Identification of a Gene from the Arbuscular Mycorrhizal Fungus Glomus intraradices Encoding for a 14-3-3 Protein that is Up-Regulated by Drought Stress during the AM Symbiosis

In the present study, a 14-3-3 protein-encoding gene from Glomus intraradices has been identified after differential hybridization of a cDNA library constructed from the fungus growing in vitro and subjected to drought stress by addition of 25% PEG 6000. Subsequently, we have studied its expression pattern under drought stress in vitro and also when forming natural symbioses with different host plants. The results obtained suggest that Gi14-3-3 gene may be involved in the protection that the arbuscular mycorrhizal (AM) symbiosis confers to the host plant against drought stress. Our findings provide new evidences that the contribution of AM fungi to the enhanced drought tolerance of the host plant can be mediated by a group of proteins (the 14-3-3) that regulate both signaling pathways and also effector proteins involved in the final plant responses.     

Xanthomonas oryzae pv. oryzae XopQ protein suppresses rice immune responses through interaction with two 14-3-3 proteins but its phospho-null mutant induces rice immune responses and interacts with another 14-3-3 protein

Many bacterial phytopathogens employ effectors secreted through the type-III secretion system to suppress plant innate immune responses. The Xanthomonas type-III secreted non-TAL effector protein Xanthomonas outer protein Q (XopQ) exhibits homology to nucleoside hydrolases. Previous work indicated that mutations which affect the biochemical activity of XopQ fail to affect its ability to suppress rice innate immune responses, suggesting that the effector might be acting through some other pathway or mechanism. In this study, we show that XopQ interacts in yeast and in planta with two rice 14-3-3 proteins, Gf14f and Gf14g. A serine to alanine mutation (S65A) of a 14-3-3 interaction motif in XopQ abolishes the ability of XopQ to interact with the two 14-3-3 proteins and to suppress innate immunity. Surprisingly, the S65A mutant gains the ability to interact with a third 14-3-3 protein that is a negative regulator of innate immunity. The XopQS65A mutant is an inducer of rice immune responses and this property is dominant over the wild-type function of XopQ. Taken together, these results suggest that XopQ targets the rice 14-3-3 mediated immune response pathway and that its differential phosphorylation might enable interaction with alternative 14-3-3 proteins.     

14-3-3 proteins and the response to abiotic and biotic stress

14-3-3 proteins function as regulators of a wide range of target proteins in all eukaryotes by effecting direct protein-protein interactions. Primarily, interactions between 14-3-3 proteins and their targets are mediated by phosphorylation at specific sites on the target protein. Hence, interactions with 14-3-3s are subject to environmental control through signalling pathways which impact on 14-3-3 binding sites. Because 14-3-3 proteins regulate the activities of many proteins involved in signal transduction, there are multiple levels at which 14-3-3 proteins may play roles in stress responses in higher plants. In this article, we review evidence which implicates 14-3-3 proteins in responses to environmental, metabolic and nutritional stresses, as well as in defence responses to wounding and pathogen attack. This evidence includes stress-inducible changes in 14-3-3 gene expression, interactions between 14-3-3 proteins and signalling proteins and interactions between 14-3-3 proteins and proteins with defensive functions.     

14-3-3 proteins are involved in the regulation of mammalian cell proliferation

Summary. The 14-3-3 proteins are a family of abundant, widely expressed acidic polypeptides. The seven isoforms interact with over 70 different proteins. 14-3-3 isoforms have been demonstrated to be involved in the control of positive as well as negative regulators of mammalian cell proliferation. Here we used the approach of inactivating 14-3-3 protein functions via overexpression of dominant negative mutants to analyse the role of 14-3-3 proteins in mammalian cell proliferation. We found 14-3-3 dominant negative mutants to downregulate the proliferation rates of HeLa cells. Overexpression of these dominant negative mutants triggers upregulation of the protein levels of the cyclin-dependent kinase inhibitor p27, a major negative cell cycle regulator. In addition, they downregulate the protein levels of the important cell cycle promoter cyclin D1. These data provide new insights into mammalian cell proliferation control and allow a better understanding of the functions of 14-3-3 proteins.     

Rice 14-3-3 protein (GF14e) negatively affects cell death and disease resistance

Plant 14-3-3 proteins regulate important cellular processes, including plant immune responses, through protein-protein interactions with a wide range of target proteins. In rice (Oryza sativa), the GF14e gene, which encodes a 14-3-3 protein, is induced during effector-triggered immunity (ETI) associated with pathogens such as Xanthomonas oryzae pv. oryzae (Xoo). To determine whether the GF14e gene plays a direct role in resistance to disease in rice, we suppressed its expression by RNAi silencing. GF14e suppression was correlated with the appearance of a lesion-mimic (LM) phenotype in the transgenic plants at 3 weeks after sowing. This indicates inappropriate regulation of cell death, a phenotype that is frequently associated with enhanced resistance to pathogens. GF14e-silenced rice plants showed high levels of resistance to a virulent strain of Xoo compared with plants that were not silenced. Enhanced resistance was correlated with GF14e silencing prior to and after development of the LM phenotype, higher basal expression of a defense response peroxidase gene (POX22.3), and accumulation of reactive oxygen species (ROS). In addition, GF14e-silenced plants also exhibit enhanced resistance to the necrotrophic fungal pathogen Rhizoctonia solani. Together, our findings suggest that GF14e negatively affects the induction of plant defense response genes, cell death and broad-spectrum resistance in rice.     

Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants

Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed.     

Identification of FAKTS as a novel 14-3-3-associated nuclear protein

Through bioinformatics and experimental approaches, we have assigned the first biochemical property to a predicted protein product in the human genome as a new 14-3-3 binding protein. 14-3-3 client proteins represent a diverse group of regulatory molecules that often function as signaling integrators in response to various environmental cues and include proteins such as Bad and Foxo. Using 14-3-3 as a probe in a yeast two-hybrid screen, we identified a novel 14-3-3 binding protein with unknown function, initially designated as clone 546. Confocal microscopy revealed that clone 546 localized to the nucleus of mammalian cells. Additional studies show that the gene encoding clone 546 is expressed in many human tissues, including the thymus, as well as a number of cancer cell lines. The interaction of clone 546 with 14-3-3 was confirmed in mammalian cells. Interestingly, this interaction was markedly enhanced by the expression of activated Akt/PKB, suggesting a phosphorylation dependent event. Mutational analysis was carried out to identify Ser479 as the predominant residue that mediates the clone 546/14-3-3 association. Phosphorylation of Ser479 by AKT/PKB further supports a critical role for Akt/PKB in regulation of the clone 546/14-3-3 interaction. On the basis of these findings, we named this undefined protein FAKTS: Fourteen-three-three associated AKT Substrate.     

A single parasite gene determines strain-specific protective immunity against malaria: The role of the merozoite surface protein I

Despite many decades of research, no registered vaccine against the pathogenic blood stages of the malaria parasite exists, translating into the loss of many hundreds of thousands of young lives each year in tropical Africa. Although many parasite proteins have been shown to induce immune responses in the host, proof for their induction of protective immunity is still lacking. We previously reported a novel genetic approach called linkage group selection (LGS) for rapid identification of target antigens of strain-specific protective immunity (SSPI) against malaria. In preliminary LGS experiments, we crossed two genetically distinct strains of Plasmodium chabaudi chabaudi and subjected their progeny to selection in strain-specifically immunised mice, measuring the effects of SSPI selection with low coverage/resolution genetic markers. In the present study, through application of high coverage/resolution, single nucleotide polymorphism (SNP) markers spanning all 14 parasite chromosomes, we analysed 35 SSPI selection events on different populations of progeny parasites. Here we report a comprehensive high resolution genome-wide analysis of the effects of strain-specific immune selection on blood stage parasites. Our analyses consistently identify a single genomic region spanning ∼79 kb on chromosome 8 as the region controlling SSPI. Within this region, one gene (that of merozoite surface protein 1, MSP-1) accounted for >60% of genetic polymorphism and was most frequently under greatest reduction under SSPI. These results, combined with those of an independent LGS analysis of a different genetic cross with different parental strains, demonstrate that more than any other locus, the gene for MSP-1 determines the effect of strain-specific protective immunity against malaria in these host-parasite combinations. Our results provide unique insight into the precise timing of the parasite killing immune response against progeny parasites carrying specific alleles of MSP-1; these findings pave the way for investigating which part(s) of this highly polymorphic molecule mediate the protective immune response.     

14-3-3蛋白家族及其临床应用研究进展

14-3-3蛋白家族是一组高度保守的可溶性酸性蛋白质,分子量在28～33kD之间,广泛分布于各种真核生物之中。该蛋白能够特异地结合含有磷酸化丝氨酸或苏氨酸的肽段,参与多种信号转导途径。14-3-3蛋白调节着许多重要细胞生命活动,如:新陈代谢、细胞周期、细胞生长发育、细胞的存活和凋亡以及基因转录,该蛋白家族异常与疾病的发生密切相关,尤其是14-3-3蛋白在脑脊液中的分布与一些神经系统疾病密切相关。14-3-3蛋白已成为一些疾病的临床诊断指标,其作为疾病治疗的靶点也在研究之中。主要阐述了14-3-3蛋白的结构、功能、及其在疾病治疗中的应用。     

14-3-3 proteins in Echinococcus: their role and potential as protective antigens

14-3-3 Proteins are a family of highly conserved proteins among all eukaryotic organisms studied so far. As basically intracellular proteins, they play a key role in basic cellular events related to cellular proliferation, including signal transduction, cell-cycle control, cell differentiation and cell survival. The 14-3-3 proteins have been described and characterized in several parasites, and mostly studied in Echinocuccus granulosus and Echinocuccus multilocularis. Here, we review the discoveries regarding this protein family in the genus Echinococcus, describing new data about specific aspects related with their implication in the parasite biology and immunology in the frame of the host-parasite relationship.     

Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.     

Phosphorylation-dependent binding of 14-3-3 to the polarity protein Par3 regulates cell polarity in mammalian epithelia

The mammalian homologs of the C. elegans partitioning-defective (Par) proteins have been demonstrated to be necessary for establishment of cell polarity. In mammalian epithelia, the Par3/Par6/aPKC polarity complex is localized to the tight junction and regulates its formation and positioning with respect to basolateral and apical membrane domains. Here we demonstrate a previously undescribed phosphorylation-dependent interaction between a mammalian homolog of the C. elegans polarity protein Par5, 14-3-3, and the tight junction-associated protein Par3. We identify phosphorylated serine 144 as a site of 14-3-3 binding. Expression of a Par3 mutant that contains serine 144 mutated to alanine (S144A) results in defects in epithelial cell polarity. In addition, overexpression of 14-3-3zeta results in a severe disruption of polarity, whereas overexpression of a 14-3-3 mutant that is defective in binding to phosphoproteins has no effect on cell polarity. Together, these data suggest a novel, phosphorylation-dependent mechanism that regulates the function of the Par3/Par6/aPKC polarity complex through 14-3-3 binding.     

Peptide-based polyclonal antibody against mosquito 14-3-3ζ recognizes 14-3-3 homolog from dipteran and lepidopteran insects

The 14-3-3 proteins are known to play an important regulatory role in apoptosis, and various cell signaling cascades. However, no investigation on mosquito 14-3-3 has been reported. To investigate the role of 14-3-3 proteins in mosquito midgut cells undergoing apoptosis, we decided to take advantage of Anopheles gambiae genome data, and were able to find Ag14-3-3ζ cDNA and protein sequences from Ensembl ( http://www.ensembl.org ). Further in silico analysis using BLAST search revealed that Ag14-3-3ζ protein is a polypeptide of 248 amino acids, and shares high identity with 14-3-3ζ homologues from Aedes aegypti (100%), Drosophila melanogaster (96%) and Bombyx mori (93%). Due to the perfect match and high homology, we hypothesized that Ag14-3-3ζ peptide antibody may recognize 14-3-3ζ homologs from other anopheline mosquitoes and insects. We thus generated 14-3-3ζ polyclonal antibody against a unique region located in the C-terminal end of Ag14-3-3ζ after in silico epitope analysis. As expected, zoo-western blot analysis of 14-3-3 proteins revealed that a polyclonal antibody against Ag14-3-3ζ peptide recognizes 14-3-3 homologs from dipteran and lepidopteran insects. To our knowledge, this is the first report on polyclonal antibody production against mosquito 14-3-3ζ. The mosquito-based 14-3-3ζ antibody will be very useful for studying the functional characterization of 14-3-3ζ in the context of host–pathogen interactions in midgut and other immune cells.