Enhancement of Heterologous Gene Expression in Flammulina velutipes Using Polycistronic Vectors Containing a Viral 2A Cleavage Sequence

Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp) gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.     

Expression of Arabidopsis thaliana xylose isomerase gene and its effect on ethanol production in Flammulina velutipes

To improve the pentose fermentation rate in Flammulina velutipes, the putative xylose isomerase (XI) gene from Arabidopsis thaliana was cloned and introduced into F. velutipes and the gene expression was evaluated in transformants. mRNA expression of the putative XI gene and XI activity were observed in two transformants, indicating that the putative gene from A. thaliana was successfully expressed in F. velutipes as a xylose isomerase. In addition, ethanol production from xylose was increased in the recombinant strains. This is the first report demonstrating the possibility of using plant genes as candidates for improving the characteristics of F. velutipes.     

Cancer-derived p53 mutants suppress p53-target gene expression--potential mechanism for gain of function of mutant p53

Tumour-derived p53 mutants are thought to have acquired ‘gain-of-function’ properties that contribute to oncogenicity. We have tested the hypothesis that p53 mutants suppress p53-target gene expression, leading to enhanced cellular growth. Silencing of mutant p53 expression in several human cell lines was found to lead to the upregulation of wild-type p53-target genes such as p21, gadd45, PERP and PTEN. The expression of these genes was also suppressed in H1299-based isogenic cell lines expressing various hot-spot p53 mutants, and silencing of mutant p53, but not TAp73, abrogated the suppression. Consistently, these hot-spot p53 mutants were able to suppress a variety of p53-target gene promoters. Analysis using the proto-type p21 promoter construct indicated that the p53-binding sites are dispensable for mutant p53-mediated suppression. However, treatment with the histone deacetylase inhibitor trichostatin-A resulted in relief of mutant p53-mediated suppression, suggesting that mutant p53 may induce hypo-acetylation of target gene promoters leading to the suppressive effects. Finally, we show that stable down-regulation of mutant p53 expression resulted in reduced cellular colony growth in human cancer cells, which was found to be due to the induction of apoptosis. Together, the results demonstrate another mechanism through which p53 mutants could promote cellular growth.     

A key ABA hydrolase gene,OsABA8ox3 is involved in rice resistance to Nilaparvata lugens by affecting callose deposition

Abscisic acid (ABA) is an important plant hormone in regulating abiotic and biotic stresses. OsABA8ox3 is the key gene in ABA hydrolase genes, and plays an important role in controlling ABA level, but little is known in rice resistance to insects. We used rice osaba8ox3 T-DNA insertion mutant (knocking down the OsABA8ox3 gene) to elucidate rice resistance to the insect. There were obvious phenotype differences between the osaba8ox3 T-DNA insertion mutant and wild-type (WT), and the relative expression of synthetase genes in the osaba8ox3 mutant was higher, while the relative expression of hydrolase genes was lower than that of WT, respectively. The electrical penetration graph (EPG) recording indicated that the osaba8ox3 mutant had the less sucking phloem sap duration compared with WT, which indicated a significant increase in rice resistance to brown planthopper (Nilaparvata lugens; BPH). The callose deposition in the osaba8ox3 mutant increased by 60.39%, 52.2%, 26.6% and 31.7% than that of WT after BPH feeding for 0, 24, 48, and 72 h, respectively. These results showed OsABA8ox3 gene played an important role in rice resistance to BPH, and also provided new insights into the mechanism of callose deposition regulation in response to the piercing-sucking pest.     

Analysis of the autonomy of imaginal disc defects in a small-disc mutant of Drosophila melanogaster.

Lethal mutations which cause imaginal disc abnormalities in Drosophila melanogaster identify genes whose function is necessary for normal disc development, and these mutant genes may be used as probes of the role of their wild-type alleles in normal development. It is crucial to the interpretation of the disc phenotype of such mutants to know which abnormalities are autonomous (caused by expression of the mutant gene in imaginal cells) and which are nonautonomous (indirectly caused, for example, by expression of the mutant gene in larval cells). We chose for study l(3)c21R (3-67.8), a late-larval lethal mutation with a complex phenotype, to test the adequacy of available techniques for assessing autonomy. We employed surgical and genetic techniques to determine the imaginal cell autonomy of the defects in cell viability, growth, and differentiation in c21R discs. The imaginal cell viability defect is nonautonomous. The disc growth and differentiation defects are autonomous; however, in genetic mosaics these two autonomous defects are separable. These results show that c21R belongs to the class of mutations which affect both larval and imaginal cells. In combination, the available methods were adequate to resolve the issue of autonomy in this complex case. However, in isolation several of the methods could have led to incomplete or misleading interpretations. This emphasizes that to analyze any developmental mutant it is necessary to examine the issue of autonomy from several points of view.     

Suppression of the transformed phenotype with retention of the viral ‘src’ gene in cell hybrids between rous sarcoma virus-transformed rat cells and untransformed mouse cells

Hybrid cell lines between untransformed mouse 3T3TK-cells and normal rat kidney (NRK) cells transformed by the B77 strain of Rous Sarcoma Virus (RSV) express a non-transformed phenotype, as determined by anchorage-dependent growth and organization of microfilament bundles. Virus rescue experiments and genetic experiments using an RSV mutant temperature-sensitive for maintenance of the transformed phenotype demonstrate that RSV is retained in the non-transformed hybrids. The action of the viral transformation gene ‘src’ therefore appears to be ‘suppressed’ in these hybrids. The suppressed hybrids generate variants in which the expression of the transformed phenotype and the ‘src’ gene is regained. This system should prove to be of value in identifying cellular genes involved in the expression of virally induced transformation.     

Loss of function of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the first committed step in the cytosolic isoprenoid biosynthesis pathway in higher plants. To understand the contribution of HMGR to plant development, we isolated T-DNA insertion mutants for HMG1 and HMG2. The hmg1 and hmg2 mutants were both more sensitive than the wild type (WT) to lovastatin, an inhibitor of HMGR. The hmg2 mutant showed no visible phenotype under normal growth conditions. In contrast, the hmg1 mutant exhibited dwarfing, early senescence, and sterility. Expression of senescence-associated genes 12 (SAG12), a marker gene for senescence, was induced in the hmg1 mutant at an earlier stage than in the WT. Levels of trans-cytokinins--hormones known to inhibit senescence--were not lower in hmg1. The mutant did not have the typical appearance of brassinosteroid (BR)-deficient mutants, except for a dwarf phenotype, because of the suppression of cell elongation. The expression of several genes involved in cell elongation was suppressed in hmg1. WT plants treated exogenously with inhibitors of sterol biosynthesis had similar gene expression and sterility characteristics as the hmg1 mutants. Pleiotropic phenotypes were rescued by feeding with squalene, the precursor of sterols and triterpenoids. The sterol levels in hmg1 mutants were lower than in the WT. These findings suggest that HMG1 plays a critical role in triterpene biosynthesis, and that sterols and/or triterpenoids contribute to cell elongation, senescence, and fertility.     

Cyanobacterial monogalactosyldiacylglycerol-synthesis pathway is involved in normal unsaturation of galactolipids and low-temperature adaptation of Synechocystis sp. PCC 6803

We characterized certain physiological functions of cyanobacterial monoglucosyldiacylglycerol using a Synechocystis sp. PCC 6803 mutant in which the gene for monoglucosyldiacylglycerol synthase had been disrupted and its function complemented by inclusion of an Arabidopsis monogalactosyldiacylglycerol synthase gene. By using this method, we prepared the first viable monoglucosyldiacylglycerol-deficient mutant of cyanobacterium and found that monoglucosyldiacylglycerol is not essential for its growth and photosynthesis under a set of “normal growth conditions” when monogalactosyldiacylglycerol is adequately supplied by the Arabidopsis monogalactosyldiacylglycerol synthase. The mutant had healthy thylakoid membranes and normal pigment content. The membrane lipid composition of the mutant was similar with that of WT except lack of monoglucosyldiacylglycerol and a slight increase in the level of phosphatidylglycerol at both normal and low temperatures. However, the ratio of unsaturated fatty acids in monogalactosyldiacylglycerol and digalactosyldiacylglycerol was reduced in the mutant compared with WT. Although the growth of the mutant was indistinguishable with that of WT at normal growth temperature, it was markedly retarded at low temperature compared with that of WT. Our data indicated the possibility that cyanobacterial monogalactosyldiacylglycerol-synthesis pathway might be required for the adequate unsaturation level of fatty acids in galactolipids and affect the low-temperature sensitivity.     

Proteome analysis of chlorotic leaves of the Arabidopsis mex1 mutant defective in the mobilization of starch degradation products

Leaf starch synthesized during the day for transient storage of photoassimilated carbon is degraded the following night to support respiration and growth in plants. Maltose is a major product of starch degradation, and is exported to the cytosol through the maltose transporter (MEX1). The Arabidopsis mex1 mutant displays growth retardation and an exceptional chlorotic phenotype that is not observed in other mutants demonstrating defective starch synthesis or degradation. Consistent with the chlorotic phenotype, proteomic analysis revealed degeneration of the photosynthetic machinery in mex1, and the down-regulation of essential components for photosynthesis was also observed. The chlorosis observed in mex1 occurs during vegetative growth period under normal growth conditions, which is distinct from general senescence-induced chlorosis. No up-regulation of senescence-related genes was found in the proteomic analysis of mex1, suggesting that the chlorotic process occurring in mex1 is likely distinct from senescence-dependent processes. On the other hand, cellular processes needed to survive stress situations caused by the blocking of maltose export are induced in mex1 by up-regulation of stress-related proteins, such as a germin-like protein and glutathione S-transferase. The increased abundance of heat shock protein 93-V participating in chloroplast biogenesis and rubisco activase, a regulatory protein of photosynthesis, likely reflects an attempt by the mex1 mutant to maintain chloroplast function to survive stress conditions.     

Characterization of the Xylella fastidiosa PD1671 Gene Encoding Degenerate c-di-GMP GGDEF/EAL Domains,and Its Role in the Development of Pierce’s Disease

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce’s disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.     

Relationship between fruiting body development and extracellular laccase production in the edible mushroom Flammulina velutipes

The biochemical mechanism underlying the development of fruiting bodies in Flammulina velutipes, an edible mushroom, was investigated using the YBLB colorimetric assay to distinguish between the normal strain (FVN-1) and the degenerate strain (FVD-1). In this assay, the color of the YBLB medium (blue-green) inoculated with FVN-1 exhibiting normal fruiting body development changed to yellow, while the color of the medium inoculated with FVD-1 changed to blue. In this study, we found that this color difference originated from extracellular laccase produced by FVN-1. Moreover, FVN-1 exhibited considerably higher extracellular laccase activity than FVD-1, under conditions facilitating fruiting body formation. Overall, these findings suggest that extracellular laccase is involved in the fruiting body development process in F. velutipes.     

Comparative genomics of the mating-type loci of the mushroom Flammulina velutipes reveals widespread synteny and recent inversions

Background

Mating-type loci of mushroom fungi contain master regulatory genes that control recognition between compatible nuclei, maintenance of compatible nuclei as heterokaryons, and fruiting body development. Regions near mating-type loci in fungi often show adapted recombination, facilitating the generation of novel mating types and reducing the production of self-compatible mating types. Compared to other fungi, mushroom fungi have complex mating-type systems, showing both loci with redundant function (subloci) and subloci with many alleles. The genomic organization of mating-type loci has been solved in very few mushroom species, which complicates proper interpretation of mating-type evolution and use of those genes in breeding programs.

Methodology/Principal Findings

We report a complete genetic structure of the mating-type loci from the tetrapolar, edible mushroom Flammulina velutipes mating type A3B3. Two matB3 subloci, matB3a that contains a unique pheromone and matB3b, were mapped 177 Kb apart on scaffold 1. The matA locus of F. velutipes contains three homeodomain genes distributed over 73 Kb distant matA3a and matA3b subloci. The conserved matA region in Agaricales approaches 350 Kb and contains conserved recombination hotspots showing major rearrangements in F. velutipes and Schizophyllum commune. Important evolutionary differences were indicated; separation of the matA subloci in F. velutipes was diverged from the Coprinopsis cinerea arrangement via two large inversions whereas separation in S. commune emerged through transposition of gene clusters.

Conclusions/Significance

In our study we determined that the Agaricales have very large scale synteny at matA (∼350 Kb) and that this synteny is maintained even when parts of this region are separated through chromosomal rearrangements. Four conserved recombination hotspots allow reshuffling of large fragments of this region. Next to this, it was revealed that large distance subloci can exist in matB as well. Finally, the genes that were linked to specific mating types will serve as molecular markers in breeding.     

Global Analysis of the Role of Autophagy in Cellular Metabolism and Energy Homeostasis in Arabidopsis Seedlings under Carbon Starvation

Germination and early seedling establishment are developmental stages in which plants face limited nutrient supply as their photosynthesis mechanism is not yet active. For this reason, the plant must mobilize the nutrient reserves provided by the mother plant in order to facilitate growth. Autophagy is a catabolic process enabling the bulk degradation of cellular constituents in the vacuole. The autophagy mechanism is conserved among eukaryotes, and homologs of many autophagy-related (ATG) genes have been found in Arabidopsis thaliana. T-DNA insertion mutants (atg mutants) of these genes display higher sensitivity to various stresses, particularly nutrient starvation. However, the direct impact of autophagy on cellular metabolism has not been well studied. In this work, we used etiolated Arabidopsis seedlings as a model system for carbon starvation. atg mutant seedlings display delayed growth in response to carbon starvation compared with wild-type seedlings. High-throughput metabolomic, lipidomic, and proteomic analyses were performed, as well as extensive flux analyses, in order to decipher the underlying causes of the phenotype. Significant differences between atg mutants and wild-type plants have been demonstrated, suggesting global effects of autophagy on central metabolism during carbon starvation as well as severe energy deprivation, resulting in a morphological phenotype.