Identification of new potential regulators of the Medicago truncatula-Sinorhizobium meliloti symbiosis using a large-scale suppression subtractive hybridization approach.

We set up a large-scale suppression subtractive hybridization (SSH) approach to identify Medicago truncatula genes differentially expressed at different stages of the symbiotic interaction with Sinorhizobium meliloti, with a particular interest for regulatory genes. We constructed 7 SSH libraries covering successive stages from Nod factor signal transduction to S. meliloti infection, nodule organogenesis, and functioning. Over 26,000 clones were differentially screened by two rounds of macroarray hybridizations. In all, 3,340 clones, corresponding to genes whose expression was potentially affected, were selected, sequenced, and ordered into 2,107 tentative gene clusters, including 767 MtS clusters corresponding to new M. truncatula genes. In total, 52 genes encoding potential regulatory proteins, including transcription factors (TFs) and other elements of signal transduction cascades, were identified. The expression pattern of some of them was analyzed by quantitative reverse-transcription polymerase chain reaction in wild-type and in Nod- M. truncatula mutants blocked before or after S. meliloti infection. Three genes, coding for TFs of the bHLH and WRKY families and a C2H2 zinc-finger protein, respectively, were found to be upregulated, following S. meliloti inoculation, in the infection-defective mutant lin, whereas the bHLH gene also was expressed in the root-hair-curling mutant hcl. The potential role of these genes in early symbiotic steps is discussed.

Godiard L ，Niebel A ，Micheli F ，Gouzy J ，Ott T ，Gamas P ... -  《MOLECULAR PLANT-MICROBE INTERACTIONS》

The Medicago truncatula N5 gene encoding a root-specific lipid transfer protein is required for the symbiotic interaction with Sinorhizobium meliloti.

The Medicago truncatula N5 gene is induced in roots after Sinorhizobium meliloti infection and it codes for a putative lipid transfer protein (LTP), a family of plant small proteins capable of binding and transferring lipids between membranes in vitro. Various biological roles for plant LTP in vivo have been proposed, including defense against pathogens and modulation of plant development. The aim of this study was to shed light on the role of MtN5 in the symbiotic interaction between M. truncatula and S. meliloti. MtN5 cDNA was cloned and the mature MtN5 protein expressed in Escherichia coli. The lipid binding capacity and antimicrobial activity of the recombinant MtN5 protein were tested in vitro. MtN5 showed the capacity to bind lysophospholipids and to inhibit M. truncatula pathogens and symbiont growth in vitro. Furthermore, MtN5 was upregulated in roots after infection with either the fungal pathogen Fusarium semitectum or the symbiont S. meliloti. Upon S. meliloti infection, MtN5 was induced starting from 1 day after inoculation (dpi). It reached the highest concentration at 3 dpi and it was localized in the mature nodules. MtN5-silenced roots were impaired in nodulation, showing a 50% of reduction in the number of nodules compared with control roots. On the other hand, transgenic roots overexpressing MtN5 developed threefold more nodules with respect to control roots. Here, we demonstrate that MtN5 possesses biochemical features typical of LTP and that it is required for the successful symbiotic association between M. truncatula and S. meliloti.

Pii Y ，Astegno A ，Peroni E ，Zaccardelli M ，Pandolfini T ，Crimi M ... -  《MOLECULAR PLANT-MICROBE INTERACTIONS》

Expression of Medicago truncatula genes responsive to nitric oxide in pathogenic and symbiotic conditions.

Nitric oxide (NO) is involved in diverse physiological processes in plants, including growth, development, response to pathogens, and interactions with beneficial microorganisms. In this work, a dedicated microarray representing the widest database available of NO-related transcripts in plants has been produced with 999 genes identified by a cDNA amplified fragment length polymorphism analysis as modulated in Medicago truncatula roots treated with two NO donors. The microarray then was used to monitor the expression of NO-responsive genes in M. truncatula during the incompatible interaction with the foliar pathogen Colletotrichum trifolii race 1 and during the symbiotic interaction with Sinorhizobium meliloti 1,021. A wide modulation of NO-related genes has been detected during the hypersensitive reaction or during nodule formation and is discussed with special emphasis on the physiological relevance of these genes in the context of the two biotic interactions. This work clearly shows that NO-responsive genes behave differently depending on the plant organ and on the type of interaction, strengthening the need to consider regulatory networks, including different signaling molecules.

Ferrarini A ，De Stefano M ，Baudouin E ，Pucciariello C ，Polverari A ，Puppo A ，Delledonne M ... -  《MOLECULAR PLANT-MICROBE INTERACTIONS》

Characterization and expression analysis of Medicago truncatula ROP GTPase family during the early stage of symbiosis.

ROPs (Rho-related GTPases of plants) are small GTPases that are plant-specific signaling proteins. They act as molecular switches in a variety of developmental processes. In this study, seven cDNA clones coding for ROP GTPases have been isolated in Medicago truncatula, and conserved and divergent domains are identified in these predicted MtROP proteins. Phylogenetic analysis has indicated that MtROPs are distributed into groups II, III, IV but group I. MtROP genes are expressed in various tissues at different levels. A quantitative reverse transcription PCR analysis indicated that these MtROP genes have different expression profiles in the roots in response to infection with rhizobia. The expression of MtROP3, MtROP5 and MtROP6 are increased, as the expression of Nod factor or rhizobial-induced marker genes--NFP, Rip1 and Enod11; MtROP10 has showed enhanced expression at a certain post-inoculation time point. No significant changes in MtROP7 and MtROP9 expression have been detected and MtROP8 expression is dramatically decreased by about 80%-90%. Additionally, ROP promoter-GUS analysis has showed that MtROP3, MtROP5 and MtROP6 have elevated expression in transgenic root hairs after rhizobial inoculation. These results might suggest a role for some ROP GTPases in the regulation of early stages during rhizobial infection in symbiosis.

Liu W ，Chen AM ，Luo L ，Sun J ，Cao LP ，Yu GQ ，Zhu JB ，Wang YZ ... -  《-》

Insights into symbiotic nitrogen fixation in Medicago truncatula.

In silico analysis of the Medicago truncatula gene index release 8.0 at The Institute for Genomic Research identified approximately 530 tentative consensus sequences (TC) clustered from 2,700 expressed sequence tags (EST) derived solely from Sinorhizobium meliloti-inoculated root and nodule tissues. A great majority (76%) of these TC were derived exclusively from nitrogen-fixing and senescent nodules. A cDNA filter array was constructed using approximately 58% of the in silico-identified TC as well as cDNAs representing selected carbon and nitrogen metabolic pathways. The purpose of the array was to analyze transcript abundance in M. truncatula roots and nodules following inoculation by a wild-type S. meliloti strain, a mutant strain that forms ineffective nodules, an uninoculated root control, and roots following nitrate or ammonium treatments. In all, 81 cDNAs were upregulated in both effective and ineffective nodules, and 78% of these cDNAs represent in silico-identified TC. One group of in silico-identified TC encodes genes with similarity to putative plant disease resistance (R) genes of the nucleotide binding site-leucine-rich repeat type. Expression of R genes was enhanced in effective nodules, and transcripts also were detected in ineffective nodules at 14 days postinoculation (dpi). Homologous R gene sequences also have been identified in the Medicago genome. However, their functional importance in nodules remains to be established. Genes for enzymes involved in organic acid synthesis along with genes involved in nitrogen metabolism were shown to be coexpressed in nitrate-fed roots and effective nodules of M. truncatula.

Tesfaye M ，Samac DA ，Vance CP 《MOLECULAR PLANT-MICROBE INTERACTIONS》