASE2 genes. Induction of P5CS1 transcription and Pro accumulation during phosphate deficiency was considerably reduced by phr1 and phl1 mutations and was impaired in the ABA-deficient aba2-3 and ABA-insensitive abi4-1 mutants. Growth and viability of phr1phl1 double mutant was significantly reduced in phosphate-depleted medium, while growth was only marginally affected in the aba2-3 mutants, suggesting that ABA is implicated in growth retardation in such nutritional stress. Our results reveal a previously unknown link between Pro metabolism and phosphate nutrition and show that Pro biosynthesis is target of cross talk between ABA signaling and regulation of phosphate homeostasis through PHR1- and PHL1-mediated transcriptional activation of the P5CS1 gene.

Novel in vivo experimental viability assays with high sensitivity and throughput capacity using a bdelloid rotifer.

Rotifers have been used in biological research as well-characterized models of aging. Their multi-organ characters and their sensitivity for chemicals and environmental changes make them useful as in vivo toxicological and lifespan models. Our aim was to create a bdelloid rotifer model to use in high-throughput viability and non-invasive assays. In order to identify our species Philodina acuticornis odiosa (PA), 18S rDNA-based phylogenetic analysis was carried out and their species-specific morphological markers identified. To execute the rotifer-based experiments, we developed an oil-covered water-drop methodology adapted from human in vitro fertilization techniques. This enables toxicological observations of individual one-housed rotifers in a closed and controllable micro-environment for up to several weeks. Hydrogen peroxide (H2O2) and sodium azide (NaN3) exposures were used as well-understood toxins. The toxicity and survival lifespan (TSL), the bright light disturbance (BLD) the mastax contraction frequency (MCF) and the cellular reduction capacity (CRC), indices were recorded. These newly developed assays were used to test the effects of lethal and sublethal doses of the toxins. The results showed the expected dose-dependent decrease in indices. These four different assays can either be used independently or as an integrated system for studying rotifers. These new indices render the PA invertebrate rotifer model a quantitative system for measuring viability, toxicity and lifespan (with TSL), systemic reaction capacity (with BLD), organic functionality (with MCF) and reductive capability of rotifers (with CRC), in vivo. This novel multi-level system is a reliable, sensitive and replicable screening tool with potential application in pharmaceutical science.

Phytosiderophore pathway response in barley exposed to iron, zinc or copper starvation.

Efficient micronutrient acquisition is a critical factor in selecting micronutrient dense crops for human consumption. Enhanced exudation and re-uptake of metal chelators, so-called phytosiderophores, by roots of graminaceous plants has been implicated in efficient micronutrient acquisition. We compared PS biosynthesis and exudation as a response mechanism to either Fe, Zn or Cu starvation. Two barley (Hordeum vulgare L.) lines with contrasting micronutrient grain yields were grown hydroponically and PS exudation (LC-MS) and root gene expression (RNAseq) were determined after either Fe, Zn, or Cu starvation. The response strength of the PS pathway was micronutrient dependent and decreased in the order Fe > Zn > Cu deficiency. We observed a stronger expression of PS pathway genes and greater PS exudation in the barley line with large micronutrient grain yield suggesting that a highly expressed PS pathway might be an important trait involved in high micronutrient accumulation. In addition to several metal specific transporters, we also found that the expression of IRO2 and bHLH156 transcription factors was not only induced under Fe but also under Zn and Cu deficiency. Our study delivers important insights into the role of the PS pathway in the acquisition of different micronutrients.

Mitochondrial complex I subunit NDUFS8.2 modulates responses to stresses associated with reduced water availability.

Mitochondria are important sources of energy in plants and are implicated in coordination of a number of metabolic and physiological processes including stabilization of redox balance, synthesis and turnover of a number of metabolites, and control of programmed cell death. Mitochondrial electron transport chain (mETC) is the backbone of the energy producing process which can influence other processes as well. Accumulating evidence suggests that mETC can affect responses to environmental stimuli and modulate tolerance to extreme conditions such as drought or salinity. Screening for stress responses of 13 Arabidopsis mitochondria-related T-DNA insertion mutants, we identified ndufs8.2-1 which has an increased ability to withstand osmotic and oxidative stresses compared to wild type plants. Insertion in ndufs8.2-1 disrupted the gene that encodes the NADH dehydrogenase [ubiquinone] fragment S subunit 8 (NDUFS8) a component of Complex I of mETC. ndufs8.2-1 tolerated reduced water availability, retained photosynthetic activity and recovered from severe water stress with higher efficiency compared to wild type plants. Several mitochondrial functions were altered in the mutant including oxygen consumption, ROS production, ATP and ADP content as well as activities of genes encoding alternative oxidase 1A (AOX1A) and various alternative NAD(P)H dehydrogenases (ND). Our results suggest that in the absence of NDUFS8.2 stress-induced ROS generation is restrained leading to reduced oxidative damage and improved tolerance to water deficiency. mETC components can be implicated in redox and energy homeostasis and modulate responses to stresses associated with reduced water availability.

A quick and simple spectrophotometric method to determine total carbon concentrations in root exudate samples of grass species.

Purpose: Root exudates are key components driving belowground interaction between plant, microbes and soil. High-end analytical approaches provide advanced insights into exudate metabolite diversity, however, the amount of total carbon (C) released by roots should always be determined as the most basic parameter when characterizing root exudation as it (i) provides quantitative information of C exuded into the surrounding soil and (ii) allows to relate the abundance of individual exudate compounds to total C released. Here we propose a simple and quick, spectrophotometry-based method to quantify total dissolved organic carbon (DOC) concentration in exudation samples that is based on measuring the absorption of a pre-filtered but otherwise untreated exudate sample at 260 nm (DOC260). Method: Exudate samples collected from different grass genotypes (Zea mays, Oryza sativa, Hordeum vulgare) grown in various experimental settings (soil, hydroponic) were analysed with the DOC260 assay and results were compared with C concentrations obtained by liquid TOC-analyser. Conclusion: We demonstrated that the DOC260 method allowed for quick and inexpensive measurements of total dissolved organic carbon concentrations in exudate samples from grass species grown under nutrient sufficient as well as under P deficient conditions. Interestingly, DOC260 failed to predict DOC concentrations in exudate samples from plants grown under Zn and Fe deficiency suggesting a strong shift in metabolite composition under micronutrient deficiency. Even though the applicability of the DOC260 method remains to be tested on exudate samples originating from dicots and plants exposed to other environmental stresses (e.g. pathogen attack, heavy metal stress, etc), it will help to increase our understanding of root exudation and related rhizosphere processes in the future.

Light Control of Salt-Induced Proline Accumulation Is Mediated by ELONGATED HYPOCOTYL 5 in Arabidopsis.

Plants have to adapt their metabolism to constantly changing environmental conditions, among which the availability of light and water is crucial in determining growth and development. Proline accumulation is one of the sensitive metabolic responses to extreme conditions; it is triggered by salinity or drought and is regulated by light. Here we show that red and blue but not far-red light is essential for salt-induced proline accumulation, upregulation of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1) and downregulation of PROLINE DEHYDROGENASE 1 (PDH1) genes, which control proline biosynthetic and catabolic pathways, respectively. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the transcription factor ELONGATED HYPOCOTYL 5 (HY5) binds to G-box and C-box elements of P5CS1 and a C-box motif of PDH1. Salt-induced proline accumulation and P5CS1 expression were reduced in the hy5hyh double mutant, suggesting that HY5 promotes proline biosynthesis through connecting light and stress signals. Our results improve our understanding on interactions between stress and light signals, confirming HY5 as a key regulator in proline metabolism.