Arbuscular Mycorrhiza Genetics of The Fungal Partner

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Arbuscular Mycorrhiza: The Challenge to Understand the Genetics of the Fungal Partner
Ian R. Sanders1 and Daniel Croll2
1 Department of Ecology and Evolution, University of Lausanne, 1015 Lausanne, Switzerland; email: ian.sanders@unil.ch 2
Annu. Rev. Genet. 2010.44:271-292. Downloaded from www.annualreviews.org by HINARI on 11/12/11. For personal use only.
Department of Plant Pathology, Institute of Integrative Biology, ETH Zurich, 8092 Zurich, Switzerland; email: daniel.croll@agrl.ethz.ch
Annu. Rev. Genet. 2010. 44:27192 First published online as a Review in Advance on September 3, 2010 The Annual Review of Genetics is online at genet.annualreviews.org This articles doi: 10.1146/annurev-genet-102108-134239 Copyright c 2010 by Annual Reviews. All rights reserved 0066-4197/10/1201-0271$20.00
Key Words
symbiosis genes, plants, recombination, genetic exchange, gene expression, transcriptomics
Abstract
Arbuscular mycorrhizal symbioses occur between fungi and the majority of plant species. They are important for plant nutrition, plant growth, protection from pathogens, plant diversity, nutrient cycling, and ecosystem processes. A key goal in research is to understand the molecular basis of the establishment, regulation, and functioning of the symbiosis. However, lack of knowledge on the genetics of the fungal side of this association has hindered progress. Here, we show how several key, recently discovered processes concerning the genetics of arbuscular mycorrhizal fungi could be essential for ultimately understanding the molecular genetics of this important symbiosis with plants.
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INTRODUCTION
A major event in the life of our planet occurred 400 to 500 mya, when plants colonized land (91). The roots of those rst plants were themselves colonized by an ancient group of fungi, the Glomeromycota or arbuscular mycorrhizal fungi (AMF) (8, 78, 97). The Glomeromycota had probably already diverged from the other fungal lineages some several hundred million years before plants emerged on land (38). Since the colonization of land, and throughout the subsequent evolution of terrestrial plants, the majority of plant species have formed intimate associations known as arbuscular mycorrhizal symbioses with these fungi (92). Plants form the basis of productivity in natural ecosystems as well as in agriculture. All the major crop plants that feed the worlds human population form the arbuscular mycorrhizal symbiosis. The symbiosis improves plant growth, most notably, by improving plant phosphorus acquisition, but also by protecting plants from pathogens (36, 93). The contribution of this symbiosis to plant growth is of enormous importance for the productivity of the major crops that humans rely on for nutrition. Additionally, the symbiosis plays a key role in nutrient cycling in natural ecosystems, in ecosystem productivity, and plant diversity (99, 100). Given its importance and thus the amount of research conducted on this symbiosis, it is clear that the genetics of this ancient symbiosis needs to be understood. This rst entails understanding the fundamental biology, genetics, and genomics of both groups of organisms involved in the symbiosis, the plants and Glomeromycotan fungi, and also the genetics, genomics, and transcriptomics of the interaction itself. Unlike much research in molecular genetics, which makes use of model organisms and is amenable to laboratory-based experimental approaches, research on the mycorrhizal symbiosis is driven by its fundamental importance for plant growth, despite the fact that the fungal partner is extremely difcult to manipulate experimentally. The basic biology and genetics of model organisms are well understood, and
this provides a foundation necessary for undertaking more complex studies in molecular genetics and cell biology. Despite the great importance of the mycorrhizal symbiosis, much of the basic biology of the fungal partner that is fundamental for understanding their genetics is lacking. Basic information, which would be taken for granted in a model organism, such as level of ploidy, number of chromosomes, and whether meiosis, genetic exchange, recombination, and segregation occur, is poorly understood for most arbuscular mycorrhizal fungi. This information is essential for understanding the basic genetics of these fungi and how the genetics of the fungus inuences the symbiosis with plants. In contrast, the basic biology and genetics of the plant partners of this symbiosis are well understood, at least for a large number of model species and for the worlds major crop species. Furthermore, full genome sequences exist for several plant species, as well as a vast array of transcriptome data and good knowledge of plant cell biology. The imbalance in our knowledge of the fungal and plant partners in the symbiosis is highlighted by the key advances that have been made in understanding the role of some of the genes involved in the establishment of the symbiosis, its regulation, and function; almost all of which are plant genes. Because of the strong disparity between what is known about the genetics of plants and genetics of AMF, we focus on current advances in understanding the genetics of the fungal partner. We rst give a brief overview of the current state of knowledge of the plant and fungal genes involved in the symbiosis, and we present new ndings regarding the biology of the fungal side of the arbuscular mycorrhizal symbiosis, which is essential for understanding their genetics. Some of these new ndings are so particular to this fungal phylum that conventional models of evolution and of Mendelian genetics are difcult to apply in studying inheritance and segregation, gene expression, and genomics of these organisms. Our conclusions are that it will be possible to develop an experimental approach to understanding the genetics of AMF, despite the complexities of
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studying these fungi, and that this represents an exciting challenge for geneticists. We argue that this complexity should not be ignored by researchers working on the mycorrhizal symbiosis, as a whole. We believe that understanding the genetics of AMF is critical for plant scientists who are trying to unravel the molecular genetics of this important symbiosis, and we suggest ways of better integrating these two elds of research in the future.
GENES INVOLVED IN THE ESTABLISHMENT AND FUNCTIONING OF THE SYMBIOSIS

AMF are obligate symbionts of plants (92). During colonization, hyphae of the fungi grow towards a root and penetrate it, passing through the rst layer of cells and then between the cells of the root cortex (Figure 1). The fungi also penetrate the cell wall of some cortical cells, where they invaginate the host cell membrane and form a highly branched structure known as an arbuscule. The arbuscule is actually the fungal membrane surrounded by the host cell membrane, and it provides the possibility for bidirectional exchange of nutrients between the host and fungus (35) (Figure 1). Hyphae of the fungi also grow out of the root and into the soil, where they can act as an extension of the root system, efciently taking up nutrients, especially phosphate, and transporting them back to the plant. The nutrients are then taken up by the plant through the arbuscule. The fungus benets from this association by obtaining carbohydrates from the plant, and these are also assumed to pass through the arbuscule. Extraradicle hyphae can also colonize other roots of the same or different plant species resulting in the formation of a below-ground hyphal network connecting many different plants within an ecosystem. Spores of the fungus can also form on the terminus of a hypha, and these spores can germinate and form a new symbiosis (Figure 1). Considerable advances have been made in identifying some of the genes involved in the
key phases of the development and functioning of the mycorrhizal symbiosis. These phases are largely divided into the presymbiotic state, penetration of the root, development inside the root cortex, development of the arbuscule, and establishment of a functioning symbiosis (70). Almost all the genes identied to date are of plant origin, not fungal. In the presymbiotic phase, the hyphae of the fungus grow toward the root, and much hyphal branching is often observed. Strigolactones, plant hormones derived from the carotenoid pathway, induce hyphal branching and unusual mitochondrial activity in the fungus (1, 6), but the genes involved are currently unknown. The fungus releases molecules called Myc factors that induce the expression of the plant gene ENOD11, which encodes putative cell wallrepetitive, proline-rich proteins in Medicago truncatula. This gene is expressed during early but also late stages of mycorrhizal development and even in arbuscule-containing cells (50). An exciting discovery was recently made showing that the plant prepares its cells before penetration in order to guide the fungus through the outer layers of cells. The plant cell constructs a tunnel (known as the prepenetration apparatus) within the plant cell, through which the fungus can grow. The plant nucleus migrates with the developing tunnel (24, 25). Two genes in M. truncatula, DMI2 and DMI3, have been shown to be necessary for induction of the prepenetration apparatus. Using mutants of Lotus japonicus, ve additional genes, CASTOR, POLLUX, NUP85, NUP133, and CYCLOPS, have been identied in legumes that are required for the development of the fungus inside the root and for development of arbuscules. All are common to the rhizobium-legume symbiosis as well as the mycorrhizal symbiosis (70), and a detailed description of their joint activity in inducing early signal transduction in the symbiosis has been presented (70). Genes that are necessary for mycorrhizal formation have also been investigated in rice (Oryza sativa). Mutations in the putative orthologs of CASTOR, POLLUX, CCAMK (DMI3), and
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Roots
f
Spore Hyphae
b
3 PO4 3 PO4
3 PO4
Spore Hypha Appressorium Prepenetration apparatus
Fusion of hyphae
Root
Arbuscule
Nucleus
Figure 1 The AMF lifestyle. (a) Spores of the fungus germinate in the soil. Traditionally, they are thought to have to nd a plant root in order to survive, but it is also possible that they could connect directly to existing hyphal networks of the fungus. Hyphae may nd a root and form a symbiosis with the plant. The fungus penetrates the plant root, after forming an appressorium, then colonizes the cortex of the plant root and develops arbuscules, structures made by the fungus inside plant cells, which allow the transfer of nutrients between the two partners. Inset (a) redrawn with permission from 71. (b) The fungus then produces new hyphae that grow out from the root into the soil and can absorb nutrients and transport them back to the plant. (c). Hyphae can produce new spores. Inset (c) shows a spore containing many nuclei labeled with a uorescent dye; courtesy of Mohamed Hijri (d ) Hyphae can also continue to grow through the soil and colonize new plants, thereby creating the hyphal network. (e) Other genetically different individuals may colonize adjacent plants or even the same plant. ( f ) Hyphae of genetically different individuals can fuse.
CYCLOPS in rice showed that these genes are necessary for mycorrhiza formation in rice as well as in legumes (33). However, a number of other genes were identied in rice that appear
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to be specic to mycorrhiza formation and are not known to have any involvement in other symbioses (33). These have been classed into two groups: AM1-3 and AM11, which are
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expressed early in the development of the symbiosis before arbuscule formation, and AM10, 11, 14, 15, 18, 20, 24, 25, 26, 29, 31, 34, 39, 42 and PT11, which are expressed during later stages of development, and some are actually in arbusculated cells. The functions encoded by most of these are currently unknown. Two genes present in AMF have been implicated in playing a possible role in the development of the fungus inside the root. Because there is no suitable knockout or transformation system for these fungi, the most successful approach has been to nd orthologs of genes in AMF that are known to be important for successful infection of roots by fungal pathogens. Complementation of mutants of the fungal pathogens Magnaporthe oryzae and Colletotrichum linemuthianum, which were unable to infect the host, was successful using two orthologs found in AMF. These studies suggested that orthologs of STE12 and a gene encoding Era-like GTPase may play a role in penetration of the root and subsequent control of hyphal growth inside the root, respectively (40, 98). Because arbuscules represent the interface between the plant and fungus and are the site where the exchange of nutrients occurs, much attention has been paid to genes expressed in arbuscule-containing cells. Given that the plant receives phosphate from the fungus, it is intriguing that at least two plant phosphate (P) transporters, PT4 and PT3, have been identied that appear to also be implicated in the maintenance and life span of arbuscules (22, 49). It is likely that these P transporters play a role in the functioning of the symbiosis, as they are expressed in the arbuscule. Several other plant P transporters and one fungal P transporter have been identied in arbusculated cells (4). The functional symbiosis also involves the transfer of carbohydrates from the plant to the fungus. NMR studies reveal that carbohydrates are received from the plant in the form of hexose (3). A novel hexose transporter has been found in the only known Glomeromycotan fungus that does not form the mycorrhizal symbiosis, Geosiphon pyriformis (90). G. pyriformis forms a symbiotic association with photosynthetic
algae, from which it receives carbohydrates. It is possible that similar transporters are used by mycorrhizal fungi to receive hexose from the plant. Other genes have been identied in the fungus that could potentially be important in the development or functioning of the symbiosis. The AMF superoxide dismutase gene GmarCuZnSOD produces a functional polypeptide that is able to increase tolerance to oxidative stress. It has been suggested that this gene may play a role as an inactivating system to localized host defense response in arbusculated cells (56). Additionally, phosphate transporters have been found in extraradicle hyphae that may be involved in transporting phosphate to the plant (37, 59).
WHY WE KNOW SO LITTLE ABOUT THE GENETICS OF THE FUNGAL PARTNER

There are several reasons why so little is known about the genetics, ploidy, genome organization, genetic variation, and population genetics of AMF. Most knowledge of fungal genetics is based on species from two phyla, the Ascomycota and Basidiomycota, that only have a common ancestor with the Glomeromycota several hundreds of millions of years ago (38, 47). Therefore, considerable divergence in their genomes, metabolism, and life history has occurred, making it difcult to perform comparative studies with other well-studied ascomycotan or basidiomycotan fungi. This is an important point, as some controversial aspects of AMF genome organization and life histories have been doubted on the basis that they are not observed in other well-studied fungal groups. Another limitation to studying AMF genetics is that the fungi have to be grown with plants, meaning that it has often been difcult to obtain clean material for molecular studies. To date, these fungi have not been successfully cultured without host roots. It is possible to culture AMF on articial media with roots transformed with Agrobacterium rhizogenes, allowing a clean culture system (5, 94). However, this axenic system
Generation: an AMF generation is a cycle of fungus growth that starts from a spore, colonizes a plant, and then produces new spores
has not been successful for culturing many different AMF species and consequently, most of our knowledge on AMF genetics is restricted to the species Glomus intraradices, which grows well in this culture system. AMF cultivation is slow compared with many other microorganisms. Generation time, dened as time from inoculation of roots with spores until production of new viable spores, is a minimum of approximately three months, making genetic experiments involving several generations very slow. However, advances in understanding the molecular genetics of AMF have probably been most greatly hindered for two reasons. As mentioned, there is no established, reliable longterm transformation system. There are reports of transformation in two species of AMF, but expression appears to be transient (34, 39). This means that alternative and innovative approaches need to be applied to understanding the possible function of AMF genes. Second, it has been assumed for a long time that AMF grow clonally, that they are ancient asexuals, and therefore, that no genetic exchange occurs among genetically different AMF (51). This has meant that there has been no experimental basis for studying AMF genetics using classical Mendelian genetic approaches.
GENOME SIZE, CHROMOSOMES, AND PLOIDY IN ARBUSCULAR MYCORRHIZAL FUNGI

Attempts at investigating genome size in AMF have been made by estimating the amount of DNA per nucleus, using ow cytometry, in different AMF species (7, 42, 43, 45). From such measurements, it is only possible to know the genome size if the ploidy of the nuclei is also known. Most fungi are haploid and have relatively small genomes compared with many other eukaryotes (11, 32). However, measurements of nuclear DNA content among AMF species range enormously from approximately 15 Mb to approximately 1058 Mb DNA per nucleus (42, 45). This has led to speculation that some AMF are highly polyploid (73). It has been
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pointed out that some of these measurements may be imprecise because of the use of inappropriate size standards and because, when most of the measurements were made, researchers were not aware of the high adenine-thymine (AT) bias that is now known to occur in some AMF genomes (42, 43). However, although corrections for these two factors would change the estimates, such changes cannot possibly account for the enormous range in nuclear DNA content among AMF species that are documented in published studies. Only two studies have actually combined measurements of nuclear DNA content with reassociation kinetics to estimate genome size and ploidy in an AMF. The genome size and ploidy have been investigated in detail in the fungus G. intraradices (42). Nuclear DNA content and the haploid genome size (as estimated by reassociation kinetics) were found to be small and also very similar. This indicates that nuclei in that species are haploid. Furthermore, there were no peaks of other sizes that could be attributed to nuclei of other ploidy levels. Given that the estimate of nuclear DNA content in G. intraradices was considerably smaller than measurements in other AMF species, it was pertinent to apply the same techniques to an AMF, Scutellospora castanea, that contained a large amount of DNA per nucleus. A reanalysis of existing data for this fungus also showed that the haploid genome size, as measured by reassociation kinetics, was very close to the nuclear DNA content (43, 44). Unfortunately, to date, there are no other published studies that have presented investigations into the ploidy of any other AMF species, and such investigations are certainly needed. In many species of plants and animals, chromosome number can be relatively easy to count. Unfortunately, this has not proved to be the case for AMF chromosomes, and there are no published studies on the observation of AMF chromosomes. Consequently, molecular approaches have been used to try to assess chromosome number in G. intraradices with limited success (41). Sequencing of the subtelomeric regions adjacent to the telomeric repeats
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revealed six different classes of sequences, indicating a minimum of three chromosomes. However, the number and sizes of fragments of genomic DNA separated by pulsed-eld gel electrophoresis suggested at least four chromosomes. The maximum chromosome number in this fungus cannot be determined from this study. Furthermore, sequence variation was unusually high in two classes of the subtelomeric sequences, indicating that there could be more chromosomes with this class of subtelomeric region. To our knowledge, there are no other published studies on chromosome number in AMF.
HETEROKARYOSIS AND ITS SIGNIFICANCE FOR UNDERSTANDING ARBUSCULAR MYCORRHIZAL FUNGI GENETICS AND GENOMICS
Many studies have shown that unusually high polymorphism exists within AMF individuals. In many cases, such studies have documented the variation within individual spores of the fungus or in material that has grown clonally from an individual spore in monoaxenic culture. Such variation was rst observed in rDNA (12, 13, 57, 82, 87, 88) and later in the coding region of genes encoding a binding protein, tubulin, H+ ATPase, and P-type II ATPase (1416, 18, 54). Such polymorphism could be explained by two features of the life history of AMF. AMF grow clonally, colonizing new roots and forming a network of hyphae in the soil connecting different plants (Figure 1). First, the hyphae are not typical of fungi in the Ascomycota and Basidiomycota as the cytoplasm is continuous rather than separated into compartments. This means that hyphae are coenocytic and that the nuclei can potentially move and migrate in the hyphal network. Second, spores develop on the termini of a single extraradical hyphae in the soil. As the spore develops, nuclei from the cytoplasm of the fungal hypha migrate into the spore until the mature spore contains hundreds, and sometimes thousands, of nuclei (48). Unlike other fungi and most, if
not all, other eukaryotes, it appears that there is never a stage during the AMF life cycle when the fungus is reduced to a propagule containing one nucleus that will go on to initiate the next generation (86, 102). There are considerable implications of these two features of the AMF life cycle for the genetics of the symbiosis. As the fungus grows, considerable nuclear division must occur. If the growth of the fungus is entirely clonal, then somatic mutations could permit divergence of the nuclei within the network, thereby creating a heterokaryotic state, a cytoplasm containing genetically different nuclei or nucleotypes. In basidiomycete and ascomycete fungi, hyphal compartments can be dikaryotic, containing two nuclei per hyphal compartment. But unlike the Glomeromycota, those fungi possess a clear sexual stage in their life cycle where the two nuclei fuse, meiosis occurs, and a new generation of spores is produced, each containing a single haploid nucleus. Direct evidence supporting the heterokaryotic state in the AMF S. castanea has been presented (54). S. castanea contains two highly divergent internal transcribed spacer (ITS2) variants in its rDNA. Fluorescence in situ hybridization was used to show that the ITS2 variants were segregated on different nuclei, and some nuclei contained both variants. The heterokaryosis hypothesis has been challenged (73). The authors proposed that if AMF contain genetically different nuclei, then not all nuclear genotypes would be inherited by every spore, assuming a random assortment of nuclei entering the spore during its development. A model predicted that such segregation of different nuclei would happen in only one generation. This was then tested by looking at inheritance of 13 variants of a marker, called POL-like sequence (PLS), that were known to coexist in the AMF Glomus etunicatum. All 13 PLS variants were inherited in all offspring after one generation. The authors concluded that all 13 variants must be present in each nucleus. Despite the fact that most fungi are haploid, they suggested that this is because G. etunicatum could be highly polyploid.
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AMF individual: an AMF that has been propagated from a single spore of the fungus Coenocytic: having a state in which nuclei are not separated into compartments by septa and coexist in a common cytoplasm Heterokaryotic: having a state in which the cytoplasm contains two or more genetically different nuclei Nucleotype: the genotype of a given nucleus
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Anastomosis: the fusion of two fungal hyphae, allowing the cytoplasm and possibly the nuclei of two individuals to mix
One additional way to explain the efcient transmission of all 13 PLS variants to the offspring would be that PLS is present in high copy number per nucleus. This issue was subsequently addressed (43). The nuclear DNA content of G. etunicatum was found to be approximately 43 Mb, meaning that high ploidy was unlikely. Combining the nuclear DNA content estimates with quantitative polymerase chain reaction (PCR) allows a prediction of the number of copies of the PLS gene per nucleus, and this was found to be approximately two, meaning that some of the 13 PLS variants must be segregated among nuclei (43). Taken together, these two studies indicate that the PLS variants were all inherited from one generation to the next and that the variants must be located on different nuclei (43, 73). This implies that the heterokaryotic state is maintained from one generation to the next, which would make AMF different from other fungi that often exist for only a transient part of their life cycle as a heterokaryon. The mycorrhizal fungus G. intraradices was chosen as a candidate for the rst whole genome sequencing project on an AMF by the U.S. Department of Energy (DoE) (55). Sequencing has been carried out at the Joint Genome Institute ( JGI). Although the whole genome of G. intraradices has not been sequenced to investigate heterokaryosis, it can shed light on the potential amount of polymorphism distributed among nuclei. A very large amount of genome sequence data now exists, although there have been considerable difculties in assembly owing to the unusually large amount of polymorphism (61). The high polymorphism is additional support for the heterokaryotic state of AMF. Owing to the high polymorphism, the predicted assembled genome size is several times larger than the amount of DNA contained per nucleus (61). Given that each nucleus contains approximately 15 Mb DNA, the total assemblable genome size will allow a prediction of the amount of polymorphism among nuclei, and further analysis will reveal how much of that variation occurs in protein-coding genes. Further sequencing is under way to complete the genome.
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SELF-SELF AND SELF-NONSELF ANASTOMOSIS AND GENETIC EXCHANGE IN ARBUSCULAR MYCORRHIZAL FUNGI Anastomosis
A fundamental aspect of fungal life cycles is the potential of fungi to undergo anastomosis or fusion among different individuals. Fungi have developed a wide range of capabilities to connect mycelia and form common structures (31). The different types of hyphal fusion could increase network connectivity and homeostasis, increase exchange of nuclei to form dikaryons during mating, and even increase higher orders of genetic complexity during the conidial fusions of germinating spores (7981). Germinating AMF spores may gain a crucial advantage by connecting primary hyphae to a larger network and getting access to already colonized host plants (29, 46), although this would mean that they mix their nuclei with another individual. In the case of AMF, anastomosis could be the mechanism that allows mixing of nuclei and potential nuclear fusion and recombination. Evidence for anastomosis in AMF is shown in a number of reports, although none of these elucidate the underlying genetic architecture controlling such fusions. Anastomoses between hyphae originating from the same isolate were shown to occur in AMF (27, 28, 66). Therefore, the potential for connecting different host plants through a web of anastomosed hyphal networks is of particular interest (46). Hyphae originating from the same isolate but growing on different host plants were shown to anastomose between layers of membrane lters (29). This indicated the potential of fungal isolates to create large hyphal networks connecting different host plants. The potential physiological signicance of anastomosis is highlighted in another study where plants, separately inoculated with the same AMF isolate, were shown to exchange nutrients (measured by movement of isotopes of phosphorus), most likely through formation of anastomoses between the two AMF hyphal networks (65). The capacity of hyphae to form underground
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networks connecting different plants was proposed as a major ecological force shaping plant resource allocation within ecosystems (46). Hyphal fusion between genetically different isolates could play a crucial role in the fungal life cycle, as it constitutes the opportunity for exchange of genetically different nuclei, similar to fusions of fungi of opposite mating types in ascomycetes and basidiomycetes. It was suggested that basic recognition mechanisms of self and nonself (i.e., vegetative incompatibility) must be present in AMF because they exist in other fungal phyla (74). Indeed, in one study, no anastomosis was observed among genetically different isolates of the same AMF species that originated from different geographic locations (30). Studies on G. intraradices from one location revealed that genetic variability among individuals was very high, using both amplied fragment length polymorphism (AFLP) and sequencebased markers (21, 53). This provided the possibility of testing whether anastomosis occurs among genetically different AMF, without the confounding factor of geographic origin. It also allowed the manipulation of genetic distance between pairings. Contacts between primary hyphae of germinating spores were observed in which hyphae originated from spores of the same isolate or two genetically different isolates (19). Four types of interactions were observed among genetically different hyphal contacts: (a) perfect fusion and cytoplasmic exchange (PF); (b) postfusion incompatibility (PFI), in which anastomosis occurred, followed by formation of septa in the two interacting hyphae, effectively blocking any further exchange; (c) prefusion incompatibility (PreFI) in which some form of initial attraction occured between the hyphae, shown by bulging in the hyphal wall of both individuals at the contact point, but no anastomosis; and (d ) no interaction (NI) of hyphae following contact. Frequency of PF between genetically different AMF was between 1% and 10%, depending on genetic distance. In all but one case, a low number of PF was observed, even between isolates that are highly differentiated genetically. The frequency of NI between genetically different hyphae was be-
tween 70% and 83% of contacts, depending on the genetic identity of the pair of isolates. In contrast, contacts between hyphae originating from spores of the same isolate gave an all or nothing response with approximately 50% PF and 50% NI and no intermediate stages such as PreFI or PFI. To date, no genes involved in the establishment of hyphal fusions have been described in AMF. However, the different reactions observed in the pairings of genetically different isolates compared with the all or nothing reactions of self versus self pairings suggest that a complex recognition and compatibility system may exist in AMF, clearly more complex than a simple switch for compatible and incompatible pairings. We speculate that this might be dependent on the relatedness of the nucleotypes at the point of contact. The presence of nuclei that are polymorphic at loci involved in compatibility within an isolate could particularly complicate the mechanisms of recognition among isolates. Identifying candidate genes with homologies to genes involved in mate recognition in ancestral fungi could yield promising results.
Vegetative incompatibility: mechanisms that prevent heterokaryons forming between two genetically different fungal individuals in the vegetative stage
Molecular Evidence for Genetic Exchange

Where PF occurred, rapid bidirectional cytoplasmic streaming was observed (http://www3. interscience.wiley.com/journal/12,1587283/ suppinfo). This could allow nuclei of two individuals to mix. However, in those experiments, no direct observations of the fate of nuclei after fusion were conducted because of the difculties in tracking live nuclei. However, direct molecular evidence for genetic exchange between AMF exists. Here we consider genetic exchange in AMF in the broadest sense. This could simply be the mixing of different nucleotypes from two genetically different parents that are then passed on jointly into spores that form the next generation. Thus, the spores exhibit biparental inheritance. Genetic exchange in AMF could also be more complex, involving nuclear fusion of different nucleotypes, following anastomosis
Genetically different parental isolates
Offspring spores
Hyphal fusion
Differential effects on plant growth
n 2n
n
Effects on plant growth
Nuclear fusion and meiosis
Frequency of different nucleotypes
Currently unknown
Figure 2 Exchange of nuclei and recombination, and their effects on plant growth. Conceptual drawing of genetic exchange and subsequent recombination. Two genetically different parental AMF are shown. The two parental AMF each contain different nucleotypes (shown in different colors). The genotype of each parental isolate is indicated by a graph next to the spore showing the frequency of the nucleotypes. Hyphae of the two parental isolates fuse at a contact zone. The enlarged box shows the fusion of different nucleotypes from the two different parents, followed by meiosis, producing recombinant daughter nuclei. Although genetic exchange has clearly been shown in AMF, at present it is unknown whether such events result in nuclear fusion, recombination, and meiosis. We see this as a priority for research on this symbiosis. The three offspring spores each show the product of nuclear exchange, indicated by the genotype graph, showing frequency of different nucleotypes after exchange. The length of the bars equals the frequency of each nuclear genotype found in the spore. However, using isolate-specic markers, offspring spores containing recombinant nuclei are not distinguishable from offspring spores not containing recombinant nuclei. Parental isolates are known to differ in their effects on host plant growth and phenotypes of offspring are known to differ from parents. Recently, offspring resulting from exchange between two AMF have been shown to induce differential effects on plant growth compared to inoculation with the parental isolates (2).
and subsequent recombination of the genomes. Fully developed hyphal networks of genetically different AMF (parental isolates) were grown together in in vitro cultures (19). This provided the potential for the parental isolates to form anastomoses in vitro. After six months of growth, newly produced spores (progeny) were individually able to colonize fresh plant roots, make a symbiosis, and produce new spores, thus completing the AMF life cycle. DNA extracted from these new cultures showed that the fungi exhibited biparental inheritance and contained parental specic molecular markers (both sequenced-based and AFLP markers) from both parental isolates. In that study, it was not possible to determine whether this represents a simple mixing of nuclei or also nuclear fusion and meiosis (see Figure 2). The parental isolates used were chosen because previous studies had shown them to have
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contrasting and highly heritable phenotypes and different symbiotic effects on plant growth (52, 53). This allowed the test of whether the AMF phenotype was altered following genetic exchange (19). Progeny exhibited a wide range of different hyphal and spore densities, some being similar to the parental isolates, others being intermediate between the two parents, and in some cases signicantly exceeded the values observed in either of the two parental isolates (19). This nding showed that exchange of nuclei among isolates leads to the emergence of novel fungal phenotypes. A more recent study has demonstrated that genetic exchange between two different AMF creates genetically novel AMF that also have different symbiotic effects on plant growth compared to the parental AMF isolates (2). In that study, O. sativa and Plantago lanceolata were inoculated with genetically different parental lines of
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G. intraradices or with lines that were crosses between two parental lines. Rice always grew worse when innoculated with mycorrhizae than if uninoculated, but inoculation with some crossed lines suppressed rice growth even more than the parental lines. Inoculation with crossed AMF lines did not have any effect on growth of P. lanceolata compared to inoculation with parental lines, but all inoculated plants grew better than uninoculated plants. What this study shows is that genetic exchange in the fungus can alter symbiotic effects in plants (Figure 2) but that this is plant species specic. The ability to perform experimental crosses among AMF lays a foundation to study the genetic architecture of phenotypic traits. Some phenotypic traits might be linked to a particular type of nucleus in a heterokaryotic isolate. Experimental crosses between different isolates could potentially yield more benecial isolates for particular host plants or be used to create novel AMF genotypes that are particularly efcient for commercial propagation in articial media.
SEGREGATION IN ARBUSCULAR MYCORRHIZAL FUNGI AND ITS EFFECT ON PLANT GROWTH

Previous studies have failed to demonstrate segregation in AMF using molecular tools. This was possibly because of the lack of enough markers. However, a recent study has demonstrated segregation of different nucleotypes in AMF during spore formation (2). Angelard and colleagues (2) hypothesized that two types of segregation could occur in AMF, thereby changing the composition of nucleotypes in spores compared with the parent. When a parental AMF hypha produces several new spores, by random processes some of the genetically different nucleotypes may not enter all spores, thus leading to genetic differences among siblings. This was dened as total segregation (Figure 3). However, Angelard and colleagues argued that by random processes sibling spores might still receive each different nucleotype but in different proportions. This
was dened as partial segregation (Figure 3). Clear molecular evidence for segregation was obtained with AFLP and also by looking at the relative frequencies of multiple alleles at one locus among siblings (2). Certainly some of this segregation was partial segregation, as shown by different relative frequencies of four co-occurring alleles among siblings. It is possible that some of the segregation observed as AFLP was also total segregation but this cannot be determined from the dataset. The segregated lines were obtained as vegetative progeny produced from crossed AMF lines that had been shown to suppress growth of rice. The parents of those crossed lines also suppressed rice growth compared to uninoculated rice plants. However, some segregated AMF lines induced up to vefold increases in rice growth compared to uninoculated plants or those inoculated with crossed lines or parental lines (2). Segregation in AMF was also shown to affect growth of P. lanceolata, with both positive and negative growth effects compared to effects of crossed AMF lines. The study by Angelard and colleagues demonstrates that it is possible to start with AMF that do not induce positive growth responses and with simple laboratory manipulations obtain AMF lines that induce positive growth responses (Figure 3). This is an exciting result, given that rice is such a globally important crop for human nutrition. Furthermore, this provides researchers with a tool to study how genetic changes in the fungus inuence the symbiosis.
Total segregation: random nucelotype segregation during spore formation so that siblings are genetically different Partial segregation: spores receiving different proportions of nucleotypes from the parent
RECOMBINATION IN ARBUSCULAR MYCORRHIZAL FUNGI

The occurrence of recombination is a fundamental characteristic shaping genomes (58). However, to identify whether recombination occurs in AMF is not only important for understanding genome evolution in AMF. The reshufing of the genome afforded by recombination could also create new genetic variants of the fungus that also vary in their symbiotic effects on plants. Therefore, we see it as essential
Parental isolate
Segregation of nucleotypes among offspring spores
Known effect on plant growth
Effects on plant growth
Figure 3 Segregation in arbuscular mycorrhizal fungi (AMF) and its effects on plant growth. Segregation of nuclei occurs during spore formation in an AMF. The parental isolate contains nuclei of three distinct nucleotypes. Each nucleotype is represented by a different color. The graph next to the parental spore shows a conceptual representation of the proportion of different nucleotypes. The length of the bars equals the frequency of each nuclear genotype found in the spore. During hyphal growth, nuclei are thought to move freely inside the cytoplasm and migrate into newly formed spores. The offspring spores show three potential outcomes of segregation. The upper spore inherited only nuclei of one nucleotype. This type of segregation would be detectable as qualitative differences using standard genotyping methods and would be seen as presence or absence of alleles. This has been referred to as total segregation (2). The middle and lower spore each inherited varying frequencies of the three nucleotypes. This form of partial segregation results in changes in the relative frequency of nucleotypes among the offspring. In such, case standard genotyping techniques detecting presence or absence of alleles would not reveal the changes in nucleotype frequency occurring, even though the offspring would be genetically different. Segregation in AMF has been shown to strongly affect plant growth (2).
to establish whether recombination occurs in order to ultimately understand the molecular genetics regulating the symbiosis between AMF and plants and their coevolution with plants. AMF have been considered to be ancient asexuals (51). The existence of organisms that lack recombination dees the classical view of evolutionary theory that predicts only shortterm advantages for asexual reproduction (62). Recombination purges deleterious mutations. In the absence of recombination, accumulation of mutations should lead to extinction (62). Even rare recombination events are thought to be enough to purge deleterious mutations in some populations. Evidence supporting the lack of recombination is mainly based on morphological stasis over hundreds of millions of years, dating back to the emergence of land plants (8, 78). Low morphological divergence is thought to be associated with a lack of recombi282 Sanders
nation and, hence, only rare emergence of novel phenotypes. Furthermore, no specialized sexual structures have been conclusively observed in AMF. However, many fungi of different phyla have been considered as asexual due to the cryptic nature of their sexual reproduction, although subsequent molecular studies revealed recombination (10, 64, 67, 69). One characteristic feature of long-term asexual organisms is the high level of genetic divergence within populations and within individuals and this is, indeed, observed in AMF. Frequent recombination would homogenize genetic differences among nuclei and, therefore, among individuals. Only in a largely clonal population would one expect the accumulation of high levels of polymorphism. Given that the long-term absence of recombination is unlikely, several studies have addressed whether recombination occurs in AMF. In many model organisms, recombination can
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be directly demonstrated experimentally in the laboratory using molecular genetics tools coupled with knowledge of the basic genetics of the organism. To demonstrate recombination in AMF, and in many other fungi that are not easy to manipulate or that might undergo recombination very rarely, is a great challenge. Even if recombination cannot be directly demonstrated experimentally, footprints of recombination can be detected within populations by applying a number of different methods. This rst requires genotyping individuals at a large number of loci, either based on fragment size, such as with AFLP, or by nding a number of different polymorphic loci within a population and sequencing the alleles of each of those loci in a representative number of individuals from the population. A population genetics approach can then be used to look for linkage disequilibrium among a given number of loci among individuals in a population. Phylogenetic methods can also be used since neighbor network algorithms can reveal reticulate patterns of evolution, which is strongly indicative of recombination (but can also be due to homoplasy). A statistical test, known as w , reliably discriminates between homoplasy and recombination occuring among different alleles (9). Three further phylogenetic tests, known as bootscanning, recombination detection program (RDP), and the partition homogeneity test, and three nucleotide substitutionbased tests, known as Geneconv, MaxChi, and Chimaera, have been shown to reliably detect footprints of recombination among polymorphic loci within a population as long as enough markers have been sequenced (60, 63, 68, 7577, 85, 89). Evidence for recombination based only on linkage of loci is largely ambiguous in AMF and was the subject of critical discussions (71, 72, 83). Several studies identied complete linkage disequilibrium in eld populations of different Glomus species (95, 96). The methodological approach consisted of isolating single spores of three different species from eld populations and performing nested PCR to directly amplify three polymorphic regions in the genome from each spore. The nested PCR strategy
has been necessary for reliable amplication of some markers from single AMF spores and from DNA of AMF in colonized roots, although the reason for this is unclear. The alleles identied at all three loci were found to be in complete linkage disequilibrium for all species (95). No potentially recombinant genotype was identied where alleles had been reshufed compared with other genotypes. The observed genotypes corresponded to the prediction of a completely clonal population with independently evolving lineages. A qualitatively similar result was found using AFLP (84). However, three fundamental criteria must be met when using genotypic data to predict clonality in populations, and these may not have been satised: (a) the level of polymorphism at the loci must reect polymorphism elsewhere in the genome, (b) there is a representative sample of the population, and (c) no cryptic species confound the identication of clonal lineages. Both unusually low polymorphism at the loci and sampling different cryptic species could lead to an erroneous conclusion of clonality. In contrast to the studies suggesting clonality of AMF species, other studies suggested the occurrence of recombination. In Glomus species, a lack of linkage disequilibrium was observed using random genetic markers, suggesting a recombining population (101). In a broad assessment of evidence for recombination in several putative ancient asexual organisms, evidence of recombination was obtained for rDNA gene sequences of several Glomus species and among different Bip sequences within one G. intraradices isolate (23). However, both rDNA and Bip have been shown to be highly polymorphic, even within a single AMF spore. Given the heterokaryotic state of AMF and the within-spore variation at these loci, neither of these regions of the genome is particularly suitable for assessing the occurrence of recombination in a population of different AMF. Multilocus sequence typing of an AMF population has provided a less ambiguous picture of the occurrence of recombination among AMF (20). Evidence for recombination was found in G. intraradices isolates originating
Nested PCR: a way of amplifying a given locus using more than one PCR reaction with different pairs of primers
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from one location, based on sequences at eleven polymorphic loci and using neighbor network algorithms, tests for linkage disequilibrium, w , bootscanning, RDP, partition homogeneity test, Geneconv, MaxChi, and Chimaera. Each locus was chosen because only one allele of the locus existed per AMF isolate, meaning that there was no within-spore variation at that locus, and all variation observed at each locus was among AMF isolates. This precluded the possibility that any detection of recombination was an artifact owing to an admixture of genetically different nuclei within AMF isolates. All the independent tests revealed recombination among the AMF isolates. Out of a total of 18 different genotypes, a core group of recombining genotypes was identied. Five genotypes showed the most unambiguous signals of recombination supported by the multiple, independent tests. In contrast, several genotypes showed a strongly clonal evolution compared to the core group. However, recombination events were predicted by some methods in almost all of these supposedly clonal lineages. While each of the above tests, used independently, has some potential alternative interpretation, the concurrent use of all these methods together excludes those alternative possibilities and makes them a powerful suite of methods for detecting the footprint of recombination in a population. Consequently, this study provides strong evidence that recombination occurs in populations of the AMF G. intraradices.
UNDERSTANDING ARBUSCULUAR MYCORRHIZAL FUNGI GENETICS IS A PRIORITY FOR UNDERSTANDING THE MOLECULAR BASIS OF THE SYMBIOSIS WITH PLANTS
In this review, we have concentrated almost entirely on the basic biology of the fungi involved in the symbiosis because we believe that is where signicant advances need to be made in order to ultimately fully understand the molecular genetics of the symbiosis. A major goal of plant molecular biologists is to understand
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the molecular basis underlying the establishment, functioning, and regulation of the arbuscular mycorrhizal symbiosis. However, the existence of high genetic variability in AMF, how it arises, and its complexity of organization have largely been ignored by plant molecular biologists working on the symbiosis. Perhaps this is because they consider it too complex, owing to the controversy surrounding some studies, or simply see it as a nuisance that makes achieving their goal of understanding the symbiosis (usually using a clearly dened model system) more difcult. However, there are several reasons why understanding the effects of genetic exchange, segregation, and recombination in AMF on plants should also be a priority for those researchers. Firstly, there are obvious potential effects of the generation of novel AMF genotypes by these processes on the symbiosis and thus plant growth. Until recently, genetic exchange and segregation have been considered absent in AMF, and no attempts have been made at manipulating the genetics of AMF in order to unravel the molecular basis of the interaction. With the nding of genetic exchange and segregation, and the ability to do it in a lab-based system, this is now possible. Secondly, because genetic exchange, segregation, and recombination occur in AMF, it means that the genotype of the fungus is much more dynamic than previously thought. Because of the low diversity of AMF (based on morphology) and the underlying assumption that they are clonal, the effect of an AMF is thought to be xed, in the sense that the fungus cannot undergo rapid genetic change. If that were the case, then the plant could easily exploit the fungus, breaking down the stability of the mutualism. The ability to change genetically could lead to a stable mutualistic situation. Plant molecular biologists have assumed that the symbiosis is regulated by highly conserved genes. This, indeed, appears to be true for some of the essential genes allowing the establishment of the symbiosis (8, 26, 70). We consider the genes involved in establishment of the symbiosis as essential for all AMF-plant
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interactions, so high conservation is not surprising. However, we predict that it is less likely to be true for genes involved in the ner scale regulation of the symbiosis between the plant and the fungus and those genes that ultimately affect the plant growth response (Figures 2 and 3). Strong clues to this already exist given that different species of AMF have differential effects on plant growth, genetic variation among isolates of an AMF species (even from one small eld) exerts different inuences on plant growth, and genetic exchange and segregation in AMF can strongly inuence plant growth. In these cases, there must be differences in gene expression in the fungus or the plant, or both, that result in the different symbiotic effects. Clearly, in these cases, the ability of the fungus to inherit or lose genetic information or recombine in the hyphal network or during spore formation could be critical to understanding regulation of the symbiosis with plants and with different hosts. There are also signs that this variation could be important for the fungal genes involved in the interaction with plants. Very few fungal genes have been identied that play a role during the development of the symbiosis. However, one gene, the superoxide dismutase, Sod1, that is up-regulated in the fungus during the development of the symbiosis has been identied in AMF (56). Interestingly, this gene shows extremely high variability in AMF, even among isolates of G. intraradices from one eld (17). What is even more striking about the variation is that it appears to be subject to diversifying selection where variants have high levels of substitutions leading to changes in the amino acid sequence of the protein. This is in contrast to the variation seen in several other AMF genes within AMF species, such as those encoding tubulin, H+ ATPase, and P-type II ATPase, where purifying selection appears to be acting to conserve the function of the gene (15, 16, 18). The Sod1 gene scavenges reactive oxygen species and is important in pathogenic fungi to allow them to cope with plant defense during infection. Variation in this gene could help AMF cope with the different environments presented by colonizing different plant hosts.
AN ALTERNATIVE APPROACH TO STUDYING GENES INVOLVED IN THE ARBUSCULAR MYCORRHIZAL SYMBIOSIS

Given the effects of genetic variation, genetic exchange, and segregation in the fungus on plant growth, we propose an alternative approach to understanding the molecular genetics of the arbuscular mychorrhizal symbiosis with plants. Using segregation and genetic exchange in AMF as a tool to manipulate the genetics of the fungus opens the way to understanding how the genetics of the fungus inuences the molecular regulation of the symbiosis. In order to nd genes involved in the symbiosis, plant molecular biologists typically compare genes expressed differentially between mycorrhizal (inoculated with one fungus) and nonmycorrhizal plants, or use plant mutants in which colonization is absent or arrested at some stage in the development of the symbiosis. Although these approaches are clearly successful in identifying genes that are essential for the formation of the symbiosis, neither of these traditional approaches will allow arbuscular mycorrhizal researchers to elucidate how genetic differences in the fungus affect the symbiosis. Indeed, in the study by Angelard and colleagues, transcription levels of both early stage and late stage symbiosis-specic rice genes AM1, AM3, AM14, and PT11 were shown to be strongly affected by segregation in the fungus (2). Given the huge advances that have occurred in plant genomics and transcriptomics and the advances that are currently being made in AMF genomics and transcriptomics, experimentally addressing this issue is immediately possible. We encourage biologists working on both plant and fungal sides of the symbiosis not to ignore the genetic variability in AMF but to use it as a tool. Plants can be inoculated with genetically different AMF, either different naturally occurring genotypes that are known to induce differences in plant growth or novel genotypes generated in the laboratory using anastomosis and genetic exchange and segregation. Their effects on plant growth and nutrition can be
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Experiment 1
Experiment 2
Effect on plant growth
Fungal colonization
Fungus A
Nonmycorrhizal
Fungus A
Fungus B
cDNA
Equally expressed genes
cDNA
Gene expression microarray data
Figure 4 Gene expression studies with genetically different AMF. Experiment 1 depicts a typical gene expression study to identify plant genes that are up- or downregulated in the mycorrhizal symbiosis. RNA is extracted from inoculated and uninoculated plants, and made into cDNA. On the microarray, red and green spots show genes that are differentially expressed in mychorrhizal and nonmicorrhizal treatments, respectively, and yellow spots show genes that are equally expressed in both treatments. In experiment 2, the plants are inoculated with two genetically different fungi (A and B) that induce differential growth responses in the plants. Genes that were expressed in both treatments in experiment 1 are still seen as yellow in experiment 2 (expressed with fungus A and B). All the mycorrhizal specic genes that were expressed in inoculated plants in experiment 1 are also expressed in both treatments and appear yellow in experiment 2. However, the plants grow differently in the two treatments in experiment 2, and there are a number of new genes discovered that are specically upregulated with fungus A (red ) or with fungus B ( green).
studied, and either microarray or deep genome and transcriptome sequencing can be used as a tool to elucidate which genes are involved in the regulation and functioning of the symbiosis that ultimately leads to better plant growth. This approach can be used to nd either plant or fungal genes involved in the symbiosis. Given the effects of segregation in AMF on rice growth and symbiosis-specic gene transcription, there is a strong rationale for doing this. We predict that this approach is likely to
lead to the discovery of many genes involved in the symbiosis that could never be identied by the traditional comparative mycorrhizal versus nonmycorrhizal plant approach (Figure 4). In the same way that AMF and plants need to cooperate with each other to form a functional symbiosis, AMF geneticists and plant and fungal molecular biologists working on the symbiosis need to pool their knowledge to elucidate the underlying molecular genetics of the symbiosis.
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SUMMARY POINTS 1. The arbuscular mycorrhizal symbiosis is important for plant nutrition, plant growth, plant diversity, and nutrient cycling in ecosystems. In order to understand the molecular genetics of this symbiosis, more knowledge about the genetics of the fungal side of this partnership is desperately needed. 2. It has been assumed that AMF are completely asexual and that they lack any form of genetic exchange or recombination. Furthermore, it has been assumed that incompatibility mechanisms prevent fusion among genetically different AMF. This dogma has been overturned by the ndings that fusion occurs, that offspring exhibit biparental inheritance and that recombination can be detected in AMF. Segregation has also recently been demonstrated in AMF. These ndings pave the way for experimental approaches to studying the molecular genetics of AMF. 3. Genetically different AMF of the same species can have signicantly different effects on plant growth. Genetic exchange and segregation can alter the genetics of AMF and therefore, alter the symbiotic effects of the fungi on plants. 4. Altering the genetic composition of AMF in the laboratory by using genetic exchange and segregation could be used as a powerful tool to better understand the molecular genetics of the symbiosis with plants. If researchers continue to use traditional approaches to nd symbiosis-related genes, such as inoculated versus noninoculated plants or mutants incapable of forming complete symbiosis, they will potentially miss a large number of genes involved in symbiosis that are altered by changes in the genetics of the fungal partners.
FUTURE ISSUES 1. The process of genetic exchange in AMF should be better understood so that the fate of nuclei after fusion can be determined. Do nuclei fuse and then undergo meiosis or does fusion simply result in a mixing of nuclei, thereby maintaining heterokaryosis? 2. Demonstrate whether the nucleotype composition of an AMF following genetic exchange and segregation is stable and whether the symbiotic effects on plants are also stable. 3. It is important to understand the molecular basis of how changes in the relative frequency of nucleotypes in AMF can so strongly affect plant growth. 4. Genetically manipulate AMF by a combination of genetic exchange and segregation and use these manipulated lines to inoculate plants. Microarrays and a reasonable amount of reference sequence data should be available for both plants and fungi. This will allow the rst investigation into how the genetics of the fungus alters expression of genes in the symbiosis. This could be achieved through ultradeep sequencing of the transcriptome and by microarrays.
DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding or nancial holdings that might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We thank all former and current members of our research group. Without their hard work and fruitful discussions, this review would not have been possible. We thank the Swiss National Science Foundation (grant numbers 3100A0-105790/2 and 31003A-127371) for supporting this work.
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Contents
New Insights into Plant Responses to the Attack from Insect Herbivores Jianqiang Wu and Ian T. Baldwin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 The Genomic Enzymology of Antibiotic Resistance Mariya Morar and Gerard D. Wright p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p25 Genetic Engineering of Escherichia coli for Biofuel Production Tiangang Liu and Chaitan Khosla p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p53 Bacterial Contact-Dependent Delivery Systems Christopher S. Hayes, Stephanie K. Aoki, and David A. Low p p p p p p p p p p p p p p p p p p p p p p p p p p p p p71 Evolution of Sex Chromosomes in Insects Vera B. Kaiser and Doris Bachtrog p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p91 Regulation of Homologous Recombination in Eukaryotes Wolf-Dietrich Heyer, Kirk T. Ehmsen, and Jie Liu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 113 Integrons Guillaume Cambray, Anne-Marie Guerout, and Didier Mazel p p p p p p p p p p p p p p p p p p p p p p p p p 141 Bacterial Antisense RNAs: How Many Are There, and What Are They Doing? Maureen Kiley Thomason and Gisela Storz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 167 Protein Homeostasis and the Phenotypic Manifestation of Genetic Diversity: Principles and Mechanisms Daniel F. Jarosz, Mikko Taipale, and Susan Lindquist p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 189 The Art of Medaka Genetics and Genomics: What Makes Them So Unique? Hiroyuki Takeda and Atsuko Shimada p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 217 Telomeric Strategies: Means to an End Devanshi Jain and Julia Promisel Cooper p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 243 Arbuscular Mycorrhiza: The Challenge to Understand the Genetics of the Fungal Partner Ian R. Sanders and Daniel Croll p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 271
Annual Review of Genetics Volume 44, 2010
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The RecQ DNA Helicases in DNA Repair Kara A. Bernstein, Serge Gangloff, and Rodney Rothstein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 393 Circadian Control of Global Gene Expression Patterns Colleen J. Doherty and Steve A. Kay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 419 Variable Tandem Repeats Accelerate Evolution of Coding and Regulatory Sequences Rita Gemayel, Marcelo D. Vinces, Matthieu Legendre, and Kevin J. Verstrepen p p p p p p p 445 Errata An online log of corrections to Annual Review of Genetics articles may be found at http:// genet.annualreviews.org/errata.shtml
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Arbuscular Mycorrhiza Genetics of The Fungal Partner

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Arbuscular Mycorrhiza Genetics of The Fungal Partner

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ANNUAL REVIEWS

GENES INVOLVED IN THE ESTABLISHMENT AND FUNCTIONING OF THE SYMBIOSIS

Spore Hypha Appressorium Prepenetration apparatus

WHY WE KNOW SO LITTLE ABOUT THE GENETICS OF THE FUNGAL PARTNER

GENOME SIZE, CHROMOSOMES, AND PLOIDY IN ARBUSCULAR MYCORRHIZAL FUNGI

Molecular Evidence for Genetic Exchange

Genetically different parental isolates

Differential effects on plant growth

Nuclear fusion and meiosis

Frequency of different nucleotypes

Frequency of different nucleotypes

SEGREGATION IN ARBUSCULAR MYCORRHIZAL FUNGI AND ITS EFFECT ON PLANT GROWTH

RECOMBINATION IN ARBUSCULAR MYCORRHIZAL FUNGI

Segregation of nucleotypes among offspring spores

Known effect on plant growth

Frequency of different nucleotypes

Effects on plant growth

Frequency of different nucleotypes

AN ALTERNATIVE APPROACH TO STUDYING GENES INVOLVED IN THE ARBUSCULAR MYCORRHIZAL SYMBIOSIS

www.annualreviews.org Arbuscular Mycorrhiza

Effect on plant growth

Gene expression microarray data

Annual Review of Genetics Volume 44, 2010

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