Molecular characterization of vegetative incompatibility genes that restrict hypovirus transmission in the chestnut blight fungus Cryphonectria parasitica

Genetic nonself recognition systems such as vegetative incompatibility operate in many filamentous fungi to regulate hyphal fusion between genetically dissimilar individuals and to restrict the spread of virulence-attenuating mycoviruses that have potential for biological control of pathogenic fungi. We report here the use of a comparative genomics approach to identify seven candidate polymorphic genes associated with four vegetative incompatibility (vic) loci of the chestnut blight fungus Cryphonectria parasitica. Disruption of candidate alleles in one of two strains that were heteroallelic at vic2, vic6, or vic7 resulted in enhanced virus transmission, but did not prevent barrage formation associated with mycelial incompatibility. Detailed characterization of the vic6 locus revealed the involvement of nonallelic interactions between two tightly linked genes in barrage formation, heterokaryon formation, and asymmetric, gene-specific influences on virus transmission. The combined results establish molecular identities of genes associated with four C. parasitica vic loci and provide insights into how these recognition factors interact to trigger incompatibility and restrict virus transmission.     

Genetic control of horizontal virus transmission in the chestnut blight fungus, Cryphonectria parasitica

Vegetative incompatibility in fungi has long been known to reduce the transmission of viruses between individuals, but the barrier to transmission is incomplete. In replicated laboratory assays, we showed conclusively that the transmission of viruses between individuals of the chestnut blight fungus Cryphonectria parasitica is controlled primarily by vegetative incompatibility (vic) genes. By replicating vic genotypes in independent fungal isolates, we quantified the effect of heteroallelism at each of six vic loci on virus transmission. Transmission occurs with 100% frequency when donor and recipient isolates have the same vic genotypes, but heteroallelism at one or more vic loci generally reduces virus transmission. Transmission was variable among single heteroallelic loci. At the extremes, heteroallelism at vic4 had no effect on virus transmission, but transmission occurred in only 21% of pairings that were heteroallelic at vic2. Intermediate frequencies of transmission were observed when vic3 and vic6 were heteroallelic (76 and 32%, respectively). When vic1, vic2, and vic7 were heteroallelic, the frequency of transmission depended on which alleles were present in the donor and the recipient. The effect of heteroallelism at two vic loci was mostly additive, although small but statistically significant interactions (epistasis) were observed in four pairs of vic loci. A logistic regression model was developed to predict the probability of virus transmission between vic genotypes. Heteroallelism at vic loci, asymmetry, and epistasis were the dominant factors controlling transmission, but host genetic background also was statistically significant, indicating that vic genes alone cannot explain all the variation in virus transmission. Predictions from the logistic regression model were highly correlated to independent transmission tests with field isolates. Our model can be used to estimate horizontal transmission rates as a function of host genetics in natural populations of C. parasitica.     

Heterokaryon incompatibility function of barrage-associated vegetative incompatibility genes (vic) in Cryphonectria parasitica

Six vegetative incompatibility (vic) loci have been identified in Cryphonectria parasitica based on barrage formation during mycelial interactions. We used hygromycin B- and benomyl-resistance as forcing markers in C. parasitica strains to test whether heteroallelism at each vic locus prevents heterokaryon formation following mycelial interactions. Paired strains that had allelic differences at any of vic1, 2, 3, 6 or 7 but not vic4 displayed heterokaryon incompatibility function, as recognized by slow growth or aberrant morphology. While clearly forming barrages in mycelial interactions, paired strains with different alleles at vic4 formed stable heterokaryons. With examples from other fungi, this inconsistency at vic4 suggests that barrage formation and heterokaryon incompatibility are not different manifestations of the same process. Rather, the evidence indicates that heterokaryon incompatibility represents a component of a vegetative incompatibility system that may also use cell-surface or extracellular factors to trigger programmed cell death to modulate nonself recognition in fungi.     

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.     

Potentiation of Mycovirus Transmission by Zinc Compounds via Attenuation of Heterogenic Incompatibility in Rosellinia necatrix

Heterogenic incompatibility is considered a defense mechanism against deleterious intruders such as mycovirus. Rosellinia necatrix shows strong heterogenic incompatibility. In the heterogenic incompatibility reaction, the approaching hyphae hardly anastomosed, a distinctive barrage line formed, and green fluorescent protein (GFP)-labeled hyphae quickly lost their fluorescence when encountering incompatible hyphae. In this study, transmission of a hypovirulence-conferring mycovirus to strains with different genetic backgrounds was attempted. Various chemical reagents considered to affect the programmed cell death pathway or cell wall modification were examined. Treatment with zinc compounds was shown to aid in transmission of mycoviruses to strains with different genetic backgrounds. In incompatible pairings, treatment with zinc compounds accelerated hyphal anastomosis; moreover, cytosolic GFP was transmitted to the newly joined hyphae. These results suggest that zinc compounds not only increase hyphal anastomosis but also attenuate heterogenic incompatibility.     

Heterokaryon incompatibility is suppressed following conidial anastomosis tube fusion in a fungal plant pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.     

Ornithine-delta-aminotransferase and proline dehydrogenase genes play a role in non-host disease resistance by regulating pyrroline-5-carboxylate metabolism-induced hypersensitive response

Non-host disease resistance involves the production of hypersensitive response (HR), a programmed cell death (PCD) that occurs at the site of pathogen infection. Plant mitochondrial reactive oxygen species (ROS) production and red-ox changes play a major role in regulating such cell death. Proline catabolism reactions, especially pyrroline-5-carboxylate (P5C) accumulation, are known to produce ROS and contribute to cell death. Here we studied important genes related to proline synthesis and catabolism in the defence against host and non-host strains of Pseudomonas syringae in Nicotiana benthamiana and Arabidopsis. Our results show that ornithine delta-aminotransferase (δOAT) and proline dehydrogenases (ProDH1 and ProDH2) are involved in the defence against non-host pathogens. Silencing of these genes in N. benthamiana delayed occurrence of HR and favoured non-host pathogen growth. Arabidopsis mutants for these genes compromised non-host resistance and showed a decrease in non-host pathogen-induced ROS. Some of the genes involved in proline metabolism were also induced by a pathogen-carrying avirulence gene, indicating that proline metabolism is influenced during effector-triggered immunity (ETI). Our results demonstrate that δOAT and ProDH enzyme-mediated steps produce ROS in mitochondria and regulate non-host HR, thus contributing to non-host resistance in plants.     

Genetics of self/nonself recognition in Serpula lacrymans

This study provides an analysis of the vegetative incompatibility system in Serpula lacrymans (Basidiomycota), a genetic system used to recognize nonself in fungi. Seventy-five worldwide isolates could be grouped into eight vegetative compatibility (VC) types, some of them distributed on different continents. Mating studies combined with vegetative incompatibility analyses revealed that the vegetative incompatibility response between isolates mainly could be explained by two biallelic vegetative incompatibility (vic) loci. The frequency distributions of the interpreted vic alleles do not seem to support the idea of frequency-dependent or balancing selection acting on the vic loci. We find little genetic variation at the vic loci and in one of the loci there was a significant heterozyote deficiency among strains in the overall material. The results may be explained by a recent worldwide dispersal of a few S. lacrymans isolates and, correspondingly, only a few vic alleles are being maintained in these populations.     

A SNARE-protein has opposing functions in penetration resistance and defence signalling pathways

Penetration resistance is often the first line of defence against fungal pathogens. Subsequently induced defences are mediated by the programmed cell death (PCD) reaction pathway and the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signalling pathways. We previously demonstrated that full penetration resistance in Arabidopsis against the non-host barley powdery mildew fungus (Blumeria graminis f.sp. hordei) requires the syntaxin SYP121 (PEN1). Here we report that SYP121, together with SYP122, functions as a negative regulator of subsequently induced defence pathways. The SA level in the syntaxin double mutant syp121-1 syp122-1 is dramatically elevated, resulting in necrosis and dwarfism. This phenotype is partially rescued by introducing the SA-signalling mutations eds1-2, eds5-3, sid2-1 and npr1-1 as well as the NahG transgene. These partially rescued triple mutants have an unknown defence to Pseudomonas syringae pv. tomato, and have increased HR-like responses to non-host and host powdery mildew fungi. The HR-like responses cause efficient resistance to the latter. These defence pathways are SA-independent. Furthermore, the JA/ET signalling marker, PDF1.2, is highly upregulated in the triple mutants. Thus SYP121 and SYP122 are negative regulators of PCD, SA, JA and ET pathways through a molecular function distinct from that of SYP121 in penetration resistance. Our data suggest that individual cells preferentially express either penetration resistance or the subsequently induced defences.     

Rapamycin mimics the incompatibility reaction in the fungus Podospora anserina

In filamentous fungi, a programmed cell death (PCD) reaction occurs when cells of unlike genotype fuse. This reaction is caused by genetic differences at specific loci termed het loci (for heterokaryon incompatibility). Although several het genes have been characterized, the mechanism of this cell death reaction and its relation to PCD in higher eukaryotes remains largely unknown. In Podospora anserina, genes induced during the cell death reaction triggered by the het-R het-V interaction have been identified and termed idi genes. Herein, we describe the functional characterization of one idi gene (idi-1) and explore the connection between incompatibility and the response to nutrient starvation. We show that IDI-1 is a cell wall protein which localizes at the septum during normal growth. We found that induction of idi-1 and of the other known idi genes is not specific of the incompatibility reaction. The idi genes are induced upon nitrogen and carbon starvation and by rapamycin, a specific inhibitor of the TOR kinase pathway. The cytological hallmarks of het-R het-V incompatibility (increased septation, vacuolization, coalescence of lipid droplets, induction of autophagy, and cell death) are also observed during rapamycin treatment. Globally the cytological alterations and modifications in gene expression occurring during the incompatibility reaction are similar to those observed during starvation or rapamycin treatment.     

Nuclear DNA degradation during heterokaryon incompatibility in Neurospora crassa

Many filamentous fungi are capable of undergoing conspecific hyphal fusion with a genetically different individual to form a heterokaryon. However, the viability of such heterokaryons is dependent upon vegetative (heterokaryon) incompatibility (het) loci. If two individuals undergo hyphal anastomosis, but differ in allelic specificity at one or more het loci, the fusion cell is usually compartmentalized and self-destructs. Many of the microscopic features associated with vegetative incompatibility resemble apoptosis in metazoans and plants. To test the hypothesis whether vegetative incompatibility results in nuclear degradation, a characteristic of apoptosis, the cytology of hyphal fusions between incompatible Neurospora crassa strains that differed at three het loci, mat, het-c and het-6, and the cytology of transformants containing incompatible het-c alleles were examined using fluorescent DNA stains and terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL). Hyphal fusion cells between het incompatible strains and hyphal segments in het-c incompatible transformants were compartmentalized by septal plugging and contained heavily degraded nuclear DNA. Hyphal fusion cells in compatible self-pairings and hyphal cells in het-c compatible transformants were not compartmentalized and rarely showed TUNEL-positive nuclei. Cell death events also were observed in senescent, older hyphae. Morphological features of hyphal compartmentation and death during vegetative incompatibility and the extent to which it is genetically controlled can best be described as a form of programmed cell death.     

Cytological analysis of mycelial incompatibility in Helicobasidium mompa

When the mycelia of Helicobasidium mompa encounter mycelia with a different genetic background, distinct demarcation lines form. The hyphae of H. mompa induce heterogenic incompatibility accompanied by active programmed cell death (PCD) process. In this study, we observed hyphal interaction between compatible and incompatible H. mompa pairs by means of light and electron microscopy. PCD started with one of the two approaching hyphae. Heterochromatin condensation and genomic DNA laddering were not observed. Moreover, cell damage began with the tonoplast and continued with the plasma membrane and nuclear membrane, suggesting that the PCD observed in heterogenic incompatibility of H. mompa is a vacuole-mediated process.     

Hypersensitive response-related death

The hypersensitive response (HR) of plants resistant to microbial pathogens involves a complex form of programmed cell death (PCD) that differs from developmental PCD in its consistent association with the induction of local and systemic defence responses. Hypersensitive cell death is commonly controlled by direct or indirect interactions between pathogen avirulence gene products and those of plant resistance genes and it can be the result of multiple signalling pathways. Ion fluxes and the generation of reactive oxygen species commonly precede cell death, but a direct involvement of the latter seems to vary with the plant-pathogen combination. Protein synthesis, an intact actin cytoskeleton and salicylic acid also seem necessary for cell death induction. Cytological studies suggest that the actual mode and sequence of dismantling the cell contents varies among plant-parasite systems although there may be a universal involvement of cysteine proteases. It seems likely that cell death within the HR acts more as a signal to the rest of the plant rather than as a direct defence mechanism.     

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.     

Autophagy–virus interplay in plants: from antiviral recognition to proviral manipulation

Autophagy is a conserved self-cleaning and renewal system required for cellular homeostasis and stress tolerance. Autophagic processes are also implicated in the response to ‘non-self’ such as viral pathogens, yet the functions and mechanisms of autophagy during plant virus infection have only recently started to be revealed. Compelling evidence now indicates that autophagy is an integral part of antiviral immunity in plants. It can promote the hypersensitive cell death response upon incompatible viral infections or mediate the selective elimination of entire particles and individual proteins from compatible viruses in a pathway similar to xenophagy in animals. Several viruses, however, have evolved measures to antagonize xenophagic degradation or utilize autophagy to suppress disease-associated cell death and other defence pathways like RNA silencing. Here, we highlight the current advances and gaps in our understanding of the complex autophagy–virus interplay and its consequences for host immunity and viral pathogenesis in plants.     

Programmed cell death in host-symbiont associations,viewed through the Gene Ontology

Manipulation of programmed cell death (PCD) is central to many host microbe interactions. Both plant and animal cells use PCD as a powerful weapon against biotrophic pathogens, including viruses, which draw their nutrition from living tissue. Thus, diverse biotrophic pathogens have evolved many mechanisms to suppress programmed cell death, and mutualistic and commensal microbes may employ similar mechanisms. Necrotrophic pathogens derive their nutrition from dead tissue, and many produce toxins specifically to trigger programmed cell death in their hosts. Hemibiotrophic pathogens manipulate PCD in a most exquisite way, suppressing PCD during the biotrophic phase and stimulating it during the necrotrophic phase. This mini-review will summarize the mechanisms that have evolved in diverse microbes and hosts for controlling PCD and the Gene Ontology terms developed by the Plant-Associated Microbe Gene Ontology (PAMGO) Consortium for describing those mechanisms.     

植物抗病分子机制研究进展

植物抗病机制是目前研究的热点。在长期的进化过程中,植物形成了一系列复杂有效的防御机制来抵御、破坏病原物的侵染。植物抗病基因在植物抗性反应中起着重要的作用,植物一旦监测到病原物马上起始防御反应,并伴随着植物体内一系列细胞和生理生化的变化。近年来,基因沉默作为一个重要的细胞内防御外源核酸的机制,越来越受到科学家重视。综述了植物抗病基因和基因沉默机制在植物抗病反应中的重要作用,并对研究植物抗病机制的前景做了展望。