A hitchhiker's guide to a crowded syconium: how do fig nematodes find the right ride?

P>1Organisms with low mobility, living within ephemeral environments,need to find vehicles that can disperse them reliably to new environments. The requirement for specificity in this passenger-vehicle relationship is enhanced within a tritrophic interaction when the environment of passenger and vehicle is provided by a third organism. Such relationships pose many interesting questions about specificity within a tritrophic framework. 2. Central to understanding how these tritrophic systems have evolved, is knowing how they function now. Determining the proximal cues and sensory modalities used by passengers to find vehicles and to discriminate between reliable and non-reliable vehicles is, therefore, essential to this investigation. 3. The ancient, co-evolved and highly species-specific nursery pollination mutualism between figs and fig wasps is host to species-specific plant-parasitic nematodes which use fig wasps to travel between figs. Since individual globular fig inflorescences, i.e. syconia, serve as incubators for hundreds of developing pollinating and parasitic wasps, a dispersal-stage nematode within such a chemically,complex and physically crowded environment is faced with the dilemma of choosing the right vehicle for dispersal into a new fig. Such a system therefore affords excellent opportunities to investigate mechanisms that contribute to the evolution of specificity between the passenger and the vehicle. 4. In this study of fig-wasp-nematode tritrophic interactions in Ficus racemosa within which seven wasp species can breed, we demonstrate using two-choice as well as cafeteria assays that plant-parasitic nematodes (Schistonchus racemosa) do not hitch rides randomly on available eclosing wasps within the fig syconium, but are specifically attracted, at close range, i.e. 3 mm distance, to only that vehicle which can quickly, within a few hours, reliably transfer it to another fig. This vehicle is the female pollinating wasp. Male wasps and female parasitic wasps are inappropriate vehicles since the former are wingless and die within the fig, while the latter never enter another fig. Nematodes distinguished between female pollinating wasps and other female parasitic wasps using volatiles and cuticular hydrocarbons. Nematodes could not distinguish between cuticular hydrocarbons of male and female pollinators but used other cues, such as volatiles, at close range, to find female pollinating wasps with which they have probably had a long history of chemical adaptation. 5. This study opens up new questions and hypotheses about the evolution and maintenance of specificity in fig-wasp-nematode tritrophic interactions

Effect of signal peptide on stability and folding of escherichia coli thioredoxin

The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner

Representative DSC scans of WT Trx, pelB Trx and malE Trx.

<p>Scans were carried out in CGH-10 buffer (pH 7.4). The scan rate was 60°C /h and protein concentration was 0.2 mg/ml. Baseline subtracted excess heat capacity data as a function of temperature are shown. The data indicate that protein stability increases in the order malE Trx</p

CD spectra of WT Trx (−), pelB Trx (–) and malE Trx (···).(A) Far UV CD spectra of WT Trx (-), pelB Trx (–) and malE Trx (···) were obtained with 10 µM protein solution in CGH-10 buffer, pH 7.4 at 25°C with a 0.1 cm path-length cuvette.

<p>(B) Near UV CD spectra were obtained using protein concentrations of 600 µM, 400 µM and 250 µM for WT Trx, pelB Trx, and malE Trx respectively. Measurements were done in CGH-10 buffer, pH 7.4 at 25°C with a 0.2 cm path-length cuvette.</p

Insulin reduction assay for redox activity.

<p>Insulin aggregation following reduction was monitored by the increase in light scattering at 650 nm. Assay conditions were 0.1 M phosphate buffer, 2 mM EDTA, 0.13 mM porcine insulin, 0.33 mM DTT, and 5 µM protein. Protein identities are adjacent to each trace. Incubation mixture without protein served as negative control and WT Trx served as a positive control.</p

Aggregation propensity profiles of various signal peptides.

<p>Panels A–C show aggregation propensity profiles for pelB (empty circles,○) and malE (empty triangles,▵) sequences calculated using (A) Zyggregator, (B) PASTA, (C) AGGRESCAN. (D) Average hydrophobicity calculated using PREDBUR. Amino acid regions with Z_agg>1 are considered to be aggregation prone, whereas regions with Z_agg<0 were assumed to have low aggregation propensities. These upper and lower cut-offs are indicated by dashed line (–) and by dash-dot lines (–) respectively in panels A,E,F,G. The regions with aggregation propensity values above −0.02 are considered as hot-spots for aggregation by AGGRESCAN algorithm. The cut-off value is indicated by a dashed line (–) in C. Panel E-G show aggregation propensity profiles calculated using Zyggregator and average hydrophobicity calculated using PREDBUR for three previously studied soluble Trx fusion systems with phoA, treA, and pcoE signal sequences. Here, Z_agg is shown in filled circles (•), and average hydrophobicity is indicated with filled triangles (▴). The amino acid sequences for all the signal peptides are given in H. The locations of the AE mutation in pelB and malE are underlined.</p

Effect of crowding agent (30% Ficoll) on Trx refolding.

<p>100 µM protein was denatured in 4 M GdmCl (CGH-10 buffer, pH 7.4), diluted and refolded into Ficoll containing buffer. Protein aggregation was monitored using the apparent absorbance at 320 nm. Bar graphs from left to right show data for proteins refolded at final protein concentrations of 2, 5, 7.5 and 10 µM each for WT Trx, pelB Trx and malE Trx respectively.</p