Figure 3.3 Technique to study translocation of 14C-sucrose by root and mycorrhizal tissue. (From Burnett, Fundamentals of Mycology (1976), London: Arnold.)

mycelium and spores. One useful technique is to grow mycorrhizal fungi on hairy roots induced by infection with the bacterium Agrobacterium rhizogenes. Hairy roots are maintained aseptically in media containing ampicillin. This technique has led to the development of an in vitro collection of AM fungi and has been particularly useful for investigating the signaling between the symbiotic partners and the metabolism of the fungus.

3.1.3 Diffusible Host and Fungal Factors

In vitro labeling with 13CO2 and NMR spectroscopic analyses shows that dark fixation of carbon dioxide occurs during spore germination (Bago et al., 1996). Therefore, CO2 emitted by the root tissue is considered as the first non-specific stimulatory compound in the establishment of symbiosis. It is generally held that for further growth and development, the AM fungus is dependent upon the presence of a host (Giovanetti et al., 1993). Certain pea mutants (Nod) insensitive to nodulation by the symbiotic nitrogen-fixing bacterium Rhizobium are also insensitive to colonization by AM fungi (Albrecht et al., 1999). Using a transgenic plant with a nodulation (Nod)-factor-gus reporter gene, it was demonstrated that symbiosis-specific Nod genes are expressed in response to a diffusible factor specific to AM fungi, indicating common controlling genes in the two symbioses (Albrecht et al., 1999; Kosuta et al., 2003).

Attempts to grow spores of AM fungi in nutrient media in the absence of any plant host have been unsuccessful. Using seedlings separated by cellophane membrane and spores of VAM fungi, it was demonstrated that hyphae from germinating spores produce a diffusible factor that is perceived by the roots of the host and stimulate hyphal attachment. Plant exudate containing flavonols stimulate hyphal growth. Though the nature and mechanism of action of these molecules is unknown, it indicates that a molecular dialogue takes place between arbuscular mycorrhizal fungi and their potential host roots. The recurrent theme in symbiosis is exchange benefits for both partners.

The lack of nuclear division correlates with the lack of incorporation of isotopically labeled precursors of DNA and RNA synthesis (Burggraff and Bernger, 1989), although Gigaspora margarita is reported to undergo nuclear division when grown in vitro even in the absence of a host plant (Becard and Pfeffer, 1993). Root exudates have a strong effect on fungal branching. Flavonols, such as quercetin, kaempferol and myrecetin, strongly stimulate Gigaspora species.

The identity of translocated solutes and the factors influencing efflux and uptake are important in understanding the bi-directional transfer of nutrients across symbiotic interfaces of the fungus and the plant. Several years ago, Harley and his colleagues found the ability of the excised beech root mycorrhiza to absorb H32PO42- was greater than that of uninfected roots and many times more radioactivity accumulated in the fungal sheath than in the core of root tissue. In short-term experiments, about 90% of the phosphate taken up is retained in the sheath; therefore, the main benefit of symbiosis to mycorrhizal plants is thought to be in phosphorus nutrition. In arbuscular mycorrhizal plants, phosphorus (as orthophosphate) can be absorbed both directly through root and through external fungal hyphae.

3.1.4 Differentially Expressed Plant Genes

Phosphates and hexoses are usually transported by means of symporters. These are sustained by an electrochemical gradient in the plasma membrane created by H+-ATPase enzymes. Their function is to generate a proton electrochemical gradient across the plasma membrane to provide the driving force for co-transport of phosphate and carbon metabolites through an interface otherwise impermeable to them. Therefore, a key role is proposed for these H+-ATPases at both the plant and fungal symbiotic interfaces for either phosphate or carbon symport. New forms of transporters are preferentially expressed in arbuscule containing plant cells in response to mycorrhizal infection.

The cloning of a high-affinity phosphate transporter from the mycorrhizal fungus Glomus versiforme and its expression in yeast revealed that the fungal phosphate transporter operates by proton-coupled symport at the arbuscular interface (Harrison and van Buuren, 1995). Using a subtractive hybridization technique to identify transcriptionally regulated genes during AM fungal development, a cDNA clone encoding H+-ATPases from the mycorrhizal fungus Glomus mosseae was isolated and its expression compared by Northern blot analysis during appressorium formation, formation of extraradical hyphae and mycorrhizal roots. A developmentally regulated expression was found during mycorrhizal symbiosis. Work is in progress to cytochemically localize the H+-ATPase in the in planta phase (Requena et al., 2003).

To determine changes in gene expression during symbiosis, the technique of in situ mRNA hybridization is used. Murphy et al. (1996) isolated polyA+ mRNA from barley roots in the early stages of colonization by Glomus intraradices and used it to construct a cDNA library. The resulting clones were screened with 32P-labelled cDNA, obtained by reverse transcription of RNA from non-colonized and mycorrhizal roots. Colonization resulted in both up- and down-regulation of genes. One of the clones that was up-regulated showed sequence homology with plant H+-ATPases. This indicates that nutrient uptake by specific carrier proteins via a proton-co-transport (symport) mechanism. Active H+-ATPases are also present in fungus membranes but how the transfer is polarized across the interface is not known. A possibility is differential activation of H+-ATPase gene in root cells (Gianinazzi-Pearson et al., 2000).

3.1.5 Multiple Genomes

Staining with 4,6-diamino-2-phenylindole (DAPI) showed that spores of AM fungi, for example, Gigaspora marginata and Scutellospora erythropa, contain approximately 20,000 nuclei per spore (Burggraff and Beringer, 1989). This number matches well with the amount of DNA extractable from crushed spores and quantifying the DNA on the basis that average DNA content per nucleus is 0.4 picogram (Viera and Glenn, 1990). Despite lack of sex and genetic exchange in the mycorrhizal fungi, surprisingly a single spore contained nuclei with different DNA sequences or several genomes. This was found when one single spore of Scutellospora castanea was crushed and the nuclear suspension diluted down to one nucleus per tube (Figure 3.4) for analysis of variation in internal transcribed spacer (ITS) sequences in ribosomal DNA. The ITS sequences are located between the 18S and 5.8S rRNA-coding regions (ITS1) and between 5.8S and 25S rRNA-coding regions (ITS2). Since ITS are flanked by highly conserved coding regions, polymerase chain reaction (PCR) amplification of ITS1 and ITS2 was done using universal primers (Hijri et al., 1999; Hosny et al., 1999). The amplified products were digested with

Spore of Scutellospora castanea

Single spores crushed and nuclear suspensions diluted to one nucleus per tube for PCR

PCR analysis shows different ribosomal DNA sequences in nuclei from the same spore

Glomus -Gigaspora Scutellospora

DNA sequences in S. castanea match genera in Gigasporaceae and Glomaceae

Figure 3.4 Demonstration of a population of discrete nuclei in a mycorrhizal fungus. Adapted from Sanders (1999).

restriction enzymes and fractionated on a gel. Fragments of different lengths (RFLP) showed several ITS sequences among the nuclei, demonstrating that the mycorrhizal spore is heter-okaryotic. ITS fragments were grouped into six types that were cloned and sequenced (Hijri et al., 1999). Most of the sequences were very similar to those of S. castanea, and a few sequences matched to different Glomales genera, Scutellospora and Glomus (Hosny et al., 1999). This observation raised the puzzling question of how such divergent nuclei have come together in an individual that lacks sexual reproduction. No evidence for sexual reproduction (and hence recombination) has been found, suggesting that they reproduce clonally for their entire association with plants. A plausible explanation is that hyphal anastomoses between mycelia of two genera occurred, followed by exchange of nuclei. Another possibility is mutations leading to creation of different nuclei (Kuhn et al., 2001). Yet another possibility is that the divergent sequences are due to contaminating microorganisms within single spores of arbuscular mycorrhizal fungi (Redecker et al., 1999).

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