scholarly journals The hrpK Operon of Pseudomonas syringae pv. tomato DC3000 Encodes Two Proteins Secreted by the Type III (Hrp) Protein Secretion System: HopB1 and HrpK, a Putative Type III Translocator

2005 ◽  
Vol 187 (2) ◽  
pp. 649-663 ◽  
Author(s):  
Tanja Petnicki-Ocwieja ◽  
Karin van Dijk ◽  
James R. Alfano
Keyword(s):  
Pseudomonas Syringae ◽  
Protein Secretion ◽  
Xanthomonas Campestris ◽  
Transmembrane Domain ◽  
Plant Cells ◽  
Secretion System ◽  
Transcriptional Unit ◽  
Tomato Dc3000 ◽  
Type Iii ◽  
Protein Secretion System

ABSTRACT Pseudomonas syringae is a gram-negative bacterial plant pathogen that is dependent on a type III protein secretion system (TTSS) and the effector proteins it translocates into plant cells for pathogenicity. The P. syringae TTSS is encoded by hrp-hrc genes that reside in a central region of a pathogenicity island (Pai). Flanking one side of this Pai is the exchangeable effector locus (EEL). We characterized the transcriptional expression of the open reading frames (ORFs) within the EEL of P. syringae pv. tomato DC3000. One of these ORFs, PSPTO1406 (hopB1) is expressed in the same transcriptional unit as hrpK. Both HopB1 and HrpK were secreted in culture and translocated into plant cells via the TTSS. However, the translocation of HrpK required its C-terminal half. HrpK shares low similarity with a putative translocator, HrpF, from Xanthomonas campestris pv. vesicatoria. DC3000 mutants lacking HrpK were significantly reduced in disease symptoms and multiplication in planta, whereas DC3000 hopB1 mutants produced phenotypes similar to the wild type. Additionally, hrpK mutants were reduced in their ability to elicit the hypersensitive response (HR), a programmed cell death associated with plant defense. The reduced HR phenotype exhibited by hrpK mutants was complemented by hrpK expressed in bacteria but not by HrpK transgenically expressed in tobacco, suggesting that HrpK does not function inside plant cells. Further experiments identified a C-terminal transmembrane domain within HrpK that is required for HrpK translocation. Taken together, HopB1 is a type III effector and HrpK plays an important role in the TTSS and is a putative type III translocator.

scholarly journals The Pseudomonas syringae HopPtoV Protein Is Secreted in Culture and Translocated into Plant Cells via the Type III Protein Secretion System in a Manner Dependent on the ShcV Type III Chaperone

2004 ◽  
Vol 186 (11) ◽  
pp. 3621-3630 ◽  
Author(s):  
Misty D. Wehling ◽  
Ming Guo ◽  
Zheng Qing Fu ◽  
James R. Alfano
Keyword(s):  
Pseudomonas Syringae ◽  
Protein Secretion ◽  
Plant Cells ◽  
Secretion System ◽  
Effector Proteins ◽  
In Planta ◽  
Type Iii ◽  
Protein Secretion System ◽  
Type Iii Protein Secretion ◽  
Plant Pathogenesis

ABSTRACT The bacterial plant pathogen Pseudomonas syringae depends on a type III protein secretion system and the effector proteins that it translocates into plant cells to cause disease and to elicit the defense-associated hypersensitive response on resistant plants. The availability of the P. syringae pv. tomato DC3000 genome sequence has resulted in the identification of many novel effectors. We identified the hopPtoV effector gene on the basis of its location next to a candidate type III chaperone (TTC) gene, shcV, and within a pathogenicity island in the DC3000 chromosome. A DC3000 mutant lacking ShcV was unable to secrete detectable amounts of HopPtoV into culture supernatants or translocate HopPtoV into plant cells, based on an assay that tested whether HopPtoV-AvrRpt2 fusions were delivered into plant cells. Coimmunoprecipitation and Saccharomyces cerevisiae two-hybrid experiments showed that ShcV and HopPtoV interact directly with each other. The ShcV binding site was delimited to an N-terminal region of HopPtoV between amino acids 76 and 125 of the 391-residue full-length protein. Our results demonstrate that ShcV is a TTC for the HopPtoV effector. DC3000 overexpressing ShcV and HopPtoV and DC3000 mutants lacking either HopPtoV or both ShcV and HopPtoV were not significantly impaired in disease symptoms or bacterial multiplication in planta, suggesting that HopPtoV plays a subtle role in pathogenesis or that other effectors effectively mask the contribution of HopPtoV in plant pathogenesis.

scholarly journals PopF1 and PopF2, Two Proteins Secreted by the Type III Protein Secretion System of Ralstonia solanacearum, Are Translocators Belonging to the HrpF/NopX Family

2006 ◽  
Vol 188 (13) ◽  
pp. 4903-4917 ◽  
Author(s):  
Damien Meyer ◽  
Sébastien Cunnac ◽  
Mareva Guéneron ◽  
Céline Declercq ◽  
Frédérique Van Gijsegem ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Ralstonia Solanacearum ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Secretion System ◽  
Effector Proteins ◽  
Experimental Conditions ◽  
Type Iii ◽  
Protein Secretion System ◽  
Type Iii Protein Secretion

ABSTRACT Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum.

scholarly journals The Xanthomonas campestris pv. vesicatoria Type III Effector Protein XopJ Inhibits Protein Secretion: Evidence for Interference with Cell Wall–Associated Defense Responses

2009 ◽  
Vol 22 (6) ◽  
pp. 655-664 ◽  
Author(s):  
Verena Bartetzko ◽  
Sophia Sonnewald ◽  
Florian Vogel ◽  
Kristina Hartner ◽  
Ruth Stadler ◽  
...  
Keyword(s):  
Cell Wall ◽  
Pseudomonas Syringae ◽  
Protein Secretion ◽  
Xanthomonas Campestris ◽  
Plant Cells ◽  
Defense Responses ◽  
Laser Scanning Microscopy ◽  
Phytopathogenic Bacterium ◽  
Type Iii ◽  
Gfp Fluorescence

The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria uses the type III secretion system (T3SS) to inject effector proteins into cells of its Solanaceous host plants. It is generally assumed that these effectors manipulate host pathways to favor bacterial replication and survival. However, the molecular mechanisms by which type III effectors suppress host defense responses are far from being understood. Based on sequence similarity, Xanthomonas outer protein J (XopJ) is a member of the YopJ/AvrRxv family of SUMO peptidases and acetyltranferases, although its biochemical activity has not yet been demonstrated. Confocal laser scanning microscopy revealed that green fluorescent protein (GFP) fusions of XopJ are targeted to the plasma membrane when expressed in plant cells, which most likely involves N-myristoylation. In contrast to a XopJ(C235A) mutant disrupted in the catalytic triad sequence, the wild-type effector GFP fusion protein was also localized in vesicle-like structures colocalizing together with a Golgi marker protein, suggesting an effect of XopJ on vesicle trafficking. To explore an effect of XopJ on protein secretion, we used a GFP-based secretion assay. When a secreted (sec)GFP marker was coexpressed with XopJ in leaves of Nicotiana benthamiana, GFP fluorescence was retained in reticulate structures. In contrast, in plant cells expressing secGFP alone or along with the XopJ(C235A) mutant, no GFP fluorescence accumulated within the cells. Moreover, coexpressing secGFP together with XopJ led to a reduced accumulation of secGFP within the apoplastic fluid of N. benthamiana leaves, further showing that XopJ affects protein secretion. Transgenic expression of XopJ in Arabidopsis suppressed callose deposition elicited by a T3SS-negative mutant of Pseudomonas syringae pv. tomato DC3000. A role of XopJ in the inhibition of cell wall–based defense responses is discussed.

scholarly journals Pseudomonas syringae pv. tomato DC3000 Type III Effector HopAA1-1 Functions Redundantly with Chlorosis-Promoting Factor PSPTO4723 to Produce Bacterial Speck Lesions in Host Tomato

2009 ◽  
Vol 22 (11) ◽  
pp. 1341-1355 ◽  
Author(s):  
Kathy R. Munkvold ◽  
Alistair B. Russell ◽  
Brian H. Kvitko ◽  
Alan Collmer
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Secretion System ◽  
Effector Proteins ◽  
Gtpase Activating Protein ◽  
Tomato Dc3000 ◽  
Bacterial Speck ◽  
Type Iii ◽  
Bacterial Speck Disease

The ability of Pseudomonas syringae pv. tomato DC3000 to cause bacterial speck disease in tomato is dependent on the injection, via the type III secretion system, of approximately 28 Avr/Hop effector proteins. HopAA1-1 is encoded in the conserved effector locus (CEL) of the P. syringae Hrp pathogenicity island. Transiently expressed HopAA1-1 acts inside Saccharomyces cerevisiae and plant cells to elicit cell death. hopAA1 homologs were cloned and sequenced from the CEL of seven P. syringae strains representing diverse pathovars. Analysis of the sequences revealed that HopAA1-1 carries a potential GTPase-activating protein (GAP) domain, GALRA, which is polymorphic (FEN instead of LRA) in HopAA1-2, a paralogous DC3000 effector. Deleting hopAA1-1 from DC3000 reduces the formation of necrotic speck lesions in dip-inoculated tomato leaves if effector-gene cluster IX or just PSPTO4723 within this region has been deleted. A HopAA1-1 mutant in which the putative catalytic arginine in the GAP-like domain has been replaced with alanine retains its ability to kill yeast and promote the formation of speck lesions by the ΔhopAA1-1ΔIX mutant, but a HopAA1-1 mutant carrying the FEN polymorphism loses both of these abilities. Unexpectedly, PSPTO4723 does not appear to encode an effector and its deletion also reduces disease-associated chlorosis.

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