Susan J. Barker

The Roles of Auxins and Cytokinins in Mycorrhizal Symbioses.

A bstractMost land plant species that have been examined exist naturally with a higher fungus living in and around their roots in a symbiotic partnership called a mycorrhiza. Several types of mycorrhizal symbiosis exist, defined by the host/partner combination and the morphology of the symbiotic structures. The arbuscular mycorrhiza (AM) is ancient and may have co-evolved with land plants. Emerging results from gene expression studies have suggested that subsets of AM genes were co-opted during the evolution of other biotrophic symbioses. Here we compare the roles of phytohormones in AM symbiosis and ectomycorrhizas (EC), a more recent symbiosis. To date, there is little evidence of physiologic overlap between the two symbioses with respect to phytohormone involvement. Research on AM has shown that cytokinin (CK) accumulation is specifically enhanced by symbiosis throughout the plant. We propose a pathway of events linking enhanced CK to development of the AM. Additional and proposed involvement of other phytohormones are also described. The role of auxin in EC symbiosis and recent research advances on the topic are reviewed. We have reflected the literature bias in reporting individual growth regulator effects. However, we consider that gradients and ratios of these molecules are more likely to be the causal agents of morphologic changes resulting from fungal associations. We expect that once the individual roles of these compounds are explained, the subtleties of their function will be more clearly addressed.

RFLP mapping of manganese efficiency in barley.

Abstract In many cropping regions of the world, yield is limited by the availability of micronutrients, and micronutrient-efficient cultivars provide a yield advantage. Traditional methods of testing cultivars for micronutrient efficiency are time-consuming and laborious. Molecular markers linked to loci controlling micronutrient efficiency will allow more rapid and efficient selection and introgression of these traits than is currently possible. Using a pot-based bioassay and bulked segregant analysis of an F2 population, we have identified several RFLPs (grouped distally on chromosome 4HS) linked to a locus for manganese efficiency in barley. This manganese efficiency locus has been designated Mel1. Pot bioassay analysis of intercrosses suggests that three useful sources of manganese efficiency are likely to be allelic at the Mel1 locus. Field evaluation of marker selected F4 progeny supports the major role of Mel1 in the genetic control of manganese efficiency. Adoption of marker assisted selection for this trait in the Southern Australian barley breeding program has occurred. This has been facilitated by the demonstration that the Mel1 allele of Amagi Nijo can be distinguished from 95 other locally useful varieties and breeder’s lines on the basis of RFLPs identified by just two molecular markers.

RFLP mapping of the Ha 2 cereal cyst nematode resistance gene in barley

Abstract The cereal cyst nematode (CCN), Heterodera avenae Woll., is an economically damaging pest of barley in many of the world’s cereal-growing areas. The development of CCN-resistant cultivars may be accelerated through the use of molecular markers. A number of resistance genes against the pest are well known; one of them, the single dominant Ha 2 resistance gene, has been shown to be effective against the Australian pathotype and maps to chromosome 2 of barley. Segregation analysis identified two restriction fragment length polymorphism (RFLP) markers flanking the resistance gene in two doubled-haploid populations of barley. AWBMA 21 and MWG 694 mapped 4.1 and 6.1 cM respectively from the Ha 2 locus in the Chebec×Harrington cross and 4.0 and 9.2 cM respectively in the Clipper×Sahara cross. Analysis of a further seven sources of CCN resistance in the form of near-isogenic lines (NILs) indicates that all available sources of resistance to the Australian pathotype of CCN in barley represent the Ha 2 locus.

Production of near-synchronous fungal colonization in tomato for developmental and molecular analyses of mycorrhiza

A method to effect rapid, near-synchronous colonization of tomato root systems by a vesicular arbuscular mycorrhizal fungus was developed. This procedure was used to compare the time course of colonization in roots of tomato that were grown under varying phosphate conditions. It was found that the primary symbiotic interaction developed through a series of regularly timed stages. Furthermore, the formation of intercellular hyphae, arbuscules and vesicles was significantly reduced in plants grown with high soil phosphate. These results show that this method is ideal for the production of material for molecular biological studies of VAM symbiosis, including investigation of the regulatory role of phosphate in the symbiosis and the processes that control the fungal life cycle.

MOLECULAR APPROACHES FOR INCREASING THE MICRONUTRIENT DENSITY IN EDIBLE PORTIONS OF FOOD CROPS

Abstract Micronutrient accumulation and uptake traits are inherited and could therefore be improved using molecular genetic techniques. Two distinct applications of molecular biological techniques are described that could be used to manipulate the micronutrient density in edible portions of food crops. One is the use of DNA markers as genetic tags for the introgression of desired traits and the second is the introduction of defined genetic material, which is the process of genetic engineering. This chapter covers concepts important in the use of molecular markers and concentrates on molecular details of micronutrient uptake by model plant and microbial species because of the recent advances in this area of research. Although micronutrient uptake processes do not all directly relate to major human nutritional deficiencies, the review of this area of research provides a conceptual framework for consideration of aspects important when developing crops for improved human health.

Isolation by differential display of three partial cDNAs potentially coding for proteins from the VA mycorrhizal Glomus intraradices.

A molecular study of the mycorrhizal symbiosis between barley and Glomus intraradices used differential display PCR and a synchronous colonization method to identify genes that are differentially expressed in symbiosis. Several PCR products were consistently differentially amplified. PCR amplification of genomic DNA from either G. intraradices or barley as templates showed that three such products were encoded by G. intraradices. Sequence analysis of the deduced amino acid sequences of the fungal fragments, following extension by 3’-RACE, revealed similarities to proteins from higher eukaryotes. One (GINMYC1) shows similarity to TRIP15, a human protein that interacts in a hormone-dependent manner with the thyroid receptor. A second (GINMYC2) is similar to O-linked N-acetylglucosamine transferases from vertebrates, and the third (GINHB1) contains a putative leucine zipper and a homeodomain which indicates that it binds DNA and may act as a transcriptional regulator. Fragments of the expected sizes were amplified by RT-PCR from mRNA of mycorrhizal barley roots for all three fungal cDNAs, which indicates that the corresponding genes are expressed during intraradical growth of G. intraradices. The results provide a promising insight to fungal gene expression early in formation of this compatible and mutualistic symbiosis.

The application of genetic approaches for investigations of mycorrhizal symbioses

Genetic analyses of mycorrhizal symbioses have been far less common to date than molecular biological investigations. This review aims to address the problem that genetic research approaches are some of the least familiar to non specialists by providing some detailed explanations of the requirements and processes involved, including concepts of genetic variation and genetic mapping. Each section includes examples of research progress which is restricted to studies of arbuscular mycorrhizal (AM) and ectomycorrhizal (EcM) symbioses. Most such research has focussed on AM hosts or EcM fungi. For AM hosts, some early work on natural genetic variation has not been exploited yet, but new research with barley and clover will enable genetic mapping of mycorrhizal associated QTLs for the first time. EcM fungal studies have shown a genetic basis for mycorrhizal capacity and quantitative genetic differences in mycorrhizal capacity. Some recent work with EcM hosts has begun genetic mapping of QTLs associated with mycorrhizal status. Most AM genetic research has focussed on analysis of nodulation-defective mutants for their AM host status. Map-based cloning and characterisation of the first genes shown by these analyses to be essential for establishment of both nodulation and mycorrhizal symbioses are anticipated shortly. Comparisons with molecular and genetic research on plant disease resistance genes and signalling pathways may prove useful as those studies are more advanced and underlying biochemical and evolutionary relationships are likely to exist.

A novel plant–fungus symbiosis benefits the host without forming mycorrhizal structures

• Most terrestrial plants form mutually beneficial symbioses with specific soil-borne fungi known as mycorrhiza. In a typical mycorrhizal association, fungal hyphae colonize plant roots, explore the soil beyond the rhizosphere and provide host plants with nutrients that might be chemically or physically inaccessible to root systems. • Here, we combined nutritional, radioisotopic ((33)P) and genetic approaches to describe a plant growth promoting symbiosis between the basidiomycete fungus Austroboletus occidentalis and jarrah (Eucalyptus marginata), which has quite different characteristics. • We show that the fungal partner does not colonize plant roots; hyphae are localized to the rhizosphere soil and vicinity and consequently do not transfer nutrients located beyond the rhizosphere. Transcript profiling of two high-affinity phosphate (Pi) transporter genes (EmPHT1;1 and EmPHT1;2) and hyphal-mediated (33)Pi uptake suggest that the Pi uptake shifts from an epidermal to a hyphal pathway in ectomycorrhizal plants (Scleroderma sp.), similar to arbuscular mycorrhizal symbioses, whereas A. occidentalis benefits its host indirectly. The enhanced rhizosphere carboxylates are linked to growth and nutritional benefits in the novel symbiosis. • This work is a starting point for detailed mechanistic studies on other basidiomycete-woody plant relationships, where a continuum between heterotrophic rhizosphere fungi and plant beneficial symbioses is likely to exist.

Position of the reduced mycorrhizal colonisation (Rmc) locus on the tomato genome map

Our research aims to investigate the molecular communication between land plants and arbuscular mycorrhizal (AM) fungi in the establishment of symbiosis. We have identified a mutation in the facultative AM host tomato, which we named rmc. Plants that are homozygous for rmc no longer host most AM fungi. The mutation also affects the interaction of tomato with root knot nematode and Fusarium wilt. However, the function/s encoded by the intact Rmc locus is/are unknown. To clone and sequence the gene or genes that comprise the Rmc locus, we have initiated a positional cloning project. In this paper, we report the construction of mapping populations and use of molecular markers from the published genome map to identify the location of Rmc on tomato chromosome 8. Nucleotide binding site-leucine rich repeat resistance genes, reported to reside in the same region of that chromosome, provided insufficient differences to develop cleaved amplified polymorphic sequence markers. Therefore, we were unable to map these sequences in relation to rmc. Our results potentiate future work to identify the Rmc function and to determine the genetic basis for the multiple plant-microbe interaction functions that the rmc mutation has defined.

The diversity of arbuscular mycorrhizas of selected Australian Fabaceae

Abstract Members of the Australian native perennial Fabaceae have been little explored with regard to their root biology and the role played by arbuscular mycorrhizal (AM) fungi in their establishment, nutrition and long-term health. The ultimate goal of our research is to determine the dependency of native perennial legumes on their co-evolved AM fungi and conversely, the impact of AM fungal species in agricultural fields on the productivity of sown native perennial legume pastures. In this paper we investigate the colonisation morphology in roots and the AMF, identified by spores extracted from rhizosphere soil, from three replicate plots of each of the native legumes, Cullen australasicum, C. tenax and Lotus australis and the exotic legumes L. pedunculatus and Medicago sativa. The plants were grown in an agricultural field. The level and density of colonisation by AM fungi, and the frequency of intraradical and extraradical hyphae, arbuscules, intraradical spores and hyphal coils all differed between host plants and did not consistently differ between native and exotic species. However, there were strong similarities between species in the same genus. The three dominant species of AM fungi in rhizosphere soil also differed with host plant, but one fungus (Glomus mosseae) was always the most dominant. Sub-dominant AM species were the same between species in the same genus. No consistent differences in dominant spores were observed between the exotic and native Fabaceae species. Our results suggest that plant host influences the mycorrhizal community in the rhizosphere soil and that structural and functional differences in the symbiosis may occur at the plant genus level, not the species level or due to provenance.