Molecular characterization of chromosome termini of the arbuscular mycorrhizal fungus Glomus intraradices (Glomeromycota).

The minimum chromosome number of Glomus intraradices was assessed through cloning and sequencing of the highly divergent telomere-associated sequences (TAS) and by pulsed field gel electrophoresis (PFGE). The telomere of G. intraradices, as in other filamentous fungi, consists of TTAGGG repeats, this was confirmed using Bal31 nuclease time course reactions. Telomere length was estimated to be roughly 0.9 kb by Southern blots on genomic DNA and a telomere probe. We have identified six classes of cloned chromosomal termini based on the TAS. An unusually high genetic variation was observed within two of the six TAS classes. To further assess the total number of chromosome termini, we used telomere fingerprinting. Surprisingly, all hybridization patterns showed smears, which demonstrate that TAS are remarkably variable in the G. intraradices genome. These analyses predict the presence of at least three chromosomes in G. intraradices while PFGE showed a pattern of four bands ranging from 1.2 to 1.5 Mb. Taken together, our results indicate that there are at least four chromosomes in G. intraradices but there are probably more. The information on TAS and telomeres in the G. intradicies will be essential for making a physical map of the G. intraradices genome and could provide molecular markers for future studies of genetic variation among nuclei in these multigenomic fungi.

Effects of genetic alterations in arbuscular mycorrhizal fungi on plant growth and on their response to a change of host

Abstract :The majority of land plants form the symbiosis with arbuscular mycorrhizal fungi (AMF). The AM symbiosis has existed for hundreds of millions of years but little or no specificity seems to have co- evolved between the partners and only about 200 morphospecies of AMF are known. The fungi supply the plants most notably with phosphate in exchange for carbohydrates. The fungi improve plant growth, protect them against pathogens and herbivores and the symbiosis plays a key role in ecosystem productivity and plant diversity. The fungi are coenocytic, grow clonally and no sexual stage in their life cycle is known. For these reasons, they are presumed ancient asexuals. Evidence suggests that AMF contain populations of genetically different nucleotypes coexisting in a common cytoplasm. Consequently, the nucleotype content of new clonal offspring could potentially be altered by segregation of nuclei at spore formation and by genetic exchange between different AMF. Given the importance of AMF, it is surprising that remarkably little is known about the genetics and genomics of the fungi.The main goal of this thesis was to investigate the combined effects of plant species differences and of genetic exchange and segregation in AMF on the symbiosis. This work showed that single spore progeny can receive a different assortment of nucleotypes compared to their parent and compared to other single spore progeny. This is the first direct evidence that segregation occurs in AMF. We then showed that both genetic exchange and segregation can lead to new progeny that differentially alter plant growth compared to their parents. We also found that genetic exchange and segregation can lead to different development of the fungus during the establishment of the symbiosis. Finally, we found that a shift of host species can differentially alter the phenotypes and genotypes of AMF progeny obtained by genetic exchange and segregation compared to their parents.Overall, this study confirms the multigenomic state of the AMF Glomus intraradices because our findings are possible only if the fungus contains genetically different nuclei. We demonstrated the importance of the processes of genetic exchange and segregation to produce, in a very short time span, new progeny with novel symbiotic effects. Moreover, our results suggest that different host species could affect the fate of different nucleotypes following genetic exchange and segregation in AMF, and can potentially contribute to the maintenance of genetic diversity within AMF individuals. This work brings new insights into understanding how plants and fungi have coevolved and how the genetic diversity in AMF can be maintained. We recommend that the intra-ir1dividual AMF diversity and these processes should be considered in future research on this symbiosis.Résumé :La majorité des plantes terrestres forment des symbioses avec les champignons endomycorhiziens arbusculaires (CEA). Cette symbiose existe depuis plusieurs centaines de millions d'années mais peu ou pas de spécificité semble avoir co-évoluée entre les partenaires et seulement 200 morpho-espèces de CEA sont connues. Le champignon fournit surtout aux plantes du phosphate en échange de carbohydrates. Le champignon augmente la croissance des plantes, les protège contre des pathogènes et herbivores et la symbiose joue un rôle clé dans la productivité des écosystèmes et de la diversité des plantes. Les CEA sont coenocytiques, se reproduisent clonalement et aucune étape sexuée n'est connue dans leur cycle de vie. Pour ces raisons, ils sont présumés comme anciens asexués. Des preuves suggèrent que les CEA ont des populations de nucleotypes différents coexistant dans un cytoplasme commun. Par conséquent, le contenu en nucleotype des nouveaux descendants clonaux pourrait être altéré par la ségrégation des noyaux lors de la fonnation des spores et par l'échange génétique entre différents CEA. Etant donné l'importance des CEA, il est surprenant que si peu soit connu sur la génétique et la génomique du champignon.Le principal but de cette thèse a été d'étudier les effets combinés de différentes espèces de plantes et des mécanismes d'échange génétique et de ségrégation chez les CEA sur la symbiose. Ce travail a montré que chaque nouvelle spore produite pouvait recevoir un assortiment différent de noyaux comparé au parent ou comparé à d'autres nouvelles spores. Ceci est la première preuve directe que la ségrégation peut se produire chez les CEA. Nous avons ensuite montré qu'à la fois l'échange génétique et la ségrégation pouvaient mener à de nouveaux descendants qui altèrent différemment la croissance des plantes, comparé à leurs parents. Nous avons également trouvé que l'échange génétique et la ségrégation pouvaient entraîner des développements différents du champignon pendant l'établissement de la symbiose. Pour finir, nous avons trouvé qu'un changement d'espèce de l'hôte pouvait altérer différemment les phénotypes et génotypes des descendants issus d'échange génétique et de ségrégation, comparé à leurs parents.Globalement, cette étude confirme l'état multigénomique du CEA Glumus intraradices car nous résultats sont possibles seulement si le champignon possède des noyaux génétiquement différents. Nous avons démontrés l'importance des mécanismes d'échange génétique et de ségrégation pour produire en très peu de temps de nouveaux descendants ayant des effets symbiotiques nouveaux. De plus, nos résultats suggèrent que différentes espèces de plantes peuvent agir sur le devenir des nucleotypes après l'échange génétique et la ségrégation chez les CEA, et pourraient contribuer à la maintenance de la diversité génétique au sein d'un même CEA. Ce travail apporte des éléments nouveaux pour comprendre comment les plantes et les champignons ont coévolué et comment la diversité génétique chez les CEA peut être maintenue. Nous recommandons de considérer la diversité génétique intra-individuelle des CEA et ces mécanismes lors de futures recherches sur cette symbiose.

The arbuscular mycorrhizal fungus Glomus intraradices is haploid and has a small genome size in the lower limit of eukaryotes.

The genome size, complexity, and ploidy of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was determined using flow cytometry, reassociation kinetics, and genomic reconstruction. Nuclei of G. intraradices from in vitro culture, were analyzed by flow cytometry. The estimated average length of DNA per nucleus was 14.07+/-3.52 Mb. Reassociation kinetics on G. intraradices DNA indicated a haploid genome size of approximately 16.54 Mb, comprising 88.36% single copy DNA, 1.59% repetitive DNA, and 10.05% fold-back DNA. To determine ploidy, the DNA content per nucleus measured by flow cytometry was compared with the genome estimate of reassociation kinetics. G. intraradices was found to have a DNA index (DNA per nucleus per haploid genome size) of approximately 0.9, indicating that it is haploid. Genomic DNA of G. intraradices was also analyzed by genomic reconstruction using four genes (Malate synthase, RecA, Rad32, and Hsp88). Because we used flow cytometry and reassociation kinetics to reveal the genome size of G. intraradices and show that it is haploid, then a similar value for genome size should be found when using genomic reconstruction as long as the genes studied are single copy. The average genome size estimate was 15.74+/-1.69 Mb indicating that these four genes are single copy per haploid genome and per nucleus of G. intraradices. Our results show that the genome size of G. intraradices is much smaller than estimates of other AMF and that the unusually high within-spore genetic variation that is seen in this fungus cannot be due to high ploidy.

Genetic variability in a population of arbuscular mycorrhizal fungi causes variation in plant growth.

Different species of arbuscular mycorrhizal fungi (AMF) alter plant growth and affect plant coexistence and diversity. Effects of within-AMF species or within-population variation on plant growth have received less attention. High genetic variation exists within AMF populations. However, it is unknown whether genetic variation contributes to differences in plant growth. In our study, a population of AMF was cultivated under identical conditions for several generations prior to the experiments thus avoiding environmental maternal effects. We show that genetically different Glomus intraradices isolates from one AMF population significantly alter plant growth in an axenic system and in greenhouse experiments. Isolates increased or reduced plant growth meaning that plants potentially receive benefits or are subject to costs by forming associations with different individuals in the AMF population. This shows that genetic variability in AMF populations could affect host-plant fitness and should be considered in future research to understand these important soil organisms.

Gene copy number polymorphisms in an arbuscular mycorrhizal fungal population.

Gene copy number polymorphism was studied in a population of the arbuscular mycorrhizal fungus Glomus intraradices by using a quantitative PCR approach on four different genomic regions. Variation in gene copy number was found for a pseudogene and for three ribosomal genes, providing conclusive evidence for a widespread occurrence of macromutational events in the population.

Significant genetic and phenotypic changes arising from clonal growth of a single spore of an arbuscular mycorrhizal fungus over multiple generations.

Arbuscular mycorrhizal fungi (AMF) are highly successful plant symbionts. They reproduce clonally producing multinucleate spores. It has been suggested that some AMF harbor genetically different nuclei. However, recent advances in sequencing the Glomus irregulare genome have indicated very low within-fungus polymorphism. We tested the null hypothesis that, with no genetic differences among nuclei, no significant genetic or phenotypic variation would occur among clonal single spore lines generated from one initial AMF spore. Furthermore, no additional variation would be expected in the following generations of single spore lines. Genetic diversity contained in one initial spore repeatedly gave rise to genetically different variants of the fungus with novel phenotypes. The genetic changes represented quantitative changes in allele frequencies, most probably as a result of changes in the frequency of genetic variation partitioned on different nuclei. The genetic and phenotypic variation is remarkable, given that it arose repeatedly from one clonal individual. Our results highlight the dynamic nature of AMF genetics. Even though within-fungus genetic variation is low, some is probably partitioned among nuclei and potentially causes changes in the phenotype. Our results are important for understanding AMF genetics, as well as for researchers and biotechnologists hoping to use AMF genetic diversity for the improvement of AMF inoculum.

Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices.

Arbuscular mycorrhizal fungi (AMF) are important symbionts of plants that improve plant nutrient acquisition and promote plant diversity. Although within-species genetic differences among AMF have been shown to differentially affect plant growth, very little is actually known about the degree of genetic diversity in AMF populations. This is largely because of difficulties in isolation and cultivation of the fungi in a clean system allowing reliable genotyping to be performed. A population of the arbuscular mycorrhizal fungus Glomus intraradices growing in an in vitro cultivation system was studied using newly developed simple sequence repeat (SSR), nuclear gene intron and mitochondrial ribosomal gene intron markers. The markers revealed a strong differentiation at the nuclear and mitochondrial level among isolates. Genotypes were nonrandomly distributed among four plots showing genetic subdivisions in the field. Meanwhile, identical genotypes were found in geographically distant locations. AMF genotypes showed significant preferences to different host plant species (Glycine max, Helianthus annuus and Allium porrum) used before the fungal in vitro culture establishment. Host plants in a field could provide a heterogeneous environment favouring certain genotypes. Such preferences may partly explain within-population patterns of genetic diversity.

Preference, specificity and cheating in the arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal symbioses are mutualistic interactions between fungi and most plants. There is considerable interest in this symbiosis because of the strong nutritional benefits conferred to plants and its influence on plant diversity. Until recently, the symbiosis was assumed to be unspecific. However, two studies have now revealed that although it can be largely unspecific with the fungal community composition changing seasonally, in certain ecosystems it can also be highly specific and might potentially allow plants to cheat the arbuscular mycorrhizal network that connects plants below ground.

High genetic variability and low local diversity in a population of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are ecologically important root symbionts of most terrestrial plants. Ecological studies of AMF have concentrated on differences between species; largely assuming little variability within AMF species. Although AMF are clonal, they have evolved to contain a surprisingly high within-species genetic variability, and genetically different nuclei can coexist within individual spores. These traits could potentially lead to within-population genetic variation, causing differences in physiology and symbiotic function in AMF populations, a consequence that has been largely neglected. We found highly significant genetic and phenotypic variation among isolates of a population of Glomus intraradices but relatively low total observed genetic diversity. Because we maintained the isolated population in a constant environment, phenotypic variation can be considered as variation in quantitative genetic traits. In view of the large genetic differences among isolates by randomly sampling two individual spores, <50% of the total observed population genetic diversity is represented. Adding an isolate from a distant population did not increase total observed genetic diversity. Genetic variation exceeded variation in quantitative genetic traits, indicating that selection acted on the population to retain similar traits, which might be because of the multigenomic nature of AMF, where considerable genetic redundancy could buffer the effects of changes in the genetic content of phenotypic traits. These results have direct implications for ecological research and for studying AMF genes, improving commercial AMF inoculum, and understanding evolutionary mechanisms in multigenomic organisms.

Evolutionary forces affect multigenomic arbuscular mycorhizal fungi : an experimental approach using Glomus intraradices

Abstract Arbuscular Mycorhizal Fungi (AMF) are important plant symbionts that can improve floristic diversity and ecosystem productivity. These important fungi are obligate biotrophs and form symbioses with roots of the majority of plant species, improving plant nutrient acquisition in exchange of photosynthates. AM fungi are successful both ecologically as they occupy a very large spectrum of environments as well as host range and evolutionarily, as this symbiosis is over 400 million years old. These fungi grow and reproduce clonally by hyphae and multinucleate spores. AMF are coenocytic and recent work has shown that they harbor genetically different nuclei and that AMF populations are genetically diverse. How AMF species diversity is maintained has been addressed theoretically and experimentally at the community level. Much less attention has been drawn to understand how genetic diversity is maintained within populations although closely related individuals are more likely to compete for the same resources and occupy similar niches. How infra-individual genetic diversity is shaped and maintained has received even less attention. In Chapter 2, we show that individuals from a field population may differ in their symbiotic efficiency under reduced phosphate availability: We show there is genetic variation in an AMF field population for fitness-related growth traits in response to different phosphate availability acid host species. Furthermore, AFLP fingerprints of the same individuals growing in contrasting environments diverged suggesting that the composition in nuclei of AMF is dynamical and affected by environmental factors. Thus environmental heterogeneity is likely to play an important role for the maintenance of genetic diversity at the population level. In Chapter 3 we show that single spores do not inherit necessarily the same genetic material. We have found genetic divergences using two different types of molecular marker, as well as phenotypic divergences among single spore lines. Our results stress the importance of considering these organisms as a multilevel hierarchical system and of better knowing their life cycle. They have important consequences for the understanding of AMF genetics, ecology and the development of commercial AMF inocculum. Résumé Les champignons endomycorhiziens arbusculaires (CEA) sont d'importants symbiontes pour les plantes, car ils augmentent la diversité et la productivité des écosystèmes. Ces importants symbiontes sont des biotrophes obligatoires et forment une symbiose avec la plupart des plantes terrestres. Ils améliorent l'acquisition de substances nutritives de leurs hôtes en échange de sucres obtenus par photosynthèse. Ces champignons ont un grand succès écologique, ils colonisent une grande rangée d'environnements ainsi que d'hôtes. Ils ont aussi un succès évolutif certain de part le fait que cette symbiose existe depuis plus de 400 millions d'années. Les CEA sont asexués et croissent clonalement en formant des hyphes et des spores multinuclées. Les CEA sont des coenocytes et des travaux de recherche récents ont montré qu'ils possèdent des noyaux génétiquement différents. D'autres travaux ont aussi révélé que les populations de CEA sont génétiquement diversifiées. Comment la diversité des CEA est maintenue a seulement été adressée par des études théoriques et expérimentalement au niveau des communautés. Très peu d'attention a été portée sur le maintien de la diversité génétique infra et inter populationnelle, or ce sont les individus les plus proches génétiquement qui vont entrer en compétition pour des ressources et niches similaires. La formation et le maintien de la diversité intra-individu des CEA a reçu très peu d'attention. Dans le chapitre 2, nous montrons que des individus CEA d'un même champ différent dans leur efficacité symbiotique lorsque la concentration en phosphoré est réduite. Nous montrons qu'il existe de la variance génétique dans une population de CEA provenant d'un même champ en réponse à différentes concentrations de phosphore, ainsi qu'en réponse à différentes espèces d'hôtes, et ceci pour des traits de croissance vraisemblablement liés au succès reproducteur. De plus grâce à des AFLP nous avons pu montrer que le génome de ces individus subissent des changements lorsqu'ils croissent dans des environnements contrastés. Ceci suggère que les noyaux génétiquement différents des CEA sont des entités dynamiques. Il est fort probable que l'hétérogénéité environnementale joue un rôle dans le maintien de la diversité génétique des populations de CEA. Dans le chapitre 3, nous montrons que toutes les spores d'un même mycélium parental de CEA ne reçoivent pas exactement le même contenu génétique. Nous avons mis en évidence des divergences entre des Lignées monosporales en utilisant deux types de marqueur moléculaires, ainsi que des différences phénotypiques. Nos résutats soulignent l'importance de considézer ces organismes comme dés systëmes hiérarchiques mufti-niveaux, ainsi que de mieux connaître leur cycle de vie. Nos résultats ont d'importantes conséquences pour la compréhension du système génétique des CEA, ainsi que de leur évolution, leur écológie, mais également des conséquences pour la production d' inoccultim commercial.

High-level molecular diversity of copper-zinc superoxide dismutase genes among and within species of arbuscular Mycorrhizal fungi.

In the ecologically important arbuscular mycorrhizal fungi (AMF), Sod1 encodes a functional polypeptide that confers increased tolerance to oxidative stress and that is upregulated inside the roots during early steps of the symbiosis with host plants. It is still unclear whether its expression is directed at scavenging reactive oxygen species (ROS) produced by the host, if it plays a role in the fungus-host dialogue, or if it is a consequence of oxidative stress from the surrounding environment. All these possibilities are equally likely, and molecular variation at the Sod1 locus can possibly have adaptive implications for one or all of the three mentioned functions. In this paper, we analyzed the diversity of the Sod1 gene in six AMF species, as well as 14 Glomus intraradices isolates from a single natural population. By sequencing this locus, we identified a large amount of nucleotide and amino acid molecular diversity both among AMF species and individuals, suggesting a rapid divergence of its codons. The Sod1 gene was monomorphic within each isolate we analyzed, and quantitative PCR strongly suggest this locus is present as a single copy in G. intraradices. Maximum-likelihood analyses performed using a variety of models for codon evolution indicated that a number of amino acid sites most likely evolved under the regime of positive selection among AMF species. In addition, we found that some isolates of G. intraradices from a natural population harbor very divergent orthologous Sod1 sequences, and our analysis suggested that diversifying selection, rather than recombination, was responsible for the persistence of this molecular diversity within the AMF population.

Genetic exchange in an arbuscular mycorrhizal fungus results in increased rice growth and altered mycorrhiza-specific gene transcription.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts with most terrestrial plants. They improve plant nutrition, particularly phosphate acquisition, and thus are able to improve plant growth. In exchange, the fungi obtain photosynthetically fixed carbon. AMF are coenocytic, meaning that many nuclei coexist in a common cytoplasm. Genetic exchange recently has been demonstrated in the AMF Glomus intraradices, allowing nuclei of different Glomus intraradices strains to mix. Such genetic exchange was shown previously to have negative effects on plant growth and to alter fungal colonization. However, no attempt was made to detect whether genetic exchange in AMF can alter plant gene expression and if this effect was time dependent. Here, we show that genetic exchange in AMF also can be beneficial for rice growth, and that symbiosis-specific gene transcription is altered by genetic exchange. Moreover, our results show that genetic exchange can change the dynamics of the colonization of the fungus in the plant. Our results demonstrate that the simple manipulation of the genetics of AMF can have important consequences for their symbiotic effects on plants such as rice, which is considered the most important crop in the world. Exploiting natural AMF genetic variation by generating novel AMF genotypes through genetic exchange is a potentially useful tool in the development of AMF inocula that are more beneficial for crop growth.