8
Chapter 26 Medicago truncatula Root Proteomics Frank Colditz Department III Plant Molecular Biology, Institute for Plant Genetics, Leibniz University of Hannover, Germany 26.1 INTRODUCTION The term Proteomics comprises all methods that are suitable for the systematic characterization of defined pro- tein fractions (Colditz and Braun, 2010). In the classical case, gel electrophoretic protein separation is combined with mass spectrometric (MS) protein identification. Nowadays, gel-free proteomic procedures, which are based on MS approaches only, are prevalently applied. As MS-based protein identification highly depends on the availability of genome sequence data, successful application of proteomics comes along with the expand- ing genomic information disposable. Thus, proteomic analyses of a certain tissue can be only as good as the accessible genomic background for the chosen organism the sample was derived from. As in many cases only little genomic information is available, researchers are well advised to refer to certain model organisms where this information is available. For legume species, which gen- erally exhibit large and often polyploid genomes difficult to capture, the two model plants Medicago truncatula and Lotus japonicus have been chosen (Cook, 1999; Udvardi et al., 2005; Young and Udvardi, 2008). For both species, a variety of genomic tools have been established during the last two decades, which qualify them as excellent sources for genomic and proteomic legume research. The galegoid legume M. truncatula exhibits a diploid genome structure and a comparatively small genome size. Very recently, the draft sequence of the M. trun- catula euchromatin was published and optical mapping allowed the assembly of 375 million base pairs (Mbp) capturing 94% of all M. truncatula genes, whereof 246 Mbp represents nonredundant sequence (Young Molecular Microbial Ecology of the Rhizosphere, Volume 1, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc. et al., 2011). Interestingly, whole-genome duplication approximately 58 million years ago and subsequent fundamental rearrangements of the M. truncatula genome are considered as the major evolutionary steps for endosymbiotic nitrogen fixation (Young et al., 2011). M. truncatula is a close relative of Medicago sativa (alfalfa), a widely cultivated crop of agronomic importance but with complex autotetraploid genetics (Young et al., 2011). By contrast, M. truncatula is self-fertile and offers further properties of a model plant such as a short regen- eration time and established transformation protocols (Colditz and Braun, 2010). Moreover, 269,238 ESTs have been deposited in Genbank (December 1, 2011; http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary .html). An Affymetrix GeneChip covers more than 50,000 M. truncatula gene probes, which allowed the establishment of a gene expression atlas (Benedito et al., 2008). For reverse genetics, large-scaled insertional mutagenesis Tnt1 populations (Tadege et al., 2008, 2009) and RNA interference (RNAi)-mediated gene silencing (Limpens et al., 2004) are well established. Investigations on plant–microbe interactions in the rhizosphere are in the center of legume biology. For those studies, legumes are the organisms of choice because of their ability to interact with a broad array of symbiotic as well as pathogenic microbes (Colditz and Braun, 2010). Figure 26.1 shows exemplary microbial interaction partners of M. truncatula. In particular, the ability to carry out endosymbiotic nitrogen fixation with Rhizobia bacteria within the characteristic root nodules (see Chapters 44, 45) represent a major task for investigations of microbial associations to legumes. This prominent Rhizobia–legume (RL) symbiosis is limited 271

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Chapter 26

Medicago truncatula Root Proteomics

Frank ColditzDepartment III Plant Molecular Biology, Institute for Plant Genetics, LeibnizUniversity of Hannover, Germany

26.1 INTRODUCTION

The term Proteomics comprises all methods that aresuitable for the systematic characterization of defined pro-tein fractions (Colditz and Braun, 2010). In the classicalcase, gel electrophoretic protein separation is combinedwith mass spectrometric (MS) protein identification.Nowadays, gel-free proteomic procedures, which arebased on MS approaches only, are prevalently applied.As MS-based protein identification highly depends onthe availability of genome sequence data, successfulapplication of proteomics comes along with the expand-ing genomic information disposable. Thus, proteomicanalyses of a certain tissue can be only as good as theaccessible genomic background for the chosen organismthe sample was derived from. As in many cases only littlegenomic information is available, researchers are welladvised to refer to certain model organisms where thisinformation is available. For legume species, which gen-erally exhibit large and often polyploid genomes difficultto capture, the two model plants Medicago truncatula andLotus japonicus have been chosen (Cook, 1999; Udvardiet al., 2005; Young and Udvardi, 2008). For both species,a variety of genomic tools have been established duringthe last two decades, which qualify them as excellentsources for genomic and proteomic legume research.

The galegoid legume M. truncatula exhibits a diploidgenome structure and a comparatively small genomesize. Very recently, the draft sequence of the M. trun-catula euchromatin was published and optical mappingallowed the assembly of 375 million base pairs (Mbp)capturing ∼94% of all M. truncatula genes, whereof246 Mbp represents nonredundant sequence (Young

Molecular Microbial Ecology of the Rhizosphere, Volume 1, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

et al., 2011). Interestingly, whole-genome duplicationapproximately 58 million years ago and subsequentfundamental rearrangements of the M. truncatula genomeare considered as the major evolutionary steps forendosymbiotic nitrogen fixation (Young et al., 2011). M.truncatula is a close relative of Medicago sativa (alfalfa),a widely cultivated crop of agronomic importance butwith complex autotetraploid genetics (Young et al.,2011). By contrast, M. truncatula is self-fertile and offersfurther properties of a model plant such as a short regen-eration time and established transformation protocols(Colditz and Braun, 2010). Moreover, 269,238 ESTshave been deposited in Genbank (December 1, 2011;http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html). An Affymetrix GeneChip covers more than50,000 M. truncatula gene probes, which allowed theestablishment of a gene expression atlas (Benedito et al.,2008). For reverse genetics, large-scaled insertionalmutagenesis Tnt1 populations (Tadege et al., 2008, 2009)and RNA interference (RNAi)-mediated gene silencing(Limpens et al., 2004) are well established.

Investigations on plant–microbe interactions in therhizosphere are in the center of legume biology. Forthose studies, legumes are the organisms of choicebecause of their ability to interact with a broad array ofsymbiotic as well as pathogenic microbes (Colditz andBraun, 2010). Figure 26.1 shows exemplary microbialinteraction partners of M. truncatula. In particular, theability to carry out endosymbiotic nitrogen fixationwith Rhizobia bacteria within the characteristic rootnodules (see Chapters 44, 45) represent a major task forinvestigations of microbial associations to legumes. Thisprominent Rhizobia–legume (RL) symbiosis is limited

271

272 Chapter 26 Medicago truncatula Root Proteomics

Arbuscular mycorrhiza

Medicago truncatula

Oomycete infection Rhizobial bacteria

2-Dimensional gel

Figure 26.1 Medicago truncatula root proteomics. Themodel legume M. truncatula can either enter into symbiotic orpathogenic microbial associations at the plants rhizosphere.Exemplarily, microscopic pictures from rhizobia bacterial,arbuscular mycorrhizal (AM) fungi and oomycetic infectionsare shown. Rhizobial bacteria: Bacterial infection thread isgrowing in an infected root hair (left picture); isolated nodulefrom Sinorhizobium meliloti infection (right picture).Arbuscular mycorrhiza: microscopic picture of AM infected M.truncatula root cortex, combined with a graphical animationfor typical cellular infection structures, the arbuscles, as well asthe infection hyphae and the appressorium (dotted lines).Oomycete infection: microscopic picture of A. euteichesinfection in M. truncatula roots, showing infection hyphae andthe typical oospores (ink staining). 2-dimensional gel:alterations in the protein profiles can be monitored bydifferential gel electrophoresis (2-D DIGE).

to legumes (Fabales) of the eurosids (I) orders formingthe nitrogen-fixing clade (Markmann and Parniske, 2009;Wang et al., 2009). Besides RL symbiosis, legume rootsalso are colonized by arbuscular mycorrhizal (AM)fungi, the presumably most widespread but certainlysignificant symbiotic association (Colditz and Braun,2010; see Chapter 43). In addition, several legume cropspecies including pea (Pisum sativum L.), common bean(Phaseolus vulgaris L.), and alfalfa represent specifichost plants for oomycete pathogens such as Aphanomyceseuteiches, which cause destructive-root rot disease (Lev-enfors et al., 2003), and important metabolic pathwayssuch as the phenylpropanoid, flavone, and phytoalexinebiosyntheses, involved in responses to biotic and abiotic(stress) factors have also been extensively investigatedin legumes, especially in Medicago (Dixon, 1999; Dixonet al., 2002).

For obvious reasons, this review will focus on theM. truncatula root proteome and the dynamic alterationsinduced by root–microbe interactions. The availabilityof the Medicago truncatula euchromatin sequence nowallows the annotation of almost the entire protein-codinggenetic sequences and therewith all putative translatedgene products, as many internal sequence gaps are nowcovered (Young et al., 2011). For proteomic applications,this represents an excellent prerequisite for protein

identification by mass spectrometry. In retrospect, defi-ciency in available protein-coding sequence informationwas often the bottleneck for successful identification ofM. truncatula proteins. And it indeed took some timeuntil the first proteomic reference map for this modellegume was presented: The M. truncatula root proteomereference map (Mathesius et al., 2001). Via classical two-dimensional (2-D) gel electrophoresis, 2500 protein spotsvia silver and 1500 protein spots via Coomassie Bluestaining were reproducibly resolved from M. truncatularoot tissue. As already mentioned, MS-based proteinidentification was a limiting factor at the beginning ofM. truncatula proteomic approaches: Of the 485 mostabundant proteins, only 179 could be identified by MSanalyses using the M. truncatula genome database asavailable in 2001. In a later proteomic approach, acomparable number of protein spots were identified in M.truncatula cell suspension cultures that derived from rootcallus, but MS analysis was already improved: using nanoliquid chromatography (nLC)-tandem mass spectrometry,1367 out of 1661 gels spots could be identified (Leiet al., 2005). Together with some further partial proteomemaps for M. truncatula root tissue (Mathesius et al.,2002, Colditz et al., 2004, 2002; Holmes et al., 2006),a basis for proteomic approaches of legume roots wasestablished.

26.2 M. truncatula Root Proteomics for the Analysis of the Rhizobia-Legume (RL) Symbiosis 273

26.2 M. truncatula ROOTPROTEOMICS FOR THE ANALYSISOF THE RHIZOBIA-LEGUME (RL)SYMBIOSIS

Rhizobial infection takes place at the root hairs of thelegume host plants. Before the initial bacterial infectionoccurs, a complex signal exchange is required betweenboth interaction partners: rhizobial lipochitin oligosaccha-ride signal molecules termed Nod factors (NF) are elicitedin response to specific flavonoids secreted by the legumehost. After host perception of NF by LysM domain recep-tor kinases, a defined signal transduction pathway is acti-vated, which results in fundamental morphological, phys-iological, and molecular changes (Limpens et al., 2003;see Chapters 45, 51). During the whole infection pro-cess, rhizobial bacteria remain outside the host cytoplasm:They differentiate into bacteroids harbored inside spe-cial organelles called the symbiosomes that are enclosedby plant plasma membrane-derived peribacteroid or sym-biosome membranes. Specialized organs are formed, theroot nodules, which function as microfermenters, provid-ing an optimal environment for bacterial cultivation andnitrogenase activity (Markmann and Parniske, 2009). Thenitrogen reduction processes allow the legume host to bemore or less independent of other nitrogen sources.

The rhizobial symbiont of M. truncatula is Sinorhi-zobium meliloti. Owing to the availability of its completegenome sequence (Galibert et al., 2001) and proteomicreference maps (Djordjevic et al., 2003; Djordjevic, 2004),discrimination of proteins belonging to the host proteomeor to the bacterial proteome is now possible.

Many proteomic studies have been carried out withthe aim of identifying alterations in protein patterns of S.meliloti-infected roots and nodules from M. truncatula.An initial comparative proteomic study of M. truncatularoots infected either with S. meliloti or Glomus mosseaeled to the identification of several symbioses-inducedproteins, whereof 56 proteins were induced during fiveweeks of nodule development (Bestel-Corre et al., 2002).Among these proteins, a specific nodulation-related hostleghemoglobin was identified next to several bacterialproteins involved in nitrogen fixation. A fractionationprocedure of proteins from M. truncatula nodules ledto the isolation of 51 proteins from the symbiosomemembrane, including an aquaporin, an H+ –ATPase,and several marker proteins of early nodulation, suchas ENOD8, ENOD16, and nodulin-25 (Catalano et al.,2004). For initiation of nodule development, the ratioof mainly two phytohormones, auxin, and cytokinin,is important. Auxin is considered to act synergistically

during rhizobia nodulation (Gonzalez-Rizzo et al., 2006).This was shown via a differential gel electrophoresis(DIGE)-based proteomic approach of early host responsesof M. truncatula to S. meliloti infection as compared withresponses to auxin treatment, which revealed a greaterthan 80% overlap in protein alterations within the first24 hours of inoculation (Van Noorden et al., 2007).Auxin-related protein induction was also identified inanother DIGE-based proteomic approach for M. truncat-ula roots already after 6 hours of S. meliloti infection,and also after Glomus intraradices mycorrhizal infection(Schenkluhn et al., 2010). By contrast, a transient plantdefence response is likely mediated by ethylene actingantagonistic to rhizobial infection (Mathesius, 2009).Consequently, the ethylene-insensitive M. truncatulamutant sickle (skl) exhibits rhizobia hypernodulation andsignificantly reduced plant defence response patterns(Penmetsa and Cook, 1997). Proteomic comparison ofM. truncatula wild type and skl mutant lines resulted inthe identification of ethylene-induced proteins, includingACC oxidase as the key enzyme that converts aminocy-clopropane carboxyl acid into ethylene (Prayitno et al.,2006). Also abiotic stress factors have consequenceson the RL symbiosis. Here, drought stress representsthe major stress condition that limits nodule symbioticnitrogen fixation (SNF) rates significantly. Via a pro-teomic shot gun approach, Larrainzar et al. (2007) coulddemonstrate that SNF rates are minimized during droughtmainly because of a reduction in metabolic enzymes,particularly that of sucrose synthase 1, leghemoglobin,and enzymes of amino acid biosynthesis pathways.Coincidently, reduced abundance of symbiosis-relatedproteins was shown for soil contaminations with heavymetals or polycyclic aromatic hydrocarbons of sewagesludge, which is used for fertilization of agricultural soil(Bestel-Corre et al., 2004).

One major challenge remains for proteomic analysisof the RL symbiosis: the identification of proteinsinvolved in the very early phases of rhizobial infectionand even pre-infection phases. At this time, a genetic pro-gram is induced in the legume host that is supposed to beconserved and common with that for AM fungal infectionsand pre-infection phases (Markmann and Parniske, 2009;Colditz and Braun, 2010; see Chapters 43, 45). Explicitly,seven symbiosis (sym) genes are involved and have beenidentified in L. japonicus, whereof four orthologues couldalso be isolated from M. truncatula: (i) DMI2 (does notmake infection) encoding a symbiotic receptor-like kinase(SYMRK); (ii) DMI1, which encodes a predicted cationchannel protein; (iii) DMI3 that codes for a calcium- andcalmodulin-dependent kinase (CCaMK); and (iv) IPD3

274 Chapter 26 Medicago truncatula Root Proteomics

encoding an interacting protein of DMI3 (Markmann andParniske, 2009). The CCaMK is a key element of sym-biotic signal transduction as it deciphers upstream signalcomponents into a nuclear calcium spiking, periodic fluc-tuations of calcium concentration occurring in responseto rhizobial bacteria, NF and also AM fungi. Downstreamof this oscillation in calcium concentrations, symbioticgenes are induced in response to either bacterial or fungalinfections (Oldroyd and Downie, 2006). The major roleof the CCaMK was shown in L. japonicus: amino acidpoint mutations in the CCaMK’s autoregulatory domainor even in the intron regions were capable of inducingspontaneous nodule development in absence of rhizobialbacteria or NF (Gleason et al., 2006; Tirichine et al.,2006). Furthermore, in vivo phosphorylation was shownfor three major proteins of this early signaling program inM .truncatula: (i) nucleoporin 133 (NUP133), (ii) IPD3,and (iii) sickle (SKL) protein (Grimsrud et al., 2010).A recent phosphoproteome analysis revealed shared anddistinct early protein phosphorylation between NF andthe pathogenic bacterial flagellin peptide (flg22) in L.japonicus (Serna-Sanz et al., 2011).

26.3 M. truncatula ROOTPROTEOMICS FOR THE ANALYSISOF THE ARBUSCULARMYCORRHIZA (AM) SYMBIOSIS

The vast majority of land plants are capable of installinga symbiotic interaction with AM fungi of the phylumGlomeromycota. This widespread plant–microbe inter-action was established more than 400 million years ago(Remy et al., 1994). Similar to the RL symbiosis, duringthe AM symbiosis also the host plant accommodatesthe microbial partner in the form of highly branchedintracellular structures termed arbuscules, but staysseparated from the plant’s cytoplasma by a specializedmembrane, the AM perifungal membrane. A transfer of(limiting) nutrients, mainly of phosphorus and nitrogen,from fungal hyphae to the plant cells occurs at thisperiarbuscular interface, which consists of the fungalarbuscular membrane, the periarbuscular matrix, andthe plant periarbuscular membrane (Parniske, 2008; seeChapter 43). In contrast to the RL symbiosis, no specialorganogenesis is induced in the host plant. During AMinfection, extraradical hyphae emerging from germinatingspores penetrate the rhizodermis via hyphopodia, crossthe outer cortical cells, and then start to proliferate inthe inner root cortex (Parniske, 2008). After their entryvia hyphopodia, fungal hyphae grow along a so-calledprepenetration apparatus (PPA), which consists of acytoplasmic channel formed after the establishment ofhyphopodia (Genre et al., 2005, 2008). Similar to the

RL symbiosis, before AM formation also a moleculardialogue is initiated by the host plant via strigolactonesthat promote the branching of fungal hyphae and activa-tion of fungal metabolism (Akiyama et al., 2005; Bessereret al., 2006; see Chapters 33, 34, 35). Recognition of AMfungi is achieved via perception of diffusible Myc signalsincluding lipochitooligosaccharides that are structurallyrelated to rhizobial Nod factors (Kosuta et al., 2003; Mail-let et al., 2011; see Chapters 43, 45). Once AM hyphaehave entered the root cortex, intracellular colonization ofroot cortical cells and subsequent formation of arbusculesin the inner root cortex are initiated (Harrison, 2005).

A proteomic reference map for the AM fungusG. intraradices was established (Dumas-Gaudot et al.,2004). This map consists of 438 protein spots, whereofonly eight proteins could be identified. Via an additionalshotgun proteomic approach, 92 different fungal proteinsof the extraradical hyphae of G. intraradices wereidentified (Recorbet et al., 2009).

A time-course-based proteomic approach of M. trun-catula roots infected with G. mosseae led to theidentification of 14 proteins induced at the AM appres-sorial stage (4 days post infection (dpi)) and of 24proteins induced during established symbiosis (3–4weeks post infection). The induced proteins are involvedin general defence responses (peroxidases, glutathione-S-transferases, PR-10 proteins), respiration (serinehydroxyl–methyl transferase, cytochrome-c oxidase), andcell wall modification (Bestel-Corre et al., 2002).

Very early signaling and infection responses in thehost plant were mainly investigated via transcriptomicapproaches (Hogekamp et al., 2011). Initial PPA for-mation was found to depend on the common symbiosisgenes DMI2 and DMI3 in M. truncatula (Genre et al.,2008; see Chapter 43). Thus, these genes were recruitedand adapted during evolution of the more recent RLsymbiosis (Kistner and Parniske, 2002). At the proteinlevel, initial AM signaling in plants has been investigatedin a comparative study of M. truncatula wild-type lineand appressorium-responsive roots of two mutants, (i)the mycorrhizal infection-defective dmi3 mutant, whichis known to arrest after formation of appressoria, and(ii) the already mentioned sunn mutant, which displayssignificant increases not only in nodulation but also inmycorrhization (Amiour et al., 2006). This approach ledto the identification of DMI3-inducable proteins (an actindepolymerising factor, a dehydroascorbate reductase, anda cyclophilin), in addition to the symbiotic signaling pro-tein MtAnn1. MtAnn1 belongs to a group of annexin-likeproteins, which are known to be localized at the nuclearperiphery of rhizobial as well as mycorrhizal-activatedcortical cells (Talukdar et al., 2009). Interestingly, a puta-tive role in the specific symbiotic calcium spiking wassuggested for these proteins because of their capability

26.4 M. truncatula Root Proteomics for the Analysis of Pathogenic Interactions 275

to bind to membrane phospholipids in a Ca2+ dependentmanner (Talukdar et al., 2009). The dmi3 mutant fromM. truncatula has also been used as a control in aproteomic investigation to identify membrane-associatedprotein regulation during AM formation (Valot et al.,2005). Comparative analysis of M. trunculata wt plantsand mutants has allowed the identification of 25 pro-teins that are differentially displayed in response to G.intraradices colonization, including a lectin, two ATPasesubunits from the F(1)-ATPase complex, a 53-kDanodulin, and an acid phosphatase (Valot et al., 2005). Alater investigation of the root plasma membrane fractionrevealed 78 proteins induced after AM infection, amongthem a predicted glycosylphosphatidylinositol-anchoredblue copper-binding protein with potential localization inthe periarbuscular membrane, the interface between thehost plant and the AM fungus (Valot et al., 2006).

AM infections are known to improve the toleranceof plants toward abiotic and biotic stress conditionsnext to the beneficial effect on plant growth. Proteomeanalyses of M. truncatula plants grown in the presenceof cadmium exhibited downregulation or nonactivationof stress-responsive proteins when plants were prein-fected with G. intraradices (Aloui et al., 2009). Similareffects of noninduced stress-responsive proteins wereidentified in M. truncatula roots secondarily infectedwith the oomycete root pathogen A. euteiches after AMpreinfection (Colditz et al., 2005; Schenkluhn et al., 2010).

26.4 M. truncatula ROOTPROTEOMICS FOR THE ANALYSISOF PATHOGENIC INTERACTIONS

A variety of pathogenic fungi and oomycetes infect theroots of legumes, where they cause severe damages anddiseases often resulting in substantial economic losses.Physiological aspects of many pathogeneses are in manycases well characterized, but often little is known aboutthe molecular mechanisms underlying disease establish-ment or the nature of host cellular responses. In com-parison to the RL and AM symbioses, there seems tobe no common basic genetic program activated duringinfection with distinct pathogens. Nevertheless, a hyper-sensitive response (HR) reaction at the infection sides asa result of substantial elevated reactive oxygen species(ROS) is frequently observed, but not obligatory, duringearly pathogenic infections (Colditz and Braun, 2010).

Large efforts have been made to generate genomicsequence information for plant pathogens. For the verydestructive oomycete plant pathogens Phytophthoraspec. (Phytophthora infestans, Phytophthora sojae, andPhytophthora ramorum) sequencing of the genomes hasbeen completed (Haas et al., 2009). The analyses revealed

an extensive expansion of the P. infestans genome incomparison to the ones of the other two Phytophthoraspecies, which is due to a repetitive proliferation of DNAregions encoding families of manifold secreted diseaseeffector proteins. Also, proteomic profiles are availablefor Phytophthora: 3897 and 2970 proteins were identifiedin mycelia and germinating cysts of P. sojae and P.ramorum, respectively, via a cross-species identificationmethod (Savidor et al., 2008). For another oomycetelegume pathogen, A. euteiches, two cDNA libraries weregenerated from mycelia isolates either from M. truncatulaor from a synthetic medium, which comprise greaterthan 18,000 ESTs assembled to nearly 8,000 unigenes,which are accessible in the “AphanoDB public database”(Madoui et al., 2008).

Concerning proteomic analyses of pathogenicinteractions with M. truncatula, there are apparentlyless investigations published as compared to the abovedescribed symbiotic interactions. M. truncatula isaffected by severe root rot diseases caused by oomycetepathogens, mainly from the genera Phytophthora,Pythium, and Aphanomyces. Oomycetes are a large class(∼600–1500 species) of fungus-like eukaryotes thatare phylogenetically distinct from true fungi, but thisclass includes the most numerous, most important, andearliest known water molds (Dick et al., 1999). Theperhaps most destructive oomycete plant pathogens arespecies from the Phytophthora genus. However, the rootpathogen A. euteiches is accountable for severe root rotin several legume crop plants, including pea as a majorhost plant, where it is regarded to be the most destructivedisease in areas with temperate climates (Levenfors et al.,2003). By contrast, most M. truncatula genotypes reactto A. euteiches infection with partial resistance (Djebaliet al., 2009; Hamon et al., 2010). In an initial proteomicstudy of M. truncatula roots infected with A. euteiches,several defence-related proteins were identified (cellwall-related proline-rich and glycine-rich proteins, anenzyme of the flavanoid pathway, and particularly aprominent protein group of PR-10-like proteins includinga PR-10-1 protein, four ABA-responsive proteins, and adisease-resistance response protein pi49) (Colditz et al.,2004). A comparative proteomic profiling of differentM. truncatula lines that are more tolerant/more suscep-tible toward A. euteiches infection indicated a positivecorrelation for specific induction of some PR-10-likeproteins because of A. euteiches infection (Colditz et al.,2005). Interestingly, further induction of proteasomeprotein subunits was detected in the more tolerant lines, afinding which could be also confirmed via identificationof a M. truncatula QTL rich in proteasome-related genes,which is involved in the establishment of partial resis-tance toward A. euteiches (Djebali et al., 2009). Usingreverse genetics, MtPR-10-1 gene silencing resulted in

276 Chapter 26 Medicago truncatula Root Proteomics

increased A. euteiches-tolerance in the transgenic rootcultures and also in a concomitant induction of other PRprotein classes (Colditz et al., 2007). Via a proteomic andphosphoproteomic approach, early protein induction anddefence-related metabolic activation were monitored forM. truncatula cell suspension cultures after applicationof A. euteiches zoospores and on elicitation with culturemedium of the pathogen (Trapphoff et al., 2009). Acomprehensive DIGE-based proteomic investigationwas carried out for M. truncatula roots inoculated withrhizobia, mycorrhizal fungi, and/or A. euteiches (singleand mixed infections), which led to the identificationof 87 differentially induced proteins at early infectiontime-points (Schenkluhn et al., 2010).

26.5 PROSPECTIVE M. truncatulaROOT PROTEOMICS

M. truncatula represents a well-developed model systemto study legume biology at a molecular level, andespecially to analyze interactions of legume roots tomicrobial infections. Surely, the major focus is set onthe molecular characterization of the symbiotic RLand AM interactions, and also for root associationsof legumes to pathogens there are requirements forin-depth molecular analyses. So far, these interactionsare consequently less investigated as compared to thesymbiotic interactions. In addition, because of mainlytechnical reasons, molecular approaches at the tran-scriptome level are still predominantly applied in thisfield. Limitations exist mainly because of insufficientavailability of plant material for large-scaled proteomicapproaches that exhibit the requested infection structures.Especially, signal transduction processes during veryearly root-microbe interaction stages are very challenging(Colditz and Braun, 2010). Therefore, novel proceduressuch as laser-microdissection of infected plant materialand of small networks of cells might overcome currentlimitations. Here, gel-free proteomic procedures will gainin importance because they can overcome problems withvery small protein quantities available. These techniquesmight also allow detection of proteins of low abundanceor difficult solubilization properties.

In future studies, identification of more symbiosisand pathogenesis-related proteins and signal transduc-tion networks will represent the major goals. Reversegenetic studies should be combined with proteomicanalyses to elucidate the specific roles of proteinsduring legume–microbe interactions. As the genomicbackground in terms of high sequence coverage is nowgiven, it can be expected that M. truncatula will maintainan exposed position in future plant–microbe interactionresearch.

ACKNOWLEDGMENTS

The author likes to thank Hans-Peter Braun from the Insti-tute for Plant Genetics, Leibniz University of Hannover,for proofreading the manuscript and for fruitful discus-sions.

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