Le risposta di difesa delle piante ai patogeni è costituita da due diversi livelli di difesa. Il primo livello di difesa, che prende il nome di “PAMP-triggered immunity” (PTI), è attivato dal riconoscimento di elicitori generici e corrisponde alla difesa basale. Il secondo livello di difesa, che è razza/cultivar specifico, è invece attivato dal riconoscimento specifico di fattori di virulenza rilasciati dal patogeno nella pianta. Il riconoscimento di questi fattori di virulenza da parte di specifiche proteine dell’ospite (R-proteins) induce lo sviluppo di una risposta ipersensibile (HR) che è caratterizzata dalla comparsa localizzata di morte cellulare al sito di infezione. Per contrastare questi meccanismi di difesa e promuovere la virulenza, molti batteri Gram-negativi rilasciano effettori nella cellula ospite in grado di modulare il macchinario di risposta della pianta e sopprimere la risposta di difesa. Tra gli altri, uno dei meccanismi utilizzati dai patogeni batterici per modulare la risposta di difesa della pianta è quello di sopprimere le cascate di trasduzione del segnale MAP-chinasiche, che sono coinvolte nella risposta di difesa sia durante la difesa basale che durante la risposta ipersensibile, nella quale sono responsabili in particolare dell’attivazione della morte cellulare. Le cascate di trasduzione chinasiche si compongono tipicamente di tre diverse chinasi, MAPKKK, MAPKK e MAPK coinvolte nell’attivazione di specifici target molecolari. L’effettore batterico HopAI1 di Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) media la defosforilazione irreversibile delle chinasi AtMPK3, AtMPK4 e AtMPK6 di Arabidopsis grazie alla sua attività fosfotreonina liasica, ed è il solo effettore in grado di spegnere la cascata MAP-chinasica agendo direttamente sulle MAPKs. In Pseudomonas DC3000 l’espressione del gene che codifica per HopAI1 è abolita a causa di una inserzione di un trasposone. L’espressione eterologa di HopAI1 in piante di Arabidopsis thaliana sopprime la risposta di difesa basale indotta dall’elicitore flag22, che richiede l’attivazione di AtMPK3 e AtMPK6, promuovendo la virulenza del patogeno. Nonostante il coinvolgimento delle MAPK anche durante la risposta ipersensibile, dati di letteratura riportano che la morte cellulare indotta nella riposta ipersensibile non viene compromessa in piante infettate con un ceppo avirulento di Pseudomonas fluorescens modificato al fine di esprimere e trasferire nella cellula vegetale anche HopAI1. Similmente, durante la risposta non-host indotta da un ceppo di Pseudomonas in grado di esprimere e rilasciare nella pianta HopAI1, si ha lo sviluppo di una normale morte cellulare, in linea con una tipica risposta non host. Una caratteristica tipica della risposta ipersensibile indotta in piante resistenti è la produzione massiva di ossido nitrico (NO). La S-nitrosilazione è una modifica post-traduzionale delle proteine che consiste nell’attacco di un gruppo NO su di un residuo di cisteina. Questo tipo di modifica post-traduzionale è un importante meccanismo nel signaling dell’ossido nitrico in pianta. Negli animali, la S-nitrosilazione mediata dall’NO prodotto dalla cellula ospite può causare l’inibizione di fattori di virulenza. Pertanto, in questo lavoro, ci siamo occupati di verificare se la S-nitrosilazione di HopAI1 da parte dell’NO potesse essere responsabile dell’inibizione della sua attività durate la risposta ipersensibile. Per verificare questa ipotesi abbiamo prima dimostrato che HopAI1 è effettivamente S-nitrosilato in vitro in seguito a trattamenti con il donatore di ossido nitrico GSNO. Inoltre, GSNO influenza l’attività di HopAI1 inibendola. Una mutazione dell’unica cisteina presente nella sequenza di HopAI1, alla posizione 138, produce una proteina insensibile alla S-nitrosilazione e all’inibizione dell’attività da parte del GSNO (HopAI1CS), confermando che l’ossido nitrico blocca l’attività di HopAI1 in vitro mediante S-nitrosilazione di questo residuo. Infine, modelling tridimensionale della proteina in presenza o assenza della modifica post-traduzionale supporta la possibilità che la S-nitrosilazione modifichi la distribuzione del potenziale elettrostatico in HopAI1, probabilmente influenzando la sua capacità di legame del substrato. Allo scopo di caratterizzare la possibile modulazione dell’attività di HopAI1 da parte dell’NO in vivo, abbiamo utilizzato quindi un sistema precedente caratterizzato, che consiste nell’induzione di una morte cellulare di tipo ipersensibile in piante di tabacco mediante espressione transiente di MKKs costitutivamente attive. La co-espressione di HopAI1 e della sua forma mutata HopAI1CS assieme alle MAP-chinasi costitutivamente attive AtMKK4 e AtMKK5 è in grado di inibire la morte cellulare ipersensibile indotta dalle MKKs attive. L’ossido nitrico blocca l’inibizione della morte cellulare mediata da HopAI1, suggerendo che l’ossido nitrico è in grado di modulare l’attività di HopAI1 anche in vivo. Al contrario, l’ossido nitrico non ha effetto sulla inibizione della morte cellulare mediate da HopAI1CS, dimostrando così che l’effetto dell’ossido nitrico dipende dalla presenza della cisteina 138, e pertanto è probabilmente dovuto alla S-nitrosilazione di HopAI1 come in vitro. Ceppi batterici avirulenti di Pst DC3000 che portano il fattore di avirulenza avrB e modificati al fine di esprimere HopAI1 o HopAI1CS sono risultati non utilizzabili per analisi in vivo in questo studio, date che la presenza di questo transgene sembra influenzare negativamente la crescita batterica. Pertanto linee transgeniche di Arabidopsis thaliana esprimenti HopAI1 o HopAI1CS sono state utilizzate, come alternativa, per investigare il possibile ruolo della S-nitrosilazione di HopAI1 in vivo durante la risposta ipersensibile. Mentre piante esprimenti HopAI1 presentano normali sintomi di morte cellulare indotta da Pst DC3000 avrRpt2 rispetto a piante controllo, piante esprimenti HopAI1CS mostrano una forte riduzione nella morte cellulare, suggerendo che HopAI1 può essere inattivato durante la risposta ipersensibile mediante un meccanismo che dipende specificamente dalla presenza della cisteina 138. Contrariamente all’attesa, tuttavia, le differenze osservate in piante esprimenti HopAI1 o HopAI1CS relativamente alla morte cellulare non sembrano tradursi in una effettiva differenza nella cinetica della crescita batterica in pianta. Nel complesso i nostri risultati suggeriscono che, in accordo con quanto osservato in vitro, HopAI1 possa essere inibito mediante S-nitrosilazione in vivo durante la risposta ipersensibile, permettendo lo sviluppo della morte cellulare mediata dalle MAPK. In conclusione, i nostri dati mostrano, per la prima volta in pianta, che l’ossido nitrico prodotto durante la risposta ipersensibile indotta da un patogeno avirulento non solo contribuisce alla trasduzione del segnale durante la difesa della pianta e a modulare l’espressione genica, ma partecipa attivamente contribuendo alla soppressione della virulenza degli effettori rilasciati dal patogeno durante l’infezione, favorendo la normale attivazione dei meccanismi di resistenza della pianta. La disattivazione dell’attività dell’effettore batterico HopAI1 mediante S-nitrosilazione rappresenta pertanto un meccanismo innovativo in pianta per la soppressione dell’attività di effettori fitopatogenici, in accordo con meccanismi già riportato nel caso di patogeni animali di tipo virale e batterico.
Active resistance of plants against potentially pathogenic microorganisms is composed of two levels of defense. The first level of resistance named PAMP-triggered immunity (PTI) is activated by general elicitors and corresponds to basal plant defense. The second one, which is race/cultivar specific, is activated by avirulent factors released by the pathogen. Their recognition by specific resistance proteins from host cells induce the so-called hypersensitive response (HR) which is characterized by cell death localized at the site of infection. To counteract such active resistance and to promote virulence many Gram-negative phytopathogenic bacteria deliver effector proteins into host cells to modulate the host signaling machinery and suppress plant defense. One of the mechanisms employed by bacterial pathogen effectors to impair active plant defense is to suppress the activity of MAPK cascades, which play a key role in the establishment of plant resistance to pathogens both during PTI and the HR, in which they are in particular involved in cell death activation. MAPK modules are typically composed of three different protein kinases, MAPKKK, MAPKK and MAPK, involved in a phosphorelay to promote the activation of specific targets. The effector HopAI1 from the model bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) been shown to mediate the irreversible dephosphorylation of the Arabidopsis MAPKs AtMPK3, AtMPK4 and AtMPK6 by its phosphothreonine lyase activity, and is the only effector able to switch off MAPK cascade by directly targeting MAPKs. In Pst DC3000 the gene encoding for HopAI1 belongs to a disrupted operon in which a transposon insertion is predicted to abolish its expression. According to its capacity to block irreversibly MAPK activity, the heterolougus expression of HopAI1 in Arabidopsis thaliana plants suppresses PTI induced by the elicitor flg22 that relies on AtMPK3/AtMPK6 activation, finally promoting pathogen virulence. Interestingly, despite the involvement of MAPK cascades also during the HR, it has been reported in the literature that hypersensitive cell death is not affected in plants infected with an avirulent Pseudomonas fluorescens strain modified to express and deliver HopAI1. Similarly the non-host resistance process induced by another Pseudomonas strain also expressing and delivering HopAI1, which typically leads to HR-like cell death, has been shown also to be not affected by the presence of HopAI1. This suggests that MAPK activation occurs normally in these plants despite the presence of HopAI1, and thus that during the HR the activity of HopAI1 could be inhibited by host cells to allow plant defense establishment. One typical feature of the HR induced in resistant plants is the massive production of nitric oxide (NO). S-nitrosylation, a post-translational modification of proteins which consists in the attachment of a NO moiety on Cys residues, has been suggested to be the most important mechanism for transduction of the NO bioactivity in plants. In animal field S-nitrosylation mediated by NO produced by host cells can cause the inhibition of virulence factors. Therefore, in this work we have investigated whether S-nitrosylation of HopAI1 by NO could be responsible for the inhibition of its activity during the HR. To prove this hypothesis we first demonstrated that HopAI1 is S-nitrosylated in vitro by the NO donor GSNO, in a dose-dependent manner. Moreover NO-treatment dramatically decreases HopAI1 activity. Mutation of the unique Cys present in the sequence of HopAI1 at position 138 (HopAI1CS) resulted in a protein insensitive to S-nitrosylation and to the inhibition by GSNO, confirming that NO blocks HopAI1 activity in vitro by S-nitrosylation at this residue. By building a 3D structure model in presence and absence of S-NO at Cys138 we showed that S-nitrosylation significantly modifies the electrostatic potential distribution in HopAI1 structure likely leading to a reduction of its binding property with the substrate. In order to characterize the possible modulation of HopAI1 activity by NO in vivo we first used a previously characterized system that consists in the induction of an HR-like cell death in tobacco plants by transiently expressing constitutively active MKKs. The co-expression of HopAI1 or the mutated HopAI1CS together with the constitutively active AtMKK4 and AtMKK5 inhibits the HR-like cell death induced by active MKKs. Interestingly, NO is able to revert HopAI1-mediated cell death inhibition, suggesting that NO can block HopAI1 activity also in vivo. On the opposite, NO has no effect on the inhibition of the cell death mediated by HopAI1CS, demonstrating therefore that the effect of NO is dependent on the presence of Cys 138, and thus likely due to the S-nitrosylation of HopAI1 as observed in vitro. An avirulent bacterial strain of Pst DC3000 carrying avrB and expressing HopAI1 or HopAI1CS proteins could not be used for in vivo analysis as the presence of the plasmids carrying HopAI1and HopAI1CS affected bacterial growth. Therefore transgenic Arabidopsis thaliana lines expressing HopAI1 and HopAI1CS were used to further enquire the possible role of HopAI1 S-nitrosylation in vivo during the HR. While plants expressing HopAI1 showed normal cell death symptoms induced by Pst DC3000 avrRpt2 as compared with control plants, plants expressing HopAI1CS showed strongly reduced cell death symptoms, suggesting that HopAI1 can be inactivated during the HR by a mechanism which is dependent on the presence of the Cys138. Unexpectedly no difference in bacterial growth was observed between the different plant lines expressing HopAI1 or HopAI1CS. Taken together our results strongly support the possibility that in agreement with the data obtained in vitro HopAI1 could be inhibited by S-nitrosylation in vivo during the HR, therefore allowing MAPK-mediated cell death development. In summary, our data demonstrate that NO produced during the HR induced by an avirulent pathogen, not only contributes to defense signal transduction and defense gene expression, but also participates in suppressing virulence activity of the effectors released by the pathogen during the infection in order to ensure plant resistance. HopAI1 S-nitrosylation would thus represent a novel mechanism for the suppression of phytopathogen effector activity, as observed in animal pathogens including viruses and bacteria.
Inhibition of HopAI1 activity during hypersensitive disease resistance response by nitric oxide-mediated S-nitrosylation
LING, Tengfang
2012-01-01
Abstract
Active resistance of plants against potentially pathogenic microorganisms is composed of two levels of defense. The first level of resistance named PAMP-triggered immunity (PTI) is activated by general elicitors and corresponds to basal plant defense. The second one, which is race/cultivar specific, is activated by avirulent factors released by the pathogen. Their recognition by specific resistance proteins from host cells induce the so-called hypersensitive response (HR) which is characterized by cell death localized at the site of infection. To counteract such active resistance and to promote virulence many Gram-negative phytopathogenic bacteria deliver effector proteins into host cells to modulate the host signaling machinery and suppress plant defense. One of the mechanisms employed by bacterial pathogen effectors to impair active plant defense is to suppress the activity of MAPK cascades, which play a key role in the establishment of plant resistance to pathogens both during PTI and the HR, in which they are in particular involved in cell death activation. MAPK modules are typically composed of three different protein kinases, MAPKKK, MAPKK and MAPK, involved in a phosphorelay to promote the activation of specific targets. The effector HopAI1 from the model bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) been shown to mediate the irreversible dephosphorylation of the Arabidopsis MAPKs AtMPK3, AtMPK4 and AtMPK6 by its phosphothreonine lyase activity, and is the only effector able to switch off MAPK cascade by directly targeting MAPKs. In Pst DC3000 the gene encoding for HopAI1 belongs to a disrupted operon in which a transposon insertion is predicted to abolish its expression. According to its capacity to block irreversibly MAPK activity, the heterolougus expression of HopAI1 in Arabidopsis thaliana plants suppresses PTI induced by the elicitor flg22 that relies on AtMPK3/AtMPK6 activation, finally promoting pathogen virulence. Interestingly, despite the involvement of MAPK cascades also during the HR, it has been reported in the literature that hypersensitive cell death is not affected in plants infected with an avirulent Pseudomonas fluorescens strain modified to express and deliver HopAI1. Similarly the non-host resistance process induced by another Pseudomonas strain also expressing and delivering HopAI1, which typically leads to HR-like cell death, has been shown also to be not affected by the presence of HopAI1. This suggests that MAPK activation occurs normally in these plants despite the presence of HopAI1, and thus that during the HR the activity of HopAI1 could be inhibited by host cells to allow plant defense establishment. One typical feature of the HR induced in resistant plants is the massive production of nitric oxide (NO). S-nitrosylation, a post-translational modification of proteins which consists in the attachment of a NO moiety on Cys residues, has been suggested to be the most important mechanism for transduction of the NO bioactivity in plants. In animal field S-nitrosylation mediated by NO produced by host cells can cause the inhibition of virulence factors. Therefore, in this work we have investigated whether S-nitrosylation of HopAI1 by NO could be responsible for the inhibition of its activity during the HR. To prove this hypothesis we first demonstrated that HopAI1 is S-nitrosylated in vitro by the NO donor GSNO, in a dose-dependent manner. Moreover NO-treatment dramatically decreases HopAI1 activity. Mutation of the unique Cys present in the sequence of HopAI1 at position 138 (HopAI1CS) resulted in a protein insensitive to S-nitrosylation and to the inhibition by GSNO, confirming that NO blocks HopAI1 activity in vitro by S-nitrosylation at this residue. By building a 3D structure model in presence and absence of S-NO at Cys138 we showed that S-nitrosylation significantly modifies the electrostatic potential distribution in HopAI1 structure likely leading to a reduction of its binding property with the substrate. In order to characterize the possible modulation of HopAI1 activity by NO in vivo we first used a previously characterized system that consists in the induction of an HR-like cell death in tobacco plants by transiently expressing constitutively active MKKs. The co-expression of HopAI1 or the mutated HopAI1CS together with the constitutively active AtMKK4 and AtMKK5 inhibits the HR-like cell death induced by active MKKs. Interestingly, NO is able to revert HopAI1-mediated cell death inhibition, suggesting that NO can block HopAI1 activity also in vivo. On the opposite, NO has no effect on the inhibition of the cell death mediated by HopAI1CS, demonstrating therefore that the effect of NO is dependent on the presence of Cys 138, and thus likely due to the S-nitrosylation of HopAI1 as observed in vitro. An avirulent bacterial strain of Pst DC3000 carrying avrB and expressing HopAI1 or HopAI1CS proteins could not be used for in vivo analysis as the presence of the plasmids carrying HopAI1and HopAI1CS affected bacterial growth. Therefore transgenic Arabidopsis thaliana lines expressing HopAI1 and HopAI1CS were used to further enquire the possible role of HopAI1 S-nitrosylation in vivo during the HR. While plants expressing HopAI1 showed normal cell death symptoms induced by Pst DC3000 avrRpt2 as compared with control plants, plants expressing HopAI1CS showed strongly reduced cell death symptoms, suggesting that HopAI1 can be inactivated during the HR by a mechanism which is dependent on the presence of the Cys138. Unexpectedly no difference in bacterial growth was observed between the different plant lines expressing HopAI1 or HopAI1CS. Taken together our results strongly support the possibility that in agreement with the data obtained in vitro HopAI1 could be inhibited by S-nitrosylation in vivo during the HR, therefore allowing MAPK-mediated cell death development. In summary, our data demonstrate that NO produced during the HR induced by an avirulent pathogen, not only contributes to defense signal transduction and defense gene expression, but also participates in suppressing virulence activity of the effectors released by the pathogen during the infection in order to ensure plant resistance. HopAI1 S-nitrosylation would thus represent a novel mechanism for the suppression of phytopathogen effector activity, as observed in animal pathogens including viruses and bacteria.File | Dimensione | Formato | |
---|---|---|---|
Thesis-Tengfang Ling1-7.pdf
accesso aperto
Tipologia:
Tesi di dottorato
Licenza:
Dominio pubblico
Dimensione
3.14 MB
Formato
Adobe PDF
|
3.14 MB | Adobe PDF | Visualizza/Apri |
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.