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SummaryXylem sap is the major transport route for nutrients from roots to shoots. Here, we investigated how variations in nitrogen (N) nutrition affected the metabolome and proteome of xylem sap, growth of the xylem endophyte Brennaria salicis and report transcriptional re-wiring of leaf defenses in poplar (Populus x canescens). We supplied poplars with high, intermediate or low concentrations of ammonium or nitrate. We identified 288 unique proteins in xylem sap. About 85% of the xylem sap proteins were shared among ammonium- and nitrate-supplied plants. The number of proteins increased with increasing N supply but the major functional categories (catabolic processes, cell wall-related enzymes, defense) were unaffected. Ammonium nutrition caused higher abundances of amino acids and carbohydrates, while nitrate caused higher malate levels in xylem sap. Pipecolic acid and N-hydroxy-pipecolic acid increased whereas salicylic acid and jasmonoyl-isoleucine decreased with increasing N nutrition. Untargeted metabolome analyses revealed 2179 features in xylem sap, of which 863 were differentially affected by N treatments. We identified 122 metabolites, mainly from specialized metabolism of the groups of salicinoids, phenylpropanoids, phenolics, flavonoids, and benzoates. Their abundances increased with decreasing N. Endophyte growth was stimulated in xylem sap of high N- and suppressed in that of low N-fed plants. The drastic changes in xylem sap composition caused massive changes in the transcriptional landscape of leaves and recruited defense pathways against leaf feeding insects and biotrophic fungi, mainly under low nitrate. Our study uncovers unexpected complexity and variability of xylem composition with consequences for plant defenses.Significance statementThis study reports the largest, currently available plant xylem sap proteome and metabolome databases and highlights novel discoveries of specialized metabolites and phytohormones in the xylem sap. This is the first multi-omics study linking differences in nitrogen supply with changes xylem sap composition, endophyte growth and transcriptional defenses in leaves.

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteome of the allodiploidVerticillium longisporumwas analyzed upon cultivation in different media. Secreted fungal proteins were identified by label free LC-MS/MS screening.V. longisporuminduced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 223 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 144 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known effectors, metallopeptidases and carbohydrate-active enzymes. The most abundant and uniquely enriched proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the CAZys Gla1, Amy1 and Cbd1. Deletion of the majority of the corresponding genes caused no phenotypic changes duringex plantagrowth or invasion and colonization of tomato plants. However, we discovered that theNLP2andNLP3deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successfulV. dahliaecolonization.Author SummaryVerticilliumspp. infect hundreds of different plants world-wide leading to enormous economic losses. Verticillium wilt is a disease of the vasculature. The fungus colonizes the xylem of its host plant where it exploits the vascular system to colonize the whole plant. Therefore, the fungus spends part of its lifetime in this nutrient-low and imbalanced environment where it is inaccessible for disease control treatments. This lifestyle as well requires the fungus to react to plant defense responses by secreting specific effector molecules to establish a successful infection. We addressed the differences in media-dependent secretion responses ofVerticillium longisporum. We identified a broad response pattern induced by several media, and a similar response (but with some distinct differences) for the plant-related environments: the pectin-rich medium SXM and xylem sap from the host rapeseed. Importantly, we show that the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3 are xylem sap-specific proteins that are required for fullV. dahliaepathogenicity on tomato. These factors play a role during the colonization phase and represent potential targets for new control strategies for Verticillium wilt.

SummaryEpichloë festucaeis an endophytic fungus that forms a mutualistic symbiotic association withLolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates ofE. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from non-infected and infectedL. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem mass spectrometry was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using the MarVis Pathway in combination with the BioCyc and KEGG databases, and an in-houseEpichloëmetabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novelEpichloë-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressedin planta. Comparative mass spectrometric analysis of apoplastic wash fluid from wild-type- versus mutant- infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium discoideum are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, its natural food substrate.

Dinucleoside 5',5"'-P1,P4-tetraphosphate hydrolase (EC 3.6.1.17) has been purified to homogeneity from tomato (Lycopersicon esculentum) cells grown in suspension. The purification procedure comprised ammonium sulphate fractionation following five standard chromatography steps and a final chromatography on Ap4A-Sepharose. The homogeneous hydrolase has a molecular mass of 20 kDa and an isoelectric point of 4.5. The enzyme hydrolyses diadenosine tetraphosphate (Ap4A) asymmetrically to AMP and ATP. Among other naturally occurring dinucleoside oligophosphates, Ap5A and Ap6A are substrates whereas Ap3A is not. Of various phosphonate analogues tested, the Ap5A analogue, AppCH2pCH2ppA, was not cleaved and the Ap3A analogue, ApCH2CH2ppA, was a very poor substrate. Enzyme activity is stimulated by 5 mM Mg2+ and inhibited by fluoride anion; I50 = 6.25 microM. The K(m) value for Ap4A is 0.8 microM. The enzyme exhibits a broad pH optimum from pH 6.5 to 9.0. In order to analyze the protein at the molecular level an internal peptide sequence from the homogeneous enzyme was identified. Within the sequence of 17 amino acids a kinase II motif as a general part of a conserved sequence of nucleotide binding sites was found. Against the internal peptide sequence a polyclonal antiserum was raised. By investigating the intracellular level of Ap4A hydrolase under different kinds of environmental stress, no changes occurred in response to heat shock. But, heavy metal stress and phosphate deprivation lead to a decrease in Ap4A hydrolase.