Expression Of Cell Wall Proteins Research Articles

The morphological and physiological basis of delayed pollination overcoming pre-fertilization cross-incompatibility in Nicotiana.

Delayed pollination is widely used to overcome pre-fertilization incompatibility, but its regulatory mechanisms are unclear. When Nicotiana tabacum was cross-pollinated with pollen of N. alata, the incompatibility occurring in the basal 1/4 region of the style (pollinated at anthesis: 0-day-old pistil) was overcome by delayed pollination (of 6-day-old pistil), and the morphological changes and corresponding physiological basis are explored here. The structure and ultrastructure of the pistil were observed under fluorescence microscopy and transmission electron microscopy. Differentially expressed proteins were screened with a monoclonal antibody chip for Nicotiana, and protein expression and distribution were analysed by immunofluorescence. Cellulase and pectinase activities were tested using enzyme-linked immunosorbent assay kits. The style of Nicotiana is solid in the basal region and pollen tubes grow in the extracellular spaces (ECM) of the transmitting tissue (TTS) cells. Seven of the 22 identified proteins were cell wall-associated proteins and were expressed at higher levels during pistil senescence. Cellulase and pectinase activities increased. The TTS cells in the basal 1/4 region of the 0-day-old style were polygonal and tightly arranged, with narrow ECM, but these were oval or partially dissolved in the 6-day-old pistil, leading to wider ECM and richer secretions. The increased expression of cell wall proteins and enhanced enzyme activity during pistil senescence might partially be responsible for the cells becoming oval and the ECM enlarged, providing the morphological basis for delayed pollination overcoming the pre-fertilization incompatibility between N. tabacum and N. alata.

Evaluation of cell wall damage by dimethyl sulfoxide in Candida species

Studies dealing with the response of microorganisms to oxidative stress require the dissolution of oxidant agents in an appropriate solvent. A commonly used medium is dimethyl sulfoxide, which has been considered as an innocuous polar solvent. However, we have observed significant differences between control, untreated cells and those receiving increasing amounts of the oxidant and hence increasing amounts of DMSO, to the maximum allowed of 1%. Here we show that, while this solvent does not influence yeast cell viability, it does affect expression of cell wall proteins as well as catalase activity. Therefore, its use in future studies of oxidative stress as an innocuous solvent should be reconsidered.

Ascorbate deficiency influences the leaf cell wall glycoproteome in Arabidopsis thaliana

The cell wall forms the first line of interaction between the plant and the external environment. Based on the observation that ascorbate-deficient vtc mutants of Arabidopsis thaliana have increased cell wall peroxidase activity, the cell wall glycoproteome of vtc2-2 was investigated. Glycoproteins were purified from fully expanded leaves by Concanavalin A affinity chromatography and analysed by liquid chromatography quadrupole time-of-flight mass spectrometry. This procedure identified 63 proteins with predicted glycosylation sites and cell wall localization. Of these, 11 proteins were differentially expressed between vtc2-2 and wild type. In particular, PRX33/34 were identified as contributing to increased peroxidase activity in response to ascorbate deficiency. This is the same peroxidase previously shown to contribute to hydrogen peroxide generation and pathogen resistance. Three fasciclin-like arabinogalactan proteins (FLA1, 2 and 8) had lower abundance in vtc2-2. Inspection of published microarray data shows that these also have lower gene expression in vtc1 and vtc2-1 and are decreased in expression by pathogen challenge and oxidative stresses. Ascorbate deficiency therefore impacts expression of cell wall proteins involved in pathogen responses and these presumably contribute to the increased resistance of vtc mutants to biotrophic pathogens.

Repression of N-glycosylation triggers the unfolded protein response (UPR) and overexpression of cell wall protein and chitin in Aspergillus fumigatus

Aspergillus fumigatus is the most common airborne fungal pathogen, causing fatal invasive aspergillosis in immunocompromised patients. The crude mortality is 60-90 % and remains around 29-42 % even with treatment. The main reason for patient death is the low efficiency of the drug therapies. As protein N-glycosylation is involved in cell wall biogenesis in A. fumigatus, a deeper understanding of its role in cell wall biogenesis will help to develop new drug targets. The Afstt3 gene encodes the essential catalytic subunit of oligosaccharyltransferase, an enzyme complex responsible for the transfer of the N-glycan to nascent polypeptides. To evaluate the role of N-glycosylation in cell wall biosynthesis, we constructed the conditional mutant strain CPR-stt3 by replacing the endogenous promoter of Afstt3 with the nitrogen-dependent niiA promoter. Repression of the Afstt3 gene in the CPR-stt3 strain led to a severe retardation of growth and a slight defect in cell wall integrity (CWI). One of the most interesting findings was that upregulation of the cell wall-related genes was not accompanied by an activation of the MpkA kinase, which has been shown to be a central element in the CWI signalling pathway in both Saccharomyces cerevisiae and A. fumigatus. Considering that the unfolded protein response (UPR) was found to be activated, which might upregulate the expression of cell wall protein and chitin, our data suggest that the UPR, instead of the MpkA-dependent CWI signalling pathway, is the major compensatory mechanism induced by repression but not abolition of N-glycosylation in A. fumigatus. Our finding is a key to understanding the complex compensatory mechanisms of cell wall biosynthesis and may provide a new strategy for drug development.

Differential expression of genes encoding cell wall proteins in vascular tissues from vertical and bent loblolly pine trees.

Differential expression of cell wall proteins during plant development and in response to biotic or abiotic stress suggests that these proteins may contribute in different ways to plant cell wall architecture. Because the wood of loblolly pine (Pinus taeda L.) is highly specialized in the formation of secondary cell walls, it is an ideal tissue for studying these proteins. The cDNAs coding for six novel cell wall associated proteins, as well as a homologue for a phytocyanin, were identified and characterized from differentiating xylem of loblolly pine. Three of these cDNAs encoded new putative loblolly pine arabinogalactan proteins, based on their structural similarity to classical arabinogalactan proteins (AGPs). In addition, one clone was related to the proline-rich protein group and the other two to the glycine-rich protein group and the mussel adhesive protein. Relative expression of these genes was examined in different tissues and organs from normal trees (needles, phloem and vertical wood), the underside of bent trees (compression wood) and the lateral sides of the bent stems (side wood). All clones, except one, were highly expressed in vascular tissues with noticeable differences among the three types of wood. Their relationships and diversity provide the first insights into concerted expression and related function for this important group of proteins in cell wall formation of wood.

Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of beta1,3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity.

Deletion of GAS1/GGP1/CWH52 results in a lower beta-glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879-1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165-170, 1995). We show here that gas1delta cells release beta1,3-glucan into the medium. Western analysis of the medium proteins with beta1,3-glucan- and beta1,6-glucan-specific antibodies showed further that at least some of the released beta1,3-glucan was linked to protein as part of a beta1,3-glucan-beta1,6-glucan-protein complex. These data indicate that Gas1p might play a role in the retention of beta1,3-glucan and/or beta-glucosylated proteins. Interestingly, the defective incorporation of beta1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in beta1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.

Dynamic expression of cell wall proteins ofCandida albicansrevealed by probes from cDNA clones

Five cDNA clones were selected from the positive clones detected by screening a germ tube expression library constructed in lambda gt11 with rabbit antisera raised against cell wall extracts of Candida albicans. The selected clones were amplified and used to obtain affinity purified antibodies by eluting from the expressed proteins that had been previously transferred onto nitrocellulose discs. The antibodies obtained were used as probes in immunoblots of the cell wall extracts separated by denaturing polyacrylamide electrophoresis. A single protein band was detected for each clone. Detection of products of the cloned sequences varied according to the extraction procedure and/or cell morphology. These products included bands exhibiting apparent molecular weights of 40, 58, 68 and 70 kDa present in beta-mercaptoethanol (beta ME) extracts from both yeast and germ tubes, and a 30 kDa beta ME extracted protein specific for germ tubes. The expression of these products at the cell surface was confirmed by indirect immunofluorescence. Expression of the mRNAs of the different cDNA clones varied according to growth- and morphology-related factors and showed no direct correlation between expression and presence in the cell wall. These observations suggest that complex mechanisms are involved in the regulation and expression of cell surface components of C. albicans.

Insertional inactivation of a chromosomal locus that modulates expression of potential virulence determinants in Staphylococcus aureus

A single insertion of transposon Tn551 into a unique chromosomal locus of Staphylococcus aureus ISP479C has resulted in a pleiotropic effect on the expression of both extracellular and cell wall proteins. In particular, the expression of cell wall protein A and clumping activity with fibrinogen were rendered undetectable in the mutant 1E3 compared with the parent. The secretion of alpha-hemolysin in mutant 1E3 was modestly increased. Southern blot and phenotypic analyses indicated that this locus is distinct from agr, xpr, and sar, three previously described global regulatory loci. Transduction experiments demonstrated that the genotype associated with mutant 1E3 could be transferred back into the parental strain ISP479C. The transductant 1E3-2 displayed a phenotypic profile similar to that of the original mutant. Northern (RNA) blot studies showed that this locus may be involved in modulating target genes at the mRNA level. In the rabbit endocarditis model, there was a significant decrease in both the infectivity rate and intravegetation bacterial density with mutant 1E3 compared with the parent at an inoculum of 10(3) CFU. Since protein A and the fibrinogen-binding protein(s) are major surface proteins that may mediate bacterial adhesion to host tissues, this locus may be an important genetic element involved in the expression of virulence determinants in S. aureus.

Tissue specific expression of cell wall proteins of seedlings of Prosopis chilensis during development and wound stress

Four cell wall proteins of seedling cotyledons of Prosopis chilensis were characterized by SDS‐polyacrylamide gel electrophoresis. The molecular masses of these proteins were 180, 126, 107 and 63 kDa. All of them immuno‐crossreacted with polyclonal antibodies raised against extensin from soybean seed coats. Immuno‐dot blot analysis demonstrated that the minimum expression of cotyledonary cell wall proteins was 48 h after seed imbibition, while 24 h after wound stress the expression of these wall proteins increased four‐fold. Tissue immuno‐prints and immuno‐histochemistry showed that the proteins are expressed in the cell wall of all tissues. However, the epidermis and vascular bundles of cotyledons, hypocotyls and roots, and the living cells surrounding the wounded areas highly expressed the wall proteins. When the primary roots of the seedlings were injured by performing cuts with razor blades, the seedlings achieved a growth three times faster than control seedlings and secondary roots developed in sites close to the injuries. Immuno‐histochemistry of secondary roots revealed that the root tips and the area of the cortical tissue of the primary roots being pressed by the the emerging root tip, highly expressed the cell wall proteins.

Tissue specific expression of cell wall proteins of seedlings of Prosopis chilensis during development and wound stress

Tissue specific expression of cell wall proteins of seedlings of Prosopis chilensis during development and wound stress

Post-transcriptional regulation of a salt-inducible alfalfa gene encoding a putative chimeric proline-rich cell wall protein.

A cDNA previously shown to identify a salt-inducible root-specific transcript in Medicago sativa was used to screen an alfalfa library for the corresponding genomic sequence. One positive clone was recovered. The nucleotide sequence of a subclone contained a 329 bp 5' region upstream of the first ATG codon, a 1143 bp coding segment, and a 447 bp 3'-untranslated region interrupted by a single 475 bp intron. Translation of the coding segment, which was designated MsPRP2, suggested it encodes a chimeric 40,569 Da cell wall protein with an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxyl-terminal sequence homologous to nonspecific lipid transfer proteins. The 3'-untranslated region of MsPRP2 contained a sequence similar to one found to destabilize mRNAs transcribed from the elicitor-regulated proline-rich protein gene PvPRP1. Transcription run-on experiments using nuclei from salt-sensitive and salt-tolerant alfalfa callus suggested that the accumulation of MsPRP2 transcripts in salt-tolerant alfalfa cells grown in the presence of salt is due primarily to increased mRNA stability. The MsPRP2 gene thus may be a useful model for studying post-transcriptional salt-regulated expression of cell wall proteins.

Role of the sar locus of Staphylococcus aureus in induction of endocarditis in rabbits

A regulatory locus on the Staphylococcus aureus chromosome, designated sar, is involved in the expression of cell wall proteins, some of which are potentially important in the pathogenesis of endocarditis. For instance, mutant 11D2 (sar::Tn917LTV1) was found to bind substantially less to matrix proteins (i.e., fibrinogen and fibronectin) than parent strain DB. Remarkably, these two strains did not differ in other phenotypes considered important in the initiation of endocarditis (e.g., binding to platelets and resistance to platelet-derived microbicidal proteins). The isogenic pair were compared for pathogenicity in a rabbit endocarditis model. There were significant differences in infectivity rates between the two strains (71 and 88% for DB versus 17 and 42% for mutant 11D2 at inocula of 10(3) and 10(4) CFU, respectively). In early adherence studies, parent DB adhered substantially better than the mutant to valvular vegetations at an inoculum of 10(6) CFU (P = 0.05). Southern blot analysis of colonies indicated that the location of the Tn917LTV1 insert in mutant 11D2 remained stable after animal passage. In vitro adherence assays revealed that mutant 11D2 was less adherent to cultured human endothelium than parent DB. These studies suggest that the sar locus is involved in the initial adherence of S. aureus to the fibrin-platelet-endothelium matrix on damaged valvular endothelium.

Expression of Cell Wall Proteins in Seeds and During Early Seedling Growth ofAraucaria araucanais a Response to Wound Stress and is Developmentally Regulated

Araucaria araucana seeds and seedlings respond to wounding after 48 h with a 3- to 4-fold increase of hydroxyproline-rich glycoproteins (HRGP) in the cell walls of the embryo and with a 15-fold increase in the cell walls of the megagametophyte. The megagametophyte walls accumulate six times more hydroxyproline per fig of cell wall protein than the embryo in this wound response. Tissue immunoprints of different parts of seeds and seedlings obtained with polyclonal antibodies raised against HRGP from carrot roots or soybean seed coats indicate that the response is due to an increase in a protein similar to the ones seen in carrot roots or soybean seed coats. Western blots of embryo and megagametophyte cell wall proteins subjected to SDS-PAGE show three bands that cross-react with these antibodies. In a native cationic gel system followed by Western blot analysis, only two bands react with these antibodies. Expression of such proteins in Araucaria araucana seeds seems to be developmental^ regulated and tissue specific, since they are present mainly in the megagametophyte and the root cap of the embryo.

Tissue-Specific Expression of Cell Wall Proteins in Developing Soybean Tissues

Tissue-Specific Expression of Cell Wall Proteins in Developing Soybean Tissues

Tissue-Specific Expression of Cell Wall Proteins in Developing Soybean Tissues.

Cell wall hydroxyproline-rich glycoproteins (HRGPs) and glycine-rich proteins (GRPs) were examined at the protein and at the mRNA levels in developing soybean tissues by tissue print immunoblots and RNA blots. In young soybean stems, HRGPs are expressed most heavily in cambium cells, in a few layers of cortex cells surrounding primary phloem, and in some parenchyma cells around the primary xylem, whereas GRPs are highly expressed in the primary xylem and also in the primary phloem. In older soybean stems, HRGP genes are expressed exclusively in cambium cells and GRP genes are most heavily expressed in newly differentiated secondary xylem cells. Similar expression patterns of HRGPs and of GRPs were found in soybean petioles, seedcoats, and young hypocotyls, and also in bean petioles and stems. HRGPs and GRPs become insolubilized in soybean stem cell walls. Three major HRGP mRNAs and two major GRP mRNAs accumulate in soybean stems. Soluble HRGPs are abundant in young hypocotyl apical regions and young root apical regions, whereas in hypocotyl and root mature regions, soluble HRGPs are found only in a few layers of cortex cells surrounding the vascular bundles. GRPs are specifically localized in primary xylem cell walls of young root. These results show that the gene expression of HRGPs and GRPs is developmentally regulated in a tissue-specific manner. In soybean tissues, HRGPs are most heavily expressed in meristematic cells and in some of those cells that may be under stress, whereas GRPs are expressed in all cells that are or are going to be lignified.

Variation in the expression of cell wall proteins of Staphylococcus aureus grown on solid and liquid media.

To evaluate the variation in the expression of cell wall antigens between Staphylococcus aureus grown in liquid medium and solid support, bacteria were harvested from liquid chemically defined medium and chemically defined medium in a 1% agar base. Cell wall proteins were then extracted by lysostaphin in a protoplast-stabilizing medium (30% raffinose). After separation of the cell wall antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots, they were probed with chicken antiserum to an S. aureus strain grown on a solid support. For each of the 15 clinical strains analyzed, high-molecular-size bands (molecular size range, 120 to 220 kilodaltons) were either enhanced or distinctly present when compared with those from the cell wall extract of the same strain grown in liquid medium. Results of enzymatic treatment of whole staphylococci grown on solid medium suggested the proteinaceous nature and the surface location of these antigens. Limited passage studies demonstrated the ability of the staphylococci to alter these surface proteins when passaged alternately on liquid and solid media. These observations suggested the importance of the microenvironment to the expression of cell wall proteins in S. aureus. Correlations with observations in vivo may help identify the determinants of microbial pathogenicity in S. aureus.