Alexander Muck

Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VII. Changes in the plant's proteome

When Manduca sexta attacks Nicotiana attenuata, fatty acid-amino acid conjugates (FACs) in the larvaes oral secretions (OS) are introduced into feeding wounds. These FACs trigger a transcriptional response that is similar to the response induced by insect damage. Using two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time of flight, and liquid chromatography-tandem mass spectrometry, we characterized the proteins in phenolic extracts and in a nuclear fraction of leaves elicited by larval attack, and/or in leaves wounded and treated with OS, FAC-free OS, and synthetic FACs. Phenolic extracts yielded approximately 600 protein spots, many of which were altered by elicitation, whereas nuclear protein fractions yielded approximately 100 spots, most of which were unchanged by elicitation. Reproducible elicitor-induced changes in 90 spots were characterized. In general, proteins that increased were involved in primary metabolism, defense, and transcriptional and translational regulation; those that decreased were involved in photosynthesis. Like the transcriptional defense responses, proteomic changes were strongly elicited by the FACs in OS. A semiquantitative reverse transcription-PCR approach based on peptide sequences was used to compare transcript and protein accumulation patterns for 17 candidate proteins. In six cases the patterns of elicited transcript accumulation were consistent with those of elicited protein accumulation. Functional analysis of one of the identified proteins involved in photosynthesis, RuBPCase activase, was accomplished by virus-induced gene silencing. Plants with decreased levels of RuBPCase activase protein had reduced photosynthetic rates and RuBPCase activity, and less biomass, responses consistent with those of herbivore-attacked plants. We conclude that the response of the plants proteome to herbivore elicitation is complex, and integrated transcriptome-proteome-metabolome analysis is required to fully understand this ubiquitous ecological interaction.

Nonuniform distribution of glucosinolates in Arabidopsis thaliana leaves has important consequences for plant defense

The spatial distribution of plant defenses within a leaf may be critical in explaining patterns of herbivory. The generalist lepidopteran larvae, Helicoverpa armigera (the cotton bollworm), avoided the midvein and periphery of Arabidopsis thaliana rosette leaves and fed almost exclusively on the inner lamina. This feeding pattern was attributed to glucosinolates because it was not evident in a myrosinase mutant that lacks the ability to activate glucosinolate defenses by hydrolysis. To measure the spatial distribution of glucosinolates in A. thaliana leaves at a fine scale, we constructed ion intensity maps from MALDI-TOF (matrix assisted laser desorption/ionization-time of flight) mass spectra. The major glucosinolates were found to be more abundant in tissues of the midvein and the periphery of the leaf than the inner lamina, patterns that were validated by HPLC analyses of dissected leaves. In addition, there were differences in the proportions of the three major glucosinolates in different leaf regions. Hence, the distribution of glucosinolates within the leaf appears to control the feeding preference of H. armigera larvae. The preferential allocation of glucosinolates to the periphery may play a key role in the defense of leaves by creating a barrier to the feeding of chewing herbivores that frequently approach leaves from the edge.

Mapping the larval midgut lumen proteome of Helicoverpa armigera, a generalist herbivorous insect

The gut lumen is the primary site of digestion and detoxification and thus presents conditions hostile to most proteins. We used 2D-gel electrophoresis and MS/MS de novo peptide sequencing to identify the major proteins stable enough to persist in the midgut lumen of caterpillars of the cotton bollworm Helicoverpa armigera, a generalist herbivorous insect and a major crop pest worldwide. As expected, we found several enzymes responsible for digestion of carbohydrates, proteins, and lipids. In addition, we identified nondigestive proteins such as a multidomain lipocalin, a protein with pathogen recognition domains, an arginine kinase related to a class of major human allergens, and abundant proteins of unknown function. Identification of the set of proteins that are secreted into the lumen will enable us to further characterize the nutritional and defensive functions of this important intraorganismal space.

MALDI Imaging of Neutral Cuticular Lipids in Insects and Plants

The spatial distribution of neutral lipids and hydrocarbons has been imaged using MALDI-TOF mass spectrometry on intact plant and insect surfaces, namely wings and legs of the gray flesh fly (Neobellieria bullata), wings of common fruit fly (Drosophila melanogaster), leaves of thale cress (Arabidopsis thaliana), and leaves of date palm tree (Phoenix sp.). The distribution of wax esters (WEs) and saturated and unsaturated hydrocarbons (HCs) was visualized. The samples were attached on a target and multiply sprayed with lithium or sodium 2,5-dihydroxybenzoate. The deposits were homogenous, consisting of small islands (50–150 µm) of matrix crystals separated by small areas (10 µm) of uncovered cuticle. Samples of N. bullata wings were found to contain HCs and WEs distributed close to their basal parts. The distribution of sodium and potassium ions was visualized on samples prepared by sublimation of 2,5-dihydroxybenzoic acid. Pheromonal dienes were detected on D. melanogaster female wings. A homogenous distribution of saturated WEs was observed on A. thaliana and Phoenix sp. leaf samples. The optimum number of laser shots per pixel was found to be higher than for polar compounds imaging.

The Coprophilous Mushroom Coprinus radians Secretes a Haloperoxidase That Catalyzes Aromatic Peroxygenation

ABSTRACT Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H2O2 concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent Km values for ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H2O2 were 49, 342, 635, 88, and 1,201 μM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.

Pathogenesis-related proteins protect extrafloral nectar from microbial infestation

Plants in more than 300 genera produce extrafloral nectar (EFN) to attract carnivores as a means of indirect defence against herbivores. As EFN is secreted at nectaries that are not physically protected from the environment, and contains carbohydrates and amino acids, EFN must be protected from infestation by micro-organisms. We investigated the proteins and anti-microbial activity in the EFN of two Central American Acacia myrmecophytes (A. cornigera and A. hindsii) and two related non-myrmecophytes (A. farnesiana and Prosopis juliflora). Acacia myrmecophytes secrete EFN constitutively at high rates to nourish the ants inhabiting these plants as symbiotic mutualists, while non-myrmecophytes secrete EFN only in response to herbivore damage to attract non-symbiotic ants. Thus, the quality and anti-microbial protection of the EFN secreted by these two types of plants were likely to differ. Indeed, myrmecophyte EFN contained significantly more proteins than the EFN of non-myrmecophytes, and was protected effectively from microbial infestation. We found activity for three classes of pathogenesis-related (PR) enzymes: chitinase, beta-1,3-glucanase and peroxidase. Chitinases and beta-1,3-glucanases were significantly more active in myrmecophyte EFN, and chitinase at the concentrations found in myrmecophyte EFN significantly inhibited yeast growth. Of the 52 proteins found in A. cornigera EFN, 28 were annotated using nanoLC-MS/MS data, indicating that chitinases and glucanases contribute more than 50% of the total protein content in the EFN of this myrmecophyte. Our study demonstrates that PR enzymes play an important role in protecting EFN from microbial infestation.

Glucanases and Chitinases as Causal Agents in the Protection of Acacia Extrafloral Nectar from Infestation by Phytopathogens

Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii × Nicotiana sanderae) contains “nectarins,” proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function.

Sialome of a Generalist Lepidopteran Herbivore: Identification of Transcripts and Proteins from Helicoverpa armigera Labial Salivary Glands

Although the importance of insect saliva in insect-host plant interactions has been acknowledged, there is very limited information on the nature and complexity of the salivary proteome in lepidopteran herbivores. We inspected the labial salivary transcriptome and proteome of Helicoverpa armigera, an important polyphagous pest species. To identify the majority of the salivary proteins we have randomly sequenced 19,389 expressed sequence tags (ESTs) from a normalized cDNA library of salivary glands. In parallel, a non-cytosolic enriched protein fraction was obtained from labial salivary glands and subjected to two-dimensional gel electrophoresis (2-DE) and de novo peptide sequencing. This procedure allowed comparison of peptides and EST sequences and enabled us to identify 65 protein spots from the secreted labial saliva 2DE proteome. The mass spectrometry analysis revealed ecdysone, glucose oxidase, fructosidase, carboxyl/cholinesterase and an uncharacterized protein previously detected in H. armigera midgut proteome. Consistently, their corresponding transcripts are among the most abundant in our cDNA library. We did find redundancy of sequence identification of saliva-secreted proteins suggesting multiple isoforms. As expected, we found several enzymes responsible for digestion and plant offense. In addition, we identified non-digestive proteins such as an arginine kinase and abundant proteins of unknown function. This identification of secreted salivary gland proteins allows a more comprehensive understanding of insect feeding and poses new challenges for the elucidation of protein function.

High-throughput ESI-MS analysis of binding between the Bombyx mori pheromone-binding protein BmorPBP1, its pheromone components and some analogues

Chip-assisted high-throughput ESI-MS analysis of the pheromone-binding protein of the silkworm moth Bombyx mori, BmorPBP1, incubated with its pheromone components bombykol, bombykal and analogues was developed. The protein bound to bombykol ((10E,12Z)-hexadecadien-1-ol) and all 3 of its geometric isomers to a lesser extent, and showed relaxed specificity toward different chain lengths possessing unsaturation. BmorPBP1 did not bind to bombykal ((10E,12Z)-hexadecadienal), demonstrating molecular recognition of the insect pheromone components.

Isolating intact chloroplasts from small Arabidopsis samples for proteomic studies.

We have established a method for the isolation of chloroplasts from Arabidopsis thaliana that allows proteomic studies in the context of biotic stress with small amounts of starting material. Employing a 50% Percoll layer to separate crude filtrates, the required leaf material was reduced to 2-3g, yielding more than 300 microg of chloroplast proteins. The quality of this fraction was confirmed by immunological, enzymatic, and gel-based assays. This protocol provides intact chloroplasts from Arabidopsis plants with a high degree of integrity and purity as well as sufficient protein recovery, thereby enabling studies of plant-herbivore or plant-pathogen interactions.