A Mutation in an Hsp90 Gene Affects the Sexual Cycle and Suppresses Vegetative Incompatibility in the Fungus Podospora Anserina

Vegetative incompatibility is widespread in fungi but its molecular mechanism and biological function are still poorly understood. A way to study vegetative incompatibility is to investigate the function of genes whose mutations suppress this phenomenon. In Podospora anserina, these genes are known as mod genes. In addition to suppressing vegetative incompatibility, mod mutations cause some developmental defects. This suggests that the molecular mechanisms of vegetative incompatibility and development pathways are interconnected. The mod-E1 mutation was isolated as a suppressor of the developmental defects of the mod-D2 strain. We show here that mod-E1 also partially suppresses vegetative incompatibility, strengthening the link between development and vegetative incompatibility. mod-E1 is the first suppressor of vegetative incompatibility characterized at the molecular level. It encodes a member of the Hsp90 family, suggesting that development and vegetative incompatibility use common steps of a signal transduction pathway. The involvement of mod-E in the sexual cycle has also been further investigated.     

Altered Mating-Type Identity in the Fungus Podospora Anserina Leads to Selfish Nuclei, Uniparental Progeny, and Haploid Meiosis

In wild-type crosses of the filamentous ascomycete Podospora anserina, after fertilization, only nuclei of opposite mating type can form dikaryons that undergo karyogamy and meiosis, producing biparental progeny. To determine the role played by the mating type in these steps, the four mat genes were mutagenized in vitro and introduced into a strain deleted for its mat locus. Genetic and cytological analyses of these mutant strains, crossed to each other and to wild type, showed that mating-type information is required for recognition of nuclear identity during the early steps of sexual reproduction. In crosses with strains carrying a mating-type mutation, two unusual developmental patterns were observed: monokaryotic cells, resulting in haploid meiosis, and uniparental dikaryotic cells providing, after karyogamy and meiosis, a uniparental progeny. Altered mating-type identity leads to selfish behavior of the mutant nucleus: it migrates alone or paired, ignoring its wild-type partner in all mutant X wild-type crosses. This behavior is nucleus-autonomous because, in the same cytoplasm, the wild-type nuclei form only biparental dikaryons. In P. anserina, mat genes are thus required to ensure a biparental dikaryotic state but appear dispensable for later stages, such as meiosis and sporulation.     

A Single Vegetative Actin Isovariant Overexpressed under the Control of Multiple Regulatory Sequences Is Sufficient for Normal Arabidopsis Development

The relative significance of gene regulation and protein isovariant differences remains unexplored for most gene families, particularly those participating in multicellular development. Arabidopsis thaliana encodes three vegetative actins, ACT2, ACT7, and ACT8, in two ancient and highly divergent subclasses. Mutations in any of these differentially expressed actins revealed only mild phenotypes. However, double mutants were extremely dwarfed, with altered cell and organ morphology and an aberrant F-actin cytoskeleton (e.g., act2-1 act7-4 and act8-2 act7-4) or totally root-hairless (e.g., act2-1 act8-2). Our studies suggest that the three vegetative actin genes and protein isovariants play distinct subclass-specific roles during plant morphogenesis. For example, during root development, ACT7 was involved in root growth, epidermal cell specification, cell division, and root architecture, and ACT2 and ACT8 were essential for root hair tip growth. Also, genetic complementation revealed that the ACT2 and ACT8 isovariants, but not ACT7, fully rescued the root hair growth defects of single and double mutants. Moreover, we synthesized fully normal plants overexpressing the ACT8 isovariant from multiple actin regulatory sequences as the only vegetative actin in the act2-1 act7-4 background. In summary, it is evident that differences in vegetative actin gene regulation and the diversity in actin isovariant sequences are essential for normal plant development.     

Cloning the Mating Types of the Heterothallic Fungus Podospora Anserina: Developmental Features of Haploid Transformants Carrying Both Mating Types

DNAs that encode the mating-type functions (mat+ and mat-) of the filamentous fungus Podospora anserina were cloned with the use of the mating-type A probe from Neurospora crassa. Cloning the full mat information was ascertained through gene replacement experiments. Molecular and functional analyses of haploid transformants carrying both mating types lead to several striking conclusions. Mat+ mat- strains are dual maters. However, the resident mat information is dominant to the mat information added by transformation with respect to fruiting body development and ascus production. Moreover, when dual mating mat+ mat- strains are crossed to mat+ or mat- testers, there is strong selection, after fertilization, that leads to the loss from the mat+ mat- nucleus of the mat information that matches that of the tester. Finally, the mat locus contains at least two domains, one sufficient for fertilization, the other necessary for sporulation.     

Vegetative Incompatibility Loci with Dedicated Roles in Allorecognition Restrict Mycovirus Transmission in Chestnut Blight Fungus

Vegetative incompatibility (vic), a form of nonself allorecognition, operates widely in filamentous fungi and restricts transmission of virulence-attenuating hypoviruses in the chestnut blight fungus Cryphonectria parasitica. We report here the use of a polymorphism-based comparative genomics approach to complete the molecular identification of the genetically defined C. parasitica vic loci with the identification of vic1 and vic3. The vic1 locus in the C. parasitica reference strain EP155 consists of a polymorphic HET-domain-containing 771-aa ORF designated vic1a-2, which shares 91% identity with the corresponding vic1a-1 allele, and a small (172 aa) idiomorphic DUF1909-domain-containing ORF designated vic1b-2 that is absent at the vic1-1 locus. Gene disruption of either vic1a-2 or vic1b-2 in strain EP155 eliminated restrictions on virus transmission when paired with a vic1 heteroallelic strain; however, only disruption of vic1a-2 abolished the incompatible programmed cell death (PCD) reaction. The vic3 locus of strain EP155 contains two polymorphic ORFs of 599 aa (vic3a-1) and 102 aa (vic3b-1) that shared 46 and 85% aa identity with the corresponding vic3a-2 and vic3b-2 alleles, respectively. Disruption of either vic3a-1 or vic3b-1 resulted in increased virus transmission. However, elimination of PCD required disruption of both vic3a and vic3b. Additional allelic heterogeneity included a sequence inversion and a 8.5-kb insertion containing a LTR retrotransposon sequence and an adjacent HET-domain gene at the vic1 locus and a 7.7-kb sequence deletion associated with a nonfunctional, pseudo vic locus. Combined gene disruption studies formally confirmed restriction of mycovirus transmission by five C. parasitica vic loci and suggested dedicated roles in allorecognition. The relevance of these results to the acquisition and maintenance of vic genes and the potential for manipulation of vic alleles for enhanced mycovirus transmission are discussed.     

A Single ClpS Monomer Is Sufficient to Direct the Activity of the ClpA Hexamer

ClpS is an adaptor protein that interacts with ClpA and promotes degradation of proteins with N-end rule degradation motifs (N-degrons) by ClpAP while blocking degradation of substrates with other motifs. Although monomeric ClpS forms a 1:1 complex with an isolated N-domain of ClpA, only one molecule of ClpS binds with high affinity to ClpA hexamers (ClpA₆). One or two additional molecules per hexamer bind with lower affinity. Tightly bound ClpS dissociates slowly from ClpA₆ with a t_½ of ∼3 min at 37 °C. Maximum activation of degradation of the N-end rule substrate, LR-GFP_Venus, occurs with a single ClpS bound per ClpA₆; one ClpS is also sufficient to inhibit degradation of proteins without N-degrons. ClpS competitively inhibits degradation of unfolded substrates that interact with ClpA N-domains and is a non-competitive inhibitor with substrates that depend on internal binding sites in ClpA. ClpS inhibition of substrate binding is dependent on the order of addition. When added first, ClpS blocks binding of both high and low affinity substrates; however, when substrates first form committed complexes with ClpA₆, ClpS cannot displace them or block their degradation by ClpP. We propose that the first molecule of ClpS binds to the N-domain and to an additional functional binding site, sterically blocking binding of non-N-end rule substrates as well as additional ClpS molecules to ClpA₆. Limiting ClpS-mediated substrate delivery to one per ClpA₆ avoids congestion at the axial channel and allows facile transfer of proteins to the unfolding and translocation apparatus.     

Encephalitogenicity of Murine,But Not Bovine,DM20 in SJL Mice Is Due to a Single Amino Acid Difference in the Immunodominant Encephalitogenic Epitope

Myelin proteolipid protein (PLP) contains 2 immunodominant encephalitogenic epitopes in SJL mice, namely PLP residues 139–151 and 178–191. DM20, a minor isoform of PLP, lacks residues 116–150 and consequently contains only the single major encephalitogenic epitope 178–191. However, it has been found previously that bovine DM20 is not encephalitogenic in SJL mice. Since residue 188 within peptide 178–191 is phenylalanine (F) in murine DM20 and alanine (A) in bovine DM20, we tested the effect of this difference on the immune responses and induction of EAE. SJL mice were immunized with either highly purified murine or bovine DM20. Residues 178–191 were found to be immunodominant for each, but only murine and not bovine DM20 was encephalitogenic. A synthetic peptide corresponding to the murine 178–191 sequence (F188) was also encephalitogenic, whereas the peptide corresponding to the bovine sequence (A 188) was not. Both F188 and A188 bind with high affinity to I-A^s and both are recognized by the SJL T cell repertoire. A188-specific T cell lines reacted to both A188 and F188, but F188-specific T cell lines were not stimulated by A188. F188-specific T cell lines produced mRNA for the Thl cytokines IL2 and IFN, and, in passive transfer experiments, were encephalitogenic upon stimulation with F188, but not A188. In contrast, A188-specific T cell lines produced mRNA for IL4, IL5 and IL10, in addition to IL2 and IFN, and were not encephalitogenic after stimulation with either F188 or A188. Cotransfer of A188-specific T cell lines with F188-specific T cell lines resulted in protection from EAE. Thus, A188 induces a functionally different phenotype of T cells from that induced by F188. Taken together these data suggest that the failure of bovine DM20 to induce EAE may be attributable to induction of protective rather than pathogenic T cells by the immunodominant epitope.     

Single Amino Acid Change in STING Leads to Constitutive Active Signaling

The production of cytokines by the immune system in response to cytosolic DNA plays an important role in host defense, autoimmune disease, and cancer immunogenicity. Recently a cytosolic DNA signaling pathway that is dependent on the endoplasmic reticulum adaptor and cyclic dinucleotide sensor protein STING has been identified. Association of cytosolic DNA with cyclic-GMP-AMP synthase (cGAS) activates its enzymatic activity to synthesize the cyclic dinucleotide second messenger cGAMP from GTP and ATP. Direct detection of cGAMP by STING triggers the activation of IRF3 and NF-kB, and the production of type I interferons and proinflammatory cytokines. The mechanism of how STING is able to mediate downstream signaling remains incompletely understood although it has been shown that dimerization is a prerequisite. Here, we identify a single amino acid change in STING that confers constitutive active signaling. This mutation appears to both enhance ability of STING to both dimerize and associate with its downstream target TBK1.     

Identification of Single Amino Acid Substitutions in Proteogenomics

An important aim of proteogenomics, which combines data of high throughput nucleic acid and protein analysis, is to reliably identify single amino acid substitutions representing a main type of coding genome variants. Exact knowledge of deviations from the consensus genome can be utilized in several biomedical fields, such as studies of expression of mutated proteins in cancer, deciphering heterozygosity mechanisms, identification of neoantigens in anticancer vaccine production, search for RNA editing sites at the level of the proteome, etc. Generation of this new knowledge requires processing of large data arrays from high–resolution mass spectrometry, where information on single–point protein variation is often difficult to extract. Accordingly, a significant problem in proteogenomic analysis is the presence of high levels of false positive results for variant–containing peptides in the produced results. Here we review recently suggested approaches of high quality proteomics data processing that may provide more reliable identification of single amino acid substitutions, especially contrary to residue modifications occurring in vitro and in vivo. Optimized methods for assessment of false discovery rate save instrumental and computational time spent for validation of interesting findings of amino acid polymorphism by orthogonal methods.     

A Short History of Small s: A Prion of the Fungus Podospora anserina

Prions are infectious proteins. In fungi, prions correspond to non-Mendelian genetic elements whose mode of inheritance has long eluded explanation. The [Het-s] cytoplasmic genetic element of the filamentous fungus Podospora anserina, was originally identified in 1952 and recognized as a prion nearly half a century later. The present chapter will attempt to describe the work on [Het-s] from a historical perspective. The initial characterization and early genetic and physiological studies of [Het-s] are described together with the isolation of the [Het-s] encoding gene. More recent work that led to the construction of a structural model for this prion is also discussed.Key Words: prion, Podospora anserina, amyloid, filamentous fungi, maternal inheritance, heterokaryon incompatibility, cell death, epigenetics     

Cytosolic Ribosomal Mutations That Abolish Accumulation of Circular Intron in the Mitochondria without Preventing Senescence of Podospora Anserina

The filamentous fungus Podospora anserina presents a degeneration syndrome called Senescence associated with mitochondrial DNA modifications. We show that mutations affecting the two different and interacting cytosolic ribosomal proteins (S7 and S19) systematically and specifically prevent the accumulation of senDNAα (a circular double-stranded DNA plasmid derived from the first intron of the mitochondrial cox1 gene or intron α) without abolishing Senescence nor affecting the accumulation of other usually observed mitochondrial DNA rearrangements. One of the mutant proteins is homologous to the Escherichia coli S4 and Saccharomyces cerevisiae S13 ribosomal proteins, known to be involved in accuracy control of cytosolic translation. The lack of accumulation of senDNAα seems to result from a nontrivial ribosomal alteration unrelated to accuracy control, indicating that S7 and S19 proteins have an additional function. The results strongly suggest that modified expression of nucleus-encoded proteins contributes to Senescence in P. anserina. These data do not fit well with some current models, which propose that intron α plays the role of the cytoplasmic and infectious Determinant of Senescence that was defined in early studies.     

Expression of a Single,Viral Oncoprotein in Skin Epithelium Is Sufficient to Recruit Lymphocytes

Established cancers are frequently associated with a lymphocytic infiltrate that fails to clear the tumour mass. In contrast, the importance of recruited lymphocytes during premalignancy is less well understood. In a mouse model of premalignant skin epithelium, transgenic mice that express the human papillomavirus type 16 (HPV16) E7 oncoprotein under a keratin 14 promoter (K14E7 mice) display epidermal hyperplasia and have a predominant infiltrate of lymphocytes consisting of both CD4 and CD8 T cells. Activated, but not naïve T cells, were shown to preferentially traffic to hyperplastic skin with an increased frequency of proliferative CD8+ T cells and CD4+ T cells expressing CCR6 within the tissue. Disruption of the interaction between E7 protein and retinoblastoma tumour suppressor protein (pRb) led to reduced epithelial hyperplasia and T cell infiltrate. Finally, while K14E7 donor skin grafts are readily accepted onto syngeneic, non-transgenic recipients, these same skin grafts lacking skin-resident lymphocytes were rejected. Our data suggests that expression of a single oncoprotein in the epidermis is sufficient for lymphocyte trafficking (including immunosuppressive lymphocytes) to premalignant skin.     

The Aurora A and Aurora B Protein Kinases: A Single Amino Acid Difference Controls Intrinsic Activity and Activation by TPX2

The Aurora A and B protein kinases are key players in mitotic control and the etiology of human cancer. Despite the near identity of amino acid sequence in the catalytic domain, monomeric Aurora B is 50 fold lower in activity than monomeric Aurora A, and previous studies have shown that TPX2 binding to the catalytic domain activates Aurora A but not Aurora B. Here we identify G205 in Aurora A as a key determinant of both intrinsic activity and regulation by TPX2. Mutation of G205 in Aurora A to N, the equivalent residue in Aurora B, had no effect on autophosphorylation of the T-loop but led to a 20-fold loss of specific activity, whereas mutation of N158 in Aurora B to G caused a 350-fold increase in specific activity. G205 N Aurora A was still activated by TPX2, but protection of pT295 from dephosphorylation by protein phosphatase 1 was abolished. Structural analysis of these effects suggests that the G198 forms a pivot point in the enzyme that results in movement of the N-terminal domain glycine-rich loop closer to the ATP binding site of the enzyme and also moves the C-helix slightly closer to the activation loop. Changes in these positions are comparable to those reported for other protein kinases and demonstrate that phosphorylation of the activation loop alone is not sufficient for enzyme activation. The generation of an activated mutant of Aurora B will be important for studying its role in cell cycle control and tumorigenesis.     

A Single Amino Acid Substitution in the Core Protein of West Nile Virus Increases Resistance to Acidotropic Compounds

West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH₄Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.     

A Hapten Generated from an Oxidation Fragment of Docosahexaenoic Acid Is Sufficient to Initiate Age-Related Macular Degeneration

The protein adduct carboxyethylpyrrole (CEP) is present in age-related macular degeneration (AMD) eye tissue and in the blood of AMD patients at higher levels than found in age-matched non-AMD tissues. Autoantibodies to CEP are also higher in AMD blood samples than in controls. To test the hypothesis that this hapten is causally involved in initiating an inflammatory response in AMD, we immunized C57BL/6J mice with mouse serum albumin (MSA) adducted with CEP. Immunized mice develop antibodies to CEP, fix complement component-3 in Bruch’s membrane, accumulate drusen below the retinal pigment epithelium during aging, show decreased a- and b-wave amplitudes in response to light, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. Inflammatory cells are present in the region of lesions and may be actively involved in the pathology observed. We conclude that early immunization of mice with CEP-adducted MSA sensitizes these animals to the ongoing production of CEP adducts in the outer retina where DHA is abundant and the conditions for oxidative damage are permissive. In response to this early sensitization, the immune system mounts a complement-mediated attack on the cells of the outer retina where CEP adducts are formed. This animal model for AMD is the first that was developed from an inflammatory signal discovered in eye tissue and blood from AMD patients. It provides a novel opportunity for dissecting the early pathology of AMD and the immune response contributing to this disorder. The availability of a mouse with a mechanistically based AMD-like disease that progresses rapidly is highly desirable. Such a model will allow for the efficient preclinical testing of the much-needed therapeutics quickly and inexpensively.     

Vegetative Incompatibility and the Mating-Type Locus in the Cellular Slime Mold DICTYOSTELIUM DISCOIDEUM

The genetic basis of vegetative incompatibility in the cellular slime mold, Dictyostelium discoideum, is elucidated. Vegetatively compatible haploid strains from parasexual diploids at a frequency of between 10^-6 and 10^-5, whereas "escaped" diploids are formed between vegetatively incompatible strains at a frequency of ~10^-8. There is probably only a single vegetative incompatibility site, which appears to be located at, or closely linked to, the mating-type locus. The nature of the vegetative incompatibility is deduced from parasexual diploid formation between wild isolates and tester strains of each mating type, examination of the frequency of formation of "escaped" diploids formed between vegetatively incompatible strains, and examination of the mating type and vegetative incompatibility of haploid segregants obtained from "escaped" diploids.     

A 25-Amino Acid Sequence of the Arabidopsis TGD2 Protein Is Sufficient for Specific Binding of Phosphatidic Acid

Genetic analysis suggests that the TGD2 protein of Arabidopsis is required for the biosynthesis of endoplasmic reticulum derived thylakoid lipids. TGD2 is proposed to be the substrate-binding protein of a presumed lipid transporter consisting of the TGD1 (permease) and TGD3 (ATPase) proteins. The TGD1, -2, and -3 proteins are localized in the inner chloroplast envelope membrane. TGD2 appears to be anchored with an N-terminal membrane-spanning domain into the inner envelope membrane, whereas the C-terminal domain faces the intermembrane space. It was previously shown that the C-terminal domain of TGD2 binds phosphatidic acid (PtdOH). To investigate the PtdOH binding site of TGD2 in detail, the C-terminal domain of the TGD2 sequence lacking the transit peptide and transmembrane sequences was fused to the C terminus of the Discosoma sp. red fluorescent protein (DR). This greatly improved the solubility of the resulting DR-TGD2C fusion protein following production in Escherichia coli. The DR-TGD2C protein bound PtdOH with high specificity, as demonstrated by membrane lipid-protein overlay and liposome association assays. Internal deletion and truncation mutagenesis identified a previously undescribed minimal 25-amino acid fragment in the C-terminal domain of TGD2 that is sufficient for PtdOH binding. Binding characteristics of this 25-mer were distinctly different from those of TGD2C, suggesting that additional sequences of TGD2 providing the proper context for this 25-mer are needed for wild type-like PtdOH binding.