Cytological and Genetic Changes During Germination and Presymbiotic Growth

Early evidence of cell cycle activation in AMF growing in the absence of the host was reported by Mosse, who described the development of dense regions containing normal cytoplasm and many dividing nuclei in spores of A. laevis prior to germination (Mosse 1970a). Also, Sward (1981b) observed a large number of nuclei with highly condensed chromatin and prominent nucleoli in G. margarita spores after 24 h of incubation on water agar. Cytological studies showed that nuclei from quiescent spores of G. versiforme were in the GO/G1 phase, whereas nuclei from mycorrhizal roots were in the synthetic and G2/M phases (Bianciotto et al. 1995). Mitotic spindles were also detected in germinated spores of G. mosseae by tubulin immunostaining, confirming the occurrence of DNA replication during pre-symbiotic growth (Requena et al. 2000). In the latter work, the gene GmTOR2, encoding a protein with high homology to the C terminus of Saccharomyces cerevisiae TOR2 (controlling cell cycle), was characterised. Under treatment with the anti-inflammatory drug rapamycin, which interferes with TOR2 by arresting S. cerevisiae cell cycle in G1 phase, G. mosseae spore germination was unaffected, whereas hyphal growth decreased, suggesting that nuclear replication in the pre-symbiotic stage is only necessary for hyphal growth (Requena et al. 2000).

EST sequencing from germinated spores of G. intraradices and G. rosea revealed putative homologues to cell cycle and meiosis-specific genes from other fungi, such as chromatin assembly factor, ubiquitin-encoding genes (Stommel et al. 2001) and Neurospora crassa NDT80, known to control exit from pachytene phase of meiosis (Jun et al. 2002). Furthermore, a putative gene involved in the biosynthesis of new nucleotides was detected in germinated spores of G. intraradices (Jun et al. 2002).

The occurrence of nuclear division was inferred in non symbiotic mycelium by using image analysis counts of the number of nuclei (Bécard and Pfeffer 1993), which decreased from 2,000 to 800 in individual spores during the early days of germination, suggesting the migration of nuclei from spores to hyphae. This was confirmed by data on the occurrence of cytoskeletal components, both microtubules and microfilaments, in the mycelium originating from germinating spores of G. mosseae and G. caJedonium (Astrom et al. 1994; Logi et al. 1998). The presence of such components is consistent with the role of cytoskeleton in the migration of nuclei and cellular organelles during active growth. Expression of ß-tubulins in germinating AM fungal spores (Franken et al. 1997; Butehorn et al. 1999) was confirmed by the detection of sequences putatively encoding other cytoskeletal proteins, such as a-tubulin, ß-actin, dynein and actin-related protein, possibly involved in nuclear and nutrient movements, in G. intraradices germinated spores (Jun et al. 2002). Recently, full-length ß-tubulin gene has been sequenced from G. gigantea and G. cJarum, showing some peculiar traits compared to fungi other than glomeromycota (Msiska and Morton 2009).

Nuclear division in G. rosea hyphae was also detected in the presence of host root exudates or of the synthetic strigolactone GR24, which induced an accumulation of nuclei in the apical area of treated hyphae (Buée et al. 2000; Besserer et al. 2008).

Early experiments showed that inhibitors of mRNA translation hindered AM fungal spore germination (Hepper 1979; Beilby 1983). Accordingly, differential display analysis of G. rosea did not show changes in RNA accumulation patterns during hyphal development, suggesting that in this phase proteins are produced only by translating transcripts synthesized prior and during spore germination (Franken et al. 2000).

Many expressed genes detected in germinating AM fungal spores showed homology to those encoding for proteins involved in translation, protein processing, primary metabolism and transport processes (Franken et al. 1997; Lammers et al. 2001; Stommel et al. 2001; Jun et al. 2002; Bago et al. 2002a, 2003). The identification of genes putatively codifying for several enzymes involved in carbon metabolism and lipid breakdown often confirmed biochemical data.

An interesting gene, G. mosseae GmGIN1, was highly and specifically expressed in non symbiotic mycelium, whereas it was silenced during the symbiosis, both in the intraradical structures and the extraradical mycelium (Requena et al. 2002). Interestingly, several genes with homology to the N-terminus of GmGIN1, sequenced from Magnaporthe grisea, N. crassa, GibbereJJa zeae and Aspergillus niduJans, encode for a family of proteins playing an essential role in polarized growth, septal formation and hyphal morphological changes in the phytopathogenic fungus Ustilago maydis and in the ectomycorrhizal fungus Suillus bovinus (Gorfer et al. 2001; Weinzierl et al. 2002).

A 14-3-3 protein encoding gene, known to be involved in modulation of cell ion pumps and channels, was detected in G. intraradices mycelium (Porcel et al. 2006). This finding suggests an important role of this gene in controlling the activity of P-type H+-ATPases, detected in G. intraradices and G. mosseae (Requena et al. 2003; Corradi and Sanders 2006), which are responsible of the maintenance of hyphal ionic gradient during polarized growth (Ramos et al. 2008).

Interestingly, a sequence showing strong similarity to an endonuclease involved in lateral transfer of an rDNA intron has been detected in G. intraradices germinated spores, suggesting the occurrence of lateral gene transfer during nuclear exchange between anastomosing hyphae belonging to genetically different AMF (Jun et al. 2002; Croll et al. 2009).

Induction of genes encoding for putative pyruvate carboxylase and mitochon-drial ADP/ATP translocase, involved in respiration enhancement activity, has been observed in G. rosea and G. intraradices during early responses to host root factors, before hyphal branching (Tamasloukht et al. 2003, 2007). The expression of the former gene could explain the stimulatory effects exerted by CO2 on AM fungal growth (Bécard and Piché 1989), whereas the expression of the latter gene could be necessary for the delivery of large quantity of ATP produced at high respiration rates (Requena et al. 2003). Activation of such genes and oxygen consumption were induced by host root exudates after 0.5-3 h, when no morphological change in hyphal growth pattern was detectable yet. On the contrary, no differences in the expression of key metabolic genes during the first 48 h after strigolactone analogue GR24 treatment were observed in G. rosea, which showed strong enhancement in transcript levels after 2 days of incubation, independently of GR24 treatment (Besserer et al. 2008). These findings suggest that other unknown signal molecules may be active and that strigolactone-induced mitochondrial activity is due to post-translational regulation of key enzymes (Delano-Frier and Tejeda-Sartorius 2008; Rani et al. 2008). The need of host-derived signals for developmental stages following spore germination can be inferred by results obtained with the pmi mutants of Solanum lycopersicum, which are regularly colonised by extraradical mycelium and mycorrhizal roots but are not susceptible to colonisation by hyphal germlings (David-Schwartz et al. 2001, 2003).

AM fungal spores germinating in the absence of host-derived factors constitu-tively release unknown compounds which are perceived as signals by host plants and are able to elicit recognition responses, such as a transient cytoplasmic calcium induction in soybean cells (Navazio et al. 2007) and the accumulation of starch in Lotus japonicus roots (Gutjahr et al. 2009). Ca2+-mediated signaling was also suggested by expression of genes involved in Ca2+-mediated signal transduction in M. truncatula roots in the presence of a diffusible factor released by G. mosseae (Weidmann et al. 2004). Previous studies had reported the release of a diffusible signal by G. mosseae, G. rosea, G. gigantea, G. margarita and G. intraradices growing in the presence of host plants (Chabaud et al. 2002; Kosuta et al. 2003). The perception of such signals by M. truncatula induced root expression of the early nodulin gene

MtENOD11, which was related, both spatially and temporally, with the appearance of hyphal branching enhancement. Moreover, factors released by G. margarita and G. intraradices mycelium growing nearby M. truncatula plant roots were able to induce lateral root formation (Olah et al. 2005) and those released by G. inraradices branching hyphae elicited root calcium-spiking responses (Kosuta et al. 2008). No information is still available on the chemical nature of AM fungal factor(s).

Although many studies reported germling growth improvement by different microorganisms, little is known about the molecular mechanisms of such phenomenon. Changes in AM fungal gene expression in response to the perception of microbial derived factors were detected by Requena et al. (1999) during co-culture of G. mosseae with a strain of the rhizobacterium B. subtilis, inducing mycelial growth increases. In particular, down-regulation of the putative gene GmFOX2, encoding a protein involved in long-chain fatty acids catabolism, was evidenced. It is not known which is the signaling pattern between bacteria and fungi, although it has been hypothesized that an increase in fungal cAMP, due to the perception of flavonoid/estrogen bacterial signals, could be responsible for the glucose repression stage that down-regulates GmFOX2 (Requena et al. 1999).

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