Bacterial phytopathogen infection disrupts belowground plant indirect defense mediated by tritrophic cascade

Plants can defend themselves against herbivores through activation of defensive pathways and attraction of third‐trophic‐level predators and parasites. Trophic cascades that mediate interactions in the phytobiome are part of a larger dynamic including the pathogens of the plant itself, which are known to greatly influence plant defenses. As such, we investigated the impact of a phloem‐limited bacterial pathogen, Candidatus Liberibacter asiaticus (CLas), in cultivated citrus rootstock on a well‐studied belowground tritrophic interaction involving the attraction of an entomopathogenic nematode (EPN), Steinernema diaprepesi, to their root‐feeding insect hosts, Diaprepes abbreviatus larvae. Using belowground olfactometers, we show how CLas infection interferes with this belowground interaction by similarly inducing the release of a C12 terpene, pregeijerene, and disconnecting the association of the terpene with insect presence. D. abbreviatus larvae that were not feeding but in the presence of a CLas‐infected plant were more likely to be infected by EPN than those near uninfected plants. Furthermore, nonfeeding larvae associated with CLas‐infected plants were just as likely to be infected by EPN as those near noninfected plants with D. abbreviatus larval damage. Larvae of two weevil species, D. abbreviatus and Pachnaeus litus, were also more attracted to plants with infection than to uninfected plants. D. abbreviatus larvae were most active when exposed to pregeijerene at a concentration of 0.1 μg/μl. We attribute this attraction to CLas‐infected plants to the same signal previously thought to be a herbivore‐induced plant volatile specifically induced by root‐feeding insects, pregeijerene, by assessing volatiles collected from the roots of infected plants and uninfected plants with and without feeding D. abbreviatus. Synthesis. Phytopathogens can influence the structuring of soil communities extending to the third trophic level. Field populations of EPN may be less effective at host‐finding using pregeijerene as a cue in citrus grove agroecosystems with high presence of CLas infection.     

Chaperone proteins as single component reagents to assess antibody nonspecificity

Early stage assays that evaluate monoclonal antibody drug-like properties serve as valuable tools for selection of lead candidates. One liability for clinical development, off-target reactivity, is often assessed by binding to a mixture or panel of noncognate proteins. While robust, these mixes are often ill-defined, and can suffer from issues such as lot-to-lot variability. In this study, we discovered in immunoprecipitation experiments that certain chaperones are present in one of these mixtures;we then explored the use of recombinant chaperone proteins as well-characterized agents to predict antibody nonspecificity. Antibody binding to the heat shock proteins HSP70, HSP90, or trigger factor all served as predictors of cross-interaction propensity, with HSP90 providing the greatest ability to predict antibody clearance rates in mouse. Individual chaperone binding correlates surprisingly closely with binding to complex cell extracts, with the exception of a few “false negatives” (assuming a complex cell extract as the “true” value). As defined reagents, these chaperone reagents present advantages for high throughput assays of nonspecificity.     

Enhanced transport of plant‐produced rabies single‐chain antibody‐RVG peptide fusion protein across an in cellulo blood–brain barrier device

The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in planta for targeted delivery across the blood–brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system. This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralizing single‐chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognizes the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv‐RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv‐RVG fusion. Both ScFv and ScFv‐RVG fusion molecules had potent neutralization activity against RABVin cellulo. The ScFv‐RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant‐produced ScFv‐RVG^P fusion could translocate across the cells. This study indicates that the plant‐produced ScFv‐RVG^P fusion protein was able to cross the in celluloBBB and neutralize RABV.     

Active hexose correlated compound enhances resistance to Klebsiella pneumoniae infection in mice in the hindlimb-unloading model of spaceflight conditions.

Previous studies have demonstrated that resistance to infection is decreased in Swiss Webster female mice maintained in the hindlimb-unloading model (Aviles H, Belay T, Fountain K, Vance M, and Sonnenfeld G. J Appl Physiol 95: 73-80, 2003; Belay T, Aviles H, Vance M, Fountain K, and Sonnenfeld G. J Allergy Clin Immunol 110: 262-268, 2002). This is a model of some of the aspects of spaceflight conditions, including lack of load bearing on hindlimbs and a fluid shift to the head. Active hexose correlated compound (AHCC), extracted from Basidiomycete mushrooms, has been shown to induce enhancement of immune responses, including enhanced natural killer activity. In the present study, AHCC was orally administered to mice to determine whether the treatment could decrease immunosuppression and mortality of mice maintained in the hindlimb-unloaded model and infected with Klebsiella pneumoniae. The results of the present study showed that administration of AHCC by gavage for 1 wk (1 g/kg body wt) before suspension and throughout the 10-day suspension period yielded significant beneficial effects for the hindlimb-unloaded group, including 1). decreased mortality, 2). increased time to death, and 3). increased ability to clear bacteria. The results suggest that AHCC can decrease the deleterious effects of the hindlimb-unloading model on immunity and resistance to infection.     

ERM proteins and NF2 tumor suppressor: the Yin and Yang of cortical actin organization and cell growth signaling.

The ERM (ezrin, radixin and moesin) family of proteins are linkers that tether actin microfilaments to the plasma membrane. Merlin, the NF2 tumor suppressor gene product, is highly homologous to ERM proteins. In ERM proteins and merlin, interdomain binding promotes auto-inhibition and homo-oligomerization or hetero-oligomerization. Recent studies have revealed that ERM proteins transduce growth signals, and have shed new light on how merlin links cell growth to the cytoskeleton.     

Response of a Sphagnum bog plant community to elevated CO2 and N supply

The factors determining herbaceous canopy architecture are poorlyunderstood, especially in natural and semi-natural plant communities. Inthis study, we tested three main hypotheses: (1) the structure of herbaceouscanopies can be explained by the vertical distribution of functional groupsdefined by leaf width and the presence/absence of leaves on upright stem;(2) the degree of canopy stratification is greater in habitats that experiencelower spatial heterogeneity in the supply of light (i.e., grasslands as opposedto forest herb layers); and (3) there is significant variation among specieswithin a growth-form, with respect to their vertical position in thecanopy. We used plant foliage height distribution data from 14 grassland and 13forest herbaceous communities to test these hypotheses. A general linear mixedmodel was applied to specify the proportions of total variance in the foliageheight, accounted for by the fixed effects of plants' basicgrowth-form properties (growth-form) and community type(forest/grassland), and by the random effects of sampling site, samplingpoint, and individual species. We were also interested in the correlation ofthedegree of the stratification with various community characteristics(productivity, other canopy properties, species richness, variation ofspecies' traits) and light availability. There was some evidence ofoverall canopy stratification according toplant growth-form, since plants with leafy stem were locatedsignificantly higher. However, such a pattern of two more or less distinctlayers (grasses + upright forbs and rosette forbs) occurred withconsistency only in grasslands (greater homogeneity in light). Thebetween-species variation within a growth-form was a highlysignificant predictor of canopy vertical structure in the 27 communities. Theproportion of total observed variance, explainable throughspecies-specific effects, was comparable to that caused bybetween-site differences. The effect of community horizontal pattern wasless obvious, but still significant. The site by site analysis revealed thatthe degree to which horizontalpatchiness explained variation in vertical canopy structure was negativelyrelated to the relative importance of species-specific effects, showingthat small between-species differences lead to a more obviouswithin-community horizontal pattern, and vice versa. The upper bound ofthe degree of foliage stratification, according to growth-form, wasrelated to the variability of species light requirements and to relative (tocommunity pool size) richness, indicating that certain aspects of canopyarchitecture might be explained through community species composition anddiversity pattern.     

Spiralin Is Not Essential for Helicity, Motility, or Pathogenicity but Is Required for Efficient Transmission of Spiroplasma citri by Its Leafhopper Vector Circulifer haematoceps

Spiralin is the most abundant protein at the surface of the plant pathogenic mollicute Spiroplasma citri and hence might play a role in the interactions of the spiroplasma with its host plant and/or its insect vector. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. A spiralin-green fluorescent protein (GFP) translational fusion was engineered and introduced into S. citri by using an oriC-based targeting vector. According to the strategy used, integration of the plasmid by a single-crossover recombination at the spiralin gene resulted in the expression of the spiralin-GFP fusion protein. Two distinct mutants were isolated. Western and colony immunoblot analyses showed that one mutant (GII3-9a5) did produce the spiralin-GFP fusion protein, which was found not to fluoresce, whereas the other (GII3-9a2) produced neither the fusion protein nor the wild-type spiralin. Both mutants displayed helical morphology and motility, similarly to the wild-type strain GII-3. Genomic DNA analyses revealed that GII3-9a5 was unstable and that GII3-9a2 was probably derived from GII3-9a5 by a double-crossover recombination between plasmid sequences integrated into the GII3-9a5 chromosome and free plasmid. When injected into the leafhopper vector Circulifer haematoceps, the spiralinless mutant GII3-9a2 multiplied to high titers in the insects (1.1 × 10⁶ to 2.8 × 10⁶ CFU/insect) but was transmitted to the host plant 100 times less efficiently than the wild-type strain. As a result, not all plants were infected, and symptom production in these plants was delayed for 2 to 4 weeks compared to that in the wild-type strain. In the infected plants however, the mutant multiplied to high titers (1.2 × 10⁶ to 1.4 × 10⁷ CFU/g of midribs) and produced the typical symptoms of the disease. These results indicate that spiralin is not essential for pathogenicity but is required for efficient transmission of S. citri by its insect vector.     

Identification by SDS PAGE of green seaweeds ( Ulva and Enteromorpha) used in the food industry

SDS PAGE was tested as an analytical tool for the identification of fourgreen algae (Ulva rigida, U. rotundata, Enteromorphaintestinalis, E. compressa) used as food ingredients. A referencepattern composed of the bands present all year long was performed foreach species. The pattern for Ulva rotundata consists of 7 bandslocated between 69.9 and 15.5 kDa with the presence of triplicate bandsat 29.5, 26.3 and 22.9 kDa. The pattern for Ulva rigida is consistsof three bands with apparent molecular weights of 68.5, 56.4 and 44.7kDa. The Enteromorpha compressa pattern is characterised by sixbands located between 65.8 and 19.8 kDa. A double band withmolecular weights of 23.1 and 23.9 distinguished this pattern from theothers. Six bands situated between 66.4 and 19.4 kDa with a specifictriplicate band of 25.9, 23.9 and 22.5 kDa constituted the specific patternof Enteromorpha intestinalis. SDS PAGE appears to be suitable forthe identification of green seaweed foods.     

Presence of NK2 binding sites in the rat brain

Attempts were made to label tachykinin NK2 binding sites in the adult rat brain using [125I]neurokinin A (NKA) as ligand in the presence of NK1 and NK3 agonist or antagonist to avoid labelling of NK1 and NK3 binding sites, respectively. A high-affinity, specifically NK2-sensitive, [125I]NKA-binding, temperature-dependent, reversible, sensitive to GTPgammaS and correspondence to a single population of binding sites (K(D) and B(max) values: 2.2 nM and 7.3 fmol/mg protein) was demonstrated on hippocampal membranes. Competition studies performed with tachykinins and tachykinin-related compounds indicated that the pharmacological properties of these NK2-sensitive [125I]NKA binding sites were identical to those identified in the rat urinary bladder and duodenum. NKA, neuropeptide K, and neuropeptide gamma, as well as the potent and selective NK2 antagonists SR 144190, SR 48968 and MEN 10627, presented a nanomolar affinity for these sites. The regional distribution of these NK2-sensitive [125I]NKA binding sites differs markedly from those of NK1 and NK3 binding sites, with the largest labeling being found in the hippocampus, the thalamus and the septum. Binding in other brain structures was low or negligible. A preliminary autoradiographic analysis confirmed [125I]NKA selective binding in hippocampal CA1 and CA3 areas, particularly, and in several thalamic nuclei.