The alternative Medicago truncatula defense proteome of ROS-defective transgenic roots during early microbial infection.

ROP-type GTPases of plants function as molecular switches within elementary signal transduction pathways such as the regulation of ROS synthesis via activation of NADPH oxidases (RBOH-respiratory burst oxidase homolog in plants). Previously, we reported that silencing of the Medicago truncatula GTPase MtROP9 led to reduced ROS production and suppressed induction of ROS-related enzymes in transgenic roots (MtROP9i) infected with pathogenic (Aphanomyces euteiches) and symbiotic microorganisms (Glomus intraradices, Sinorhizobium meliloti). While fungal infections were enhanced, S. meliloti infection was drastically impaired. In this study, we investigate the temporal proteome response of M. truncatula MtROP9i transgenic roots during the same microbial interactions under conditions of deprived potential to synthesize ROS. In comparison with control roots (Mtvector), we present a comprehensive proteomic analysis using sensitive MS protein identification. For four early infection time-points (1, 3, 5, 24 hpi), 733 spots were found to be different in abundance: 213 spots comprising 984 proteins (607 unique) were identified after S. meliloti infection, 230 spots comprising 796 proteins (580 unique) after G. intraradices infection, and 290 spots comprising 1240 proteins (828 unique) after A. euteiches infection. Data evaluation by GelMap in combination with a heatmap tool allowed recognition of key proteome changes during microbial interactions under conditions of hampered ROS synthesis. Overall, the number of induced proteins in MtROP9i was low as compared with controls, indicating a dual function of ROS in defense signaling as well as alternative response patterns activated during microbial infection. Qualitative analysis of induced proteins showed that enzymes linked to ROS production and scavenging were highly induced in control roots, while in MtROP9i the majority of proteins were involved in alternative defense pathways such as cell wall and protein degradation.

The Mitochondrial Complexome of Medicago truncatula.

Legumes (Fabaceae, Leguminosae) are unique in their ability to carry out an elaborate endosymbiotic nitrogen fixation process with rhizobia proteobacteria. The symbiotic nitrogen fixation enables the host plants to grow almost independently of any other nitrogen source. Establishment of symbiosis requires adaptations of the host cellular metabolism, here foremost of the energy metabolism mainly taking place in mitochondria. Since the early 1990s, the galegoid legume Medicago truncatula Gaertn. is a well-established model for studying legume biology, but little is known about the protein complement of mitochondria from this species. An initial characterization of the mitochondrial proteome of M. truncatula (Jemalong A17) was published recently. In the frame of this study, mitochondrial protein complexes were characterized using Two-dimensional (2D) Blue native (BN)/SDS-PAGE. From 139 detected spots, the "first hit" (=most abundant) proteins of 59 spots were identified by mass spectrometry. Here, we present a comprehensive analysis of the mitochondrial "complexome" (the "protein complex proteome") of M. truncatula via 2D BN/SDS-PAGE in combination with highly sensitive MS protein identification. In total, 1,485 proteins were identified within 158 gel spots, representing 467 unique proteins. Data evaluation by the novel GelMap annotation tool allowed recognition of protein complexes of low abundance. Overall, at least 36 mitochondrial protein complexes were found. To our knowledge several of these complexes were described for the first time in Medicago. The data set is accessible under http://www.gelmap.de/medicago/. The mitochondrial protein complex proteomes of Arabidopsis available at http://www.gelmap.de/arabidopsis/ and Medicago are compared.

Proteomic and histological analyses of endosperm development in Cyclamen persicum as a basis for optimization of somatic embryogenesis.

The endosperm plays an important role for the development of zygotic embryos, while somatic embryos lack a seed coat and endosperm and often show physiological disorders. This study aims at elucidating the cellular and physiological processes within the endosperm of the ornamental species Cyclamen persicum Mill. Histological analyses were performed from 0 to 11 weeks after pollination (WAP). At 3WAP, a syncytium was clearly visible with a globular zygotic embryo. From 4WAP, cellularization of the endosperm, at 5WAP a small torpedo shaped embryo, and from 7WAP cell expansion was observed. By 11WAP the endosperm appeared fully differentiated. Total soluble proteins were extracted from the endosperm at 4, 5, 7, 9 and 11WAP and resolved using two dimensional isoelectric focussing/sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2D IEF/SDS-PAGE). A shift from high-molecular-mass proteins to low-molecular-mass proteins during endosperm development was observed. A total of 1137proteinspots/gel were detected in the three protein fractions extracted at 7, 9 and 11WAP. Mass spectrometry analysis of the 48 predominant protein spots in endosperm at 7, 9 and 11WAP resulted in the identification of 62 proteins, ten of which were described for the first time in Cyclamen. Additionally, 186 proteins were identified using the C. persicum embryo proteome reference map. Proteins involved in abscisic acid signalling and oxidative stress responsive proteins were found to be important for seed development in Cyclamen. The new insights into endosperm physiology including storage compounds are discussed.

Silencing of the Rac1 GTPase MtROP9 in Medicago truncatula stimulates early mycorrhizal and oomycete root colonizations but negatively affects rhizobial infection.

RAC/ROP proteins (ρ-related GTPases of plants) are plant-specific small G proteins that function as molecular switches within elementary signal transduction pathways, including the regulation of reactive oxygen species (ROS) generation during early microbial infection via the activation of NADPH oxidase homologs of plants termed RBOH (for respiratory burst oxidase homolog). We investigated the role of Medicago truncatula Jemalong A17 small GTPase MtROP9, orthologous to Medicago sativa Rac1, via an RNA interference silencing approach. Composite M. truncatula plants (MtROP9i) whose roots have been transformed by Agrobacterium rhizogenes carrying the RNA interference vector were generated and infected with the symbiotic arbuscular mycorrhiza fungus Glomus intraradices and the rhizobial bacterium Sinorhizobium meliloti as well as with the pathogenic oomycete Aphanomyces euteiches. MtROP9i transgenic lines showed a clear growth-reduced phenotype and revealed neither ROS generation nor MtROP9 and MtRBOH gene expression after microbial infection. Coincidently, antioxidative compounds were not induced in infected MtROP9i roots, as documented by differential proteomics (two-dimensional differential gel electrophoresis). Furthermore, MtROP9 knockdown clearly promoted mycorrhizal and A. euteiches early hyphal root colonization, while rhizobial infection was clearly impaired. Infected MtROP9i roots showed, in part, extremely swollen noninfected root hairs and reduced numbers of deformed nodules. S. meliloti nodulation factor treatments of MtROP9i led to deformed root hairs showing progressed swelling of its upper regions or even of the entire root hair and spontaneous constrictions but reduced branching effects occurring only at swollen root hairs. These results suggest a key role of Rac1 GTPase MtROP9 in ROS-mediated early infection signaling.

DIGE analysis of plant tissue proteomes using a phenolic protein extraction method.

Two-dimensional difference gel electrophoresis is an invaluable technique for the analysis of plant proteomes. However, preparation of protein fractions from plant tissues is challenging due to the special features of plant cells: a robust cell wall, large vacuoles which often contain high concentrations of organic acids and a broad range of secondary metabolites like phenolic compounds and pigments. Therefore, protein preparation for difference gel electrophoresis (DIGE) analyses has to be adapted. Here, we describe both a phenolic protein extraction method for plant tissues and an adapted protocol for DIGE labeling of the generated fractions.

The mitochondrial proteome of the model legume Medicago truncatula.

Legumes carry out special biochemical functions, e.g. the fixation of molecular nitrogen based on a symbiosis with proteobacteria. At the cellular level, this symbiosis has to be implemented into the energy metabolism of the host cell. To provide a basis for future analyses, we have characterized the protein complement of mitochondria of the model legume Medicago truncatula using two-dimensional isoelectric focussing (IEF) and blue-native (BN)-SDS-PAGE. While the IEF reference map resulted mainly in resolution of those proteins associated with the mitochondrial matrix, the BN proteomic map allowed separation of protein subunits from the respiratory chain protein complexes, which are located in the organelle's inner membrane. The M. truncatula mitochondrial BN reference map revealed some striking similarities to the one from Arabidopsis thaliana but at the same time exhibited also some special features: complex II is of increased abundance and additionally represented by a low molecular mass form not reported for Arabidopsis. Furthermore three highly abundant forms of prohibitin complexes are present in the mitochondrial proteome of M. truncatula. Special features with respect to mitochondrial protein complexes might reflect adaptations of legumes to elevated cellular energy requirements enabling them to develop symbiotic interactions with rhizobial bacteria.

Medicago truncatula proteomics.

Legumes (Fabaceae) are unique in their ability to enter into an elaborate symbiosis with nitrogen-fixing rhizobial bacteria. Rhizobia-legume (RL) symbiosis represents one of the most productive nitrogen-fixing systems and effectively renders the host plants to be more or less independent of other nitrogen sources. Due to high protein content, legumes are among the most economically important crop families. Beyond that, legumes consist of over 16,000 species assigned to 650 genera. In most cases, the genomes of legumes are large and polyploid, which originally did not predestine these plants as genetic model systems. It was not until the early 1990 th that Medicago truncatula was selected as the model plant for studying Fabaceae biology. M. truncatula is closely related to many economically important legumes and therefore its investigation is of high relevance for agriculture. Recently, quite a number of studies were published focussing on in depth characterizations of the M. truncatula proteome. The present review aims to summarize these studies, especially those which focus on the root system and its dynamic changes induced upon symbiotic or pathogenic interactions with microbes.

Differential gel electrophoresis (DIGE) to quantitatively monitor early symbiosis- and pathogenesis-induced changes of the Medicago truncatula root proteome.

Symbiosis- and pathogenesis-related early protein induction patterns in the model legume Medicago truncatula were analysed with two-dimensional differential gel electrophoresis. Two symbiotic soil microorganisms (Glomus intraradices, Sinorhizobium meliloti) were used in single infections and in combination with a secondary pathogenic infection by the oomycete Aphanomyces euteiches. Proteomic analyses performed 6 and 24h after inoculations led to identification of 87 differentially induced proteins which likely represent the M. truncatula root 'interactome'. A set of proteins involved in a primary antioxidant defense reaction was detected during all associations investigated. Symbiosis-related protein induction includes a typical factor of early symbiosis-specific signalling (CaM-2), two Ran-binding proteins of nucleocytoplasmic signalling, and a set of energy-related enzymes together with proteins involved in symbiosis-initiated C- and N-fixation. Pathogen-associated protein induction consists of mainly PR proteins, Kunitz-type proteinase inhibitors, a lectin, and proteins related to primary carbohydrate metabolism and phytoalexin synthesis. Absence of PR proteins and decreased pathogen-induced protein patterns during mixed symbiotic and pathogenic infections indicate bioprotective effects due to symbiotic co-infection. Several 14-3-3 proteins were found as predominant proteins during mixed infections. With respect to hormone-regulation, A. euteiches infection led to induction of ABA-related pathways, while auxin-related pathways are induced during symbiosis.

Induction of distinct defense-associated protein patterns in Aphanomyces euteiches (Oomycota)-elicited and -inoculated Medicago truncatula cell-suspension cultures: a proteome and phosphoproteome approach.

A comprehensive proteomic approach was applied to investigate molecular events occurring upon inoculation of Medicago truncatula cell-suspension cultures with the oomycete root pathogen Aphanomyces euteiches. Establishment of an inoculation assay in the cell cultures allowed a direct comparison between proteins induced by elicitation with a crude culture extract of the oomycete and by inoculation with A. euteiches zoospores representing the natural infection carrier. Oxidative burst assays revealed responsiveness of the cell cultures for perception of elicitation and inoculation signals. The plant "elicitation proteome" resembles the "inoculation proteome" in early incubation stages and includes proteins induced following initial oxidative burst and defense reactions, but also proteins involved in the antioxidative system. However, approximately 2 days after incubation, the inoculation proteome differs drastically from the proteome of elicited cultures, where a cessation of responses assignable to A. euteiches elicitation occurred. The specific protein induction patterns of zoospore-inoculated cells appeared consistent with the protein induction identified in recent studies for an A. euteiches infection in planta and consist of three functional groups: i) pathogenesis-related proteins, ii) proteins associated with secondary phenylpropanoid or phytoalexin metabolism, and, particularly, iii) proteins assigned to carbohydrate metabolism and energy-related cellular processes. Phosphoproteomic analyses revealed consistent and specific activation of these defense-related pathways already at very early timepoints of inoculation, providing evidence that the identified protein profiles are representative for an established A. euteiches infection of M. truncatula.

Silencing of PR-10-like proteins in Medicago truncatula results in an antagonistic induction of other PR proteins and in an increased tolerance upon infection with the oomycete Aphanomyces euteiches.

Recent studies on the root proteome of Medicago truncatula (Gaertn.) showed an induction of pathogenesis-related (PR) proteins of the class 10 after infection with the oomycete pathogen Aphanomyces euteiches (Drechs.). To get insights into the function of these proteins during the parasitic root-microbe association, a gene knockdown approach using RNAi was carried out. Agrobacterium rhizogenes-mediated transformation of M. truncatula roots led to a knockdown of the Medicago PR10-1 gene in transgenic in vitro root cultures. Proteomic analyses of the MtPr10-1i root cultures showed that MtPr10-1 was efficiently knocked down in two MtPr10-1i lines. Moreover, five additional PR-10-type proteins annotated as abscisic acid responsive proteins (ABR17s) revealed also an almost complete silencing in these two lines. Inoculation of the root cultures with the oomycete root pathogen A. euteiches resulted in a clearly reduced colonization and thus in a suppressed infection development in MtPr10-1i roots as compared to that in roots of the transformation controls. In addition, MtPr10-1 silencing led to the induction of a new set of PR proteins after infection with A. euteiches including the de novo induction of two isoforms of thaumatin-like proteins (PR-5b), which were not detectable in A. euteiches-infected control roots. Thus, antagonistic induction of other PR proteins, which are normally repressed due to PR-10 expression, is supposed to cause an increased resistance of M. truncatula upon an A. euteiches in vitro infection. The results were also further confirmed by detecting increased PR-5b induction levels in 2-D gels of a previously analyzed M. truncatula line (F83.005-9) exhibiting increased A. euteiches tolerance associated with reduced PR-10 induction levels.

Proteomic profiling unravels insights into the molecular background underlying increased Aphanomyces euteiches-tolerance of Medicago truncatula.

To investigate the molecular mechanisms underlying susceptibility of legumes to the root pathogen Aphanomyces euteiches (oomycota), comparative proteomic studies have been carried out. In a first approach, we have analysed two Medicago truncatula lines of the French CORE collection (F83.005-5 (R2002) and F83.005-9 (R2002)), which showed either increased or decreased susceptibility to A. euteiches as compared to the widely adopted line A17. Several proteins were identified to be differentially induced after pathogen challenge in the two M. truncatula accessions with altered disease susceptibility, whereof proteins with increased abundances in the more resistant line F83.005-9 could be involved in mechanisms that lead to an improved disease resistance. Among these proteins, we identified two proteasome alpha subunits, which might be involved in defense response. To broaden our studies on A. euteiches-tolerance of M. truncatula, we investigated two other phenomena that lead to an either increased A. euteiches-resistance or to an enhanced susceptibility. The topic of an enhanced plant resistance to A. euteiches was studied in plants showing a bioprotective effect of a pre-established arbuscular mycorrhiza (AM) symbiosis. Evaluation of root fresh weights and pathogen spreading in the root system clearly indicate that mycorrhizal plants show increased A. euteiches-resistance as compared to non-mycorrhizal plants. Proteome analyses revealed the induction of similar protein patterns as in the M. truncatula accessions with comparatively high resistance level to A. euteiches. In a third approach, increased A. euteiches susceptibility was effected by exogenous abscisic acid (ABA) application prior to root infection. Evaluation of the abundance levels of a group of pathogenesis related class 10 (PR10)-like proteins, which were previously identified to be regulated after A. euteiches infection, revealed a correlation between the abundance levels of these proteins and the A. euteiches infection level or severity.

Proteomic approach: identification of Medicago truncatula proteins induced in roots after infection with the pathogenic oomycete Aphanomyces euteiches.

The legume root rot disease caused by the oomycete pathogen Aphanomyces euteiches is one major yield reducing factor in legume crop production. A comparative proteomic approach was carried out in order to identify proteins of the model legume Medicago truncatula which are regulated after an infection with A. euteiches . Several proteins were identified by two dimensional gel electrophoresis to be differentially expressed after pathogen challenge. Densitometric evaluation of expression values showed different regulation during the time-course analysed. Proteins regulated during the infection were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Among the differentially expressed proteins, two encoded putative cell wall proteins and two were designated as small heat shock proteins. Furthermore, an isoform of the chalcone-O-methyltransferase was found to be increased in infected roots. The majority of induced proteins belonged to the family of class 10 of pathogenesis related proteins (PR10). Previously, various PR10-like proteins have been shown to be regulated by general stress or abscisic acid (ABA). Therefore, these proteins were further investigated concerning their regulation in response to drought stress and exogenous ABA-application. Complex regulation patterns were identified: three of the A. euteiches -induced PR10-like proteins were also induced by exogenous ABA- but none of them is induced after drought stress. In contrast, three of these proteins are down-regulated by drought stress. Hence, the strong expression of different PR10-family members and their regulation profiles indicates that this set of proteins plays a major role during root adaptations to various stress conditions.