Multicopy Suppressor Research Articles

Ssams2, a Gene Encoding GATA Transcription Factor, Is Required for Appressoria Formation and Chromosome Segregation in Sclerotinia sclerotiorum.

AMS2, a multicopy suppressor for the cpn1 (SpCENP-A) mutant, functions to specifically regulate histone genes transcription and chromosome segregation. As a cell-cycle-regulated GATA transcription factor in eukaryotic organisms, little research has been done on the role of AMS2 protein in pathogenic fungi. In Sclerotinia sclerotiorum, Ssams2 (SS1G_03252) encodes a protein which has been predicted to contain GATA-box domain. Here, Ssams2-silenced strains with significantly reduced Ssams2 gene expression levels exhibited defect in hyphal growth, hyphal branching patterns, compound appressoria differentiation and the oxalic acid production compared to the wild-type (WT) strain. By common bean leaves infection assays, we identified the role of Ssams2 in full virulence. Furthermore, the numbers of cell nucleus in the same length of mycelium in Ssams2-silenced transformants were significantly less than that in the WT strain. The expression levels of histone genes and cell cycle genes in transformants were down-regulated significantly in the RNAi strains. Taken together, our work suggests that the TF SsAMS2 is required for growth, appressoria formation, virulence, and chromosome segregation in S. sclerotiorum.

Interaction network of the mitochondrial outer membrane protein Mcp3.

Mitochondria are organelles containing two membranes that are distinct in composition and function. A role of the mitochondrial outer membrane (MOM) is to mediate contact of the organelle with the rest of the cell. In yeast, the MOM contains about 40 different integral proteins. Recently, we described the MOM protein Mcp3, which can serve as a multicopy suppressor of loss of ERMES complex that mediates mitochondria-endoplasmic reticulum contacts. To shed further light on the role of Mcp3 in the MOM, we analyzed its physical interaction with other proteins. We show that Mcp3 interacts with the MOM protein Om45 and the inner membrane protein Aim19. Our observations hint toward a potential involvement of Mcp3 in a structural and/or functional link between both mitochondrial membranes.

Mannosylinositol phosphorylceramides and ergosterol coodinately maintain cell wall integrity in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group, and breakdown of their normal composition causes several cellular dysfunctions. Previously we found that loss of biosynthesis of mannosylinositol phosphorylceramide (MIPC) causes a defect in cell wall integrity (CWI). In this study, we screened for multicopy suppressor genes that rescue the defect in CWI in cells lacking MIPC synthases (Sur1 and Csh1), and found that the defect is partly suppressed by upregulation of ergosterol biosynthesis. In addition, repression of expression of ERG9, which encodes squalene synthase in the ergosterol biosynthesis pathway, in sur1∆ csh1∆ cells caused a strong growth defect and enhancement of the defect in CWI. The repression of ERG9 and/or the deletion of SUR1 and CSH1 caused an increase in the phosphorylated form of Slt2, a mitogen-activated protein kinase activated through impairment of CWI. Moreover, the deletion of SLT2 or WSC1/2, encoding a sensor protein recognizing CWI, enhanced the growth defect in the ERG9-repressed sur1∆ csh1∆ cells. On the other hand, the ERG9-repressed sur1∆ csh1∆ cells also exhibited an increase in the cell wall chitin level in a Slt2- and Wsc1/2-independent manner. These results suggested that MIPC and ergosterol are coordinately involved in maintenance of CWI, and the activation of Slt2 suppressed the CWI defect caused by these metabolic defects.

Increased Activity of Cystathionine β-Lyase Suppresses 2-Aminoacrylate Stress in Salmonella enterica.

Reactive enamine stress caused by intracellular 2-aminoacrylate accumulation leads to pleiotropic growth defects in a variety of organisms. Members of the well-conserved RidA/YER057c/UK114 protein family prevent enamine stress by enhancing the breakdown of 2-aminoacrylate to pyruvate. In Salmonella enterica, disruption of RidA allows 2-aminoacrylate to accumulate and to inactivate a variety of pyridoxal 5'-phosphate-dependent enzymes by generating covalent bonds with the enzyme and/or cofactor. This study was initiated to identify mechanisms that can overcome 2-aminoacrylate stress in the absence of RidA. Multicopy suppressor analysis revealed that overproduction of the methionine biosynthesis enzyme cystathionine β-lyase (MetC) (EC 4.4.1.8) alleviated the pleiotropic consequences of 2-aminoacrylate stress in a ridA mutant strain. Degradation of cystathionine by MetC was not required for suppression of ridA phenotypes. The data support a model in which MetC acts on a noncystathionine substrate to generate a metabolite that reduces 2-aminoacrylate levels, representing a nonenzymatic mechanism of 2-aminoacrylate depletion.IMPORTANCE RidA proteins are broadly conserved and have been demonstrated to deaminate 2-aminoacrylate and other enamines. 2-Aminoacrylate is generated as an obligatory intermediate in several pyridoxal 5'-phosphate-dependent reactions; if it accumulates, it damages cellular enzymes. This study identified a novel mechanism to eliminate 2-aminoacrylate stress that required the overproduction, but not the canonical activity, of cystathionine β-lyase. The data suggest that a metabolite-metabolite interaction is responsible for quenching 2-aminoacrylate, and they emphasize the need for emerging technologies to probe metabolism in vivo.

Investigating the Role of DNA Polymerase IV in the Resolution of R‐loops

As the replication fork progresses through the DNA template, it may encounter a number of obstacles, one of which is the RNA transcription machinery. This encounter occurs because both processes utilize the same template and because the rate of replication (~1000 nt/s) is much faster than the rate of transcription (40–80 nt/s). The encounter of the two machineries, in either a co‐directional or a head‐on orientation, results in replication‐transcription collisions (RTCs). Failure to resolve these collisions leads to replication fork stalling and eventually cell death. One of the events required to resolve RTCs is the removal of RNA: DNA hybrids, known as R‐loops. In E. coli, RNase H1, coded by rnhA, is a member of a family of highly conserved endonucleases mediating resolution of R‐loops by cleaving the RNA strand on RNA: DNA hybrids. If not resolved, R‐loops cause genomic instability in the form of double‐stranded breaks through the collapse of the replication fork and accumulation of DNA lesions.In E. coli, both replication stalling and accumulation of DNA lesions leads to induction of the SOS response. Among the first genes to be upregulated is dinB, encoding DNA polymerase IV (DinB), becoming the most abundant DNA polymerase in the cell (~2500 nM). DinB main role is to synthesize DNA over template lesions. There is evidence to support the notion that DinB is involved in recombination, a mechanism that is used to repair double‐stranded breaks. In stressful conditions, DinB, due to its abundance, outcompetes other polymerases for the recombination intermediate and synthesizes DNA from it. Because of its abundance during SOS induction and its role in recombination, DinB may also be involved in the resolution of R‐loops. Here, we investigate the possible role of DinB in R‐loop resolution in a rnhA‐deletion strain. Loss of rnhA results in slow growth that is rescued by expression of DinB from a low copy plasmid. This data suggest DinB is multicopy suppressor of the rnhA deletion and that DinB plays a role in the resolution of R‐loops.Support or Funding InformationNational Institute of General Medical Sciences (RO1GM088230 to VG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phospholipid flippases and Sfk1p, a novel regulator of phospholipid asymmetry, contribute to low permeability of the plasma membrane.

Phospholipid flippase (type 4 P-type ATPase) plays a major role in the generation of phospholipid asymmetry in eukaryotic cell membranes. Loss of Lem3p-Dnf1/2p flippases leads to the exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell surface in yeast, resulting in sensitivity to PS- or PE-binding peptides. We isolated Sfk1p, a conserved membrane protein in the TMEM150/FRAG1/DRAM family, as a multicopy suppressor of this sensitivity. Overexpression of SFK1 decreased PS/PE exposure in lem3Δ mutant cells. Consistent with this, lem3Δ sfk1Δ double mutant cells exposed more PS/PE than the lem3Δ mutant. Sfk1p was previously implicated in the regulation of the phosphatidylinositol-4 kinase Stt4p, but the effect of Sfk1p on PS/PE exposure in lem3Δ was independent of Stt4p. Surprisingly, Sfk1p did not facilitate phospholipid flipping but instead repressed it, even under ATP-depleted conditions. We propose that Sfk1p negatively regulates transbilayer movement of phospholipids irrespective of directions. In addition, we showed that the permeability of the plasma membrane was dramatically elevated in the lem3Δ sfk1Δ double mutant in comparison with the corresponding single mutants. Interestingly, total ergosterol was decreased in the lem3Δ sfk1Δ mutant. Our results suggest that phospholipid asymmetry is required for the maintenance of low plasma membrane permeability.

Coupling next-generation sequencing to dominant positive screens for finding antibiotic cellular targets and resistance mechanisms in Escherichia coli.

In order to expedite the discovery of genes coding for either drug targets or antibiotic resistance, we have developed a functional genomic strategy termed Plas-Seq. This technique involves coupling a multicopy suppressor library to next-generation sequencing. We generated an Escherichia coli plasmid genomic library that was transformed into E. coli. These transformants were selected step by step using 0.25× to 2× minimum inhibitory concentrations for ceftriaxone, gentamicin, levofloxacin, tetracycline or trimethoprim. Plasmids were isolated at each selection step and subjected to Illumina sequencing. By searching for genomic loci whose sequencing coverage increased with antibiotic pressure we were able to detect 48 different genomic loci that were enriched by at least one antibiotic. Fifteen of these loci were studied functionally, and we showed that 13 can decrease the susceptibility of E. coli to antibiotics when overexpressed. These genes coded for drug targets, transcription factors, membrane proteins and resistance factors. The technique of Plas-Seq is expediting the discovery of genes associated with the mode of action or resistance to antibiotics and led to the isolation of a novel gene influencing drug susceptibility. It has the potential for being applied to novel molecules and to other microbial species.

Protective role of the HOG pathway against the growth defect caused by impaired biosynthesis of complex sphingolipids in yeast Saccharomyces cerevisiae.

Complex sphingolipids play critical roles in various cellular events in the yeast Saccharomyces cerevisiae. To identify genes that are related to the growth defect caused by disruption of complex sphingolipid biosynthesis, we screened for suppressor mutations and multicopy suppressor genes that confer resistance against repression of AUR1 encoding inositol phosphorylceramide synthase. From the results of this screening, we found that the activation of high-osmolarity glycerol (HOG) pathway is involved in suppression of growth defect caused by impaired biosynthesis of complex sphingolipids. Furthermore, it was found that transcriptional regulation via Msn2, Msn4 and Sko1 is involved in the suppressive effect of the HOG pathway. Lack of the HOG pathway did not enhance the reductions in complex sphingolipid levels or the increase in ceramide level caused by the AUR1 repression, implying that the suppressive effect of the HOG pathway on the growth defect is not attributed to restoration of impaired biosynthesis of complex sphingolipids. On the contrary, the HOG pathway and Msn2/4-mediated transcriptional activation was involved in suppression of aberrant reactive oxygen species accumulation caused by the AUR1 repression. These results indicated that the HOG pathway plays pivotal roles in maintaining cell growth under impaired biosynthesis of complex sphingolipids.

Overexpression of PDR16 confers resistance to complex sphingolipid biosynthesis inhibitor aureobasidin A in yeast Saccharomyces cerevisiae.

Sphingolipids are essential for normal cell growth of yeast Saccharomyces cerevisiae. Aureobasidin A (AbA), an antifungal drug, inhibits Aur1, an enzyme catalyzing the synthesis of inositol phosphorylceramide, and induces a strong growth defect in yeast. In this study, we screened for multicopy suppressor genes that confer resistance to AbA, and identified PDR16. In addition, it was found that PDR17, a paralog of PDR16, also functions as a multicopy suppressor. Pdr16 and Pdr17 belong to a family of phosphatidylinositol transfer proteins; however, cells overexpressing the other members of the family hardly exhibited resistance to AbA. Overexpression of a lipid-binding defective mutant of Pdr16 did not confer the resistance to AbA, indicating that the lipid-binding activity is essential for acquiring resistance to AbA. When expression of the AUR1 gene was repressed by a tetracycline-regulatable promoter, the overexpression of PDR16 or PDR17 did not suppress the growth defect caused by the AUR1 repression. Quantification analysis of complex sphingolipids revealed that in AbA-treated cells, but not in cells in which AUR1 was repressed by the tetracycline-regulatable promoter, the reductions of complex sphingolipid levels were suppressed by the overexpressed PDR16. Thus, it was indicated that the overexpression of PDR16 reduces the effectiveness of AbA against intracellular Aur1 activity.

Phenotypes of dnaXE145A Mutant Cells Indicate that the Escherichia coli Clamp Loader Has a Role in the Restart of Stalled Replication Forks.

The Escherichia colidnaXE145A mutation was discovered in connection with a screen for multicopy suppressors of the temperature-sensitive topoisomerase IV mutation parE10 The gene for the clamp loader subunits τ and γ, dnaX, but not the mutant dnaXE145A , was found to suppress parE10(Ts) when overexpressed. Purified mutant protein was found to be functional in vitro, and few phenotypes were found in vivo apart from problems with partitioning of DNA in rich medium. We show here that a large number of the replication forks that initiate at oriC never reach the terminus in dnaXE145A mutant cells. The SOS response was found to be induced, and a combination of the dnaXE145A mutation with recBC and recA mutations led to reduced viability. The mutant cells exhibited extensive chromosome fragmentation and degradation upon inactivation of recBC and recA, respectively. The results indicate that the dnaXE145A mutant cells suffer from broken replication forks and that these need to be repaired by homologous recombination. We suggest that the dnaX-encoded τ and γ subunits of the clamp loader, or the clamp loader complex itself, has a role in the restart of stalled replication forks without extensive homologous recombination.IMPORTANCE The E. coli clamp loader complex has a role in coordinating the activity of the replisome at the replication fork and loading β-clamps for lagging-strand synthesis. Replication forks frequently encounter obstacles, such as template lesions, secondary structures, and tightly bound protein complexes, which will lead to fork stalling. Some pathways of fork restart have been characterized, but much is still unknown about the actors and mechanisms involved. We have in this work characterized the dnaXE145A clamp loader mutant. We find that the naturally occurring obstacles encountered by a replication fork are not tackled in a proper way by the mutant clamp loader and suggest a role for the clamp loader in the restart of stalled replication forks.

TsNAC1 Is a Key Transcription Factor in Abiotic Stress Resistance and Growth.

NAC proteins constitute one of the largest families of plant-specific transcription factors, and a number of these proteins participate in the regulation of plant development and responses to abiotic stress. T. HALOPHILA STRESS RELATED NAC1 (TsNAC1), cloned from the halophyte Thellungiella halophila, is a NAC transcription factor gene, and its overexpression can improve abiotic stress resistance, especially in salt stress tolerance, in both T. halophila and Arabidopsis (Arabidopsis thaliana) and retard the growth of these plants. In this study, the transcriptional activation activity of TsNAC1 and RD26 from Arabidopsis was compared with the target genes' promoter regions of TsNAC1 from T. halophila, and the results showed that the transcriptional activation activity of TsNAC1 was higher in tobacco (Nicotiana tabacum) and yeast. The target sequence of the promoter from the target genes also was identified, and TsNAC1 was shown to target the positive regulators of ion transportation, such as T. HALOPHILA H+-PPASE1, and the transcription factors MYB HYPOCOTYL ELONGATION-RELATED and HOMEOBOX12 In addition, TsNAC1 negatively regulates the expansion of cells, inhibits LIGHT-DEPENDENT SHORT HYPOCOTYLS1 and UDP-XYLOSYLTRANSFERASE2, and directly controls the expression of MULTICOPY SUPPRESSOR OF IRA14 Based on these results, we propose that TsNAC1 functions as an important upstream regulator of plant abiotic stress responses and vegetative growth.

The stress-regulatory transcription factors Msn2 and Msn4 regulate fatty acid oxidation in budding yeast

The transcription factors Msn2 and Msn4 (multicopy suppressor of SNF1 mutation proteins 2 and 4) bind the stress-response element in gene promoters in the yeast Saccharomyces cerevisiae However, the roles of Msn2/4 in primary metabolic pathways such as fatty acid β-oxidation are unclear. Here, in silico analysis revealed that the promoters of most genes involved in the biogenesis, function, and regulation of the peroxisome contain Msn2/4-binding sites. We also found that transcript levels of MSN2/MSN4 are increased in glucose-depletion conditions and that during growth in nonpreferred carbon sources, Msn2 is constantly localized to the nucleus in wild-type cells. Of note, the double mutant msn2Δmsn4Δ exhibited a severe growth defect when grown with oleic acid as the sole carbon source and had reduced transcript levels of major β-oxidation genes. ChIP indicated that Msn2 has increased occupancy on the promoters of β-oxidation genes in glucose-depleted conditions, and in vivo reporter gene analysis indicated reduced expression of these genes in msn2Δmsn4Δ cells. Moreover, mobility shift assays revealed that Msn4 binds β-oxidation gene promoters. Immunofluorescence microscopy with anti-peroxisome membrane protein antibodies disclosed that the msn2Δmsn4Δ strain had fewer peroxisomes than the wild type, and lipid analysis indicated that the msn2Δmsn4Δ strain had increased triacylglycerol and steryl ester levels. Collectively, our data suggest that Msn2/Msn4 transcription factors activate expression of the genes involved in fatty acid oxidation. Because glucose sensing, signaling, and fatty acid β-oxidation pathways are evolutionarily conserved throughout eukaryotes, the msn2Δmsn4Δ strain could therefore be a good model system for further study of these critical processes.

Involvement of the exomer complex in the polarized transport of Ena1 required for Saccharomyces cerevisiae survival against toxic cations.

Exomer is an adaptor complex required for the direct transport of a selected number of cargoes from the trans-Golgi network (TGN) to the plasma membrane in Saccharomyces cerevisiae However, exomer mutants are highly sensitive to increased concentrations of alkali metal cations, a situation that remains unexplained by the lack of transport of any known cargoes. Here we identify several HAL genes that act as multicopy suppressors of this sensitivity and are connected to the reduced function of the sodium ATPase Ena1. Furthermore, we find that Ena1 is dependent on exomer function. Even though Ena1 can reach the plasma membrane independently of exomer, polarized delivery of Ena1 to the bud requires functional exomer. Moreover, exomer is required for full induction of Ena1 expression after cationic stress by facilitating the plasma membrane recruitment of the molecular machinery involved in Rim101 processing and activation of the RIM101 pathway in response to stress. Both the defective localization and the reduced levels of Ena1 contribute to the sensitivity of exomer mutants to alkali metal cations. Our work thus expands the spectrum of exomer-dependent proteins and provides a link to a more general role of exomer in TGN organization.

Mitochondrial Cochaperone Mge1 Is Involved in Regulating Susceptibility to Fluconazole in Saccharomyces cerevisiae and Candida Species.

ABSTRACTMGE1 encodes a yeast chaperone involved in Fe-S cluster metabolism and protein import into the mitochondria. In this study, we identified MGE1 as a multicopy suppressor of susceptibility to the antifungal fluconazole in the model yeast Saccharomyces cerevisiae. We demonstrate that this phenomenon is not exclusively dependent on the integrity of the mitochondrial DNA or on the presence of the drug efflux pump Pdr5. Instead, we show that the increased dosage of Mge1 plays a protective role by retaining increased amounts of ergosterol upon fluconazole treatment. Iron metabolism and, more particularly, Fe-S cluster formation are involved in regulating this process, since the responsible Hsp70 chaperone, Ssq1, is required. Additionally, we show the necessity but, by itself, insufficiency of activating the iron regulon in establishing the Mge1-related effect on drug susceptibility. Finally, we confirm a similar role for Mge1 in fluconazole susceptibility in the pathogenic fungi Candida glabrata and Candida albicans.

GSK-3β Homolog Rim11 and the Histone Deacetylase Complex Ume6-Sin3-Rpd3 Are Involved in Replication Stress Response Caused by Defects in Dna2.

Lagging strand synthesis is mechanistically far more complicated than leading strand synthesis because it involves multistep processes and requires considerably more enzymes and protein factors. Due to this complexity, multiple fail-safe factors are required to ensure successful replication of the lagging strand DNA. We attempted to identify novel factors that are required in the absence of the helicase activity of Dna2, an essential enzyme in Okazaki-fragment maturation. In this article, we identified Rim11, a GSK-3β-kinase homolog, as a multicopy suppressor of dna2 helicase-dead mutant (dna2-K1080E). Subsequent epistasis analysis revealed that Ume6 (a DNA binding protein, a downstream substrate of Rim11) also acted as a multicopy suppressor of the dna2 allele. We found that the interaction of Ume6 with the conserved histone deacetylase complex Sin3-Rpd3 and the catalytic activity of Rpd3 were indispensable for the observed suppression of the dna2 mutant. Moreover, multicopy suppression by Rim11/Ume6 requires the presence of sister-chromatid recombination mediated by Rad52/Rad59 proteins, but not vice versa. Interestingly, the overexpression of Rim11 or Ume6 also suppressed the MMS sensitivity of rad59Δ. We also showed that the lethality of dna2 helicase-dead mutant was attributed to checkpoint activation and that decreased levels of deoxynucleotide triphosphates (dNTPs) by overexpressing Sml1 (an inhibitor of ribonucleotide reductase) rescued the dna2 mutant. We also present evidence that indicates Rim11/Ume6 works independently but in parallel with that of checkpoint inhibition, dNTP regulation, and sister-chromatid recombination. In conclusion, our results establish Rim11, Ume6, the histone deacetylase complex Sin3-Rpd3 and Sml1 as new factors important in the events of faulty lagging strand synthesis.

Developmental patterning of the sub-epidermal integument cell layer in Arabidopsis seeds.

Angiosperm seed development is a paradigm of tissue cross-talk. Proper seed formation requires spatial and temporal coordination of the fertilization products – embryo and endosperm – and the surrounding seed coat maternal tissue. In early Arabidopsis seed development, all seed integuments were thought to respond homogenously to endosperm growth. Here, we show that the sub-epidermal integument cell layer has a unique developmental program. We characterized the cell patterning of the sub-epidermal integument cell layer, which initiates a previously uncharacterized extra cell layer, and identified TRANSPARENT TESTA 16 and SEEDSTICK MADS box transcription factors as master regulators of its polar development and cell architecture. Our data indicate that the differentiation of the sub-epidermal integument cell layer is insensitive to endosperm growth alone and to the repressive mechanism established by FERTILIZATION INDEPENDENT ENDOSPERM and MULTICOPY SUPPRESSOR OF IRA1 Polycomb group proteins. This work demonstrates the different responses of epidermal and sub-epidermal integument cell layers to fertilization.

Identification of multi-copy suppressors for endoplasmic reticulum-mitochondria tethering proteins inSaccharomyces cerevisiae

In yeast, the endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) tethers the ER to mitochondria, but its primary function remains unclear. To gain insight into ERMES functions, we screened multi-copy suppressors of the growth-defective phenotype of mmm1∆ cells, which lack a core component of ERMES, and identified MCP1, MGA2, SPT23, and YGR250C (termed RIE1). Spt23 and Mga2 are homologous transcription factors known to activate transcription of the OLE1 gene, which encodes the fatty acid ∆9 desaturase. We found that Ole1 partially relieves the growth defects of ERMES-lacking cells, thus uncovering a relationship between fatty acid metabolism and ERMES functions.

Dual Fatty Acid Elongase Complex Interactions in Arabidopsis.

Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase 3 hydroxyacyl-CoA dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFAs elongation but not the plant pas2-1 defects. PTPLA interacted with elongase subunits in the Endoplasmic Reticulum (ER) and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function increased VLCFA levels, an effect that was dependent on the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted to the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes based on PAS2 or PTPLA dehydratase that are functionally interacting.

Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress

Human Nedd4 ubiquitin ligase, or its variants, inhibit yeast cell growth by disturbing the actin cytoskeleton organization and dynamics, and lead to an increase in levels of ubiquitinated proteins. In a screen for multicopy suppressors which rescue growth of yeast cells producing Nedd4 ligase with an inactive WW4 domain (Nedd4w4), we identified a fragment of ATG2 gene encoding part of the Atg2 core autophagy protein. Expression of the Atg2-C1 fragment (aa 1074-1447) improved growth, actin cytoskeleton organization, but did not significantly change the levels of ubiquitinated proteins in these cells. The GFP-Atg2-C1 protein in Nedd4w4-producing cells primarily localized to a single defined structure adjacent to the vacuole, surrounded by an actin filament ring, containing Hsp42 and Hsp104 chaperones. This localization was not affected in several atg deletion mutants, suggesting that it might be distinct from the phagophore assembly site (PAS). However, deletion of ATG18 encoding a phosphatidylinositol-3-phosphate (PI3P)-binding protein affected the morphology of the GFP-Atg2-C1 structure while deletion of ATG14 encoding a subunit of PI3 kinase suppressed toxicity of Nedd4w4 independently of GFP-Atg2-C1. Further analysis of the Atg2-C1 revealed that it contains an APT1 domain of previously uncharacterized function. Most importantly, we showed that this domain is able to bind phosphatidylinositol phosphates, especially PI3P, which is abundant in the PAS and endosomes. Together our results suggest that human Nedd4 ubiquitinates proteins in yeast and causes proteotoxic stress and, with some Atg proteins, leads to formation of a perivacuolar structure, which may be involved in sequestration, aggregation or degradation of proteins.

Polycomb-group protein SlMSI1 represses the expression of fruit-ripening genes to prolong shelf life in tomato.

Polycomb-group (PcG) protein MULTICOPY SUPPRESSOR OF IRA1 (MSI1) protein is an evolutionarily conserved developmental suppressor and plays a crucial role in regulating epigenetic modulations. However, the potential role and function of MSI1 in fleshy fruits remain unknown. In this study, SlMSI1 was cloned and transformed into tomato to explore its function. The quantitative real-time PCR results showed that SlMSI1 was highly expressed in flowers and fruits and that its transcript and protein levels were significantly decreased in fruits after the breaker stage. Additionally, SlMSI1-overexpressing transgenic tomatoes displayed abnormal non-ripening fruit formation, whereas its suppression promoted fruit ripening in transgenic tomatoes. Quantitative real-time PCR assays also showed that RIN and its regulons were decreased in SlMSI1 overexpression transgenic tomato fruits. Furthermore, RNA-seq analysis demonstrated that SlMSI1 inhibits fruit ripening by negatively regulating a large set of fruit-ripening genes in addition to RIN and its regulons. Finally, genetic manipulation of SlMSI1 and RIN successfully prolonged the fruit shelf life by regulating the fruit-ripening genes in tomato. Our findings reveal a novel regulatory function of SlMSI1 in fruit ripening and provide a new regulator that may be useful for genetic engineering and modification of fruit shelf life.