Proteolytic proteoforms : elusive components of hormonal pathways?

Hormonal pathways often converge on transcriptional repressors that can be degraded by the proteasome to initiate a response. We wish to draw attention to developments in a less-explored proteolytic branch called 'limited proteolysis' that, in addition to the classical proteolytic pathways, seems to regulate auxin and ethylene signaling

Molecular architecture of type III secretion system proteins from Pseudomonas syringae

Type III secretion system (T3SS) is a conserved in structure and function macromolecular multiprotein complex, built from over 20 proteins encoded by conserved pathogenicity islands (PAIs) found in many Gram negative bacteria with known pathogenic, symbiotic or commensal associations with eykaryotic organisms. Νine of the T3SS proteins are similar to proteins of the flagellar basal body, which reflects the distant evolutionary relationship between these two systems. The present study concerns the characterization of the T3S protein interaction network in the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola, using new molecular and biophysical methods. A bicistronic plasmid vector, pPROPET, was used for the heterologous co-expression of TTSS proteins in E.coli. For the HrpG protein, the genomic location next to the gene coding for the HrcC secretin suggested that it is a putative class I T3SS chaperone and the protein was isolated as a complex with the regulatory protein HrpJ, after co-expression in E. coli. The HrpG, HrpV and HrpJ proteins were also isolated as a ternary complex. The binding region of HrpV and HrpJ on HrpG appear to be overlapping. The HrpG-HrpJ interaction provides a missing link in the T3SS regulatory network which so far involved proteins HrpG, HrpV, HrpS & HrpR and HrpL, coded by the hrp/hrc PAI. Further, HrpG was shown to interact with the carboxy-terminal, cytoplasmic domain of the transmembrane core protein HrcU and, less strongly, with the cytoplasmic protein HrpO. The interactions with predicted components of the P.syringae T3SS Cring further support a T3SS chaperone role for protein HrpG. Moreover, a complex between the T3SS ATPase, HrcN, and its negative regulator HrpE was isolated at pH 11 but not at pH 8 from E. coli overexpressing the two proteins. Gel filtration experiments revealed two distinct forms of the complex, each with different apparent molecular size and different subunit stoichiometry in gel filtration experiments. The physicochemical properties of selected T3SS soluble proteins were studied at pH 8 & 11 by gel filtration, small-angle X-ray scattering and circular dichroism methods. These studies included the following proteins: a) harpin (HrpZ), b) HrpP (putative substrate specificity switch protein, by analogy to the Yersinia YscP and the flagellum FliK proteins), c) HrpE (negative regulator of HrcN ATPase) and its carboxy-terminal domain and d) the HrpG-HrpV complex, isolated after coexpression from the bicistronic vector pPROPET. Extreme alkaline pH conditions: a) resulted in the solubilization of large aggregates of the co-expressed proteins, b) promoted a more extended form for secreted proteins HrpZ, HrpP without disturbing the homodimer formation, c) disrupted the putative HrpE homotetramer, favoring its dimeric form, d) did not disrupt the HrcN-HrpE complex, e) lead to a reduction of the α-helix content in favor of β-strand in all cases, although it did not result in a collapse of the secondary structure, f) had a little or no effect on the tertiary structure of the proteins investigated, as judged by circular dichroism studies in near UV, g) reduced the thermal stability for almost all proteins examined, as judged by the reduction in the TM value during thermal denaturation and h) did not lead to formation of intermediates during the thermal transition from the folded to the unfolded state for any of the proteins investigated.Το εκκριτικό σύστημα τύπου ΙΙΙ (Τ3SS, type three secretion system) είναι ένα δομικά και λειτουργικά συντηρημένο υπερμοριακό σύμπλοκο δομούμενο από περισσότερες από 20 διαφορετικές πρωτεΐνες που κωδικοποιούνται από συντηρημένες γονιδιακές «νησίδες» οι οποίες απαντώνται σε πολλά αρνητικά κατά Gram βακτήρια, κατά κανόνα είτε συμβιωτικά είτε παθογόνα ευκαρυωτικών οργανισμών. Εννέα από τις πρωτεΐνες του Τ3SS έχουν σημαντικό βαθμό ομολογίας, σε επίπεδο πρωτοταγούς αλληλουχίας, με πρωτεΐνες που δομούν στοιχεία του βασικού σωματίου του βακτηριακού μαστιγίου, με το οποίο το Τ3SS έχει κοινές εξελικτικές ρίζες. Στην παρούσα διατριβή μελετήθηκε το δίκτυο αλληλεπιδράσεων μεταξύ πρωτεϊνών που δομούν το T3SS του φυτοπαθογόνου βακτηρίου Pseudomonas syringae pv. phaseolicola, αξιοποιώντας κυρίως ένα δικιστρονικό πλασμιδιακό φορέα, τον pPROPET ο οποίος κατασκευάστηκε στην πορεία της διατριβής, για την ετερόλογη συνέκφραση πρωτεϊνών στην Escherichia coli και συγκαθαρισμό συμπλόκων μεταξύ επιλεγμένων ζευγών πρωτεϊνών. Για την πρωτεΐνη HrpG, η θέση του κωδικού της πλαισίου σε σχέση με της σεκρετίνης HrcC υπέδειξε ρόλο πιθανήs σαπερόνηs τάξης Ι. Περεταίρω ένδειξη ενός τέτοιου ρόλου προέκυψε από την κατάδειξη αλληλεπίδρασης της με τη ρυθμιστική του συστήματος πρωτεΐνη HrpJ μετά από συνέκφραση και συγκαθαρισμό σε ετερόλογο βακτηριακό σύστημα. Οι πρωτεΐνες HrpG-HrpV-HrpJ ανιχνεύτηκαν ως σύμπλοκο μετά από συνέκφραση από τρικιστρονική κασέτα στην E. coli. Οι θέσεις πρόσδεσης των HrpV και HrpJ επί της HrpG πιθανόν να είναι αλληλεπικαλυπτόμενες. Η αλληλεπίδραση HrpG-HrpJ συμπληρώνει τον ελλειπή κρίκο της αλυσίδας πρωτεϊνικών αλληλεπιδράσεων που εμπλέκονται στη μεταγραφική ρύθμιση του TTSS, στην οποία, σύμφωνα με προηγούμενες μελέτες, συμμετέχουν οι πρωτεΐνες HrpG, HrpV, HrpS, HrpR και ΗrpL που κωδικοποιούνται από τη νησίδα hrp/hrc. Πειράματα συνέκφρασης/συγκαθαρισμού έδειξαν ομοίως ότι η πρωτεΐνη HrpG αλληλεπιδρά ισχυρά με την καρβοξυτελική κυτταροπλασματική επικράτεια της μεμβρανικής πρωτεΐνης HrcU, συστατικού του κεντρικού πυρήνα του ενυοσώματος, και λιγότερο ισχυρά με την κυτταροπλασματική πρωτεΐνη HrpO. Οι αλληλεπιδράσεις της HrpG με μέλη του C-δακτυλίου υποστηρίζουν τον ρόλο σαπερόνης του T3SS για την πρωτεΐνη. Συμπληρωματικά, απομονώθηκε μετά από ετερόλογη έκφραση το σύμπλοκο HrcN-HrpE, ATPάσης του Τ3SS και αρνητικού ρυθμιστή της αντίστοιχα, αλλά μόνο σε ακραία αλκαλικό pH. Πειράματα μοριακής διήθησης κατέδειξαν την ύπαρξη δυο διαφορετικών μορφών του συμπλόκου HrcNHrpE με διαφορετική μοριακή μάζα και πιθανή στοιχειομετρία. Οι φυσικοχημικές ιδιότητες μιας σειράς επιλεγμένων διαλυτών πρωτεϊνών και πρωτεϊνικών συμπλόκων του T3SS της P. syringae pv. phaseolicola μελετήθηκαν σε τιμές pΗ 8 και 11 με μεθόδους μοριακής διήθησης, σκέδασης ακτίνων Χ σε μικρές γωνίες (SAXS) και κυκλικού διχρωισμού (CD): Οι πρωτεΐνες που μελετήθηκαν ήταν: α) η χαρπίνη HrpZ, β) η πρωτεΐνη HrpP, φερόμενη ως διακόπτης αλλαγής ειδίκευσης έκκρισης υποστρώματος κατ’ αναλογία με τις ανάλογές της πρωτεΐνες YscP στη Yersinia και FliK του μαστιγιακού TTSS, γ) η πρωτεΐνη HrpE (αρνητικός ρυθμιστής της ATPάσης του συστήματος HrcN) και η καρβοξυτελική της επικράτεια, ξεχωριστά και δ) το σύμπλοκο HrpG-HrpV, απομονωμένο μετά από υπερέκφραση από τον αντίστοιχο δικιστρονικό φορέα. Το ακραίο αλκαλικό pΗ: α) προώθησε την αποσυσσωμάτωση των μεγαλομοριακών φυσικών πληθυσμών των πρωτεϊνών, όπως εκτιμήθηκε από την υποχώρηση του εντοπιζόμενου στο νεκρό όγκο των στηλών μοριακής διήθησης ποσοστού της κάθε πρωτεΐνης, β) οδήγησε στην εκτεταμένη διαμόρφωση των εκκρινόμενων πρωτεϊνών HrpZ, HrpP αφήνοντας άθικτα τα διμερή τους, γ) διέσπασε το πιθανό τετραμερές της πρωτεΐνης HrpE στην κατάσταση πιθανώς του διμερούς, δ) δεν διέσπασε το σύμπλοκο HrcN-HrpE, ε) οδήγησε σε μείωση των ποσοστών α-έλικας προς όφελος των ποσοστών β-κλώνων σε όλες τις περιπτώσεις αλλά σε καμία περίπτωση δεν επέφερε κατάρρευση της πρωτεϊνικής δευτεροταγούς δομής, στ) είχε μικρή ή καμία επίδραση στην τριτοταγή διαμόρφωση των υπό εξέταση μορίων, όπως έδειξαν μελέτες κυκλικού διχρωισμού στο εγγύς υπεριώδες ζ) κατά κανόνα οδήγησε σε μείωση της θερμοσταθερότητας των εξετασθέντων πρωτεϊνών, όπως αυτή εκφράζεται από την μείωση του σημείου μετάπτωσης (ΤΜ), και η) σε καμία περίπτωση δεν οδήγησε σε εμφάνιση ενδιάμεσων καταστάσεων κατά τη μετάβαση των μορίων από την αναδιπλωμένη στην αποδιπλωμένη μορφή

The RsRlpA Effector Is a Protease Inhibitor Promoting Rhizoctonia solani Virulence through Suppression of the Hypersensitive Response

Rhizoctonia solani (Rs) is a soil-borne pathogen with a broad host range. This pathogen incites a wide range of disease symptoms. Knowledge regarding its infection process is fragmented, a typical feature for basidiomycetes. In this study, we aimed at identifying potential fungal effectors and their function. From a group of 11 predicted single gene effectors, a rare lipoprotein A (RsRlpA), from a strain attacking sugar beet was analyzed. The RsRlpA gene was highly induced upon early-stage infection of sugar beet seedlings, and heterologous expression in Cercospora beticola demonstrated involvement in virulence. It was also able to suppress the hypersensitive response (HR) induced by the Avr4/Cf4 complex in transgenic Nicotiana benthamiana plants and functioned as an active protease inhibitor able to suppress Reactive Oxygen Species (ROS) burst. This effector contains a double-psi beta-barrel (DPBB) fold domain, and a conserved serine at position 120 in the DPBB fold domain was found to be crucial for HR suppression. Overall, R. solani seems to be capable of inducing an initial biotrophic stage upon infection, suppressing basal immune responses, followed by a switch to necrotrophic growth. However, regulatory mechanisms between the different lifestyles are still unknown

Structural Insights of Shigella Translocator IpaB and Its Chaperone IpgC in Solution

International audienceBacterial Type III Secretion Systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or deliver Type III Secretion effectors directly into eukaryotic host cell cytoplasm. Shigella, the causing agent of bacillary dysentery or shigellosis, bears a set of T3SS proteins termed translocators that form a pore in the host cell membrane. IpaB, the major translocator of the system, is a key factor in promoting Shigella pathogenicity. Prior to secretion, IpaB is maintained inside the bacterial cytoplasm in a secretion competent folding state thanks to its cognate chaperone IpgC. IpgC couples T3SS activation to transcription of effector genes through its binding to MxiE, probably after the delivery of IpaB to the secretion export gate. Small Angle X-ray Scattering experiments and modeling reveal that IpgC is found in different oligomeric states in solution, as it forms a stable heterodimer with fulllength IpaB in contrast to an aggregation-prone homodimer in the absence of the translocator. These results support a stoichiometry of interaction 1:1 in the IpgC/IpaB complex and the multi-functional nature of IpgC under different T3SS states

Phylogenetic analysis of a gene cluster encoding an additional, rhizobial-like type III secretion system that is narrowly distributed among Pseudomonas syringae strains

International audienceBackgroundThe central role of Type III secretion systems (T3SS) in bacteria-plant interactions is well established, yet unexpected findings are being uncovered through bacterial genome sequencing. Some Pseudomonas syringae strains possess an uncharacterized cluster of genes encoding putative components of a second T3SS (T3SS-2) in addition to the well characterized Hrc1 T3SS which is associated with disease lesions in host plants and with the triggering of hypersensitive response in non-host plants. The aim of this study is to perform an in silico analysis of T3SS-2, and to compare it with other known T3SSs.ResultsBased on phylogenetic analysis and gene organization comparisons, the T3SS-2 cluster of the P. syringae pv. phaseolicola strain is grouped with a second T3SS found in the pNGR234b plasmid of Rhizobium sp. These additional T3SS gene clusters define a subgroup within the Rhizobium T3SS family. Although, T3SS-2 is not distributed as widely as the Hrc1 T3SS in P. syringae strains, it was found to be constitutively expressed in P. syringae pv phaseolicola through RT-PCR experiments.ConclusionsThe relatedness of the P. syringae T3SS-2 to a second T3SS from the pNGR234b plasmid of Rhizobium sp., member of subgroup II of the rhizobial T3SS family, indicates common ancestry and/or possible horizontal transfer events between these species. Functional analysis and genome sequencing of more rhizobia and P. syringae pathovars may shed light into why these bacteria maintain a second T3SS gene cluster in their genome

The Role of Proteases in Determining Stomatal Development and Tuning Pore Aperture: A Review

Plant proteases, the proteolytic enzymes that catalyze protein breakdown and recycling, play an essential role in a variety of biological processes including stomatal development and distribution, as well as, systemic stress responses. In this review, we summarize what is known about the participation of proteases in both stomatal organogenesis and on the stomatal pore aperture tuning, with particular emphasis on their involvement in numerous signaling pathways triggered by abiotic and biotic stressors. There is a compelling body of evidence demonstrating that several proteases are directly or indirectly implicated in the process of stomatal development, affecting stomatal index, density, spacing, as well as, size. In addition, proteases are reported to be involved in a transient adjustment of stomatal aperture, thus orchestrating gas exchange. Consequently, the proteases-mediated regulation of stomatal movements considerably affects plants' ability to cope not only with abiotic stressors, but also to perceive and respond to biotic stimuli. Even though the determining role of proteases on stomatal development and functioning is just beginning to unfold, our understanding of the underlying processes and cellular mechanisms still remains far from being completed

Phylogenetic analysis of a gene cluster encoding an additional, rhizobial-like type III secretion system that is narrowly distributed among <it>Pseudomonas syringae</it> strains

Abstract Background The central role of Type III secretion systems (T3SS) in bacteria-plant interactions is well established, yet unexpected findings are being uncovered through bacterial genome sequencing. Some Pseudomonas syringae strains possess an uncharacterized cluster of genes encoding putative components of a second T3SS (T3SS-2) in addition to the well characterized Hrc1 T3SS which is associated with disease lesions in host plants and with the triggering of hypersensitive response in non-host plants. The aim of this study is to perform an in silico analysis of T3SS-2, and to compare it with other known T3SSs. Results Based on phylogenetic analysis and gene organization comparisons, the T3SS-2 cluster of the P. syringae pv. phaseolicola strain is grouped with a second T3SS found in the pNGR234b plasmid of Rhizobium sp. These additional T3SS gene clusters define a subgroup within the Rhizobium T3SS family. Although, T3SS-2 is not distributed as widely as the Hrc1 T3SS in P. syringae strains, it was found to be constitutively expressed in P. syringae pv phaseolicola through RT-PCR experiments. Conclusions The relatedness of the P. syringae T3SS-2 to a second T3SS from the pNGR234b plasmid of Rhizobium sp., member of subgroup II of the rhizobial T3SS family, indicates common ancestry and/or possible horizontal transfer events between these species. Functional analysis and genome sequencing of more rhizobia and P. syringae pathovars may shed light into why these bacteria maintain a second T3SS gene cluster in their genome.</p

Polyamine homeostasis in tomato biotic/abiotic stress cross-tolerance

Adverse conditions and biotic strain can lead to significant losses and impose limitations on plant yield. Polyamines (PAs) serve as regulatory molecules for both abiotic/biotic stress responses and cell protection in unfavourable environments. In this work, the transcription pattern of 24 genes orchestrating PA metabolism was investigated in Cucumber Mosaic Virus or Potato Virus Y infected and cold stressed tomato plants. Expression analysis revealed a differential/pleiotropic pattern of gene regulation in PA homeostasis upon biotic, abiotic or combined stress stimuli, thus revealing a discrete response specific to diverse stimuli: (i) biotic stress-influenced genes, (ii) abiotic stress-influenced genes, and (iii) concurrent biotic/abiotic stress-regulated genes. The results support different roles for PAs against abiotic and biotic stress. The expression of several genes, significantly induced under cold stress conditions, is mitigated by a previous viral infection, indicating a possible priming-like mechanism in tomato plants pointing to crosstalk among stress signalling. Several genes and resulting enzymes of PA catabolism were stimulated upon viral infection. Hence, we suggest that PA catabolism resulting in elevated H2O2 levels could mediate defence against viral infection. However, after chilling, the activities of enzymes implicated in PA catabolism remained relatively stable or slightly reduced. This correlates to an increase in free PA content, designating a per se protective role of these compounds against abiotic stress