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Involvement of auxin and LTP proteins in the regulation of root nodule formation in Medicago truncatula - Sinorhizobium meliloti Symbiosis
Full text linkQuesto progetto di dottorato ha avuto come obiettivi: i) valutazione del ruolo del’auxina di derivazione batterica nella simbiosi rizobio-leguminosa, che dà origine a noduli di tipo indeterminato, ii) lo studio funzionale di MtN5, una proteina di tipo “Pathogenesis Related”, che viene indotta precocemente durante la nodulazione e che presenta omologie di sequenza con membri della famiglia delle Lipid Transfer Protein vegetali.
L’auxina (acido indol-3-acetico, IAA) è un ormone vegetale implicato in molti aspetti che riguardano la vita e lo sviluppo delle piante; un suo coinvolgimento nello sviluppo del nodulo radicale era stato ipotizzato già all’inizio del secolo scorso. Studi successivi hanno dimostrato un’inibizione del trasporto acropeto di IAA nella radice a seguito dell’infezione con rizobi, con un conseguente accumulo di fitormone a livello del sito di infezione. E’ stato dimostrato che la maggior parte dei batteri della rizosfera che producono effetti di promozione sulla crescita della pianta, rizobi inclusi, possiedono vie biosintetiche per IAA. I rizobi sono in grado di sintetizzare auxina in coltura liquida e, molto probabilmente, mantengono questa capacità anche durante la nodulazione. Ad oggi, però, i dati riguardanti il ruolo dell’auxina batterica nello sviluppo dei noduli sono ancora controversi; sono stati infatti documentati sia effetti stimolatori che inibitori.
Molti degli eventi che stanno alla base dell’associazione simbiotica tra rizobi e leguminose devono ancora essere chiariti. Ad esempio, la natura e la funzione dei segnali ormonali scambiati tra ospite e simbionte non sono ancora stati compresi nel dettaglio, così come le differenze e i parallelismi nella risposta delle leguminose verso il simbionte e verso i patogeni della radice. A tal riguardo, recenti osservazioni hanno dimostrato che la repressione della via di segnalazione intracellulare dell’auxina risulta in una maggiore resistenza innata delle piante verso microrganismi patogeni.
Piante di Medicago truncatula, specie modello per le leguminose che producono noduli di tipo indeterminato, e Medicago sativa (erba medica), specie correlata di interesse agronomico, sono state nodulate sia con rizobi wild-type e che con rizobi in grado di iper-produrre IAA (S. meliloti IAA). I risultati ottenuti hanno dimostrato che piante nodulate con S. meliloti IAA producevano un numero maggiore di noduli (aumento del 50% in M. sativa e
aumento del 100% in M. truncatula) e un apparato radicale più ramificato. Inoltre il contenuto di auxina nei noduli prodotti da rizobi IAA è mediamente 10 volte superiore alla concentrazione dei noduli prodotti da rizobi wild-type. I livelli di espressione dei geni responsabili del trasporto di auxina è stato valutato mediante RT-PCR quantitativa (qRT-PCR) e il carrier di efflusso MtPIN2 è risultato significativamente più espresso (circa 2 volte) nel tessuto radicale di piante nodulate con rizobi IAA rispetto alle radici infettate con il rizobio di controllo. Questi risultati suggeriscono che l’effetto di promozione osservato sulla nodulazione e sull’accrescimento della radice laterale siano dovuti alla produzione di IAA nel nodulo e ad una sua redistribuzione all’interno dell’apparato radicale.
E’ stato ampiamente dimostrato che l’ossido nitrico (NO) agisce come secondo messaggero nell’induzione di radici laterali e avventizie stimolata da auxina. Considerando la comune organogenesi tra radici laterali e avventizie e noduli indeterminati, in questo lavoro abbiamo dimostrato che esiste un collegamento tra auxina e NO nella via di segnalazione che porta all’induzione del nodulo.
Per mezzo di uno screening preliminare, condotto mediante qRT-PCR e volto ad individuare geni differenzialmente espressi in piante nodulate con rizobi IAA e piante nodulate con rizobi wild-type, fu osservato che il gene MtN5 era più espresso negli apparati radicali di piante infettate con rizobi iperproduttori di auxina. Il prodotto genico di MtN5 è stato annotato come una Lipid Transfer Protein (LTP) putativa. Le LTP vegetali sono caratterizzate dalla capacità sia di legare lipidi in vitro che di inibire la crescita di microrganismi. In questo progetto di tesi è stato dimostrato che MtN5 possiede la capacità di legare lisolipidi e che, come molti altri membri di questa famiglia di proteine, possiede attività antimicrobica in vitro, in particolare contro Fusarium semitectum, Xanthomonas campestris e S. meliloti.
Lo studio del profilo di espressione conferma che MtN5 viene precocemente indotta durante la nodulazione e che è specificamente localizzata all’interno del nodulo radicale. Inoltre, l’infezione di piante con F. semitectum provoca l’accumulo di MtN5 nel tessuto radicale.
La funzione di MtN5 nella nodulazione è stata studiata mediante la generazione di radici avventizie transgeniche, sia overesprimenti che silenziante per il gene di interesse. Le radici
silenziate per MtN5 sviluppano circa la metà dei noduli rispetto a radici di controllo, mentre in radici transgeniche over-esprimenti MtN5 il numero di noduli è risultato incrrementato del 300% rispetto al controllo. I risultati ottenuti dimostrano che MtN5 facilita l’interazione simbiotica tra M. truncatula e S. meliloti, agendo probabilmente negli stadi precoci dell’infezione, e suggeriscono che MtN5 potrebbe avere un ruolo nella protezione dei noduli verso patogeni della radice. Ulteriori studi sono comunque necessari per ottenere una immagine più chiara del ruolo di MtN5 sia nella simbiosi che nella risposta verso i patogeni.The present thesis has had two main focuses: i) the evaluation of the role of bacteria-derived auxin in the symbiosis between rhizobia and legumes that bear indeterminate nodules, ii) the functional study of MtN5, a pathogenesis related protein which presents sequence homology with the members of the plant Lipid Transfer Proteins (LTP) family and is precociously induced during nodulation.
Auxin (indol-3-acetic acid, IAA) is a phytohormone involved in many aspects of plants growth and development; The role of auxin in the development of the rhizobia-legumes symbiosis was first hypothesised at the beginning of the twentieth century. More recent studies have demonstrated that auxin is accumulated at the site of infection as a consequence of the inhibition of the acropetal auxin transport in roots upon rhizobia inoculation. The production of IAA has also been documented in plant-associated rhizobacteria, including rhizobia, that have promoting effects on plants growth. When grown in liquid media, rhizobia can synthesize auxin and most likely they retain the same capability also during the nodule development. However, up to date, the data concerning the role of bacteria-derived auxin in the establishment of the symbiotic association are still contradictory, since both stimulatory and inhibitory effects have been documented.
Thus, there are still open questions in the understanding of the events that result in the establishment of the symbiosis. First of all the nature and the function of the hormonal signal(s) exchanged between the host and the symbiont are not thoroughly unfolded, as well as the parallelisms and the differences in the responses of legumes against root pathogens and root symbiont. In these regards, recent findings have pointed out that plants innate immunity results, at least in part, from the down-regulation of the auxin signalling pathway.
Medicago truncatula and Medicago sativa plants were nodulated with both wild-type and auxin hyper-synthesising rhizobia (Sinorhizobium meliloti IAA). The results obtained showed that plants nodulated with S. meliloti IAA produced a higher number of root nodules (50% more nodules in M. sativa and 100% more nodules in M. truncatula) and a more branched root apparatus. The root nodules elicited by S. meliloti IAA had a higher IAA content (at least 10-fold) when compared to control nodules. The expression levels of the auxin carriers were
evaluated and the efflux facilitator MtPIN2 resulted more abundant (about 2-fold) in the root tissue of IAA plants when compared to wild-type plants These data suggested that such promoting effects on nodulation and lateral root growth might be due to the increased auxin content detected in IAA nodule produced by auxin hyper-synthesising rhizobia, as well as to a redistribution of the phytohormone in the root tissue.
It has been largely demonstrated that nitric oxide (NO) acts as second messenger in the auxin-induced pathway that leads to formation of lateral and adventitious roots. Since root nodules have the same organogenetic origin of lateral and adventitious roots, the possible connection between NO and root nodule induction was investigated and we demonstrated that NO participate in the signalling pathway for root nodule induction.
During a preliminary screening carried out by means of qRT-PCR, it has been found that N5 gene of M. truncatula was more abundantly expressed in roots nodulated with S. meliloti IAA with respect to roots infected by wild-type rhizobia. The gene product of MtN5 was annotated as putative Lipid Transfer Protein (LTP). LTPs are characterized by their ability to bind lipids in vitro and the majority of them exhibits antimicrobial activity. In this thesis, it has been demonstrated that the recombinant MtN5 protein is able to bind lysolipids and possesses inhibitory activity against Fusarium semitectum, Xanthomonas campestris and S. meliloti.
The studies of the expression pattern of both MtN5 transcript and MtN5 protein confirmed that it is precociously induced during nodulation and revealed that it is specifically localized in the root nodule. In addition, when M. truncatula plants are infected with the root pathogenic fungus F. semitectum, MtN5 protein is accumulated in the root apparatus.
The function of MtN5 in nodulation has been studied through the generation of transgenic adventitious roots, both over-expressed and silenced for the gene of interest. MtN5-silenced roots developed approximately 50% fewer nodules as compared to control roots, whereas in hairy roots over-expressing MtN5 the nodule number was increased by about 300% with respect to control roots. Collectively the data indicate that MtN5 facilitates the symbiotic interaction between M. truncatula and S. meliloti, probably acting in the early stages of rhizobia infection, and suggest that it might have a role in the protection of nodules against
root pathogen. However, further studies are needed to have a clear picture of the role played by MtN5 in both symbiosis and defence response against pathogens
Involvement of Medicago truncatula lipid transfer protein N5 in epidermal phases of rhizobia-host interaction.
No full textThe early nodulin N5 of Medicago truncatula is a lipid transfer
protein that has been proven to positively regulate nodulation, although it displays in vitro inhibitory activity against Sinorhizobium meliloti. We utilized a RNAi-based hairpin construct to down-regulate MtN5 expression in M. truncatula hairy roots.
MtN5-silenced roots inoculated with rhizobia displayed an increased number of root hair curling events and a reduced number of invaded primordia and mature nodules
as compared to wild type roots. Nodule primordia formation appeared unaltered in MtN5-silenced roots. This phenotype was associated with the stimulation of ENOD11 expression, an early marker of infection, and with the down-regulation of Flotillin 4(FLOT4), a protein involved in rhizobia entry pathway
Study of Mtn5 transcriptional control and of its involvement in Medicago truncatula nodulation pathway
No full textThe symbiosis between legumes and rhizobia starts with an exchange of molecular signals between the two partners. In response to the plant-derived flavonoids, bacteria synthesize Nod Factors (NFs), which are able to induce a series of events, such as ion fluxes, root hair deformation and the expression of the early nodulin genes, that eventually lead to the formation of root nodules. We previously demonstrated that MtN5 is an early nodulin, required for the establishment of the symbiosis and also present in mature nodules. In order to investigate the role of MtN5 in root nodules induction pathway, its expression profile during the early stages of infection was studied. MtN5promoter::GUS fusion showed that the promoter was active in epidermis and root hairs a few hours after inoculation, whilst in mature nodules, GUS was observed in the distal zone.In a time course nodulation experiment, MtN5 showed to be co-expressed with early markers of rhizobia infection, such as RIP1, NIN and ENOD11, and resulted to be more precocious than ENOD20 and MtN6. In transgenic adventitious root silenced for MtN5 expression (MtN5hp roots), we observed that upon rhizobia infection the nodulin MtNIN was not induced, whilst ENOD11 was strongly upregulated with respect to control roots. Furthermore, in MtN5hp roots the expression of FLOT4, a nodulin gene known to be involved in the infection thread growth, was unaffected by the inoculation with symbiotic bacteria, in contrast with what observed in control roots
Analysis of Arabidopsis thaliana Aucsia mutants
No full textTomato Aucsia genes have been shown to control fruit initiation and affect auxin-related processes. The Aucsia homologues of Arabidopsis, AtAucsia-1 and AtAucsia-2 are expressed in all plant organs and preferentially in inflorescences. Reporter gene studies have been performed with either Aucsia-1p:GUS or with Aucsia-2p:GUS to visualize the expression pattern of the two genes during seedlings and plant development. Furthermore, we have analysed two Arabidopsis Salk lines: SALK_117986 a T-DNA insertion AtAucsia-1 mutant and SALK_065659 a T-DNA insertion AtAucsia-2 mutant. In plants homozygous for the insertion in AtAucsia-2, AtAucsia-2 mRNA level is 50% lower than in wild-type. Homozygous ataucsia-1 mutant shows no detectable expression of AtAucsia-1 mRNA. Homozygous ataucsia-1 mutant seedlings show no visible differences in growth compared with wild-type, but when cultivated with growth-inhibiting concentrations of IAA, they are less sensitive indicating that ataucsia-1 mutants might be affected in auxin response. Homozygous ataucsia-2 mutant seedlings display a slight reduction in primary root growth compared to wild-type, whereas their sensitivity to auxin is not altered
Effect of increased IAA synthesis in root nodules of Medicago plants
No full textIn plants auxin modulates diverse processes such as tropic response to light and gravity, general root and shoot growth and vascular development. IAA (indol-3-acetic acid) is produced largely in shoot apex. Auxin is transported basipetally in shoots, while both acropetal (in the central cylinder) and basipetal (via epidermaland cortical cells) transport occur in roots [4]. Most Rhizobacteria synthesize IAA, and in symbiotic Rhizobia have been identified different auxin biosyntheticpathways. It has been proposed that IAA plays a role in nodule formation, and it has been also demonstrated that nodulated roots have a phytormone content higherthan root tissue [1]. Sinorhizobium meliloti induces nodule formation in Medicago species; S. meliloti 1021 has been engineered by introducing promintron-iaaM-tms2gene construct (S. meliloti 1021 IAA), which increases auxin content in the nodules
Comparison of changes in root transcriptome of two maize inbred lines in response to nitrate treatment
No full textLo5 line responded to NO3
-‐ treatment (induction) faster than T250 line at each concentration assayed. Even if
the two inbred lines reached highest induction at different times (2 h for 500 μM and 4 h for 200 μM for Lo5 and 8
h for 500 μM and 12 h for 200 μM for T250), they showed similar values of NO3
-‐ uptake rate. On the basis of this
different behaviour, roots were sampled at different times (1, 2 and 4 h for Lo5 and 5, 11 and 12 h for T250) after
NO3
-‐ treatment (200 μM) in order to perform a “genome-‐wide” transcriptional characterization. Data analysis of
microarray experiments, performed with a chip that allows monitoring the expression of about 60000 maize
transcripts, showed that roots of Lo5 and T250 inbred lines responded to different periods of NO3
-‐ contact through
a modulation of a different number of transcripts. Changes in root Lo5 transcriptome involved a higher number of
transcripts (about 5000 at 2 h) relative to T250 (about 1900 at 12 h). After the peak, transcripts modulated by the
treatment in Lo5 decreased, with the behaviour different from T250 where the number increased linearly up to 12
h. In addition, NO3
-‐ treatment affected the Lo5 and T250 Gene Ontology (GO) terms in a different way. PAGE
analysis performed in Lo5 suggests that processes related to protein modifications (i.e. protein phosphorylation)
and translation were positively affected at 1 h and 4 h, passing through a negative phase of regulation at 2 h. On
the contrary, “protein metabolic process” terms were down-‐regulated at all sampling time points in T250 relative
to Lo5 and only in T250 was a positive modulation of transcripts related to nitrogen metabolism (“nitrogen
compound of metabolic process”) recorded at 5 and 11 h. Furthermore, in the two lines only about 400 transcripts
were modulated in common in response to NO3
-‐
A proteomic approach to study the autoregulation of nodulation in Medicago truncatula.
No full textPlant belonging to Leguminosae family can establish a symbiotic relationship with nitrogen-fixing bacteria, termed rhizobia, that lead to the formation of a new organ, the root nodule. This organogenetic pathway starts as a consequence of a molecular cross-talk between plants and bacteria. The systemic pathway, also termed Autoregulation Of Nodulation (AON), is a mechanism by which a plant controls the number of nodules inhibiting further nodule formation after the first few nodules have been formed, as a consequence of a molecular communication between root and shoot.With the aim of gaining a deeper insight in the signalling cascade of AON, we compared the root and shoot proteomes of Medicago truncatula plants inoculated with the symbiotic rhizobium, Sinorhizobium meliloti, with those of uninoculated plants
Local and Systemic Proteomic Changes in Medicago truncatula at an Early Phase of Sinorhizobium meliloti Infection.
No full textThe symbiotic association with N-fixing bacteria facilitates the growth of leguminous plants under nitrogen-limiting conditions. The establishment of the symbiosis requires signal exchange between the host and the bacterium, which leads to the formation of root nodules, inside which bacteria are hosted. The formation of nodules is controlled through local and systemic mechanisms which involves root-shoot communication. Our study was aimed at investigating the proteomic changes occurring in shoots and concomitantly in roots of Medicago truncatula at an early stage of Sinorhizobium meliloti infection. The principal systemic effects consisted in alteration of chloroplast proteins, induction of proteins responsive to biotic stress and changes in proteins involved in hormonal signaling and metabolism. The most relevant local effect was the induction of proteins involved in the utilization of photosynthates and C-consuming processes (such as sucrose synthase and fructose-bisphosphate aldolase). In addition, some redox enzymes such as peroxiredoxin and ascorbate peroxydase showed an altered abundance. The analysis of local and systemic proteome changes suggests the occurrence of a stress response in the shoots and the precocious alteration of energy metabolism in roots and shoots. Furthermore, our data support the hypothesis that ABA and ethylene participate in the communicative network between root and shoot in the control of rhizobial infection
Auxin and nitric oxide control indeterminate nodule formation
Full text linkAbstract Background Rhizobia symbionts elicit root nodule formation in leguminous plants. Nodule development requires local accumulation of auxin. Both plants and rhizobia synthesise auxin. We have addressed the effects of bacterial auxin (IAA) on nodulation by using Sinorhizobium meliloti and Rhizobium leguminosarum bacteria genetically engineered for increased auxin synthesis. Results IAA-overproducing S. meliloti increased nodulation in Medicago species, whilst the increased auxin synthesis of R. leguminosarum had no effect on nodulation in Phaseolus vulgaris, a legume bearing determinate nodules. Indeterminate legumes (Medicago species) bearing IAA-overproducing nodules showed an enhanced lateral root development, a process known to be regulated by both IAA and nitric oxide (NO). Higher NO levels were detected in indeterminate nodules of Medicago plants formed by the IAA-overproducing rhizobia. The specific NO scavenger cPTIO markedly reduced nodulation induced by wild type and IAA-overproducing strains. Conclusion The data hereby presented demonstrate that auxin synthesised by rhizobia and nitric oxide positively affect indeterminate nodule formation and, together with the observation of increased expression of an auxin efflux carrier in roots bearing nodules with higher IAA and NO content, support a model of nodule formation that involves auxin transport regulation and NO synthesis.</p
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