GENE EXPRESSION AND MOLECULAR MODIFICATIONS ASSOCIATED WITH PLANT RESPONSES TO INFECTION BY ARBUSCULAR MYCORRHIZAL FUNGI

Vivienne Gianinazzi-Pearson, Armelle Gollotte, Eliane Dumas-Gaudot, Philipp Franken and Silvio Gianinazzi Laboratoire de Phytoparasitologie INRAlCNRS, INRA, SGAP, BV 1540,21034 Dijon cedex, France

Introduction

Microorganisms that colonize living plant tissues generally show a relatively restricted host range, but the striking feature of arbuscular mycorrhizal fungi is their ability to form a symbiotic association with roots of the large majority of terrestrial plant species. Likewise, the ancestral nature of arbuscular mycorrhiza [1] makes them a remarkable example of reciprocal cellular and physiological compatibility between plant and fungal taxa that must have been established early during land colonization and maintained through evolution. In spite of the widespread occurrence of arbuscular mycorrhiza, analyses into the molecular mechanisms and genetic determinants involved are still in their infancy due to the complexity of symbiont interactions and the incalcitrance of the fungal partner to pure culture. We have adopted two strategies to investigate plant processes regulating symbiotic fungus-root interactions: firstly, by probing tissues for molecular events that may be in common with other plant-microbe associations and, secondly, by characterizing of genes and gene products specific to arbuscular mycorrhiza. In the present paper we discuss recent data and speculate about the plant's role in establishment of the symbiotic state and in control over fungal development in arbuscular mycorrhiza.

Comparisons with other plant-microbe interactions

PLANT DEFENCE EXPRESSION AND MYCORRHIZAL FUNGI

Since we first reported that mycorrhizal fungi elicit the production oflow amounts of phytoalexins and related compounds in roots [2], several other aspects of defence responses have been analyzed (Fig. 1). Results vary depending on the biological system and method of analysis, but the overall conclusion that can be drawn is that only weak or very local activation of plant defence mechanisms occurs during mycorrhizal interactions [3], in contrast to the extensive responses of plants to pathogens.

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M.J. Daniels et al. (eds.), Advances in Molecular Genetics of Plant-Microbe Interactions, Vol. 3, 179-186. © 1994 Kluwer Academic Publishers.

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Phytoalexins

Enzymes: Phenylpropanoid (PAL, CHS, CHI) Chitinase ~ (1,3) Glucanase Peroxidase

'PRI' protein

Callose HRGP Phenolics

Figure 1. Plant defence responses investigated in arbuscular mycorrhiza

In addition to confirmation of low activation of the phenylpropanoid pathway [4], phenolics and callose have not been detected to any significant extent [5, 6, 7], peroxidase and chitinase are little or transiently induced [8, 9, 10], synthesis ofHRGP and protein of the PRI group is weak and very localized at the cellular level [3, 11, 12, 13], and glucanase has not been found to increase [10, 14]. This low priming of defence reactions in mycorrhiza is not due to symbiotic fungi lacking essential signal molecules since we have observed that they can elicit resistance responses in mutated host plants [6].

It appears therefore that the plant defence response must be somehow controlled or suppressed during establishment of the functional compatibility between mycorrhizal symbionts. The relationship between defence molecules and regulation of fungal development in arbuscular mycorrhiza is not, however, straightforward. Our detailed studies of increased chitinase activities accompagnying mycorrhiza formation have shown that there is also induction of new isoforms, one of which is associated with the very early stages of root colonization [14, 15]. These are different from those activated during a pathogenic fungal infection of roots [16], indicating a differential plant response to the two types of root infection. Furthermore, root colonization by a mycorrhizal fungus is not affected by the constitutive over production of defence-related molecules either in transgenic tobacco [3, 17, unpublished results] or in a Nicotiana hybrid which shows a general resistance to pathogens, including root­infecting fungi [18] (Table 1). We are presently investigating whether this could be the result of a localized suppression of the synthesis of such molecules where mycelium develops or, alternatively, due to the symbiotic fungi being insensitive to them.

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Table 1. Development of arbuscular mycorrhiza in N. glutinosa, N. debneyi and an amphidiploid N. glutinosa x N. debneyi hybrid

N. glutinosa N. debneyi Amphidiploid hybrid

Mycorrhizal infection

Colonization intensity

34.0 47.7 48.0

Arbuscule frequency

22.3 41.8 36.5

NODULE SYMBIOSIS EVENTS IN ARBUSCULAR MYCORRHIZA

Although at first sight arbuscular mycorrhizal and nodule symbioses may appear very different, closer consideration of cellular events reveals that several features are common to the two symbioses. Both involve root penetration phases where plant wall material isolates the microsymbiont from the host cell (infection thread or wall apposition layer) and then proliferation of intracellular forms (bacteroids, arbuscules-Fig. 1) which develop an extended interface with the plant protoplast, where metabolite flow between the symbionts preferentially takes place. Evidence that some steps in the infection processes by arbuscular mycorrhizal fungi and rhizobia are controlled by common genetic determinants has been provided by the isolation and characterization of symbiotically-defective pea mutants (see below). Mycorrhiza-resistant (myc-) mutants have been identified amongst non­nodulating (nod-) genotypes, and genetical analyses indicate that in mutants defective for both symbiosis (myc-nod-) the two phenotypes are determined by the same mutated gene [19, 20]. Furthermore, similar features have been observed in comparisons of molecular events associated with plant-microbe interactions during arbuscular mycorrhiza and nodule formation. We have localized several antigenic glycoconjugate components (Table 2), characteristic of the infection thread or peribacteroid compartment of nodules, in the plant derived material or the specialized periarbuscular interface associated with different phases of the mycorrhizal symbiosis [21]. As may be expected we have also found differences between the two types of root-microbe interactions. For example, although late nodulin-related polypeptides have been reported in mycorrhizal tissues, we could not localize expression of two early nodulin genes nor certain nodulation glycoconjugates (Table 2). Moreover, the existence of myc+nod- phenotypes amongst pea mutants [19] confirms that formation of the two types of root symbiosis involves also different plant genes.

More information about the extent to which similarities and differences exist between mycorrhiza and nodule symbiosis could lead to a better understanding of the genetical and molecular bases of symbiotic root-microbe interactions. Moreover, seeing the ancestral nature of arbuscular mycorrhiza, it is interesting to speculate that part of the plant processes leading to nodulation may have evolved from those already established for mycorrhiza.

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Table 2. Nodulin gene expression and nodule glycoconjugate localization in arbuscular mycorrhiza

Nodulins

PsENOD5 PsENOD12 Four unknown Nod26

Glycoconjugate antigens

Peribacteroid compartment: MAC 64 MAC 206

MAC 268 MAC 266 MAC 207

Infection thread MAC 236 MAC 265

+ enhanced

periarbuscular membrane + interface periarbuscular membrane periarbuscular interface periarbuscular interface

host wall deposit

References

6 6

21 22,23

24 24

25 25

25,26

26 25,26

Mycorrhiza.related genes and gene products

PLANT GENETIC CONTROL OF ARBUSCULAR MYCORRHlZA FORMATION

We have obtained proof that specific plant genes are essential to arbuscular mycorrhiza establishment and development following the isolation of two types of pea mutants obtained by chemical mutagenesis. One phenotype, described above as myc·nod-, is completely resistant to mycorrhizal fungi and stops fungal development at the root surface (early mutants) whilst in a second phenotype, found amongst nod+fix- mutants, root colonization occurs but arbuscule formation is prevented (late mutants) [20]. Early mutants are the most frequent and have been most studied. The same phenotype is induced by at least four mutated loci and properties of the mycorrhiza-resistant character can be summarized as follows: monogenic, recessive, genetically stable, and indissociable from the nod­character [19]. Mycorrhiza formation is therefore governed by dominant host genes and since infection is stopped at a very early stage in the mutants, the mutated genes must be upstream in the plant control processes. The mutations affect plant genes that are specific to interactions with symbiotic microorganisms since they do not alter the root phenotype towards other root-infecting or pathogenic organisms (Table 3). Nevertheless, they must be wide-action genes

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because the mutants are resistant to different species of mycorrhizal fungi, whether inoculated under laboratory or field conditions (unpublished data).

Table 3. Susceptible (S), resistant (R) and hypersensitive (H) behaviour of wild-type (myc+nod+) and early mutant (myc-nod-) peas to different root-infecting or pathogenic organisms

Mycorrhizal fungi Rhizobium leguminosarum Chalara elegans Aphanomyces euteiches Rhizoctonia species Agrobacterium tumefaciens Meloidogyne species

myc+nod+ S S H S S S S

myc-nod-R R H S S S S

References 19,20

19 27 27 28 6 6

In analyses of resistance to arbuscular mycorrhizal fungi in the early mutants, we have observed cellular responses that recall those in incompatible plant-pathogen interactions. Wall appositions develop in epidermal and hypodermal cells in contact with fungal appressoria and these are characterized by the accumulation of defence-associated molecules like phenolics, callose and 'PR1' protein [6, 7]. Elicitation of a strong resistance reaction by symbiotic fungi in these genetically altered pea plants contrasts with the weak defence response normally encountered in hosts, suggesting that symbiosis-specific genes somehow regulate defence gene expression during mycorrhiza establishment.

GENE EXPRESSION SPECIFIC TO MYCORRHIZA FORMATION

Studies of the expression of plant genes specific to the establishment of arbuscular mycorrhiza are hampered by the fact that the fungal symbionts cannot be grown in pure culture and the difficulty in establishing synchronized infection events. Analyses have been mainly limited to comparisons of proteins or polypeptides extracted from mycorrhizal and nonmycorrhizal tissues. Using 2D PAGE in time course analyses, we have clearly shown that new proteins (endomycorrhizins) are synthesized during mycorrhiza development [29], and de novo gene expression has recently been confirmed by in vivo mRNA translation [30] and by DDRT-PCR [31]. However, it has not yet been possible to conclusively distinguish which of the mycorrhiza-induced modifications are part of the host response and which are of fungal origin. Such studies and analyses of mRNA translation products have also revealed more subtle modifications with additional quantitative changes and disappearance of plant proteins [29, 30, 32]. Although it is evident that protein modifications accompany mycorrhiza formation from its earliest stages and onwards, probes to detect specific plant gene activation have not yet been generated. Our present research is aimed at isolating and characterizing

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mycorrhiza-specific cDNA sequences of both plant and fungal origin in order to study in more detail the fmely tuned regulation processes involved in the symbiosis.

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