Lotus Cell Walls and the Genes Involved in its Synthesis and Modification

The lotus genome (Nelumbo nucifera (Gaertn.)) lacks the paleo-triplication found in other eudicots and has evolved remarkably slowly with fewer nucleotide mutations. It is thought to have greater retention of duplicated genes than other angiosperms. We evaluated the potential genes involved in cell wall synthesis and its modification, and ethylene synthesis and response. In many cell wall transferases and hydrolases families, lotus had fewer members in most families when compared to Arabidopsis. Lotus had similar or fewer members in each family as found in poplar, grape and papaya. The exceptions were in the sialyl and beta-glucuronsyl transferases where similar number were found as in the core eudicots. Lotus had similar numbers of polygalacturonase and pectin methyl esterases as found in Arabidopsis but fewer in all other hydrolases families. For starch degradation, lotus had only two alpha amylases predicted genes versus eight to ten in other eudicots, with similar numbers of beta amylase genes predicted. Lotus also had less than half the number of genes predicted for the enzymes involved in lignin and tannin synthesis compared to Arabidopsis. The stress plant growth regulator ethylene’s synthesis, reception and response predicted genes were fewer in lotus than other eudicots. Only two ethylene receptor genes were predicted in lotus with five reported for Arabidopsis and six for tomato. Our analysis does not supports the conclusion that this species has greater retention of duplicated genes though our data does support the conclusion that lotus split occurred at the base of the eudicots.     

Turgor Regulation via Cell Wall Adjustment in White Spruce

Turgor regulation at reduced water contents was closely associated with changes in the elastic quality of the cell walls of individual needles and shoots of naturally drought-resistant seedlings of white spruce (Picea glauca [Moench] Voss.) and of seedlings of intermediate resistance that had been pretreated with paclobutrazol, a stress-protecting, synthetic plant-growth regulator. Paclobutrazol-treated seedlings showed marked increases in drought resistance, and pressure-volume analysis combined with Chardakov measurements confirmed observations that water stress was ameliorated during prolonged drought. Turgor was maintained in the paclobutrazol-treated and in the naturally resistant drought-stressed seedlings despite water contents near or below the turgor-loss volumes of well-watered controls. The maintenance of turgor in these seedlings was in large part a function of the dynamic process of cell wall adjustment, as reflected by marked reductions in both the saturated and turgor-loss volumes and by large increases in the elastic coefficients of the tissues. Shear and Young's moduli, calculated from pressure-volume curves and the radii and wall thicknesses of mesophyll cells, also confirmed observed changes in the elastic qualities of the cell walls. Elastic coefficients of well-watered, paclobutrazol-treated seedlings were consistently larger than those in well-watered controls and several times larger than the values in untreated plants, which succumbed rapidly to drought. In contrast, untreated seedlings that withstood prolonged drought without wilting displayed elastic coefficients similar to those in seedlings that had been treated with paclobutrazol but that had not been exposed to drought.     

Identification of New Genes Involved in the Regulation of Yeast Alcohol Dehydrogenase II

Recessive mutations in two negative control elements, CRE1 and CRE2, have been obtained that allow the glucose-repressible alcohol dehydrogenase (ADHII) of yeast to escape repression by glucose. Both the cre1 and cre2 alleles affected ADHII synthesis irrespective of the allele of the positive effector, ADR1. However, for complete derepression of ADHII synthesis, a wild-type ADR1 gene was required. Neither the cre1 nor cre2 alleles affected the expression of several other glucose-repressible enzymes. A third locus, CCR4, was identified by recessive mutations that suppressed the cre1 and cre2 phenotypes. The ccr4 allele blocked the derepression of ADHII and several other glucose-repressible enzymes, indicating that the CCR4 gene is a positive control element. The ccr4 allele had no effect on the repression of ADHII when it was combined with the ADR1-5c allele, whereas the phenotypically similar ccr1 allele, which partially suppresses ADR1-5c, did not suppress the cre1 or cre2 phenotype. Complementation studies also indicated that ccr1 and snf1 are allelic. A model of ADHII regulation is proposed in which both ADR1 and CCR4 are required for ADHII expression. CRE1 and CRE2 negatively control CCR4, whereas CCR1 is required for ADR1 function.     

An Extensive Circuitry for Cell Wall Regulation in Candida albicans

Protein kinases play key roles in signaling and response to changes in the external environment. The ability of Candida albicans to quickly sense and respond to changes in its environment is key to its survival in the human host. Our guiding hypothesis was that creating and screening a set of protein kinase mutant strains would reveal signaling pathways that mediate stress response in C. albicans. A library of protein kinase mutant strains was created and screened for sensitivity to a variety of stresses. For the majority of stresses tested, stress response was largely conserved between C. albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. However, we identified eight protein kinases whose roles in cell wall regulation (CWR) were not expected from functions of their orthologs in the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe. Analysis of the conserved roles of these protein kinases indicates that establishment of cell polarity is critical for CWR. In addition, we found that septins, crucial to budding, are both important for surviving and are mislocalized by cell wall stress. Our study shows an expanded role for protein kinase signaling in C. albicans cell wall integrity. Our studies suggest that in some cases, this expansion represents a greater importance for certain pathways in cell wall biogenesis. In other cases, it appears that signaling pathways have been rewired for a cell wall integrity response.     

Regulation of Cell Wall Synthesis by the Clathrin Light Chain Is Essential for Viability in Schizosaccharomyces pombe

The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, β(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization.     

Genes Involved in Cell Wall Localization and Side Chain Formation of Rhamnose-Glucose Polysaccharide in Streptococcus mutans

We identified in Streptococcus mutans six new genes (rgpA through rgpF), whose disruption results in a loss of serotype-specific antigenicity, specified by the glucose side chains of rhamnose-glucose polysaccharide from the cell wall. Rhamnose and glucose content of the cell wall decreased drastically in all these disruption mutants, except that in the rgpE mutant only the glucose content decreased. RgpC and RgpD are homologous to ATP-binding cassette transporter components and may be involved in polysaccharide export, whereas RgpE may be a transferase of side chain glucose.     

Identification of a New Stromal Cell Type Involved in the Regulation of Inflamed B Cell Follicles

Lymph node (LN) stromal cells provide survival signals and adhesive substrata to lymphocytes. During an immune response, B cell follicles enlarge, questioning how LN stromal cells manage these cellular demands. Herein, we used a murine fate mapping system to describe a new stromal cell type that resides in the T cell zone of resting LNs. We demonstrated that upon inflammation, B cell follicles progressively trespassed into the adjacent T cell zone and surrounded and converted these stromal cells into CXCL13 secreting cells that in return delineated the new boundaries of the growing follicle. Acute B cell ablation in inflamed LNs abolished CXCL13 secretion in these cells, while LT-β deficiency in B cells drastically affected this conversion. Altogether, we reveal the existence of a dormant stromal cell subset that can be functionally awakened by B cells to delineate the transient boundaries of their expanding territories upon inflammation.     

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see^1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.     

Possible Role of Hexosamine-Containing Cell Wall Component in Growth Regulation of Rice Coleoptiles

The cell wall of rice coleoptile was found to contain severalhundred microgram hexosamine per gram dry wt with the pectic,hemicellulosic, and -cellulose fractions containing 50%, 40%,and 10%, respectively. The cell wall hexosamine content increasedseveralfold with coleoptile growth and was higher in air-typecoleoptiles (grown on the surface of water) than water-typeones (grown under water). Rice coleoptiles were cultured in glucosamine, NH₄⁺, glutamine,or asparagine solution and growth was inhibited at 10^–4M and above. Coleoptile growth capacity in glucosamine or NH₄⁺solution correlated inversely with the cell wall hexosaminecontent. Both of these solutions also inhibited elongation ofsubmerged air-type coleoptile sections. Azaserine promoted thegrowth of both intact and excised coleoptiles at 10^–6to 10^–5 M and halved the cell wall hexosamine contentof intact ones. 6-Diazo-5-oxo-L-norleucine promoted the elongationof sections. These results suggest that the hexosamine-containingcell wall component is an important growth suppression factorin rice coleoptiles. (Received April 25, 1983; Accepted August 30, 1983)     

Large Scale Identification of Genes Involved in Cell Surface Biosynthesis and Architecture in Saccharomyces Cerevisiae

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.     

Targeting and Regulation of Cell Wall Synthesis During Tip Growth in Plants

Root hairs and pollen tubes are formed through tip growth, a process requiring synthesis of new cell wall material and the precise targeting and integration of these components to a selected apical plasma membrane domain in the growing tips of these cells. Presence of a tip-focused calcium gradient, control of actin cytoskeleton dynamics, and formation and targeting of secretory vesicles are essential to tip growth. Similar to cells undergoing diffuse growth, cellulose, hemicelluloses, and pectins are also deposited in the growing apices of tip-growing cells. However, differences in the manner in which these cell wall components are targeted and inserted in the expanding portion of tip-growing cells is reflected by the identification of elements of the plant cell wall synthesis machinery which have been shown to play unique roles in tip-growing cells. In this review, we summarize our current understanding of the tip growth process, with a particular focus on the subcellular targeting of newly synthesized cell wall components, and their roles in this form of plant cell expansion.     

Use of the Plant Defense Protein Osmotin To Identify Fusarium oxysporum Genes That Control Cell Wall Properties

Fusarium oxysporum is the causative agent of fungal wilt disease in a variety of crops. The capacity of a fungal pathogen such as F. oxysporum f. sp. nicotianae to establish infection on its tobacco (Nicotiana tabacum) host depends in part on its capacity to evade the toxicity of tobacco defense proteins, such as osmotin. Fusarium genes that control resistance to osmotin would therefore reflect coevolutionary pressures and include genes that control mutual recognition, avoidance, and detoxification. We identified FOR (Fusarium Osmotin Resistance) genes on the basis of their ability to confer osmotin resistance to an osmotin-sensitive strain of Saccharomyces cerevisiae. FOR1 encodes a putative cell wall glycoprotein. FOR2 encodes the structural gene for glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting step in the biosynthesis of hexosamine and cell wall chitin. FOR3 encodes a homolog of SSD1, which controls cell wall composition, longevity, and virulence in S. cerevisiae. A for3 null mutation increased osmotin sensitivity of conidia and hyphae of F. oxysporum f. sp. nicotianae and also reduced cell wall β-1,3-glucan content. Together our findings show that conserved fungal genes that determine cell wall properties play a crucial role in regulating fungal susceptibility to the plant defense protein osmotin.Studies of plant-pathogen interactions strongly suggest that under the pressure to survive, plants and pathogens continuously react to one another''s defense arsenal and evolve to overcome these defenses (13). Plants recognize pathogen-associated molecular patterns, such as fungal cell wall fragments composed of chitin, glucans, oligosaccharides, or glycoprotein peptides (32). It has been established that pathogens evolved effector proteins to avoid plant surveillance mechanisms that recognize pathogen-associated molecular patterns and this in turn led to the evolution of plant surveillance mechanisms that recognize pathogen-specific effector proteins. All pathogen recognition mechanisms induce intracellular signaling that culminates in the synthesis of factors, such as antimicrobial plant proteins, that help in limiting the severity of infection (74). The antimicrobial proteins are therefore among the ultimate effectors of plant defense. There is evidence of recognition between plant antimicrobial proteins and pathogen-specific molecules (74). Therefore, pathogen mechanisms of resistance to the antimicrobial proteins and the antimicrobial proteins themselves must have coevolved. Consequently, we postulated that a screen for fungal genes that alter the sensitivity of a phytopathogen to an antifungal protein of the host plant (that is, a cognate plant defense effector) would lead to identification of genes involved in controlling pathogenicity, in controlling access of the antifungal protein to its target fungal molecules (such as genes controlling cell surface composition), and in controlling detoxification mechanisms.The plant antifungal protein selected to test this hypothesis was osmotin. Osmotin is an antifungal protein that is overexpressed in and secreted by salt-adapted cultured tobacco (Nicotiana tabacum) cells (63). It is a member of a family of ubiquitous plant proteins, referred to as plant pathogenesis-related proteins of family 5 (PR-5), that are implicated in defense against fungi (74). Osmotin gene and protein expression is induced by biotic stresses, and overexpression of osmotin delays development of disease symptoms in transgenic plants (41, 42, 43, 84). The genetic bases of the susceptibility and resistance of Saccharomyces cerevisiae to osmotin have been explored in our laboratory (49, 50). The results show that specific interactions of osmotin with the plasma membrane are responsible for cell death signaling. However, because the cell wall governs access of osmotin to the plasma membrane, differences in cell wall composition largely account for the differential osmotin sensitivity of various S. cerevisiae strains, and specific cell wall components play a significant role in modulating osmotin toxicity (30, 31, 49, 50, 81, 82). These studies in the model nonpathogenic fungus, S. cerevisiae, support our hypothesis that a screen for genes that alter the sensitivity of a phytopathogenic fungus to an antifungal defense effector protein of the host plant will uncover genes involved in controlling access of the antifungal protein to its target fungal molecules.Osmotin, like other plant defense antifungal proteins, has specific but broad-spectrum antifungal activity (74). One of the most osmotin-sensitive phytopathogenic fungi is Fusarium oxysporum. F. oxysporum is an ascomycete fungus, like S. cerevisiae, and has been touted as an appropriate multihost model for studying fungal virulence (53). It is a soilborne plant pathogen of economic significance, because it causes vascular wilt disease on a large variety of crop plants and produces toxic food contaminants (17, 58). In humans it also causes skin, nail, and eye disease that can become serious or life-threatening illnesses in immunocompromised patients (52, 69). F. oxysporum f. sp. lycopersici, F. oxysporum f. sp. nicotianae, and F. oxysporum f. sp. meloni, like S. cerevisiae, are quite sensitive to osmotin (1, 51; M. L. Narasimhan, unpublished data). Furthermore, it was recently shown that overexpression in F. oxysporum f. sp. nicotianae of an S. cerevisiae cell wall glycoprotein that increases the osmotin resistance of S. cerevisiae also increases the osmotin resistance of the plant pathogen and its virulence on tobacco, the osmotin-producing host plant (51). This suggested that osmotin resistance mechanisms may be conserved between S. cerevisiae and F. oxysporum and that S. cerevisiae could be used as a tool to uncover F. oxysporum genes that control osmotin sensitivity or resistance.In the current study, we expressed an F. oxysporum f. sp. nicotianae cDNA library in the osmotin-sensitive S. cerevisiae strain BWG1-7a and selected genes for their ability to increase osmotin tolerance. We report here the identification and characterization of three FOR (Fusarium Osmotin Resistance) genes that affect the cell wall in S. cerevisiae. The product of FOR1 has homology with a putative cell surface glycoprotein; FOR2 encodes glutamine:fructose-6-phosphate amidotransferase (GFAT), an enzyme that catalyzes the first step in the biosynthetic pathway leading to amino sugar-containing macromolecules, such as glycoproteins and chitin (64); and FOR3 has high homology with S. cerevisiae SSD1, a gene that controls cell wall composition and virulence (31, 78). FOR2 and FOR3 are the functional equivalents of the corresponding S. cerevisiae genes. Our parallel analysis using two model fungi verifies the notion that cell wall proteins play a critical role in determining the sensitivity/resistance of fungi to osmotin. In addition, these results implicate that the tobacco defense protein, osmotin, can serve as an effective/useful tool in identifying genes that control cell wall composition not only in a model fungus, such as S. cerevisiae, but also in phytopathogenic fungi, such as F. oxysporum.     

Cloning of Nicotianamine Synthase Genes, Novel Genes Involved in the Biosynthesis of Phytosiderophores

Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.     

Role of the Fungal Cell Wall in Pathogenesis and Antifungal Resistance

Study of the fungal cell wall is currently an area of very active research. The relevance of the fungal cell wall for cell survival, and pathogenicity has been well established. The view of the cell wall as a tough and impenetrable structure has been left behind, and it is now conceived as a plastic shield that undergoes structural changes depending on the surrounding environmental conditions and morphological states. The fungal cell wall is also the source of most of the pathogen-associated molecular patterns that immune cells recognize, and thus facilitates establishment of a protective antifungal immunity. Paradoxically, fungi, through their cell wall, possess disguising mechanisms to avoid immune recognition. This review gathers the current knowledge about the cell wall of Candida albicans, Aspergillus fumigatus and Paracoccidioides brasiliensis, stressing the importance of the fungal cell wall for pathogenesis, immune recognition, and as a source of targets for antifungal drugs.