Synthase Catalytic Subunit Research Articles

A Novel Gene, OsRLCK191, Involved in Culm Strength Improving Lodging Resistance in Rice.

Lodging is one of the major problems in rice production. However, few genes that can explain the culm strength within the temperate japonica subspecies have been identified. In this study, we identified OsRLCK191, which encodes receptor-like cytoplasmic kinase and plays critical roles in culm strength. OsRLCK191 mutants were produced by the CRISPR-Cas9 DNA-editing system. Compared with wild types (WTs), the bending moment of the whole plant (WP), the bending moment at breaking (BM), and the section modulus (SM) were decreased in rlck191 significantly. Although there is no significant decrease in the culm length of rlck191 compared with the WT; in the mutant, except the length of the fourth internode being significantly increased, the lengths of other internodes are significantly shortened. In addition, the yield traits of panicle length, thousand-seed weight, and seed setting rate decreased significantly in rlck191. Moreover, RNA-seq experiments were performed at an early stage of rice panicle differentiation in shoot apex. The differentially expressed genes (DEGs) are mainly involved in cell wall biogenesis, cell wall polysaccharide metabolic processes, cellar component biogenesis, and DNA-binding transcription factors. Transcriptome analysis of the cell wall biological process pathways showed that major genes that participated in the cytokinin oxidase/dehydrogenase family, cellulose synthase catalytic subunit genes, and ethylene response factor family transcription factor were related to culm strength. Our research provides an important theoretical basis for analyzing the lodging resistance mechanism and lodging resistance breeding of temperate japonica.

Key Proteins in Rat Cerebral Cortex: Application of Cornu aspersum Extract as a Neuroprotective Agent in Alzheimer's Type Dementia.

Alzheimer's disease (AD) is the most widespread neurodegenerative disorder. Recently, it was found that mucus extract from Cornu aspersum has beneficial effects on memory and cognitive processes in a rat scopolamine model of AD. The present study elucidated the mechanisms of action of standardized mucus snail extract (SE) enriched with a fraction above 20 kDa on Alzheimer-type dementia in rats. Using proteomic analysis on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) on rat cortex extracts, we compared protein expression in both groups: the first group was treated intraperitoneally with scopolamine (Sco, 2 mg/kg, 11 days) and the second (Sco + SE) group was treated intraperitoneally with Sco (Sco, 2 mg/kg) and protected by SE (0.5 mL/100 g bw) applied daily orally for 11 days. Brain cortex was separated and the expressions of various proteins related to memory and cognitive functions were identified. We found that the expression of Ubiquitin carboxyl-terminal hydrolase isozyme L1, Calbindin, Vacuolar ATP synthase catalytic subunit A, Tropomyosin beta chain, 14-3-3 zeta/delta, Kinesin-1 heavy chain, and Stathmin-4 significantly differs in SE-protected rats as compared to dement animals treated only by Sco, and these brain proteins might be potential therapeutic targets for Alzheimer's-type dementia treatment.

A cellulose synthesis inhibitor affects cellulose synthase complex secretion and cortical microtubule dynamics.

P4B (2-phenyl-1-[4-(6-(piperidin-1-yl) pyridazin-3-yl) piperazin-1-yl] butan-1-one) is a novel cellulose biosynthesis inhibitor (CBI) discovered in a screen for molecules to identify inhibitors of Arabidopsis (Arabidopsis thaliana) seedling growth. Growth and cellulose synthesis inhibition by P4B were greatly reduced in a novel mutant for the cellulose synthase catalytic subunit gene CESA3 (cesa3pbr1). Cross-tolerance to P4B was also observed for isoxaben-resistant (ixr) cesa3 mutants ixr1-1 and ixr1-2. P4B has an original mode of action as compared with most other CBIs. Indeed, short-term treatments with P4B did not affect the velocity of cellulose synthase complexes (CSCs) but led to a decrease in CSC density in the plasma membrane without affecting their accumulation in microtubule-associated compartments. This was observed in the wild type but not in a cesa3pbr1 background. This reduced density correlated with a reduced delivery rate of CSCs to the plasma membrane but also with changes in cortical microtubule dynamics and orientation. At longer timescales, however, the responses to P4B treatments resembled those to other CBIs, including the inhibition of CSC motility, reduced growth anisotropy, interference with the assembly of an extensible wall, pectin demethylesterification, and ectopic lignin and callose accumulation. Together, the data suggest that P4B either directly targets CESA3 or affects another cellular function related to CSC plasma membrane delivery and/or microtubule dynamics that is bypassed specifically by mutations in CESA3.

Bioinformatics characterization of BcsA-like orphan proteins suggest they form a novel family of pseudomonad cyclic-β-glucan synthases.

Bacteria produce a variety of polysaccharides with functional roles in cell surface coating, surface and host interactions, and biofilms. We have identified an 'Orphan' bacterial cellulose synthase catalytic subunit (BcsA)-like protein found in four model pseudomonads, P. aeruginosa PA01, P. fluorescens SBW25, P. putida KT2440 and P. syringae pv. tomato DC3000. Pairwise alignments indicated that the Orphan and BcsA proteins shared less than 41% sequence identity suggesting they may not have the same structural folds or function. We identified 112 Orphans among soil and plant-associated pseudomonads as well as in phytopathogenic and human opportunistic pathogenic strains. The wide distribution of these highly conserved proteins suggest they form a novel family of synthases producing a different polysaccharide. In silico analysis, including sequence comparisons, secondary structure and topology predictions, and protein structural modelling, revealed a two-domain transmembrane ovoid-like structure for the Orphan protein with a periplasmic glycosyl hydrolase family GH17 domain linked via a transmembrane region to a cytoplasmic glycosyltransferase family GT2 domain. We suggest the GT2 domain synthesises β-(1,3)-glucan that is transferred to the GH17 domain where it is cleaved and cyclised to produce cyclic-β-(1,3)-glucan (CβG). Our structural models are consistent with enzymatic characterisation and recent molecular simulations of the PaPA01 and PpKT2440 GH17 domains. It also provides a functional explanation linking PaPAK and PaPA14 Orphan (also known as NdvB) transposon mutants with CβG production and biofilm-associated antibiotic resistance. Importantly, cyclic glucans are also involved in osmoregulation, plant infection and induced systemic suppression, and our findings suggest this novel family of CβG synthases may provide similar range of adaptive responses for pseudomonads.

Recombinase Polymerase Amplification Assay with Lateral Flow Strips for Rapid Detection of Candidiasis Due to Candida parapsilosis.

Candida parapsilosis is a common cause of candidiasis among hospitalized patients, often surpassing Candida albicans. Due to the recent increase in C. parapsilosis infections, there is an urgent need for rapid, sensitive, and real-time on-site detection of nucleic acids for timely diagnosis of candidiasis. We developed an assay for detection of C. parapsilosis by combining recombinase polymerase amplification (RPA) with a lateral flow strip (LFS). The RPA-LFS assay was used to amplify the beta-1,3-glucan synthase catalytic subunit 2 (FKS2) gene of C. parapsilosis with a primer-probe set optimized by introducing base mismatches (four bases modified by the probe and one by the reverse primer) to achieve specific and sensitive detection of clinical samples. The RPA assays can rapidly amplify and visualize a target gene within 30min, while the entire process can be completed within 40min by pre-processing the sample. The product of RPA has two chemical labels, FITC and Biotin, of the amplification product can be carefully on the strip. The sensitivity and specificity of the RPA-LFS assay were determined by analysis of 35 common clinical pathogens and 281 clinical samples against quantitative PCR. The results confirmed that the proposed RPA-LFS assay is a reliable molecular diagnostic method for the detection of C. parapsilosis to meet the urgent need for rapid, specific, sensitive, and portable field testing.

CEF3 is involved in membrane trafficking and essential for secondary cell wall biosynthesis and its mutation enhanced biomass enzymatic saccharification in rice

BackgroundAs one of the most important staple food crops, rice produces large of agronomic biomass residues that contain lots of secondary cell walls (SCWs). Membrane trafficking plays key roles in SCWs biosynthesis, but information association membrane trafficking and SCWs formation in plants is limited.ResultsIn this study, we report the function characterization of a rice mutant, culm easily fragile 3 (cef3), that exhibits growth retardation and fragile culm phenotype with significantly altered cell wall composition and reduced secondary wall thickness. Map-based cloning revealed that CEF3 encodes a homologous protein of Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE2 (SCD2). The saccharification assays revealed that CEF3 mutation can improve biomass enzymatic saccharification. Expression pattern analysis indicated that CEF3 is ubiquitously expressed in many organs at different developmental stages. Subcellular localization revealed that CEF3 is a Golgi-localized protein. The FM4-64 uptake assay revealed CEF3 is involved in endocytosis. Furthermore, mutation of CEF3 not only affected cellulose synthesis-related genes expression, but also altered the abundance of cellulose synthase catalytic subunit 9 (OsCESA9) in the PM and in the endomembrane systems.ConclusionsThis study has demonstrated that CEF3 participates in the membrane trafficking that is essential for normal cellulose and other polysaccharides biosynthesis of the secondary cell wall, thereby manipulation of CEF3 could alter cellulose content and enhance biomass enzymatic saccharification in rice plants. Therefore, the study of the function of CEF3 can provide a strategy for genetic modification of SCWs in bioenergy crops.

Phosphoregulation of the ATP synthase beta subunit stimulates mitochondrial activity for G2/M progression.

Mitochondrial ATP synthase is a multifunctional enzyme complex involved in ATP production. We previously reported that the ATP synthase catalytic beta subunit (Atp2p in yeast) is regulated by the 2A-like protein phosphatase Sit4p, which targets Atp2p at T124/T317 impacting on ATP synthase levels and mitochondrial respiration. Here we report that Atp2-T124/T317 is also potentially regulated by Cdc5p, a polo-like mitotic kinase. Since both Cdc5p and Sit4p have established roles in cell cycle regulation, we investigated whether Atp2-T124/T317 phosphorylation was cell cycle-related. We present evidence that Atp2p levels and phosphorylation vary during cell cycle progression, with an increase at G2/M phase. Atp2-T124/T317 phosphorylation stimulates mitochondrial membrane potential, respiration and ATP levels at G2/M phase, indicating that dynamic Atp2p phosphorylation contributes to mitochondrial activity at this specific cell cycle phase. Preventing Atp2p phosphorylation delays G2/M to G1 transition, suggesting that enhanced bioenergetics at G2/M may help meet the energetic demands of cell cycle progression. However, mimicking constitutive T124/T317 phosphorylation or overexpressing Atp2p leads to mitochondrial DNA instability, indicating that reversible Atp2p phosphorylation is critical for homeostasis. These results indicate that transient phosphorylation of Atp2p, a protein at the core of the ATP production machinery, impacts on mitochondrial bioenergetics and supports cell cycle progression at G2/M.

Comparative Transcriptome Analysis Reveals Candidate Genes and Pathways for Potential Branch Growth in Elm (Ulmus pumila) Cultivars.

Simple SummaryElm (Ulmus pumila) is a strong essential wood, and it is widely used in cabinets, sculptures, and ship making. In the present study, phenotypic and comparative transcriptomic analyses were performed in elm fast- (UGu17 and UZuantian) and slow-growing cultivars (U81-07 and U82-39). Phenotypic observation showed that the thickness of secondary xylem of 2-year-old fast-growing branches was greater compared with slow-growing cultivars. Comparative transcriptome analysis predicted that many pathways were involved in vascular development and transcriptional regulation in elm, such as “plant-type secondary cell wall biogenesis”, “cell wall thickening”, and “phenylpropanoid biosynthesis”. NAC domain transcriptional factors (TFs) and their master regulators, cellulose synthase catalytic subunits (CESAs), xylan synthesis, and secondary wall thickness were presumed to function in the thickening mechanism of elm branches. Our results indicated that the general phenylpropanoid pathway and lignin metabolism had vital functions in the growth of elm branches.Wood plays a vital role in human life. It is important to study the thickening mechanism of tree branches and explore the mechanism of wood formation. Elm (Ulmus pumila) is a strong essential wood, and it is widely used in cabinets, sculptures, and ship making. In the present study, phenotypic and comparative transcriptomic analyses were performed in U. pumila fast- (UGu17 and UZuantian) and slow-growing cultivars (U81-07 and U82-39). Phenotypic observation showed that the thickness of secondary xylem of 2-year-old fast-growing branches was greater compared with slow-growing cultivars. A total of 9367 (up = 4363, down = 5004), 7159 (3413/3746), 7436 (3566/3870), and 5707 (2719/2988) differentially expressed genes (DEGs) were identified between fast- and slow-growing cultivars. Moreover, GO and KEGG enrichment analyses predicted that many pathways were involved in vascular development and transcriptional regulation in elm, such as “plant-type secondary cell wall biogenesis”, “cell wall thickening”, and “phenylpropanoid biosynthesis”. NAC domain transcriptional factors (TFs) and their master regulators (VND1/MYB26), cellulose synthase catalytic subunits (CESAs) (such as IRX5/IRX3/IRX1), xylan synthesis, and secondary wall thickness (such as IRX9/IRX10/IRX8) were supposed to function in the thickening mechanism of elm branches. Our results indicated that the general phenylpropanoid pathway (such as PAL/C4H/4CL) and lignin metabolism (such as HCL/CSE/CCoAOMT/CCR/F5H) had vital functions in the growth of elm branches. Our transcriptome data were consistent with molecular results for branch thickening in elm cultivars.

Mechanism study on the regulation of metabolite flux for producing promising bioactive substances in microalgae Desmodesmus sp.YT through salinity stress

Desmodesmus sp.YT rich in bioactive substances exhibited impressive tolerance to various environmental conditions. To investigate the metabolism transformation influenced by salt stress in Desmodesmus sp.YT, biochemical compositions and comparative transcriptome were thoroughly explored in this research. Results showed that normal treatment (0‰ salinity) was beneficial for the production of biomass (up to 1.87 times) and protein (up to 1.46 times), compared with salt treatment. Furthermore, differentially expressed genes analysis revealed that vital genes involved in photosynthesis (light-harvesting complexs, LHCs; photosystem II oxygen-evolving enhancer proteins, Psbs), C3 photosynthetic pathway (fructose-bisphosphate aldolase, ALDO; fructose-1,6-bisphosphatase, PFK; phosphoglycerate kinase, PGK) and chlorophyll synthesis (coproporphyrinogen III oxidase, CPOX; porphobilinogen synthase, HemB) were significantly up-regulated in 0‰ salinity, leading to enhanced cell growth. Interestingly, salt stress stimulated the expression of cellulose synthase catalytic subunit A2 (CesA2) and starch synthase (GLGA), increasing the biosynthesis of cellulose (up to 3.23 times) and starch (up to 1.05 times). Results showed that Desmodesmus sp.YT cultured at freshwater could be applied as feed additives while microalgae grown in seawater had the potential for biofuel production for further mass cultivation.

Identification and Fine Mapping of the Recessive Gene BK-5, Which Affects Cell Wall Biosynthesis and Plant Brittleness in Maize.

The cellulose of the plant cell wall indirectly affects the cell shape and straw stiffness of the plant. Here, the novel brittleness mutant brittle stalk-5 (bk-5) of the maize inbred line RP125 was characterized. We found that the mutant displayed brittleness of the stalk and even the whole plant, and that the brittleness phenotype existed during the whole growth period from germination to senescence. The compressive strength was reduced, the cell wall was thinner, and the cellulose content was decreased compared to that of the wild type. Genetic analysis and map-based cloning indicated that bk-5 was controlled by a single recessive nuclear gene and that it was located in a 90.2-Kb region on chromosome 3 that covers three open reading frames (ORFs). Sequence analysis revealed a single non-synonymous missense mutation, T-to-A, in the last exon of Zm00001d043477 (B73: version 4, named BK-5) that caused the 951th amino acid to go from leucine to histidine. BK-5 encodes a cellulose synthase catalytic subunit (CesA), which is involved with cellulose synthesis. We found that BK-5 was constitutively expressed in all tissues of the germinating stage and silking stage, and highly expressed in the leaf, auricula, and root of the silking stage and the 2-cm root and bud of the germinating stage. We found that BK-5 mainly localized to the Golgi apparatus, suggesting that the protein might move to the plasma membrane with the aid of Golgi in maize. According to RNA-seq data, bk-5 had more downregulated genes than upregulated genes, and many of the downregulated genes were enzymes and transcription factors related to cellulose, hemicellulose, and lignin biosynthesis of the secondary cell wall. The other differentially expressed genes were related to metabolic and cellular processes, and were significantly enriched in hormone signal transduction, starch and sucrose metabolism, and the plant–pathogen interaction pathway. Taken together, we propose that the mutation of gene BK-5 causes the brittle stalk phenotype and provides important insights into the regulatory mechanism of cellulose biosynthesis and cell wall development in maize.

Mutation of glucan synthase catalytic subunit in Beauveria bassiana affects fungal growth and virulence

Beauveria bassiana is a well-known entomopathogenic fungus that parasitizes on a variety of insect species. Glucan in the cell wall of B. bassiana plays a crucial role in its structure and growth and is also involved in the activation of the host insect’s immune system. Glucan biosynthesis is primarily regulated by glucan synthase, however, it is unclear if the glucan synthase catalytic subunit gene (GluS) affects the growth and virulence of B. bassiana. In this study, we constructed the mutant of the B. bassiana glucan synthase catalytic subunit (ΔGluS) by homologous recombination and observed that glucan synthase knockout affects both spore germination and cell area. Further enzyme-based assays along with gene expression analysis of glucan synthase revealed a significant downregulation in the mutant strains compared to the wild type of B. bassiana. Moreover, the virulence of ΔGluS strains against gypsy moth (Lymantria dispar) showed no significant difference compared to the wild-type strains when injected, while the spraying gypsy moths with the conidia of ΔGluS was significantly more lethal than spraying the conidia of the wild type. Altogether, our study constructed a new, highly efficient B. bassiana mutant that can be used for pest control and provides a readily transferable method for other insect-entomopathogenic interaction studies.

Regulation of sphingolipid biosynthesis in the endoplasmic reticulum via signals from the plasma membrane in budding yeast.

Saccharomyces cerevisiae LIP1 encodes a regulatory subunit that forms a complex with the ceramide synthase catalytic subunits, Lag1/Lac1, which is localized on the membrane of endoplasmic reticulum. To understand the underlying regulatory mechanism of sphingolipid biosynthesis, we generated strains upon replacing the chromosomal LIP1 promoter with a Tet-off promoter, which enables the expression in Dox-dependent manner. The lip1-1 strain, obtained through the promoter substitution, exhibits severe growth inhibition and remarkable decrease in sphingolipid synthesis in the presence of Dox. Using this strain, we investigated the effect of a decrease in ceramide synthesis on TOR complex 2 (TORC2)-Ypk1 signaling, which senses the complex sphingolipid level at the plasma membrane and promotes sphingolipid biosynthesis. In lip1-1 cells, Ypk1 was activated via both upstream kinases, TORC2 and yeast PDK1 homologues, Pkh1/2, thereby inducing hyperphosphorylation of Lag1, but not of another Ypk1-substrate, Orm1, which is a known negative regulator of the first step of sphingolipid metabolism, in the presence of Dox. Therefore, our data suggest that the metabolic enzyme activities at each step of the sphingolipid biosynthetic pathway are controlled through a fine regulatory mechanism.

Differential Regulation of Echinocandin Targets Fks1 and Fks2 in Candida glabrata by the Post-Transcriptional Regulator Ssd1

Invasive infections caused by the opportunistic pathogen Candida glabrata are treated with echinocandin antifungals that target β-1,3-glucan synthase, an enzyme critical for fungal cell wall biosynthesis. Echinocandin resistance develops upon mutation of genes (FKS1 or FKS2) that encode the glucan synthase catalytic subunits. We have analyzed cellular factors that influence echinocandin susceptibility and here describe effects of the post-transcriptional regulator Ssd1, which in S. cerevisiae, can bind cell wall related gene transcripts. The SSD1 homolog in C. glabrata was disrupted in isogenic wild type and equivalent FKS1 and FKS2 mutant strains that demonstrate echinocandin resistance (MICs > 0.5 µg/mL). A reversal of resistance (8- to 128-fold decrease in MICs) was observed in FKS1 mutants, but not in FKS2 mutants, following SSD1 deletion. Additionally, this phenotype was complemented upon expression of SSD1 from plasmid (pSSD1). All SSD1 disruptants displayed susceptibility to the calcineurin inhibitor FK506, similar to fks1∆. Decreases in relative gene expression ratios of FKS1 to FKS2 (2.6- to 4.5-fold) and in protein ratios of Fks1 to Fks2 (2.7- and 8.4-fold) were observed in FKS mutants upon SSD1 disruption. Additionally, a complementary increase in protein ratio was observed in the pSSD1 expressing strain. Overall, we describe a cellular factor that influences Fks1-specific mediated resistance and demonstrates further differential regulation of FKS1 and FKS2 in C. glabrata.

Screening of Bombyx mori brain proteins interacting with protein tyrosine phosphatase of BmNPV.

In this study, glutathione-S-transferase pull-down combined with mass spectrometry techniques were used to identify the candidate proteins interacting with protein tyrosine phosphatase of theBombyx Mori nucleopolyhedrovirus in theB. mori (BmNPV-PTP) brain. A total of 36 proteins were identified from BmNPV-PTP coprecipitate samples by searching the NCBI_Bombyx Mori database with the original mass spectrum data. Among those proteins, the interaction between BmNPV-PTP and B. mori cyclophilin A may acceleratethe apoptosis of certain nerve cells involvedin regulating behavior, and thus may be an inducer of enhanced locomotor activity (ELA). After the BmNPV invasion, BmNPV-PTP binding to peripheral-type benzodiazepine receptors may initiate a series of abnormal cascades of the nervous system, which resultsin abnormal hyperactive behavior in B. mori. Besides this, vacuolar ATP synthase catalytic subunit A, annexin, and several enzymes for energy conversion were identified, which may play a rolein enhancing viral entry and infectivity and provide energy for enhancing the locomotor activity of B. mori. In general, the results of this study will facilitate the understanding of the molecular mechanisms underlying the ELA of B. mori larva induced by BmNPV.

Analysis of a cellulose synthase catalytic subunit from the oomycete pathogen of crops Phytophthora capsici

Phytophthora capsici Leonian is an important oomycete pathogen of crop vegetables, causing significant economic losses each year. Its cell wall, rich in cellulose, is vital for cellular integrity and for interactions with the host organisms. Predicted cellulose synthase (CesA) proteins are expected to catalyze the polymerization of cellulose, but this has not been biochemically demonstrated in an oomycete. Here, we present the properties of the four newly identified CesA proteins from P. capsici and compare their domain organization with that of CesAs from other lineages. Using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity, we have achieved successful heterologous expression and biochemical characterization of a CesA protein from P. capsici (PcCesA1). Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the major reaction product. Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to form a complex whose multiproteic nature is most likely required for cellulose microfibril formation. In addition to providing important insights into cellulose synthesis in the oomycetes, our data may assist the longer term identification of cell wall biosynthesis inhibitors to control infection by pathogenic oomycetes.

Features of a novel protein, rusticalin, from the ascidian Styela rustica reveal ancestral horizontal gene transfer event

BackgroundThe transfer of genetic material from non-parent organisms is called horizontal gene transfer (HGT). One of the most conclusive cases of HGT in metazoans was previously described for the cellulose synthase gene in ascidians.ResultsIn this study we identified a new protein, rusticalin, from the ascidian Styela rustica and presented evidence for its likely origin by HGT. Discernible homologues of rusticalin were found in placozoans, coral, and basal Chordates. Rusticalin was predicted to consist of two distinct regions, an N-terminal domain and a C-terminal domain. The N-terminal domain comprises two cysteine-rich repeats and shows remote similarity to the tick carboxypeptidase inhibitor. The C-terminal domain shares significant sequence similarity with bacterial MD peptidases and bacteriophage A500 L-alanyl-D-glutamate peptidase. A possible transfer of the C-terminal domain by bacteriophage was confirmed by an analysis of noncoding sequences of C. intestinalis rusticalin-like gene, which was found to contain a sequence similar to the bacteriophage A500 recombination site. Moreover, a sequence similar to the bacteriophage recombination site was found to be adjacent to the cellulose synthase catalytic subunit gene in the genome of Streptomices sp., the donor of ascidian cellulose synthase.ConclusionsThe C-terminal domain of rusticalin and rusticalin-like proteins is likely to be horizontally transferred by the bacteriophage A500. A common mechanism involving bacteriophage mediated gene transfer can be proposed for at least two HGT events in ascidians.

Arabidopsis Group IIId ERF proteins positively regulate primary cell wall-type CESA genes

The cell wall determines morphology and the environmental responses of plant cells. The primary cell wall (PCW) is produced during cell division and expansion, determining the cell shape and volume. After cell expansion, specific types of plant cells produce a lignified wall, known as a secondary cell wall (SCW). We functionally analyzed Group IIId Arabidopsis AP2/EREBP genes, namely ERF34, ERF35, ERF38, and ERF39, which are homologs of a rice ERF gene previously proposed to be related to SCW biosynthesis. Expression analysis revealed that these four genes are expressed in regions related to cell division and/or cell differentiation in seedlings (i.e., shoot apical meristems, the primordia of leaves and lateral roots, trichomes, and central cylinder of primary roots) and flowers (i.e., vascular tissues of floral organs and replums and/or valve margins of pistils). Overexpression of ERF genes significantly upregulated PCW-type, but not SCW-type, CESA genes encoding cellulose synthase catalytic subunits in Arabidopsis seedlings. Transient co-expression reporter analysis indicated that ERF35, ERF38, and ERF39 possess transcriptional activator activity, and that ERF34, ERF35, ERF38, and ERF39 upregulated the promoter activity of CESA1, a PCW-type CESA gene, through the DRECRTCOREAT elements, the core cis-acting elements known to be recognized by AP2/ERF proteins. Together, our findings show that Group IIId ERF genes are positive transcriptional regulators of PCW-type CESA genes in Arabidopsis and are possibly involved in modulating cellulose biosynthesis in response to developmental requirements and environmental stimuli.

Site-Directed Mutagenesis of the 1,3-β-Glucan Synthase Catalytic Subunit of Pneumocystis jirovecii and Susceptibility Assays Suggest Its Sensitivity to Caspofungin.

The echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthesizing 1,3-β-glucan, an essential compound of the fungal wall. Studies with rodents showed that caspofungin is effective against Pneumocystis asci. However, its efficacy against asci of Pneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of the P. jirovecii Gsc1 subunit, as well as of those of Pneumocystis carinii and Pneumocystis murina infecting, respectively, rats and mice. In the absence of an established in vitro culture method for Pneumocystis species, we used functional complementation of the Saccharomyces cerevisiae gsc1 deletant. In the fungal pathogen Candida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into the Pneumocystis gsc1 genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The MICs of caspofungin determined by Etest and YeastOne for strains complemented with Pneumocystis enzymes (respectively, 0.125 and 0.12 μg/ml) were identical to those upon complementation with the enzyme of C. albicans, for which caspofungin presents low MICs. However, they were lower than the MICs upon complementation with the enzyme of the resistant species Candida parapsilosis (0.19 and 0.25 μg/ml). Sensitivity levels of Gsc1 enzymes of the three Pneumocystis species were similar. Our results suggest that P. jirovecii is sensitive to caspofungin during infections, as are P. carinii and P. murina.

Transcript Profiling of Non‐Target‐Site Imidazolinone Resistance in Imisun Sunflower

Imidazolinone resistance found in a wild sunflower (Helianthus annuus L.) population was successfully transferred to a cultivated inbred line developing ‘Imisun’ sunflowers. Genetic regulation of this trait has been reported to involve two genes: Imr1, an allelic variant of ahasl1 locus that codes for acetohydroxyacid synthase catalytic subunit, and the modifier Imr2, whose identity remains unknown, but it could be related to non‐target‐site resistance such as xenobiotic metabolism. The aim of the present study was to characterize the gene expression of resistant and susceptible sunflower lines in response to imazethapyr herbicide by complementary DNA amplified fragment‐length polymorphism (cDNA‐AFLP). Three assays were performed to determine (i) optimal herbicide treatment concentration, (ii) duration of herbicide treatment, and (iii) in vitro acetohydroxyacid synthase activity to assess enzyme inhibition levels. An important number of genes related to metabolism of xenobiotics and stress was found: cytochrome P450 monooxygenases, UDP‐glucuronosyl/UDP‐glucosyltransferases, glycosyltransferases, and ATP‐binding cassette transporters, among others. These results suggest that non‐target‐site resistance mechanisms may contribute to herbicide resistance in Imisun sunflower and could be related to the modifier gene Imr2. Using cDNA‐AFLP, we were able to detect candidate detoxification‐related genes potentially involved in imidazolinone resistance in sunflower.

Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition.

The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the β-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.