SUMMARYMost terrestrial plants engage into arbuscular mycorrhizal (AM) symbiosis with fungi of the phylum Glomeromycota. The initial recognition of the fungal symbiont results in the activation of a symbiosis signalling pathway that is shared with the root nodule symbiosis (common SYM pathway). The subsequent intracellular accommodation of the fungus, and the elaboration of its characteristic feeding structures, the arbuscules, depends on a genetic programme in the plant that has recently been shown to involve the VAPYRIN gene in Medicaco truncatula. We have previously identified a mutant in Petunia hybrida, penetration and arbuscule morphogenesis 1 (pam1), that is defective in the intracellular stages of AM development. Here, we report on the cloning of PAM1, which encodes a VAPYRIN homologue. PAM1 protein localizes to the cytosol and the nucleus, with a prominent affinity to mobile spherical structures that are associated with the tonoplast, and are therefore referred to as tonospheres. In mycorrhizal roots, tonospheres were observed in the vicinity of intracellular hyphae, where they may play an essential role in the accommodation and morphogenesis of the fungal endosymbiont.

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Nodule formation induced by nitrogen-fixing rhizobia depends on bacterial nodulation factors (NFs), modified chitin oligosaccharides with a fatty acid moiety. Certain NFs can be cleaved and inactivated by plant chitinases. However, the most abundant NF of Sinorhizobium meliloti, an O-acetylated and sulfated tetramer, is resistant to hydrolysis by all plant chitinases tested so far. Nevertheless, this NF is rapidly degraded in the host rhizosphere. Here, we identify and characterize MtNFH1 (for Medicago truncatula Nod factor hydrolase 1), a legume enzyme structurally related to defense-related class V chitinases (glycoside hydrolase family 18). MtNFH1 lacks chitinase activity but efficiently hydrolyzes all tested NFs of S. meliloti. The enzyme shows a high cleavage preference, releasing exclusively lipodisaccharides from NFs. Substrate specificity and kinetic properties of MtNFH1 were compared with those of class V chitinases from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), which cannot hydrolyze tetrameric NFs of S. meliloti. The Michaelis-Menten constants of MtNFH1 for NFs are in the micromolar concentration range, whereas nonmodified chitin oligosaccharides represent neither substrates nor inhibitors for MtNFH1. The three-dimensional structure of MtNFH1 was modeled on the basis of the known structure of class V chitinases. Docking simulation of NFs to MtNFH1 predicted a distinct binding cleft for the fatty acid moiety, which is absent in the class V chitinases. Point mutation analysis confirmed the modeled NF-MtNFH1 interaction. Silencing of MtNFH1 by RNA interference resulted in reduced NF degradation in the rhizosphere of M. truncatula. In conclusion, we have found a novel legume hydrolase that specifically inactivates NFs.

The Medicago truncatula (Gaertn.) ecotypes Jemalong A17 and R108-1 differ in Sinorhizobium meliloti-induced chitinase gene expression. The pathogen-inducible class IV chitinase gene, Mtchit 4, was strongly induced during nodule formation of the ecotype Jemalong A17 with the S. meliloti wild-type strain 1021. In the ecotype R108-1, the S. meliloti wild types Sm1021 and Sm41 did not induce Mtchit 4 expression. On the other hand, expression of the putative class V chitinase gene, Mtchit 5, was found in roots of M. truncatula cv. R108-1 nodulated with either of the rhizobial strains. Mtchit 5 expression was specific for interactions with rhizobia. It was not induced in response to fungal pathogen attack, and not induced in roots colonized with arbuscular mycorrhizal (AM) fungi. Elevated Mtchit 5 gene expression was first detectable in roots forming nodule primordia. In contrast to Mtchit 4, expression of Mtchit 5 was stimulated by purified Nod factors. Conversely, Mtchit 4 expression was strongly elevated in nodules formed with the K-antigen-deficient mutant PP699. Expression levels of Mtchit 5 were similarly increased in nodules formed with PP699 and its parental wild-type strain Sm41. Phylogenetic analysis of the deduced amino acid sequences of Mtchit 5 (calculated molecular weight = 41,810 Da, isoelectric point pH 7.7) and Mtchit 4 (calculated molecular weight 30,527 Da, isoelectric point pH 4.9) revealed that the putative Mtchit 5 chitinase forms a separate clade within class V chitinases of plants, whereas the Mtchit 4 chitinase clusters with pathogen-induced class IV chitinases from other plants. These findings demonstrate that: (i) Rhizobium-induced chitinase gene expression in M. truncatula occurs in a plant ecotype-specific manner, (ii) Mtchit 5 is a putative chitinase gene that is specifically induced by rhizobia, and (iii) rhizobia-specific and defence-related chitinase genes are differentially influenced by rhizobial Nod factors and K antigens.Keywords Class IV and V chitinase AE K antigen AE Medicago truncatula ecotypes R108-1 and Jemalong A17 AE Nod factors AE Sinorhizobium AE Suppression of plant defence

Arbuscular mycorrhiza (AM) is a symbiosis between plants and AM fungi that requires the intracellular accommodation of the fungal partner in the host. For reciprocal nutrient exchange, AM fungi form intracellular arbuscules that are surrounded by the peri-arbuscular membrane. This membrane, together with the fungal plasma membrane, and the space in between, constitute the symbiotic interface, over which nutrients are exchanged. Intracellular establishment of AM fungi requires the VAPYRIN protein which is induced in colonized cells, and which localizes to numerous small mobile structures of unknown identity (Vapyrin-bodies). In order to characterize the identity and function of the Vapyrin-bodies we pursued a dual strategy. First, we co-expressed fluorescently tagged VAPYRIN with a range of subcellular marker proteins, and secondly, we employed biochemical tools to identify interacting partner proteins of VAPYRIN. As an important tool for the quantitative analysis of confocal microscopic data sets from co-expression of fluorescent proteins, we developed a semi-automated image analysis pipeline that allows for precise spatio-temporal quantification of protein co-localization and of the dynamics of organelle association from movies. Taken together, these experiments revealed that Vapyrin-bodies have an endosomal identity with trans-Golgi features, and that VAPYRIN interacts with a symbiotic R-SNARE of the VAMP721 family, that localizes to the same compartment.

Different arbuscular mycorrhizal fungi (AMF) alter growth and nutrition of a given plant differently. Plant gene expression patterns in response to fungal colonization show a certain overlap when colonized by fungi of the Glomeraceae. However, little is known of plant responses to fungi of different fungal taxa, e.g. the Gigasporaceae. We therefore compared the impact of colonization by three taxonomically different AMF species (Glomus intraradices, Glomus mosseae and Scutellospora castanea) on Medicago truncatula at the physiological and transcriptional level using quantitative-PCR. Each AMF developed a species-typical colonization pattern, with a colonization degree of 60% for G. intraradices and 30% for G. mosseae. Both species developed appressoria, intraradical hyphae, arbuscules and vesicles. S. castanea showed a colonization degree of 10% and developed appressoria, intraradical hyphae, arbuscules and arbusculate coils. All AMF enhanced the plant biomass accumulation and nutritional status although not in correlation with the colonization degree. The expression of 10 mycorrhiza-specific or mycorrhiza-associated plant genes could be separated into two clusters. The first cluster, containing arbuscule-induced genes, was highly induced in interactions with G. intraradices and G. mosseae but also slightly induced by S. castanea. The second cluster of genes contained genes that were induced primarily by S. castanea. In conclusion, genes that respond to colonization by fungi of the genus Glomus also respond to Scutellospora. However, there is also a group of genes that is significantly induced only by Scutellospora and not by Glomus species in this study. Our data indicate that genes may be differentially regulated in response to the different AM fungi.

Plant VAPYRINS are required for the establishment of arbuscular mycorrhiza (AM) and root nodule symbiosis (RNS). In vapyrin mutants, the intracellular accommodation of AM fungi and rhizobia is blocked, and in the case of AM, the fungal endosymbiont cannot develop arbuscules which serve for nutrient exchange. VAPYRINs are plant-specific proteins that consists of a major sperm protein (MSP) domain and an ankyrin domain. Comparison of VAPYRINS of dicots, monocots, and the moss Physcomitrella patens reveals a highly conserved domain structure. We focused our attention on the ankyrin domain, which closely resembles the D34 domain of human ankyrin R. Conserved residues within the petunia VAPYRIN cluster to a surface patch on the concave side of the crescent-shaped ankyrin domain, suggesting that this region may represent a conserved binding site involved in the formation of a protein complex with an essential function in intracellular accommodation of microbial endosymbionts.

Summary• Expression of Mtchit 3-3 , a class III chitinase gene, is specifically induced by arbuscular mycorrhizal (AM) fungi in roots of the model legume Medicago truncatula and its transcripts accumulate in cells containing arbuscules.• Agrobacterium rhizogenes -transformed roots and root-organ cultures of M. truncatula were used to study effects of Mtchit 3-3 on AM fungi.• This work provides evidence for enzymatic activity of the Mtchit 3-3 gene product and shows with promoter: gus fusions that a 2 kb fragment located 5 ′ upstream from the translational start codon of Mtchit 3-3 is sufficient to confer arbuscule-dependent gene expression. By fusing the Mtchit 3-3 coding region to the CaMV 35S promoter the expression pattern was disrupted. Surprisingly, disruption stimulated spore germination of Glomus intraradices and Glomus constrictum , and in the case of G. intraradices resulted in a higher probability of root colonization and spore formation. However, no effect on the abundance of arbuscules within colonized roots became apparent.• These observations demonstrate that disruption of the tight arbuscule-dependent expression pattern of Mtchit 3-3 has effects on the early interaction between roots and AM fungi.