Oviposition by Spodoptera exigua on Solanum dulcamara Alters the Plant's Response to Herbivory and Impairs Larval Performance.

Plant resistance traits against insect herbivores are extremely plastic. Plants respond not only to the herbivory itself, but also to oviposition by herbivorous insects. How prior oviposition affects plant responses to larval herbivory is largely unknown. Combining bioassays and defense protein activity assays with microarray analyses and metabolite profiling, we investigated the impact of preceding oviposition on the interaction of Solanum dulcamara with the generalist lepidopteran herbivore Spodoptera exigua at the levels of the plant's resistance, transcriptome and metabolome. We found that oviposition increased plant resistance to the subsequent feeding larvae. While constitutive and feeding-induced levels of defensive protease inhibitor activity remained unaffected, pre-exposure to eggs altered S. dulcamara's transcriptional and metabolic response to larval feeding in leaves local and systemic to oviposition. In particular, genes involved in phenylpropanoid metabolism were more strongly expressed in previously oviposited plants, which was reflected by reciprocal changes of primary metabolites upstream and within these pathways. Our data highlight that plants integrate signals from non-threatening life stages of their natural enemies to optimize their response when they become actually attacked. The observed transcriptional and metabolic reshaping of S. dulcamara's response to S. exigua herbivory suggests a role of phenylpropanoids in oviposition-primed plant resistance.

The Sporisorium reilianum Effector Vag2 Promotes Head Smut Disease via Suppression of Plant Defense Responses.

Genome comparison between the maize pathogens Ustilago maydis and Sporisorium reilianum revealed a large diversity region (19-1) containing nearly 30 effector gene candidates, whose deletion severely hampers virulence of both fungi. Dissection of the S. reilianum gene cluster resulted in the identification of one major contributor to virulence, virulence-associated gene 2 (vag2; sr10050). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) experiments revealed high expression of vag2 during biotrophic growth of S. reilianum. Using the yeast secretion trap assay, we confirmed the existence of a functional signal peptide allowing protein secretion via the conventional secretory pathway. We identified the cytoplasmic maize chorismate mutase ZmCM2 by yeast two-hybrid screening as a possible interaction partner of Vag2. Interaction of the two proteins in planta was confirmed by bimolecular fluorescence complementation. qRT-PCR experiments revealed vag2-dependent downregulation of salicylic acid (SA)-induced genes, which correlated with higher SA levels in plant tissues colonized by Δvag2 deletion strains relative to S. reilianum wildtype strains. Metabolite analysis suggested rewiring of pathogen-induced SA biosynthesis by preferential conversion of the SA precursor chorismate into the aromatic amino acid precursor prephenate by ZmCM2 in the presence of Vag2. Possibly, the binding of Vag2 to ZmCM2 inhibits the back reaction of the ZmCM2-catalyzed interconversion of chorismate and prephenate, thus contributing to fungal virulence by lowering the plant SA-induced defenses.

Induced, Imprinted, and Primed Responses to Changing Environments: Does Metabolism Store and Process Information?

Metabolism is the system layer that determines growth by the rate of matter uptake and conversion into biomass. The scaffold of enzymatic reaction rates drives the metabolic network in a given physico-chemical environment. In response to the diverse environmental stresses, plants have evolved the capability of integrating macro- and micro-environmental events to be prepared, i.e., to be primed for upcoming environmental challenges. The hierarchical view on stress signaling, where metabolites are seen as final downstream products, has recently been complemented by findings that metabolites themselves function as stress signals. We present a systematic concept of metabolic responses that are induced by environmental stresses and persist in the plant system. Such metabolic imprints may prime metabolic responses of plants for subsequent environmental stresses. We describe response types with examples of biotic and abiotic environmental stresses and suggest that plants use metabolic imprints, the metabolic changes that last beyond recovery from stress events, and priming, the imprints that function to prepare for upcoming stresses, to integrate diverse environmental stress histories. As a consequence, even genetically identical plants should be studied and understood as phenotypically plastic organisms that continuously adjust their metabolic state in response to their individually experienced local environment. To explore the occurrence and to unravel functions of metabolic imprints, we encourage researchers to extend stress studies by including detailed metabolic and stress response monitoring into extended recovery phases.

Primed primary metabolism in systemic leaves: a functional systems analysis.

Plants evolved mechanisms to counteract bacterial infection by preparing yet uninfected systemic tissues for an enhanced defense response, so-called systemic acquired resistance or priming responses. Primed leaves express a wide range of genes that enhance the defense response once an infection takes place. While hormone-driven defense signalling and defensive metabolites have been well studied, less focus has been set on the reorganization of primary metabolism in systemic leaves. Since primary metabolism plays an essential role during defense to provide energy and chemical building blocks, we investigated changes in primary metabolism at RNA and metabolite levels in systemic leaves of Arabidopsis thaliana plants that were locally infected with Pseudomonas syringae. Known defense genes were still activated 3-4 days after infection. Also primary metabolism was significantly altered. Nitrogen (N)-metabolism and content of amino acids and other N-containing metabolites were significantly reduced, whereas the organic acids fumarate and malate were strongly increased. We suggest that reduction of N-metabolites in systemic leaves primes defense against bacterial infection by reducing the nutritional value of systemic tissue. Increased organic acids serve as quickly available metabolic resources of energy and carbon-building blocks for the production of defense metabolites during subsequent secondary infections.

Extrafloral nectar secretion from wounds of Solanum dulcamara.

Plants usually close wounds rapidly to prevent infections and the loss of valuable resources such as assimilates(1). However, herbivore-inflicted wounds on the bittersweet nightshade Solanum dulcamara appear not to close completely and produce sugary wound secretions visible as droplets. Many plants across the plant kingdom secrete sugary nectar from extrafloral nectaries(2) to attract natural enemies of herbivores for indirect defence(3,4). As ants forage on wound edges of S. dulcamara in the field, we hypothesized that wound secretions are a form of extrafloral nectar (EFN). We show that, unlike EFN from known nectaries, wound secretions are neither associated with any specific structure nor restricted to certain locations. However, similar to EFN, they are jasmonate-inducible and the plant controls their chemical composition. Wound secretions are attractive for ants, and application of wound secretion mimics increases ant attraction and reduces herbivory on S. dulcamara plants in a natural population. In greenhouse experiments, we reveal that ants can defend S. dulcamara from two of its native herbivores, slugs and flea beetle larvae. Since nectar is defined by its ecological function as a sugary secretion involved in interactions with animals(5), such 'plant bleeding' could be a primitive mode of nectar secretion exemplifying an evolutionary origin of structured extrafloral nectaries.

Priming and memory of stress responses in organisms lacking a nervous system.

Experience and memory of environmental stimuli that indicate future stress can prepare (prime) organismic stress responses even in species lacking a nervous system. The process through which such organisms prepare their phenotype for an improved response to future stress has been termed 'priming'. However, other terms are also used for this phenomenon, especially when considering priming in different types of organisms and when referring to different stressors. Here we propose a conceptual framework for priming of stress responses in bacteria, fungi and plants which allows comparison of priming with other terms, e.g. adaptation, acclimation, induction, acquired resistance and cross protection. We address spatial and temporal aspects of priming and highlight current knowledge about the mechanisms necessary for information storage which range from epigenetic marks to the accumulation of (dormant) signalling molecules. Furthermore, we outline possible patterns of primed stress responses. Finally, we link the ability of organisms to become primed for stress responses (their 'primability') with evolutionary ecology aspects and discuss which properties of an organism and its environment may favour the evolution of priming of stress responses.

Profiling methods to identify cold-regulated primary metabolites using gas chromatography coupled to mass spectrometry.

This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of single leaves from plants that were exposed to temperature stress experiments. The described method is modular. The modules include a rapid sampling and metabolic inactivation protocol for samples in a wide size range, a sample extraction procedure, a chemical derivatization step that is required to make the metabolite fraction amenable to gas chromatographic analysis, a routine GC-MS method, and finally the procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The method has two applications: (1) the rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification of cold-regulated metabolites by exact quantification using a GC-MS protocol with extended internal and external standardization.

The Metabolic Interplay between Plants and Phytopathogens.

Plant diseases caused by pathogenic bacteria or fungi cause major economic damage every year and destroy crop yields that could feed millions of people. Only by a thorough understanding of the interaction between plants and phytopathogens can we hope to develop strategies to avoid or treat the outbreak of large-scale crop pests. Here, we studied the interaction of plant-pathogen pairs at the metabolic level. We selected five plant-pathogen pairs, for which both genomes were fully sequenced, and constructed the corresponding genome-scale metabolic networks. We present theoretical investigations of the metabolic interactions and quantify the positive and negative effects a network has on the other when combined into a single plant-pathogen pair network. Merged networks were examined for both the native plant-pathogen pairs as well as all other combinations. Our calculations indicate that the presence of the parasite metabolic networks reduce the ability of the plants to synthesize key biomass precursors. While the producibility of some precursors is reduced in all investigated pairs, others are only impaired in specific plant-pathogen pairs. Interestingly, we found that the specific effects on the host's metabolism are largely dictated by the pathogen and not by the host plant. We provide graphical network maps for the native plant-pathogen pairs to allow for an interactive interrogation. By exemplifying a systematic reconstruction of metabolic network pairs for five pathogen-host pairs and by outlining various theoretical approaches to study the interaction of plants and phytopathogens on a biochemical level, we demonstrate the potential of investigating pathogen-host interactions from the perspective of interacting metabolic networks that will contribute to furthering our understanding of mechanisms underlying a successful invasion and subsequent establishment of a parasite into a plant host.

Plant-growth promoting effect of newly isolated rhizobacteria varies between two Arabidopsis ecotypes.

Various rhizobacteria are known for their beneficial effects on plants, i. e. promotion of growth and induction of systemic resistance against pathogens. These bacteria are categorized as plant growth promoting rhizobacteria (PGPR) and are associated with plant roots. Knowledge of the underlying mechanisms of plant growth promotion in vivo is still very limited, but interference of bacteria with plant hormone metabolism is suggested to play a major role. To obtain new growth promoting bacteria, we started a quest for rhizobacteria that are naturally associated to Arabidopsis thaliana. A suite of native root-associated bacteria were isolated from surface-sterilized roots of the Arabidopsis ecotype Gol-1 derived from a field site near Golm (Berlin area, Germany). We found several Pseudomonas and a Microbacterium species and tested these for growth promotion effects on the Arabidopsis ecotypes Gol-1 and Col-0, and for growth-promotion associated traits, such as auxin production, ACC deaminase activity and phosphate solubilization capacity. We showed that two of the bacteria strains promote plant growth with respect to rosette diameter, stalk length and accelerate development and that the effects were greater when bacteria were applied to Col-0 compared with Gol-1. Furthermore, the capability of promoting growth was not explained by the tested metabolic properties of the bacteria, suggesting that further bacterial traits are required. The natural variation of growth effects, combined with the extensive transgenic approaches available for the model plant Arabidopsis, will build a valuable tool to augment our understanding of the molecular mechanisms involved in the natural Arabidopsis - PGPR association.

A naturally associated rhizobacterium of Arabidopsis thaliana induces a starvation-like transcriptional response while promoting growth.

Plant growth promotion by rhizobacteria is a known phenomenon but the underlying mechanisms are poorly understood. We searched for plant growth-promoting rhizobacteria that are naturally associated with Arabidopsis thaliana to investigate the molecular mechanisms that are involved in plant growth-promotion. We isolated a Pseudomonas bacterium (Pseudomonas sp. G62) from roots of field-grown Arabidopsis plants that has not been described previously and analyzed its effect on plant growth, gene expression and the level of sugars and amino acids in the host plant. Inoculation with Pseudomonas sp. G62 promoted plant growth under various growth conditions. Microarray analysis revealed rapid changes in transcript levels of genes annotated to energy-, sugar- and cell wall metabolism in plants 6 h after root inoculation with P. sp. G62. The expression of several of these genes remained stable over weeks, but appeared differentially regulated in roots and shoots. The global gene expression profile observed after inoculation with P. sp. G62 showed a striking resemblance with previously described carbohydrate starvation experiments, although plants were not depleted from soluble sugars, and even showed a slight increase of the sucrose level in roots 5 weeks after inoculation. We suggest that the starvation-like transcriptional phenotype - while steady state sucrose levels are not reduced - is induced by a yet unknown signal from the bacterium that simulates sugar starvation. We discuss the potential effects of the sugar starvation signal on plant growth promotion.

Reverse genetics in ecological research.

By precisely manipulating the expression of individual genetic elements thought to be important for ecological performance, reverse genetics has the potential to revolutionize plant ecology. However, untested concerns about possible side-effects of the transformation technique, caused by Agrobacterium infection and tissue culture, on plant performance have stymied research by requiring onerous sample sizes. We compare 5 independently transformed Nicotiana attenuata lines harboring empty vector control (EVC) T-DNA lacking silencing information with isogenic wild types (WT), and measured a battery of ecologically relevant traits, known to be important in plant-herbivore interactions: phytohormones, secondary metabolites, growth and fitness parameters under stringent competitive conditions, and transcriptional regulation with microarrays. As a positive control, we included a line silenced in trypsin proteinase inhibitor gene (TPI) expression, a potent anti-herbivore defense known to exact fitness costs in its expression, in the analysis. The experiment was conducted twice, with 10 and 20 biological replicates per genotype. For all parameters, we detected no difference between any EVC and WT lines, but could readily detect a fitness benefit of silencing TPI production. A statistical power analyses revealed that the minimum sample sizes required for detecting significant fitness differences between EVC and WT was 2-3 orders of magnitude larger than the 10 replicates required to detect a fitness effect of TPI silencing. We conclude that possible side-effects of transformation are far too low to obfuscate the study of ecologically relevant phenotypes.

SNF1-related kinases allow plants to tolerate herbivory by allocating carbon to roots.

Herbivore attack elicits costly defenses that are known to decrease plant fitness by using resources that are normally slated for growth and reproduction. Additionally, plants have evolved mechanisms for tolerating attack, which are not understood on a molecular level. Using 11C-photosynthate labeling as well as sugar and enzyme measurements, we found rapid changes in sink-source relations in the annual Nicotiana attenuata after simulated herbivore attacks, which increased the allocation of sugars to roots. This herbivore-induced response is regulated by the beta-subunit of an SnRK1 (SNF1-related kinase) protein kinase, GAL83, transcripts of which are rapidly down-regulated in source leaves after herbivore attack and, when silenced, increase assimilate transport to roots. This C diversion response is activated by herbivore-specific elicitors and is independent of jasmonate signaling, which regulates most of the plant's defense responses. Herbivore attack during early stages of development increases root reserves, which, in turn, delays senescence and prolongs flowering. That attacked GAL83-silenced plants use their enhanced root reserves to prolong reproduction demonstrates that SnRK1 alters resource allocation so that plants better tolerate herbivory. This tolerance mechanism complements the likely defensive value of diverting resources to a less vulnerable location within the plant.