Inhibition of HopAI1 activity during hypersensitive disease resistance response by nitric oxide-mediated S-nitrosylation

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.