V. Gianinazzi-Pearson

Plant Cell Responses to Arbuscular Mycorrhizal Fungi: Getting to the Roots of the Symbiosis.

Since their colonization of terrestrial ecosystems, plants have developed numerous strategies to cope with the diverse bi? otic and abiotic challenges that are a consequence of their sedentary life cycle. One of the most successful strategies is the ability of root systems to establish mutualistic and reciprocally beneficial symbiotic relationships with microorganisms. Mycorrhizas, the intricate associations roots form with specific fungal groups, are by far the most frequent of these and rep? resent the underground absorbing organs of most plants in nature (Gianinazzi-Pearson, 1984). Through their function in the efficient exploitation of soil mineral resources and their bioprotective role against a number of common soilborne pathogens, mycorrhizas are instrumental in the survival and fitness of many plant taxa in diverse ecosystems, including many crop species (reviewed in Allen, 1991; Bethlenfalvay and Linderman, 1992). Several kinds of mycorrhizal associations can be distin? guished according to their morphology and the plant and fungal taxa concerned. They fail almost exclusively into two broad groups: (1) the ectomycorrhizas of woody Angiosperms and Gymnosperms, in which Basidiomycetes, Ascomycetes, or Zygomycetes develop intercellular hyphae from a mycelial sheath covering the surface of short lateral roots; and (2) the endomycorrhizas, characterized by intraradical mycelium growth and intracellular fungal proliferation, which are formed by Basidiomycetes in the Orchidaceae (orchidoid mycorrhiza), Ascomycetes in the Ericales (ericoid mycorrhiza), and Zygomy? cetes in most other terrestrial plant taxa (arbuscular mycorrhiza; reviewed in Harley and Smith, 1983). Plant compatibility with mycorrhizal fungi is a generalized and ancient phenomenon. Species in >80?/o of extant plant families are capable of establishing arbuscular mycorrhiza (AM), and fossil evidence suggests that symbioses of this kind existed >400 million years ago in the tissues of the first land plants (Pirozynski and Dalpe, 1989; Remy et al., 1994). As such, the ability of plants to form AM must be under the control of mechanisms that have been conserved in new plant taxa as they appeared during evolution. This compatibility also implies that selective recognition processes in plants discriminate be? tween beneficial and harmful microorganisms and that the essential genetic determinants for AM establishment are com? mon to an extensive part of the plant kingdom. In contrast to their extremely wide host range and despite their ancient origins, only six genera of fungi belonging to the order Glomales of the Zygomycetes have evolved the ability to form AM (Morton and Benny, 1990). Interactions between an AM fungus and a plant begin when a hypha from a ger? minating soilborne spore comes into contact with a host root. Thi step is followed by induction of an appressorium, from which an infection hypha penetrates deep into the parenchyma cortex (Figure 1A), where interand intracellular proliferation of mycelium is intense. Here, fungal development culminates in the differentiation of intracellular haustoria, known as arb scules (Figure 1B). These fungal structures, which establish large surface of contact with the plant protopiast, are attrib? uted a key role in reciprocal nutrient exchange between the plant cells and the AM fungal symbionts (Smith and Smith, 1990). However, arbuscules are ephemeral structures, and an individual arbuscule reaches full development within several days, after which it begins to senesce (Alexander et al., 1988). AM development continues within a root system as the fungus spre ds to newly emerging roots. In this way, fungal coloniza? tion occurs concomitantly in different roots in an unsynchronized manner.

First report of non-mycorrhizal plant mutants (Myc^-) obtained in pea (Pisum sativum L.) and fababean (Vicia faba L.)

Abstract Genetic resistance to vesicular-arbuscular (VA) mycorrhiza formation has been obtained in spontaneous or chemically induced mutants of two mycorrhiza-forming species (Pisum sativum L. and Vicia faba L.). The eight mutants, termed myc−, are characterized by aborted infections limited to one or two host cells. Expression of the myc− character is associated with that of the nod− character in both legumes, and is likewise under recessive genetic control. Preliminary analysis of the genetic behaviour of the myc− mutants in diallel crosses has shown that at least three genes are involved in VA mycorrhiza infection.

Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions.

Suppression subtractive hybridization (SSH), expression profiling and EST sequencing identified 12 plant genes and six fungal genes that are expressed in the arbuscular mycorrhizal symbiosis between Medicago truncatula and Glomus mosseae. All the plant genes and three of the fungal genes were up-regulated in symbiotic tissues. Expression of 15 of the genes is described for the first time in mycorrhizal roots and two are novel sequences. Six M. truncatula genes were also activated during appressorium formation at the root surface, suggesting a role in this early stage of mycorrhiza establishment, whilst the other six plant genes were only induced in the late stages of mycorrhization and could be involved in the development or functioning of the symbiosis. Phosphate fertilization had no significant influence on expression of any of the plant genes. Expression profiling of G. mosseae genes indicated that two of them may be associated with appressorium development on roots and one with arbuscule formation or function. The other three fungal genes were expressed throughout the life-cycle of G. mosseae.

Genetic and Cellular Analysis of Resistance to Vesicular Arbuscular (VA) Mycorrhizal Fungi in Pea Mutants

Screening of nodulation mutants of Pisum sativum has yielded mutants showing resistance to VA fungi (termed myc-). Most of these have aborted infections (myc-(1) phenotype) which are characterised by host cell reactions recalling those in certain pathogen infections. Mutants affected in later steps of mycorrhiza development (myc-(2) phenotype), with blocking of arbuscule formation, were less frequent. The myc-(1) character is recessive, segregates monogenically and occurs on at least five different, independently mutated loci, indicating that VA endomycorrhiza formation is under multiple gene control. Expression of the myc-(1) character is indissociable from that of nod’ in mutants and appears to be the result of pleiotropic effects of single genes. In late mutants where arbuscule formation is blocked, nodules develop but are inefficient (nod+,fix-). Coincidences between myc and nod characters may reflect common mechanisms in plant control over some step(s) in endomycorrhiza and nodule symbioses. Since chemical mutagenesis generally causes loss of gene function, inactivation of symbiosis-specific susceptibility genes in the mutants could affect production of signal molecules and somehow lead to stronger expression of plant resistance mechanisms to the symbionts.

Effect of vesicular-arbuscular mycorrhizal fungi and phosphate-solubilizing bacteria on growth and nutrition of soybean in a neutral-calcareous soil amended with32P-45Ca-tricalcium phosphate

SummaryThe interactions between vesicular-arbuscular (VA) mycorrhiza and phosphate-solubilizing bacteria (PSB) on the utilization of the sparingly soluble32P-45Ca-tricalcium phosphate byGlycine max-Rhizobium japonicum was studied in a neutral calcareous soil. Two mycorrhizal endophytes,Glomus mosseae and aGlomus sp. (E3), were assayed. Both of them increased plant growth and nutrition, E3 being the most efficient. Mycorrhizal inoculation, especially with E3, improved plant utilization of the sparingly soluble tricalcium phosphate. However, the proportion of P in the plant shoots derived from the fertilizer was lower in mycorrhizal plants. Although PSB inoculation did not improve fertilizer utilization by either roots or mycorrhizas, it did produce an increase in shoot N concentration and content, and in the shoot to root ratio. The amount of mycorrhizal infection was also increased by PSB at all levels of added phosphate.This experiment confirms the significance of VA mycorrhizas on growth and P uptake by soybeans, and provides evidence of a key role of this symbiosis in P-cyccling in neutral calcareous soils.

Modulation of Host Defence Systems

Arbuscular mycorrhizal (AM) fungi extensively invade host root tissues. This raises the question of how host plants contend with them; they must exert some kind of control over fungal proliferation since it is confined to a specific root tissue, the parenchymal cortex. Defence processes, which are triggered as a general plant response to microbial invasion, are modulated during root-fungus interactions in arbuscular mycorrhizas. This chapter presents an up-dated review of data on plant defence elicitation in these symbiotic systems and discusses possible mechanisms whereby defence reactions are maintained at a low level, as well as their implication in the phenomenon of bioprotection by AM fungi against soil-borne pathogens.

Cytochemical modifications in the host-fungus interface during intracellular interactions in vesicular- arbuscular mycorrhizae

Abstract The cytochemical modifications occuring in the host-fungus interface during intracellular interactions in two different vesicular-arbuscular mycorrhizae (VAM) ( Allium cepa/Glomus mosseae and Ornithogalum umbellatum/ Glomus fasciculatus ) are very similar; marked differences were only found in the wall cytochemistry of the two fungi involved. In both mycorrhizae, host primary wall material which is deposited around hyphae penetrating cells and which seems to contain a glycoprotein complex, strongly diminishes with development of the finer arbuscule branches and appears to change in nature. Host plasmalemma is not structurally or cytochemically modified with its invagination during arbuscule development. The analogies and differences in these observations to those reported for pathogenic infections are discussed in relation to the hypothesis that the arbuscule is the preferential site of bi-directional nutrient transfer between the fungus and plant in VAM.

Ultrastructural Cytochemistry of the Host-Fungus Interfaces in the Endornycorrhizal Association Glomus mosseae / Allium cepa

Summary The structure of the host-fungus interface in the VA mycorrhizal association Glomus mosseae / Allium cepa has been investigated cytochemically (Thiery test; differential DMSO and EDTA extraction; phosphotungstic acid staining) and using different fixation procedures (glutaraldehyde and OsO 4 post-fixation; simultaneous glutaraldehyde — OSO 4 fixation). The continous, highly invaginated host plasmalemma surrounding the intracellular fungal structures retains its affinity for phosphotungstic acid and continues to produce polysaccharide fibrils, but in the presence of the endophyte it progressively loses the capacity to organise these into well-structured wall material. At the point of cell penetration and in the primary arbuscule branches the fungal wall, which is stained by the Thiery reaction and phosphotungstic acid but unaffected by DMSO or EDTA, is surrounded by a collar of host wall material which has the same cytochemical properties as the primary walls of the plant cell. The fine arbuscule branches are surrounded by a layer of condensed matrical fibrils. When the arbuscule branches senesce and collapse, they become encased by the polysaccharide fibrillar material derived from the host. The appearance of the interfacial matrix separating the host plasmalemma from the fungal wall is always the same, no matter which fixation procedure is used. It contains membranous vesicles and scattered polysaccharide fibrils, both of which are elaborated by the host plasmalemma. The importance of the host and endophyte plasmalemma formations is emphasised. The vesicles arising from the host plasmalemma are often in contact with the walls of the endophytic hyphae. In the fungus, these bulbous or tubular configurations of the plasmalemma can become very numerous and can occupy the whole hyphal width. Although these structures could be involved in host-fungus exchanges, their eventual participation in wall synthesis is also orooosed.

RNA accumulation and genes expressed in spores of the arbuscular mycorrhizal fungus, Gigaspora rosea

RNA could be extracted from ungerminat- ed spores of the arbuscular mycorrhizal fungus Gigas- pora rosea with a maximum of yield 7 days after isola- tion from soil. cDNA was synthesized and used for li- brary construction or as template for the amplification by polymerase chain reaction of fragments corre- sponding to several genes. Analysis of cDNA clones corresponding to fragments of ribosomal RNA genes indicated that the extracted RNA as starting material was of arbuscular mycorrhizal fungal origin. Polymer- ase chain reaction showed the presence of mRNA from genes encoding glyceraldehyde-3-phosphate de- hydrogenase, fi-tubulin and two types of ATPases; mRNA accumulation of the glyceraldehyde-3-phos-

Conceptual approaches for the rational use of VA endomycorrhizae in agriculture: Possibilities and limitations

Abstract A strategy is proposed of plant inoculation with vesicular-arbuscular mycorrhizal (VAM) fungi based on appropriate biological tests (soil endomycorrhizal potential and fungal effectiveness/soil receptivity test) that provide information on whether it is necessary to inoculate and which fungi to use. It is suggested that the inoculum production methods should be chosen in function of the techniques used for plant production and of the economic criterion. Examples of successful inoculation of micropropagated apple root-stocks and ornamental plants sown in commercial nurseries are presented.