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Although it is now well-established that decorated lipo-chitooligosaccharide Nod factors are the key rhizobial signals which initiate infection/nodulation in host legume species, the identity of the equivalent microbial signaling molecules in the Frankia/actinorhizal association remains elusive. With the objective of identifying Frankia symbiotic factors we present a novel approach based on both molecular and cellular pre-infection reporters expressed in the model actinorhizal species Casuarina glauca. By introducing the nuclear-localized cameleon Nup-YC2.1 into Casuarina glauca we show that cell-free culture supernatants of the compatible Frankia CcI3 strain are able to elicit sustained high frequency Ca(2+) spiking in host root hairs. Furthermore, an excellent correlation exists between the triggering of nuclear Ca(2+) spiking and the transcriptional activation of the ProCgNIN:GFP reporter as a function of the Frankia strain tested. These two pre-infection symbiotic responses have been used in combination to show that the signal molecules present in the Frankia CcI3 supernatant are hydrophilic, of low molecular weight and resistant to chitinase degradation. In conclusion, the biologically active symbiotic signals secreted by Frankia appear to be chemically distinct from the currently known chitin-based rhizobial/arbuscular mycorrhizal signaling molecules. Convenient bioassays in Casuarina glauca are now available for their full characterization.

The primary objective of this study was to identify the molecular signals present in arbuscular mycorrhizal (AM) germinated spore exudates (GSEs) responsible for activating nuclear Ca(2+) spiking in the Medicago truncatula root epidermis. Medicago truncatula root organ cultures (ROCs) expressing a nuclear-localized cameleon reporter were used as a bioassay to detect AM-associated Ca(2+) spiking responses and LC-MS to characterize targeted molecules in GSEs. This approach has revealed that short-chain chitin oligomers (COs) can mimic AM GSE-elicited Ca(2+) spiking, with maximum activity observed for CO4 and CO5. This spiking response is dependent on genes of the common SYM signalling pathway (DMI1/DMI2) but not on NFP, the putative Sinorhizobium meliloti Nod factor receptor. A major increase in the CO4/5 concentration in fungal exudates is observed when Rhizophagus irregularis spores are germinated in the presence of the synthetic strigolactone analogue GR24. By comparison with COs, both sulphated and nonsulphated Myc lipochito-oligosaccharides (LCOs) are less efficient elicitors of Ca(2+) spiking in M. truncatula ROCs. We propose that short-chain COs secreted by AM fungi are part of a molecular exchange with the host plant and that their perception in the epidermis leads to the activation of a SYM-dependent signalling pathway involved in the initial stages of fungal root colonization.

Ca(2+) spiking is a central component of a common signaling pathway that is activated in the host epidermis during initial recognition of endosymbiotic microbes. However, it is not known to what extent Ca(2+) signaling also plays a role during subsequent root colonization involving apoplastic transcellular infection. Live-tissue imaging using calcium cameleon reporters expressed in Medicago truncatula roots has revealed that distinct Ca(2+) oscillatory profiles correlate with specific stages of transcellular cortical infection by both rhizobia and arbuscular mycorrhizal fungi. Outer cortical cells exhibit low-frequency Ca(2+) spiking during the extensive intracellular remodeling that precedes infection. This appears to be a prerequisite for the formation of either pre-infection threads or the pre-penetration apparatus, both of which are fully reversible processes. A transition from low- to high-frequency spiking is concomitant with the initial stages of apoplastic cell entry by both microbes. This high-frequency spiking is of limited duration in the case of rhizobial infection and is completely switched off by the time transcellular infection by both microsymbionts is completed. The Ca(2+) spiking profiles associated with both rhizobial and arbuscular mycorrhizal cell entry are remarkably similar in terms of periodicity, suggesting that microbe specificity is unlikely to be encoded by the Ca(2+) signature during this particular stage of host infection in the outer cortex. Together, these findings lead to the proposal that tightly regulated Ca(2+) -mediated signal transduction is a key player in reprogramming root cell development at the critical stage of commitment to endosymbiotic infection.

A 3300-base segment of Escherichia coli chromosomal DNA, cloned into pBR322, will complement a methionine auxotroph in which the lesion is a defective methionyl-tRNA synthetase with a much reduced affinity for methionine. Crude extracts of these transformants contain elevated levels of a protein which has a subunit molecular weight of 66000, methionyl-tRNA synthetase aminoacylation activity in vitro and which cross-reacts with anti-(methionyl-tRNA synthetase) antibodies. This polypeptide is very slightly larger than the well-characterised and crystallised tryptic fragment of methionyl-tRNA synthetase. A DNA sequence of 1750 residues at one end of the cloned insert codes for a non-terminated open reading frame in which we can locate a large number of methionyl-tRNA synthetase tryptic and chymotryptic peptides. We have also sequenced 300 nucleotides upstream of this coding segment where we find a large invert repeat in the putative methionyl-tRNA synthetase promoter region.

In order to study the spatio-temporal patterns of lectin gene expression throughout the various developmental stages of the Rhizobium—legume interaction, promoter-gusA fusions for each of three lectin genes from Medicago truncatula (Mtlec1, 2 and 3) have been introduced into transgenic Medicago varia plants. Histochemical staining for β-glucuronidase activity has shown that Mtlec1 and Mtlec3 are expressed independently of symbiosis in inner cortical and vascular tissues of the root, respectively. Transcription of Mtlec3 in the root stele is markedly enhanced following inoculation with Rhizobium meliloti. Furthermore, both Mtlec1 and Mtlec3 are transcribed in dividing cells of the root nodule primordium, whether elicited by R. meliloti or by purified nodulation (Nod) factors. These findings suggest that these two lectins are involved in developmental processes early during nodule ontogeny. In contrast, Mtlec2 is not expressed in uninoculated roots or during early stages of the symbiotic interaction. At later stages, all three lectin promoters are functional in mature nodules. In this context, lectin gene expression is restricted to non-infected cells of both central and certain peripheral tissues and appears to be correlated with the nitrogen-fixing capacity of the nodule.

We have sequenced a 4200-base-pair fragment of Schizosaccharomyces pombe DNA which encompasses the entire DNA ligase gene, CDC17. S1 mapping has enabled us to identify two small introns (40 and 62 nucleotides) at the 5' end of the coding region of the gene and their 3' internal conserved sequences match the CTRAY consensus found in other S. pombe introns. The major transcription initiation and 3' polyadenylation sites have been mapped and are preceded by higher eukaryotic-like TATA and AATAAA sequences respectively. Furthermore, the CDC17 mRNA carries a poly(A) tail whose length (approximately 250 nucleotides) is typical of that found in higher eukaryotic mRNAs, and is in contrast to the much shorter polyadenylated sequences found for the mRNAs of the budding yeast, Saccharomyces cerevisiae. The deduced amino acid sequence of the S. pombe DNA ligase predicts a protein of 86182 daltons, and an overall 53% homology with the same enzyme from S. cerevisiae. In particular, a stretch of 24 amino acids with 100% sequence homology spans the putative ATP-binding region which is also conserved in T4 and T7 bacteriophage DNA ligases.

The primary structure of the tyrosy1-RNA synthetase(Tyr Ts) of Bacillus stearothermophilus has been dedced from the nucleotide sequence of the cloned gene and from the amino acid sequence of peptides isolated from the purified enzyme. TyrTS (B. stearothermophilus) has a molecular weight of 47316 and the sequence is 56% homologous with that of TyrTS (Escherichia coli). The binding domain for the substrate intermediate tyrosy1 adenylate is located in the N-terminal protion of the polypeptide and is highly conserved in both enzymes. Several Iysine residues, which are shielded from acetylation in the TyrTS-tRNAtyr complex, are also located in a stretch of highly conserved sequence.

The Saccharomyces cerevisiae cdc9 gene has been cloned in the vector YRp12 by complementation of the temperature-sensitive lesion in vivo. The gene is contained within a 3300-base-pair fragment of DNA, which maps to the chromosomal locus of cdc9 and which is able to complement a DNA-ligase-deficient mutant of the fission yeast Schizosaccharomyces pombe.