Early developmental stages of AM
In this work a new system to cultivate mycorrhizal seedlings under gnotobiotic conditions was established. Mini-mycorrhizotrons allow natural growth of the symbiotic partners during the early stages, because, in contrast to root cultures, in mini-mycorrhizotrons the plants are photosynthetically active and it is not necessary to add additional sources of carbohydrates. Also, the chambers permit the continuous and noninvasive observation of the development of plant and fungus under a microscope, without disturbing the system. In particular, it was possible to observe the development of the symbiosis at any time over a period of at least 20 d, and to count fungal structures along the root without staining it, confirming that the establishment of the AM symbiosis in the chambers was highly reproducible. Furthermore, roots grown in mini-mycorrhizotrons can be directly observed by ESEM, avoiding washing and fixation procedures that can detach fungal structures and create artefacts. Observation of each chamber allows the selection of samples at one specific developmental stage and harvesting of them for further molecular analysis, allowing the establishment of correlations between plant gene expression and mycorrhiza development. Any interaction with unwanted organisms can be excluded because of the aseptic growth conditions. Moreover, in addition to the interaction with mycorrhizal fungi, plants also interact normally with rhizobia or pathogenic fungi when grown in the mini-mycorrhizotrons, allowing comparison of the reactions of the plant to the different organisms.
Some previous attempts to establish a similar system were made by different authors (reviewed in Hepper, 1981); however, all of these approaches were extremely labour-intensive and visual observation of the symbiosis often led to the loss of the aseptic conditions.
Induction of a defence response before appressoria formation
Chalcone synthase (EC 220.127.116.11) is the enzyme catalysing the first step committed to the flavonoid biosynthesis. Flavonoids act as antimicrobial compounds as medicarpin in M. sativa (Harrison & Dixon, 1994), but also play a role as signalling molecules which can stimulate AM fungi spore germination in vitro (Tsai & Philipps, 1991). The activation of the flavonoid pathway by CHS is well studied in other plants, such as P. sativum, Phaseolus vulgaris, Nicotiana tabacum, Petunia hybrida, Petroselinum crispum and many others (Block et al., 1990; van der Meer et al., 1992; An et al., 1993; Bate et al., 1994; Blee & Anderson, 1996). Legumes contain a family of between three and 12 CHS genes, as reported for M. sativa, G. max and P. sativum (Junghans et al., 1993). However, it is not known how many genes code for CHS in M. truncatula. The CHSs share 82–90% sequence identity among the genera and are highly conserved (McKhann & Hirsch, 1994).
In this study, CHS expression was used to monitor the response of M. truncatula to G. intraradices in mini-mycorrhizotrons during the ealry stages of the interaction. One gene encoding CHS in M. truncatula, Mt-chs1, was identified by differential display comparing mRNA of seedlings grown with and without the AM fungus. RT-PCR analysis was used to confirm that Mt-chs1 is induced in plants grown in mini-mycorrhizotrons after 5–10 dpi, when the fungus for the first time contacts the root surface. An enhanced expression of CHS in M. truncatula mycorrhized with Glomus versiforme was described earlier (Harrison & Dixon, 1993). As a hybridization probe the M. sativachs2 gene, which is 94% similar to Mt-chs1, was used and therefore the same gene as described here may have been detected. Moreover, CHS expression was detected by in situ hybridization in the same system at later stages in arbuscule-containing cells (Harrison & Dixon, 1994). The less similar (89%) heterologus probe Ms-chs1 used in this case may have also detected Mt-chs1, indicating that this gene, or a homologue, is continuously up to the arbuscular stage of the symbiosis.
Our experiments showed that Mt-chs1 is expressed at a low level also in noninoculated plants. This could have led some authors to conclude that the transcription of CHS, if at all, is only slightly enhanced (Blee & Anderson, 1996; Mohr et al., 1998). In particular, the presence of the highest levels of CHS transcripts in young elongating roots (McKhann & Hirsch, 1994), localized mainly in the cortical cells (Harrison & Dixon, 1994), could explain the presence of background expression in the noninoculated plants cultivated in the mini-mycorrhizotrons. Alternatively, the low and fluctuating level of CHS in the noninoculated plants may be due to an as yet unidentified environmental factor. Since the expression pattern of Mt-chs1 was similar in plants grown in Multiflor plates and in mini-mycorrhizotrons, it can be excluded that the gene expression pattern is an artefact.
The enhanced expression level of CHS mRNA after treatment of M. truncatula with F. solani f. sp. phaesoli, which also produces symptoms, and the inability to induce the transcript by F. solani f. sp. pisi, which does not produce symptoms, provides evidence that induction of Mt-chs1 is also part of the defence pathway. In fact, F. solani f. sp. phaesoli also induced the expression of the pathogen-related M. truncatula chitinases classes I, II, III-1 and IV, whereas the F. solani f. sp. pisi induced only the class IV chitinase (Salzer et al., 2000). Similarly, F. solani f. sp. pisi could not induce a defence response in the closely related P. sativum (Mohr et al., 1998). Both this and the lack of induction of Mt-chs1 by R. solani indicate that Mt-chs2 is only induced in compatible interactions. Along this line, Mt-chs1 was strongly induced in the mutant dmi1. This mutant exhibits a massive defence response when brought into contact with AM fungi, an event that might prevent a successful fungal penetration, as shown in pea mutants (Gianinazzi-Pearson et al., 1996).
Our study describes the development and validation of a novel culture system for mycorrhizal symbiosis. Mini-mycorrhizotrons represent an attractive tool for molecular and developmental studies of the early stages of symbioses in a gnotobiotic environment. The mini-mycorrhizotrons allowed identification and cloning of a symbiosis-related gene, and the correlation between its expression and the developmental stage of the symbiosis could be established. This tool might be useful to identify further early symbiosis genes.