Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina

In filamentous fungi, a cell death reaction occurs when cells of unlike genotype fuse. This cell death reaction, known as incompatibility reaction, is genetically controlled by a set of loci termed het loci (for heterokaryon incompatibility loci). In Podospora anserina, genes induced during this cell death reaction (idi genes) have been identified. The idi-6/pspA gene encodes a serine protease that is the orthologue of the vacuolar protease B of Saccharomyces cerevisiae involved in autophagy. We report here that the PSPA protease participates in the degradative autophagic pathway in Podospora. We have identified the Podospora orthologue of the AUT7 gene of S. cerevisiae involved in the early steps of autophagy in yeast. This gene is induced during the development of the incompatibility reaction and was designated idi-7. We have used a GFP-IDI7 fusion protein as a cytological marker of the induction of autophagy. Relocalization of this fusion protein and detection of autophagic bodies inside the vacuoles during the development of the incompatibility reaction provide cytological evidence of induction of autophagy during this cell death reaction. Therefore, cell death by incompatibility in fungi appears to be related to type II programmed cell death in metazoans. In addition, we found that pspA and idi-7 null mutations confer differentiation defects such as the absence of female reproductive structures, indicating that autophagy is required for differentiation in Podospora.     

Heteromorphism for Heterokaryon Incompatibility Genes in Natural Populations of NEUROSPORA CRASSA

Five Neurospora crassa isolates from each of three sites in Louisiana were compared for genotype at five heterokaryon incompatibility (het) loci. The comparisons were made using duplications (partial diploids), based on the fact that duplications heterozygous for het loci have strikingly abnormal phenotypes which greatly facilitate the study of such genes. Duplications were synthesized in crosses between the wild strains (normal chromosome sequence) and testers of defined het genotype and having duplication-producing chromosome rearrangements. Crosses segregating for phenotypes characteristic of duplications heterozygous for het loci indicated allelic differences between testers and wild strains for specific het genes. Whenever a wild strain differed from a tester for a specific het locus, but another wild strain did not, the two wild strains could be inferred to differ from each other.—No two isolates from any site were heterokaryon compatible (of identical het genotype), despite the fact that all isolates from each of two sites occurred within several meters of each other. Heteromorphism was found for all five genes studied at one site, four genes at another site, and three at another. Intra- and interpopulation differences between strains were approximately the same.—Confirmation is also provided that two het genes originally detected in duplications are in fact heterokaryon incompatibility loci.     

The Use of Duplication-Generating Rearrangements for Studying Heterokaryon Incompatibility Genes in Neurospora

Heterokaryon (vegetative) incompatibility, governing the fusion of somatic hyphal filaments to form stable heterokaryons, is of interest because of its widespread occurrence in fungi and its bearing on cellular recognition. Conventional investigations of the genetic basis of heterokaryon incompatibility in N. crassa are difficult because in commonly used stocks differences are present at several het loci, all with similar incompatibility phenotypes. This difficulty is overcome by using duplications (partial diploids) that are unlikely to contain more than one het locus. A phenotypically expressed incompatibility reaction occurs when unlike het alleles are present within the same somatic nucleus, and this parallels the heterokaryon incompatibility reaction that occurs when unlike alleles in different haploid nuclei are introduced into the same somatic hypha by mycelial fusion.—Nontandem duplications were used to confirm that the incompatibility reactions in heterokaryons and in duplications are alternate expressions of the same genes. This was demonstrated for three loci which had previously been established by conventional heterokaryon tests—het-e, het-c and mt. These were each obtained in duplications as recombinant meiotic segregants from crosses heterozygous for duplication-generating chromosome rearrangements. The particular method of producing the duplications is irrelevant so long as the incompatibility alleles are heterozygous.—The duplication technique has made it possible to determine easily the het-e and het-c genotypes of numerous laboratory and wild strains of unknown constitution. In laboratory strains both loci are represented simply by two alleles. Analysis of het-c is more complicated in some wild strains, where differences have been demonstrated at one or more additional het loci within the duplication used and multiple allelism is also possible.—The results show that the duplication method can be used to identify and map additional vegetative incompatibility loci, without the necessity of heterokaryon tests.     

Molecular genetics of heterokaryon incompatibility in filamentous ascomycetes.

Filamentous fungi spontaneously undergo vegetative cell fusion events within but also between individuals. These cell fusions (anastomoses) lead to cytoplasmic mixing and to the formation of vegetative heterokaryons (i.e., cells containing different nuclear types). The viability of these heterokaryons is genetically controlled by specific loci termed het loci (for heterokaryon incompatibility). Heterokaryotic cells formed between individuals of unlike het genotypes undergo a characteristic cell death reaction or else are severely inhibited in their growth. The biological significance of this phenomenon remains a puzzle. Heterokaryon incompatibility genes have been proposed to represent a vegetative self/nonself recognition system preventing heterokaryon formation between unlike individuals to limit horizontal transfer of cytoplasmic infectious elements. Molecular characterization of het genes and of genes participating in the incompatibility reaction has been achieved for two ascomycetes, Neurospora crassa and Podospora anserina. These analyses have shown that het genes are diverse in sequence and do not belong to a gene family and that at least some of them perform cellular functions in addition to their role in incompatibility. Divergence between the different allelic forms of a het gene is generally extensive, but single-amino-acid differences can be sufficient to trigger incompatibility. In some instances het gene evolution appears to be driven by positive selection, which suggests that the het genes indeed represent recognition systems. However, work on nonallelic incompatibility systems in P. anserina suggests that incompatibility might represent an accidental activation of a cellular system controlling adaptation to starvation.     

Molecular Genetics of Heterokaryon Incompatibility in Filamentous Ascomycetes

Filamentous fungi spontaneously undergo vegetative cell fusion events within but also between individuals. These cell fusions (anastomoses) lead to cytoplasmic mixing and to the formation of vegetative heterokaryons (i.e., cells containing different nuclear types). The viability of these heterokaryons is genetically controlled by specific loci termed het loci (for heterokaryon incompatibility). Heterokaryotic cells formed between individuals of unlike het genotypes undergo a characteristic cell death reaction or else are severely inhibited in their growth. The biological significance of this phenomenon remains a puzzle. Heterokaryon incompatibility genes have been proposed to represent a vegetative self/nonself recognition system preventing heterokaryon formation between unlike individuals to limit horizontal transfer of cytoplasmic infectious elements. Molecular characterization of het genes and of genes participating in the incompatibility reaction has been achieved for two ascomycetes, Neurospora crassa and Podospora anserina. These analyses have shown that het genes are diverse in sequence and do not belong to a gene family and that at least some of them perform cellular functions in addition to their role in incompatibility. Divergence between the different allelic forms of a het gene is generally extensive, but single-amino-acid differences can be sufficient to trigger incompatibility. In some instances het gene evolution appears to be driven by positive selection, which suggests that the het genes indeed represent recognition systems. However, work on nonallelic incompatibility systems in P. anserina suggests that incompatibility might represent an accidental activation of a cellular system controlling adaptation to starvation.     

Heterokaryon incompatibility in fungi—more than just another way to die

In filamentous fungi heterokaryon (vegetative) compatibility is regulated by a number of different loci. Vegetative incompatibility is most often detected as the inability to form a prototrophic heterokaryon under forcing conditions, or as the formation of a barrage when two incompatible strains interact. Vegetative compatibility has been used as a multilocus phenotype in analysis of fungal populations. In some highly clonal populations the vegetative-compatibility phenotype is correlated with pathogenicity. The molecular basis for vegetative compatibility is not well understood. Fourhet loci have been cloned fromNeurospora crasset orPodospora anserina, inch but no two are alike and it is clear that thehet genes themselves do not encode the gene products that are directly responsible for cell death. We suggest that a broader view of vegetative compatibility would include genes that are responsible for prefusion, fusion, and postfusion activities. Postfusion activities could include the fungal apoptotic apparatus since microscopic observations of cell death resemble those in higher plants and animals.     

Evolution and diversity of a fungal self/nonself recognition locus

Background

Self/nonself discrimination is an essential feature for pathogen recognition and graft rejection and is a ubiquitous phenomenon in many organisms. Filamentous fungi, such as Neurospora crassa, provide a model for analyses of population genetics/evolution of self/nonself recognition loci due to their haploid nature, small genomes and excellent genetic/genomic resources. In N. crassa, nonself discrimination during vegetative growth is determined by 11 heterokaryon incompatibility (het) loci. Cell fusion between strains that differ in allelic specificity at any of these het loci triggers a rapid programmed cell death response.

Methodology/Principal Findings

In this study, we evaluated the evolution, population genetics and selective mechanisms operating at a nonself recognition complex consisting of two closely linked loci, het-c (NCU03493) and pin-c (NCU03494). The genomic position of pin-c next to het-c is unique to Neurospora/Sordaria species, and originated by gene duplication after divergence from other species within the Sordariaceae. The het-c pin-c alleles in N. crassa are in severe linkage disequilibrium and consist of three haplotypes, het-c1/pin-c1, het-c2/pin-c2 and het-c3/pin-c3, which are equally frequent in population samples and exhibit trans-species polymorphisms. The absence of recombinant haplotypes is correlated with divergence of the het-c/pin-c intergenic sequence. Tests for positive and balancing selection at het-c and pin-c support the conclusion that both of these loci are under non-neutral balancing selection; other regions of both genes appear to be under positive selection. Our data show that the het-c2/pin-c2 haplotype emerged by a recombination event between the het-c1/pin-c1 and het-c3/pin-c3 approximately 3–12 million years ago.

Conclusions/Significance

These results support models by which loci that confer nonself discrimination form by the association of polymorphic genes with genes containing HET domains. Distinct allele classes can emerge by recombination and positive selection and are subsequently maintained by balancing selection and divergence of intergenic sequence resulting in recombination blocks between haplotypes.     

Accelerated cell death in Podospora autophagy mutants

Although autophagy is characteristic of type II programmed cell death (PCD), its role in cell death is currently debated. Both cell death-promoting and prosurvival roles of autophagy have been reported depending on the organism and the cell type. In filamentous fungi, a cell death reaction known as an incompatibility reaction occurs when cells of unlike genotype fuse. Cell death by incompatibility is characterized by a dramatic vacuolar enlargement and cell lysis. In Podospora anserina, autophagy is induced early during this cell death reaction. Cell death by incompatibility in Podospora is a model of type II PCD used here to assess the role of autophagy in this type of cell death. We have inactivated PaATG1, the Podospora ortholog of the Saccharomyces cerevisiae ATG1 gene involved in the early steps of autophagy in yeast. The DeltaPaATG1 mutant displays developmental defects characteristic of abrogated autophagy in Podospora. Using the green fluorescent protein-PaATG8 autophagosome marker, we show that autophagy is abolished in this mutant. Neither cell death by incompatibility nor vacuolization are suppressed in DeltaPaATG1 and DeltaPaATG8 autophagy mutants, indicating that a vacuolar cell death reaction without autophagy occurs in Podospora. Our results thus provide a novel example of a type II PCD reaction in which autophagy is not the cause of cell death. In addition, we found that cell death is accelerated in DeltaPaATG null mutants, suggesting that autophagy has a protective role in this type II PCD reaction.     

Regulation of gene expression during the vegetative incompatibility reaction in Podospora anserina. Characterization of three induced genes.

Vegetative incompatibility in fungi limits the formation of viable heterokaryons. It results from the coexpression of incompatible genes in the heterokaryotic cells and leads to a cell death reaction. In Podospora anserina, a modification of gene expression takes place during this reaction, including a strong decrease of total RNA synthesis and the appearance of a new set of proteins. Using in vitro translation of mRNA and separation of protein products by two-dimensional gel electrophoresis, we have shown that the mRNA content of cells is qualitatively modified during the progress of the incompatibility reaction. Thus, gene expression during vegetative incompatibility is regulated, at least in part, by variation of the mRNA content of specific genes. A subtractive cDNA library enriched in sequences preferentially expressed during incompatibility was constructed. This library was used to identify genomic loci corresponding to genes whose mRNA is induced during incompatibility. Three such genes were characterized and named idi genes for genes induced during incompatibility. Their expression profiles suggest that they may be involved in different steps of the incompatibility reaction. The putative IDI proteins encoded by these genes are small proteins with signal peptides. IDI-2 protein is a cysteine-rich protein. IDI-2 and IDI-3 proteins display some similarity in a tryptophan-rich region.     

Defence gene expression in soybean is linked to the status of the cell death program

Soybean cell cultures (cv. Williams 82) respond to Pseudomonas syringae bacteria expressing the avirulence gene AvrA with a hypersensitive reaction, a programmed cell death (PCD) of plant cells to pathogen attack. This PCD is under control of salicylic acid (SA) via an unknown mechanism. In the presence of low concentrations of SA, the cells undergo a very rapid cell death, which needs only half of the time required for the normal hypersensitive reaction (HR). Northern blot studies for defence-related genes show that the expression of many of these genes is tightly linked to the status of the cell death program rather than to pathogen-derived elicitors. Thus the expression is much faster in the SA-accelerated PCD than in the normal hypersensitive reaction. In contrast, other pathogen-responsive genes are induced independently of the speed of PCD, indicating a divergent signalling mechanism. The production of reactive oxygen species during the oxidative burst of bacteria-inoculated soybean cells is slightly enhanced in the presence of SA but occurs at the same time as in untreated cells, suggesting that SA exhibits the control of the PCD downstream of the oxidative burst. Consistent with these findings a HR-specific marker gene is neither directly induced by H₂O₂ or SA. However, this gene shows a high expression in the regular HR and is induced much faster in the SA-accelerated PCD.     

Cell death in wheat roots induced by the powdery mildew fungus Blumeria graminis f. sp. tritici

Inoculation of wheat (Triticum aestivum L. cv. Huamai 8) leaves with wheat powderly mildew fungus (Blumeria graminis f. sp. tritici) induced cell death in wheat adventitious roots, where no fungal structures were observed. The cytological and molecular characterization of this cell death was shown as following: cell nuclei were TUNEL positive labeled; genomic DNA was fragmented and showed DNA laddering; chromatin condensed and formed peripheral conglomeration in nuclei; and perinuclear spaces partly dilated. These results suggested that, without pathogen spread, the infection could induce systemic PCD in adventitious roots. Comparison with a leaf-cutting experiment (LC)enabled us to speculate that lack of assimilates was not the only reason for the systemic PCD in wheat roots in powdery mildew experiment and that such systemic PCD might be mediated by long-distance signals. In addition, reactive oxygen species (ROS) and Ca²⁺ were related to the systemic PCD.     

Entamoeba histolytica: Differential gene expression during programmed cell death and identification of early pro- and anti-apoptotic signals

We have demonstrated that programmed cell death (PCD) in Entamoeba histolytica is induced in vitro by G418 aminoglycoside antibiotic. To ascertain if biochemical and morphological changes previously observed are paired to molecular changes that reflect a genetic program, we looked here for early differential gene expression during the induction of PCD.Using cDNA-amplified fragment length polymorphisms (AFLPs) and in silico derived analysis we showed in E. histolytica a differential gene expression during PCD induced by G418. The genes identified encoded for proteins homologous to Glutaminyl-tRNA synthase, Ribosomal Subunit Proteins 40S and 18S, Saposin-like, Silent Information Regulator-2 (Sir-2), and Grainins 1 and 2. Using real-time quantitative PCR (RT Q-PCR), we found that glutaminyl-tRNA synthetase, sir-2, grainins and saposin-like genes were strongly overexpressed after 30 min of PCD induction, while its expression dramatically decreased up to 60 min. On the other hand, overexpression of ribosomal genes increased only 7-fold of basal expression, showing a progressive down-regulation up to 90 min. glutaminyl-tRNA synthetase, sir-2 and grainins could act as negative regulators of PCD, trying to control the biochemical changes related to PCD activation. Overexpression of saposin-like gene could act as up-regulator of some cell death pathways. Our results give evidence of the first genes identified during the early stage of PCD in E. histolytica that could be implicated in regulation of apoptotic pathways.     

Programmed cell death correlates with virus transmission in a filamentous fungus

Programmed cell death (PCD) is an essential part of the defence response in plants and animals against pathogens. Here, we report that PCD is also involved in defence against pathogens of fungi. Vegetative incompatibility is a self/non-self recognition system in fungi that results in PCD when cells of incompatible strains fuse. We quantified the frequency of cell death associated with six vegetative incompatibility (vic) genes in the filamentous ascomycete fungus Cryphonectria parasitica. Cell death frequencies were compared with the effects of vic genes on transmission of viruses between the same strains. We found a significant negative correlation between cell death and virus transmission. We also show that asymmetry in cell death correlates with asymmetry in virus transmission; greater transmission occurs into vic genotypes that exhibit delayed or infrequent PCD after fusion with an incompatible strain. Furthermore, we found that virus infection can have a significant, strain-specific, positive or negative effect on PCD. Specific interactions between vic gene function and viruses, along with correlations between cell death and transmission, strongly implicate PCD as a host-mediated pathogen defence strategy in fungi.     

Temporal and metabolic overlap between lipid accumulation and programmed cell death due to nitrogen starvation in the unicellular chlorophyte Chlamydomonas reinhardtii

Lipid accumulation due to nitrogen depletion has been studied extensively in Chlamydomonas reinhardtii and the metabolic changes that lead to triacylglycerol biosynthesis have been of particular interest to researchers in the biodiesel industry. The induction of programmed cell death (PCD) in response to nitrogen starvation has also been documented in related chlorophytes. Here, we examined the temporal and metabolic overlap of lipid accumulation and PCD in response to nitrogen starvation in the important model organism C. reinhardtii. Nitrogen starvation induced physiological stress, measured by the progressive decline in chlorophyll a fluorescence, reduced photosynthetic efficiency and decreased growth. In keeping with previous reports, cells accumulated lipids reaching a peak after 2–3 days. At the same time, DNA nicking and caspase‐like protease activity was observed in a proportion of cells, and ultrastructural observations confirmed that death was via PCD. Our results demonstrate that DNA nicking and caspase‐like activity are observed during PCD in C. reinhardtii in response to nitrogen starvation, and that death occurs at the same time as lipid biosynthesis. Microalgal lipid production due to nitrogen depletion in C. reinhardtii is limited by the decrease in culture growth and knowing that the loss of culture density is, at least in part, due to PCD is important for the biotechnology industry.     

Involvement of phospholipase D-related signal transduction in chemical-induced programmed cell death in tomato cell cultures

Phospholipase D (PLD) and its product phosphatidic acid (PA) are incorporated in a complex metabolic network in which the individual PLD isoforms are suggested to regulate specific developmental and stress responses, including plant programmed cell death (PCD). Despite the accumulating knowledge, the mechanisms through which PLD/PA operate during PCD are still poorly understood. In this work, the role of PLDα1 in PCD and the associated caspase-like proteolysis, ethylene and hydrogen peroxide (H₂O₂) synthesis in tomato suspension cells was studied. Wild-type (WT) and PLDα1-silenced cell lines were exposed to the cell death-inducing chemicals camptothecin (CPT), fumonisin B1 (FB1) and CdSO₄. A range of caspase inhibitors effectively suppressed CPT-induced PCD in WT cells, but failed to alleviate cell death in PLDα1-deficient cells. Compared to WT, in CPT-treated PLDα1 mutant cells, reduced cell death and decreased production of H₂O₂ were observed. Application of ethylene significantly enhanced CPT-induced cell death both in WT and PLDα1 mutants. Treatments with the PA derivative lyso-phosphatidic acid and mastoparan (agonist of PLD/PLC signalling downstream of G proteins) caused severe cell death. Inhibitors, specific to PLD and PLC, remarkably decreased the chemical-induced cell death. Taken together with our previous findings, the results suggest that PLDα1 contributes to caspase-like-dependent cell death possibly communicated through PA, reactive oxygen species and ethylene. The dead cells expressed morphological features of PCD such as protoplast shrinkage and nucleus compaction. The presented findings reveal novel elements of PLD/PA-mediated cell death response and suggest that PLDα1 is an important factor in chemical-induced PCD signal transduction.