[Prokaryotic expression and purification of Chlamydomonas reinhardtii intraflagellar transport protein 46(IFT46) and preparation of polyclonal antibody].

IFT46 is one of the important components of intraflagellar transport complex B in Chlamydomonas reinhardtii, and plays important roles in the assembly, movement and perception of ciliary. To study its functional mechanism, a GST-tagged and an MBP-tagged prokaryotic expression plasmid, pGEX-2T-ift46 and pMAL-C2X-ift46 were constructed, respectively, by inserting ift46 into the pGEX-2T and pMAL-C2X vector, and then transformed into Escherichia coli BL21 (DE3) for protein expression. SDS-PAGE (15%) analysis results showed that the molecular weights of the fusion protein GST-IFT46 and MBP-IFT46 were 70 kDa and 86 kDa, respectively. We used the fusion protein GST-IFT46 purified by affinity adsorption purification (more than 95% purity) for immunity to New Zealand white rabbits. The 5th immune serum was collected and the antibody titer was determined to be 256 000 by ELISA. The antiserum was purified by Protein A affinity adsorption purification and immobilized MBP-IFT46 purification, and the specificity of polyclonal antibodies was evaluated by Western blotting and immunofluorescence. Results showed that the polyclonal antibody prepared could specifically and precisely bind IFT46 in C. reinhardtii, and IFT46 was mainly concentrated at basal body regions and few localized along the entire length of the flagellum as punctuated dots, which will make a foundation to further study the mechanism of IFT46 in cilia related diseases such as obesity, diabetes and polycystic kidney disease.

Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii.

Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI-TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae.

Repression of essential chloroplast genes reveals new signaling pathways and regulatory feedback loops in chlamydomonas.

Although reverse genetics has been used to elucidate the function of numerous chloroplast proteins, the characterization of essential plastid genes and their role in chloroplast biogenesis and cell survival has not yet been achieved. Therefore, we developed a robust repressible chloroplast gene expression system in the unicellular alga Chlamydomonas reinhardtii based mainly on a vitamin-repressible riboswitch, and we used this system to study the role of two essential chloroplast genes: ribosomal protein S12 (rps12), encoding a plastid ribosomal protein, and rpoA, encoding the α-subunit of chloroplast bacterial-like RNA polymerase. Repression of either of these two genes leads to the arrest of cell growth, and it induces a response that involves changes in expression of nuclear genes implicated in chloroplast biogenesis, protein turnover, and stress. This response also leads to the overaccumulation of several plastid transcripts and reveals the existence of multiple negative regulatory feedback loops in the chloroplast gene circuitry.

γδ T cells recognize a microbial encoded B cell antigen to initiate a rapid antigen-specific interleukin-17 response.

γδ T cells contribute uniquely to immune competence. Nevertheless, how they function remains an enigma. It is unclear what most γδ T cells recognize, what is required for them to mount an immune response, and how the γδ T cell response is integrated into host immune defense. Here, we report that a noted B cell antigen, the algae protein phycoerythrin (PE), is a murine and human γδ T cell antigen. Employing this specificity, we demonstrated that antigen recognition activated naive γδ T cells to make interleukin-17 and respond to cytokine signals that perpetuate the response. High frequencies of antigen-specific γδ T cells in naive animals and their ability to mount effector response without extensive clonal expansion allow γδ T cells to initiate a swift, substantial response. These results underscore the adaptability of lymphocyte antigen receptors and suggest an antigen-driven rapid response in protective immunity prior to the maturation of classical adaptive immunity.

Highly dynamic exon shuffling in candidate pathogen receptors ... what if brown algae were capable of adaptive immunity?

Pathogen recognition is the first step of immune reactions. In animals and plants, direct or indirect pathogen recognition is often mediated by a wealth of fast-evolving receptors, many of which contain ligand-binding and signal transduction domains, such as leucine-rich or tetratricopeptide repeat (LRR/TPR) and NB-ARC domains, respectively. In order to identify candidates potentially involved in algal defense, we mined the genome of the brown alga Ectocarpus siliculosus for homologues of these genes and assessed the evolutionary pressures acting upon them. We thus annotated all Ectocarpus LRR-containing genes, in particular an original group of LRR-containing GTPases of the ROCO family, and 24 NB-ARC-TPR proteins. They exhibit high birth and death rates, while a diversifying selection is acting on their LRR (respectively TPR) domain, probably affecting the ligand-binding specificities. Remarkably, each repeat is encoded by an exon, and the intense exon shuffling underpins the variability of LRR and TPR domains. We conclude that the Ectocarpus ROCO and NB-ARC-TPR families are excellent candidates for being involved in recognition/transduction events linked to immunity. We further hypothesize that brown algae may generate their immune repertoire via controlled somatic recombination, so far only known from the vertebrate adaptive immune systems.

Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1.

Fungal and oomycete plant pathogens translocate effector proteins into host cells to establish infection. However, virulence targets and modes of action of their effectors are unknown. Effector AVR3a from potato blight pathogen Phytophthora infestans is translocated into host cells and occurs in two forms: AVR3a(KI), which is detected by potato resistance protein R3a, strongly suppresses infestin 1 (INF1)-triggered cell death (ICD), whereas AVR3a(EM), which evades recognition by R3a, weakly suppresses host ICD. Here we show that AVR3a interacts with and stabilizes host U-box E3 ligase CMPG1, which is required for ICD. In contrast, AVR3a(KI/Y147del), a mutant with a deleted C-terminal tyrosine residue that fails to suppress ICD, cannot interact with or stabilize CMPG1. CMPG1 is stabilized by the inhibitors MG132 and epoxomicin, indicating that it is degraded by the 26S proteasome. CMPG1 is degraded during ICD. However, it is stabilized by mutations in the U-box that prevent its E3 ligase activity. In stabilizing CMPG1, AVR3a thus modifies its normal activity. Remarkably, given the potential for hundreds of effector genes in the P. infestans genome, silencing Avr3a compromises P. infestans pathogenicity, suggesting that AVR3a is essential for virulence. Interestingly, Avr3a silencing can be complemented by in planta expression of Avr3a(KI) or Avr3a(EM) but not the Avr3a(KI/Y147del) mutant. Our data provide genetic evidence that AVR3a is an essential virulence factor that targets and stabilizes the plant E3 ligase CMPG1, potentially to prevent host cell death during the biotrophic phase of infection.

The 74-kilodalton immunodominant antigen of the pathogenic oomycete Pythium insidiosum is a putative exo-1,3-beta-glucanase.

The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the causative agent of pythiosis, a life-threatening infectious disease of humans and animals living in tropical and subtropical areas of the world. Common sites of infection are the arteries, eyes, cutaneous/subcutaneous tissues, and gastrointestinal tract. Diagnosis of pythiosis is time-consuming and difficult. Radical excision of the infected organs is the main treatment for pythiosis because conventional antifungal drugs are ineffective. An immunotherapeutic vaccine prepared from P. insidiosum crude extract showed limited efficacy in the treatment of pythiosis patients. Many pythiosis patients suffer lifelong disabilities or die from an advanced infection. Recently, we identified a 74-kDa major immunodominant antigen of P. insidiosum which could be a target for development of a more effective serodiagnostic test and vaccines. Mass spectrometric analysis identified two peptides of the 74-kDa antigen (s74-1 and s74-2) which perfectly matched a putative exo-1,3-ss-glucanase (EXO1) of Phytophthora infestans. Using degenerate primers derived from these peptides, a 1.1-kb product was produced by PCR, and its sequence was found to be homologous to that of the P. infestans exo-1,3-ss-glucanase gene, EXO1. Enzyme-linked immunosorbent assays targeting the s74-1 and s74-2 synthetic peptides demonstrated that the 74-kDa antigen was highly immunoreactive with pythiosis sera but not with control sera. Phylogenetic analysis using part of the 74-kDa protein-coding sequence divided 22 Thai isolates of P. insidiosum into two clades. Further characterization of the putative P. insidiosum glucanase could lead to new diagnostic tests and to antimicrobial agents and vaccines for the prevention and management of the serious and life-threatening disease of pythiosis.

Antibodies in the sera of host species with pythiosis recognize a variety of unique immunogens in geographically divergent Pythium insidiosum strains.

Studies by Western blot analyses have shown that antibodies in the sera of host species infected by Pythium insidiosum recognized several prominent proteins expressed by this fungus-like pathogen. Although these studies have utilized sera from infected patients and relevant local strains of P. insidiosum, the results are difficult to compare because of the lack of method standardization. In an effort to resolve this issue, we have utilized standardized methodologies to evaluate six P. insidiosum strains from Asia and the Americas and 15 serum samples from cattle, cats, dogs, horses, and humans with pythiosis from the same geographical regions. Our data show that the antibodies present in these sera recognize a wide variety of unique P. insidiosum immunogenic proteins. Although some of the prominent proteins in this study have been previously reported, several others have yet to be described. For instance, a approximately 28-kDa-molecular-mass antigen was detected by the antibodies in all serum samples evaluated. However, this antigen was strongly expressed by only one of the strains evaluated. A diffuse approximately 51-kDa protein was not detected by the antibodies in the human sera; but it was recognized by the antibodies in the sera of cattle, cats, dogs, and horses. This antigen was expressed by only two of the strains investigated. Several other similar examples were also observed. The variation of the P. insidiosum protein profile identified by the antibodies in the sera evaluated indicates that some geographically diverged P. insidosum strains expressed some unique immunogens in vitro and that during natural infection (in vivo) P. insidiosum might express a broader number of antigens variably detected by individuals within the same species but especially across species.

Induction of a small heat shock protein and its functional roles in Nicotiana plants in the defense response against Ralstonia solanacearum.

In tobacco (Nicotiana tabacum), Ralstonia solanacearum OE1-1 (RsOE1-1) is pathogenic, whereas R. solanacearum 8107 (Rs8107) is nonpathogenic and induces the hypersensitive response (HR). To elucidate the molecular mechanisms of plant-R. solanacearum interactions, we used differential display to isolate a cDNA fragment, A6, regulated in tobacco by inoculation with RsOE1-1. The deduced amino acid sequence predicted from full-length A6-cDNA showed similarity to small heat shock proteins from Arabidopsis (Arabidopsis thaliana; hypothetical protein), Medicago truncatula, and Cucumis melo; we therefore designated A6 to correspond to Ntshsp17 (for tobacco small heat shock protein 17). Recombinant Ntshsp17 overproduced in Escherichia coli exhibited molecular chaperone function. Expression of Ntshsp17 was increased in tobacco leaves inoculated with both RsOE1-1 and Rs8107. Expression was induced by heat treatment and by treatment with aminocyclopropane carboxylic acid, hydrogen peroxide, methyl jasmonate, and salicylic acid. Ntshsp17 expression was induced by inoculation with a HR and pathogenicity gene mutant of Rs8107 that does not induce the HR, but not by Agrobacterium-mediated transient expression of INF1, an HR elicitor. In Nbshsp17-silenced plants (an Ntshsp17 ortholog in Nicotiana benthamiana), expression of ETHYLENE-RESPONSE ELEMENT-BINDING PROTEIN, PATHOGENESIS-RELATED1a (PR1a), and PR4 genes was compromised, but expression of ELONGATION FACTOR1alpha was scarcely affected. Appearance of the HR was not affected in the silenced plants. In the silenced plants, growth of Rs8107 was accelerated. Bacterial growth and wilt symptoms elicited by RsOE1-1 were also accelerated in the silenced plants. These results indicate that this small heat shock protein might have a role in HR-independent defenses in Nicotiana plants.

The two divergent PEP-carboxylase catalytic subunits in the green microalga Chlamydomonas reinhardtii respond reversibly to inorganic-N supply and co-exist in the high-molecular-mass, hetero-oligomeric Class-2 PEPC complex.

Our recent molecular studies revealed two divergent PEP-carboxylase (PEPC [Ppc]) encoding genes in the green microalga Chlamydomonas reinhardtii, CrPpc1 and CrPpc2, which are coordinately responsive to changes in inorganic-N and -C supply at the transcript level [Mamedov, T.G., Moellering, E.R. and Chollet, R. (2005) Identification and expression analysis of two inorganic C- and N-responsive genes encoding novel and distinct molecular forms of eukaryotic phosphoenolpyruvate carboxylase in the green microalga C. reinhardtii, Plant J. 42, 832-843]. Here, we report the distribution of these two encoded catalytic subunits in the minor Class-1 and predominant Class-2 PEPC enzyme-forms, the latter of which is a novel high-molecular-mass, hetero-oligomeric complex containing both CrPpc1 (p109) and CrPpc2 (p131) polypeptides. The Class-1 enzyme, however, is a typical PEPC homotetramer comprised solely of p109. We also document that the amount of both CrPpc1/2 catalytic subunits is up-/down-regulated by varying levels of NH(4)(+) supplied to the culture medium.

The chaotrope-soluble glycoprotein GP2 is a precursor of the insoluble glycoprotein framework of the Chlamydomonas cell wall.

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyproline-containing glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyproline-containing polypeptides of the insoluble wall fraction. Matrix-assisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64- and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.

Isolation of a novel carotenoid-rich protein in Cyanophora paradoxa that is immunologically related to the light-harvesting complexes of photosynthetic eukaryotes.

Novel eukaryotic chlorophyll-carotenoid proteins have evolved at least twice following the origin of the plastid and include the widely distributed integral membrane light-harvesting complexes (LHCs) and the dinoflagellate-specific soluble peridinin-chlorophyll proteins. In the glaucophytes, homologs of these proteins are reportedly absent. We have identified a novel carotenoid-rich protein (CRP) in the glaucophyte Cyanophora paradoxa that is 28 kDa and immunologically related to the family of LHCs. CRP is associated with the thylakoid membrane, though it can be removed by stringent washes, suggesting that there are probably significant structural differences between CRP and the LHCs. CRP co-localizes with a zeaxanthin-rich thylakoid membrane fraction that also contains beta-carotene, chlorophyll and an unidentified carotenoid. Despite this, we found no evidence for carotenoid-chlorophyll energy transfer in the isolated complex, suggesting that light harvesting may not be a primary function. The presence of CRP in C. paradoxa is evidence for the evolution of a novel pigment-binding protein in the glaucophytes.

Production of monoclonal antibodies against peripheral-vesicle proteins in zoospores of Phytophthora nicotianae.

A coimmunisation protocol using microsomal fractions from Phytophthora nicotianae cells has enhanced the production of monoclonal antibodies directed towards proteins produced during asexual sporulation. Over 40% of the antibodies targeted three categories of zoospore peripheral vesicles. Five antibodies label the contents of dorsal vesicles, with three of these reacting with two P. nicotianae polypeptides with a relative molecular mass of approximately 100 kDa. Two antibodies label the contents of large peripheral vesicles and react with two very high-molecular-weight polypeptides in extracts of P. nicotianae cells. These antibodies cross-react with the contents of large peripheral vesicles in P. cinnamomi zoospores. Ten antibodies label the contents of P. nicotianae zoospore ventral vesicles and react with a single polypeptide with a relative molecular mass of 230 kDa. A number of these antibodies against the contents of ventral vesicles in P. nicotianae zoospores cross-react with ventral-vesicle proteins in P. cinnamomi cells in immunofluorescence and immunoblot assays. The study illustrates the value of the coimmunisation protocol and has produced antibodies that could be instrumental in the cloning of genes encoding peripheral-vesicle proteins.

Plant recognition of microbial patterns.

Animals express an innate immune system against pathogens through receptor-mediated recognition of conserved microbial structures called pathogen-associated molecular patterns (PAMPs). In plants, resistance to invading microorganisms is often governed by specific recognition between plant and pathogen proteins. Perception of more broadly conserved 'general' pathogen elicitors constitutes another layer of plant resistance and prompts questions of where, mechanistically and evolutionarily, this mode of non-self discrimination fits within known systems of microbial surveillance in animals and plants.

Localization of p210-related proteins in green flagellates and analysis of flagellar assembly in the green alga Dunaliella bioculatawith monoclonal anti-p210.

Recently, p210 was identified as a component of the flagellar basal apparatus in the green flagellate Spermatozopsis similis. In a search for potential homologues to p210, isolated cytoskeletons of several green flagellates were probed with a monoclonal antibody, BAS4.13, against p210. In Western blots, cross-reacting bands in the molecular-mass range of 210 kDa were detected only in the quadriflagellate Spermatozopsis exsultans. As described earlier for S. similis, the flagellar transition region was decorated in Chlamydomonas reinhardtii and several other green flagellates, whereas in the marine alga Dunaliella bioculata the antigen was present in the proximal part of the axoneme. Double immunofluorescence of D. bioculata with an antitubulin antibody further revealed dotlike signals at sites where the probasal bodies are located. Since most of the antigen in D. bioculata was located in the axoneme, deflagellation offered a possibility to study the kinetics of its incorporation during flagellar regeneration. The antigen was only detected after a flagellum reached a length of 3-4 microm and its integration into the growing flagellar proceeded from proximal to distal. A similar delay in the incorporation of the antigen was also observed during flagellar assembly on new basal bodies during cell division. Thus, the antigen of BAS4.13 was incorporated late and from proximal to distal into the growing flagellum. We conclude that the pace and site by which individual proteins are integrated into the flagellum differ greatly.

Simple assay for antitumour immunoactive glycoprotein derived from Chlorella vulgaris strain CK22 using ELISA.

A quantitative ELISA system was developed using a monoclonal antibody (MAb) specific for an antitumour immunoactive glycoprotein (CVS) derived from C. vulgaris strain CK22. The full measuring range of the assay extends from 0.63 to 10.0 ng/mL of CVS. Although no cross-reaction was observed to proteins tested or other biological response modifiers (BRMs) derived from different sources, cross-reactions were found with culture supernatants from two other strains of C. vulgaris having a strong antitumour immunoactivity. Treatment of CVS with protease, acid or alkali weakened or completely eliminated the reactivity against the MAb and also its antitumour immunoactivities. This ELISA system is suitable for the biologically active form of CVS derived from C. vulgaris strain CK22 and related immunoactive strains.

Pleuralins are involved in theca differentiation in the diatom Cylindrotheca fusiformis.

Diatom cells are encased within a silica-based cell wall (frustule) that serves as armour-like protection for the enclosed protoplast. Maintaining the integrity of the frustule requires a precise coupling between the biogenesis of new frustule components and the cell cycle. Thus far, the molecular mechanisms by which this coupling is achieved are unknown. This study demonstrates that pleuralins (formerly HEPs), a previously characterized family of diatom cell wall proteins, are involved in cell cycle-dependent frustule development. The frustule is made up of two, overlapping half-shells termed the epitheca and hypotheca. Both thecae are morphologically identical, yet immunolocalisation with anti-pleuralin antibodies demonstrates that their protein composition is clearly different. During interphase, pleuralins are associated only with the epitheca, where they are confined to the inner surface of the terminal elements (pleural bands) in the region of overlap with the hypotheca. At cell division, pleuralins also become associated with the newly formed pleural bands of the hypotheca. Remarkably, this process is concomitant with the functional conversion of the parental hypotheca into the epitheca of one of the progeny cells. These results indicate that developmentally controlled association of pleuralins with the frustule is involved in hypotheca-epitheca differentiation, which is a crucial process to ensure proper frustule development.

Flagellar membrane proteins of Tetraselmis striata butcher (Chlorophyta).

Highly purified flagella of the green alga Tetraselmis striata (Chlorophyta) were extracted by Triton X-114 phase partitioning. SDS-PAGE analysis revealed that most proteins were present in the aqueous phase, only two prominent flagellar membrane proteins (fmp) of apparent molecular weight 145 and 57 kDa (fmp145 and fmp57) were enriched in the detergent phase. Fmp145 was purified by gel permeation chromatography. Glycosidase treatment in combination with lectin blot analysis showed that fmp145 is a glycoprotein containing 3-5 N-glycans of the high mannose and/or hybrid type. A polyclonal antibody (anti-fmp136) was raised against the deglycosylated form of fmp145 and used to localize fmp145 by immunofluorescence and immunoelectron microscopy. Immunogold labeling showed fmp145 to be present between the scale layers and the flagellar membrane. During flagellar regeneration fmp145 is incorporated evenly and rapidly into the newly developing flagella. Anti-fmp136 specifically cross-reacted with flagella of only a subgroup of Tetraselmis strains characterized by a specific flagellar hair type (type II according to Marin et al. 1993) and thus could be a useful immunomarker for the identification of Tetraselmis strains by fluorescence microscopy.