Serine phosphorylation of cortactin is required for maximal host cell invasion by <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background

Campylobacter jejuni causes acute disease characterized by severe diarrhea containing blood and leukocytes, fever, and abdominal cramping. Disease caused by C. jejuni is dependent on numerous bacterial and host factors. C. jejuni invasion of the intestinal epithelial cells is seen in both clinical samples and animal models indicating that host cell invasion is, in part, necessary for disease. C. jejuni utilizes a flagellar Type III Secretion System (T3SS) to deliver the Campylobacter invasion antigens (Cia) to host cells. The Cia proteins modulate host cell signaling leading to actin cytoskeleton rearrangement necessary for C. jejuni host cell invasion, and are required for the development of disease.

Results

This study was based on the hypothesis that the C. jejuni CiaD effector protein mediates Erk 1/2 dependent cytoskeleton rearrangement. We showed that CiaD was required for the maximal phosphorylation of Erk 1/2 by performing an immunoblot with a p-Erk 1/2 specific antibody and that Erk 1/2 participates in C. jejuni invasion of host cells by performing the gentamicin protection assay in the presence and absence of the PD98059 (a potent inhibitor of Erk 1/2 activation). CiaD was also found to be required for the maximal phosphorylation of cortactin S405 and S418, as judged by immunoblot analysis. The response of human INT 407 epithelial cells to infection with C. jejuni was evaluated by confocal microscopy and scanning electron microscopy to determine the extent of membrane ruffling. This analysis revealed that CiaD, Erk 1/2, and cortactin participate in C. jejuni-induced membrane ruffling. Finally, cortactin and N-WASP were found to be involved in C. jejuni invasion of host cells using siRNA to N-WASP, and siRNA to cortactin, coupled with the gentamicin protection assay.

Conclusion

We conclude that CiaD is involved in the activation of Erk 1/2 and that activated Erk 1/2 facilitates C. jejuni invasion by phosphorylation of cortactin on serine 405 and 418. This is the first time that cortactin and N-WASP have been shown to be involved in C. jejuni invasion of host cells. These data also provide a mechanistic basis for the requirement of Erk 1/2 in C. jejuni-mediated cytoskeletal rearrangement.

     

Identification and characterization of insect-specific proteins by genome data analysis

Background

Previous studies have sought to identify a link between the distribution of variable genes amongst isolates of Campylobacter jejuni and particular host preferences. The genomic sequence data available currently was obtained using only isolates from human or chicken hosts. In order to identify variable genes present in isolates from alternative host species, five subtractions between C. jejuni isolates from different sources (rabbit, cattle, wild bird) were carried out, designed to assess genomic variability within and between common multilocus sequence type (MLST) clonal complexes (ST-21, ST-42, ST-45 and ST-61).

Results

The vast majority (97%) of the 195 subtracted sequences identified had a best BLASTX match with a Campylobacter protein. However, there was considerable variation within and between the four clonal complexes included in the subtractions. The distributions of eight variable sequences, including four with putative roles in the use of alternative terminal electron acceptors, amongst a panel of C. jejuni isolates representing diverse sources and STs, were determined.

Conclusion

There was a clear correlation between clonal complex and the distribution of the metabolic genes. In contrast, there was no evidence to support the hypothesis that the distribution of such genes may be related to host preference. The other variable genes studied were also generally distributed according to MLST type. Thus, we found little evidence for widespread horizontal gene transfer between clonal complexes involving these genes.     

Production and characterization of anti-<Emphasis Type="Italic">Campylobacter jejuni</Emphasis> IgY derived from egg yolks

Background

Campylobacter jejuni is a major cause of foodborne disease having chickens as an important reservoir. Its control at the farm would lower the contamination of the final products and therefore also lower the risk of transmission to humans. At the farm, C. jejuni is rarely found in chickens before they reach 2 weeks of age. Past studies have shown that maternal antibodies could hamper C. jejuni gut colonization. The objective of this study was to compare protocols to use in order to produce anti-C. jejuni antibodies derived from egg yolks in the perspective to be used as feed additives for the control of chicken C. jejuni colonization. Laying hens were naturally contaminated with four well-characterized strains or injected with either outer membrane proteins or formalin-killed whole bacteria derived from these same strains. Eggs were collected and IgYs present in the yolks were extracted. The amount and the specificity of the recovered antibodies were characterized.

Results

It was observed that injection yielded eggs with superior concentrations of both total and anti-C. jejuni antibodies. Equivalent performances for antibodies recovered from all protocols were observed for the ability of the antibodies to agglutinate the live C. jejuni homologous strains, to hinder their motility or to lyse the bacteria. Western blot analyses showed that proteins from all strains could be recognized by all IgY extracts. All these characteristics were strain specific. The characterization assays were also made for heterologous strains and weaker results were observed when compared to the homologous strains.

Conclusions

Based on these results, only an IgY quantitative based selection can be made in regards to which protocol would give the best anti-C. jejuni IgY enriched egg-yolks as all tested protocols were equivalent in terms of the recovered antibody ability to recognized the tested C. jejuni strains.

     

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients

Background

Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence.

Methods

In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni.

Results

Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI–TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro.

Conclusion

We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients.

General significance

This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.     

Formation of VEGF isoform-specific spatial distributions governing angiogenesis: computational analysis

Background

Cellular interaction networks can be used to analyze the effects on cell signaling and other functional consequences of perturbations to cellular physiology. Thus, several methods have been used to reconstitute interaction networks from multiple published datasets. However, the structure and performance of these networks depends on both the quality and the unbiased nature of the original data. Due to the inherent bias against membrane proteins in protein-protein interaction (PPI) data, interaction networks can be compromised particularly if they are to be used in conjunction with drug screening efforts, since most drug-targets are membrane proteins.

Results

To overcome the experimental bias against PPIs involving membrane-associated proteins we used a probabilistic approach based on a hypergeometric distribution followed by logistic regression to simultaneously optimize the weights of different sources of interaction data. The resulting less biased genome-scale network constructed for the budding yeast Saccharomyces cerevisiae revealed that the starvation pathway is a distinct subnetwork of autophagy and retrieved a more integrated network of unfolded protein response genes. We also observed that the centrality-lethality rule depends on the content of membrane proteins in networks.

Conclusions

We show here that the bias against membrane proteins can and should be corrected in order to have a better representation of the interactions and topological properties of protein interaction networks.     

Evolutionary conservation of domain-domain interactions

Background

Recently, there has been much interest in relating domain-domain interactions (DDIs) to protein-protein interactions (PPIs) and vice versa, in an attempt to understand the molecular basis of PPIs.

Results

Here we map structurally derived DDIs onto the cellular PPI networks of different organisms and demonstrate that there is a catalog of domain pairs that is used to mediate various interactions in the cell. We show that these DDIs occur frequently in protein complexes and that homotypic interactions (of a domain with itself) are abundant. A comparison of the repertoires of DDIs in the networks of Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens shows that many DDIs are evolutionarily conserved.

Conclusion

Our results indicate that different organisms use the same 'building blocks' for PPIs, suggesting that the functionality of many domain pairs in mediating protein interactions is maintained in evolution.     

Data mining of cancer vaccine trials: a bird's-eye view

Background

Details of the mechanisms and selection pressures that shape the emergence and development of complex biological systems, such as the human immune system, are poorly understood. A recent definition of a reference set of proteins essential for the human immunome, combined with information about protein interaction networks for these proteins, facilitates evolutionary study of this biological machinery.

Results

Here, we present a detailed study of the development of the immunome protein interaction network during eight evolutionary steps from Bilateria ancestors to human. New nodes show preferential attachment to high degree proteins. The efficiency of the immunome protein interaction network increases during the evolutionary steps, whereas the vulnerability of the network decreases.

Conclusion

Our results shed light on selective forces acting on the emergence of biological networks. It is likely that the high efficiency and low vulnerability are intrinsic properties of many biological networks, which arise from the effects of evolutionary processes yet to be uncovered.     

Sialylation of Campylobacter jejuni lipo-oligosaccharides: impact on phagocytosis and cytokine production in mice

Background

Guillain-Barré syndrome (GBS) is a post-infectious polyradiculoneuropathy, frequently associated with antecedent Campylobacter jejuni (C. jejuni) infection. The presence of sialic acid on C. jejuni lipo-oligosaccharide (LOS) is considered a risk factor for development of GBS as it crucially determines the structural homology between LOS and gangliosides, explaining the induction of cross-reactive neurotoxic antibodies. Sialylated C. jejuni are recognised by TLR4 and sialoadhesin; however, the functional implications of these interactions in vivo are unknown.

Methodology/Principal Findings

In this study we investigated the effects of bacterial sialylation on phagocytosis and cytokine secretion by mouse myeloid cells in vitro and in vivo. Using fluorescently labelled GM1a/GD1a ganglioside-mimicking C. jejuni strains and corresponding (Cst-II-mutant) control strains lacking sialic acid, we show that sialylated C. jejuni was more efficiently phagocytosed in vitro by BM-MΦ, but not by BM-DC. In addition, LOS sialylation increased the production of IL-10, IL-6 and IFN-β by both BM-MΦ and BM-DC. Subsequent in vivo experiments revealed that sialylation augmented the deposition of fluorescent bacteria in splenic DC, but not macrophages. In addition, sialylation significantly amplified the production of type I interferons, which was independent of pDC.

Conclusions/Significance

These results identify novel immune stimulatory effects of C. jejuni sialylation, which may be important in inducing cross-reactive humoral responses that cause GBS.     

A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory

Background

The study of biological interaction networks is a central theme of systems biology. Here, we investigate the relationships between two distinct types of interaction networks: the metabolic pathway map and the protein-protein interaction network (PIN). It has long been established that successive enzymatic steps are often catalyzed by physically interacting proteins forming permanent or transient multi-enzymes complexes. Inspecting high-throughput PIN data, it was shown recently that, indeed, enzymes involved in successive reactions are generally more likely to interact than other protein pairs. In our study, we expanded this line of research to include comparisons of the underlying respective network topologies as well as to investigate whether the spatial organization of enzyme interactions correlates with metabolic efficiency.

Results

Analyzing yeast data, we detected long-range correlations between shortest paths between proteins in both network types suggesting a mutual correspondence of both network architectures. We discovered that the organizing principles of physical interactions between metabolic enzymes differ from the general PIN of all proteins. While physical interactions between proteins are generally dissortative, enzyme interactions were observed to be assortative. Thus, enzymes frequently interact with other enzymes of similar rather than different degree. Enzymes carrying high flux loads are more likely to physically interact than enzymes with lower metabolic throughput. In particular, enzymes associated with catabolic pathways as well as enzymes involved in the biosynthesis of complex molecules were found to exhibit high degrees of physical clustering. Single proteins were identified that connect major components of the cellular metabolism and may thus be essential for the structural integrity of several biosynthetic systems.

Conclusion

Our results reveal topological equivalences between the protein interaction network and the metabolic pathway network. Evolved protein interactions may contribute significantly towards increasing the efficiency of metabolic processes by permitting higher metabolic fluxes. Thus, our results shed further light on the unifying principles shaping the evolution of both the functional (metabolic) as well as the physical interaction network.     

Multiple origins of endosymbiosis within the Enterobacteriaceae (γ-Proteobacteria): convergence of complex phylogenetic approaches

Background

Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels.

Results

Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners.

Conclusion

Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.     

A rapid method to map mutations in Drosophila

Background

Genetic screens in Drosophila have provided a wealth of information about a variety of cellular and developmental processes. It is now possible to screen for mutant phenotypes in virtually any cell at any stage of development by performing clonal screens using the flp/FRT system. The rate-limiting step in the analysis of these mutants is often the identification of the mutated gene, however, because traditional mapping strategies rely mainly on genetic and cytological markers that are not easily linked to the molecular map.

Results

Here we describe the development of a single-nucleotide polymorphism (SNP) map for chromosome arm 3R. The map contains 73 polymorphisms between the standard FRT chromosome, and a mapping chromosome that carries several visible markers (rucuca), at an average density of one SNP per 370 kilobases (kb). Using this collection, we show that mutants can be mapped to a 400 kb interval in a single meiotic mapping cross, with only a few hundred SNP detection reactions. Discovery of further SNPs in the region of interest allows the mutation to be mapped with the same recombinants to a region of about 50 kb.

Conclusion

The combined use of standard visible markers and molecular polymorphisms in a single mapping strategy greatly reduces both the time and cost of mapping mutations, because it requires at least four times fewer SNP detection reactions than a standard approach. The use of this map, or others developed along the same lines, will greatly facilitate the identification of the molecular lesions in mutants from clonal screens.     

Daily temperature profiles in and around Western Kenyan larval habitats of Anopheles gambiae as related to egg mortality

Background

Erythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis. Erythrocyte binding antigen-181 (EBA-181) is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R) was recently demonstrated using phage display technology. In the current study, recombinant proteins were utilized to define and characterize the precise molecular interaction between the two proteins.

Methods

4.1R structural domains (30, 16, 10 and 22 kDa domain) and the 4.1R binding region in EBA-181 were synthesized in specific Escherichia coli strains as recombinant proteins and purified using magnetic bead technology. Recombinant proteins were subsequently used in blot-overlay and histidine pull-down assays to determine the binding domain in 4.1R.

Results

Blot overlay and histidine pull-down experiments revealed specific interaction between the 10 kDa domain of 4.1R and EBA-181. Binding was concentration dependent as well as saturable and was abolished by heat denaturation of 4.1R.

Conclusion

The interaction of EBA-181 with the highly conserved 10 kDa domain of 4.1R provides new insight into the molecular mechanisms utilized by P. falciparum during erythrocyte entry. The results highlight the potential multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite's life cycle.     

Beyond DNA binding - a review of the potential mechanisms mediating quinacrine's therapeutic activities in parasitic infections,inflammation, and cancers

Background

Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin^-/-, integrin-beta1^-/-, focal adhesion kinase (FAK)^-/- and Src/Yes/Fyn^-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake.

Results

Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2^-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry.

Conclusion

Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.     

Different mutation patterns of atovaquone resistance to Plasmodium falciparum in vitro and in vivo : rapid detection of codon 268 polymorphisms in the cytochrome b as potential in vivo resistance marker

Background

Anopheles gambiae is the main vector of Plasmodium falciparum in Africa. The mosquito midgut constitutes a barrier that the parasite must cross if it is to develop and be transmitted. Despite the central role of the mosquito midgut in the host/parasite interaction, little is known about its protein composition. Characterisation of An. gambiae midgut proteins may identify the proteins that render An. gambiae receptive to the malaria parasite.

Methods

We carried out two-dimensional gel electrophoresis of An. gambiae midgut proteins and compared protein profiles for midguts from males, sugar-fed females and females fed on human blood.

Results

Very few differences were detected between male and female mosquitoes for the approximately 375 silver-stained proteins. Male midguts contained ten proteins not detected in sugar-fed or blood-fed females, which are therefore probably involved in male-specific functions; conversely, female midguts contained twenty-three proteins absent from male midguts. Eight of these proteins were specific to sugar-fed females, and another ten, to blood-fed females.

Conclusion

Mass spectrometry analysis of the proteins found only in blood-fed female midguts, together with data from the recent sequencing of the An. gambiae genome, should make it possible to determine the role of these proteins in blood digestion or parasite receptivity.     

DNA damage and cell cycle events implicate cerebellar dentate nucleus neurons as targets of Alzheimer's disease

Background

The low-density lipoprotein receptor related protein 1 (LRP1) has been implicated in Alzheimer's disease (AD) but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1.

Results

We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP), which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b). Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression.

Conclusions

These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered early in disease. Genetic and functional studies have implicated both LRP1 and NYGGF4 in obesity and cardiovascular disease and the physical association of these proteins may reflect a common mechanism. This is particularly interesting in light of the dual role of ApoE in both cardiovascular risk and AD. The results support further studies on the functional relationship between NYGGF4 and LRP1.     

Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface

Background

Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis. Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein.

Results

In this study we demonstrate that the N-terminus of LcrG is required for interaction with LcrV. The interaction likely occurs within a predicted amphipathic coiled-coil domain within LcrG. Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction. Additionally, we demonstrate that the LcrG homolog, PcrG, is incapable of blocking type III secretion in Y. pestis. A genetic selection was utilized to obtain a PcrG variant capable of blocking secretion. This PcrG variant allowed us to locate a region of LcrG involved in secretion blocking.

Conclusion

Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif. We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.     

Microbial-mediated feedbacks of leaf litter on invasive plant growth and interspecific competition

Background and Aims

In line with the Stress Gradient Hypothesis, studies of facilitation have tended to focus on plant–plant interactions (biotic nurses), while the relative role of abiotic nurses has been little studied. We assessed the role of biotic and abiotic nurses, and their interaction, on soil enhancement and the consequential performance of a native annual grass, Dactyloctenium radulans.

Methods

We used a growth chamber study with two levels of water application to compare the performance of D. radulans growing in soil from foraging pits of the Short-beaked echidna (Tachyglossus aculeatus; abiotic nurse) and non-pit soil from either under tree canopies (biotic nurse) or surrounding open areas.

Results

All measures of plant performance were more pronounced under the high than the low water treatment. The greatest differences between pit and surface Microsites occurred under the low water application, reinforcing our view that facilitatory effects are greater in resource-limited environments. Despite tree canopy soil having greater N, there was no significant effect on plant performance, nor any significant interaction with Microsite.

Conclusions

Our study provides strong evidence that foraging pits enhance soil properties and this soil, in turn, facilitates plant growth; and supports previous work documenting the positive effect of nurse-protégé interactions under greater levels of abiotic stress.     

Proteomic changes of Citrus roots in response to long-term manganese toxicity

Key message

Fifty-three and thirty-nine differentially expressed protein spots were isolated from Mn-toxic Citrus sinensis and Citrus grandis roots, respectively. Mn-toxicity-induced changes in protein profiles greatly differed between the two species.

Abstract

Limited information is available on the manganese (Mn)-toxicity-responsive proteins in plant roots. ‘Sour pummelo’ (Citrus grandis) and ‘Xuegan’ (Citrus sinensis) seedlings were irrigated for 17 weeks with 2 (control) or 600 μM (Mn-toxic) MnSO₄. C. sinensis displayed more tolerance to Mn-toxicity than C. grandis, which may be related to more Mn accumulation in roots and less Mn distribution in shoots. Using two-dimensional electrophoresis (2-DE), we isolated 11 up-regulated and 42 down-regulated protein spots from Mn-toxic C. sinensis roots, and 25 up-regulated and 14 down-regulated protein spots from Mn-toxic C. grandis roots. This indicated more metabolic flexibility in C. sinensis roots, thus contributing to the Mn-tolerance of C. sinensis. According to the biological functional properties, these differentially expressed proteins in the two species were classified into the following categories: protein metabolism, nucleic acid metabolism, carbohydrate and energy metabolism, stress responses, cell wall and cytoskeleton, cell transport, signal transduction and fatty acid metabolism. Under Mn-toxicity, proteins involved in nucleic acid metabolism, glycolysis and cell transport were up-regulated in nontolerant C. grandis roots, and down-regulated in tolerant C. sinensis roots. The notable down-regulation of proteins in Mn-toxic C. sinensis roots with less accumulation of carbohydrates may provide an advantage to the net carbon balance by lowering related metabolic processes, and enhancing the Mn-tolerance of C. sinensis. To conclude, there are many important differences in Mn-toxicity-induced changes in protein profiles and metabolic responses between the two species.