Two
            Pantoea agglomerans
            type III effectors can transform nonpathogenic and phytopathogenic bacteria into host‐specific gall‐forming pathogens

HsvG and HsvB, two paralogous type III effectors of the gall-forming bacteria Pantoea agglomerans pv. gypsophilae and P. agglomerans pv. betae, determine host specificity on gypsophila and beet, respectively. They were previously shown to be DNA-binding proteins imported into host and non-host nuclei and might act as transcriptional activators. Sequence analysis of these effectors did not detect canonical nuclear localization signals (NLSs), but two basic amino acid clusters designated putative NLS1 and NLS2 were detected in their N-terminal and C-terminal regions, respectively. pNIA assay for nuclear import in yeast and bombardment of melon leaves with each of the NLSs fused to a 2xYFP reporter indicated that putative NLS1 and NLS2 were functional in transport of HsvG into the nucleus. A yeast two-hybrid assay showed that HsvB, HsvG, putative NLS1, putative NLS2, HsvG converted into HsvB, or HsvB converted into HsvG by exchanging the repeat domain, all interacted with AtKAP-α and importin-α3 of Arabidopsis thaliana. Deletion analysis of the NLS domains in HsvG suggested that putative NLS1 or NLS2 were required for pathogenicity on gypsophila cuttings and presumably for import of HsvG into the nucleus. This study demonstrates the presence of two functional NLSs in the type III effectors HsvG and HsvB.

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Quorum-Sensing System Affects Gall Development Incited by Pantoea agglomerans pv. gypsophilae

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-8-1094 ◽

2008 ◽

Vol 21 (8) ◽

pp. 1094-1105 ◽

Cited By ~ 15

Author(s):

Laura Chalupowicz ◽

Shulamit Manulis-Sasson ◽

Maxim Itkin ◽

Ayelet Sacher ◽

Guido Sessa ◽

...

Keyword(s):

Quorum Sensing ◽

Regulatory Gene ◽

Regulatory System ◽

Homoserine Lactone ◽

Culture Collection ◽

Pantoea Agglomerans ◽

Type Iii Effectors ◽

Mass Spectral ◽

Type Iii ◽

A Minor

The quorum-sensing (QS) regulatory system of the gall-forming Pantoea agglomerans pv. gypsophilae was identified. Mass spectral analysis, together with signal-specific biosensors, demonstrated that P. agglomerans pv. gypsophilae produced N-butanoyl-l-homoserine lactone (C4-HSL) as a major and N-hexanoyl-l-homoserine lactone (C6-HSL) as a minor QS signal. Homologs of luxI and luxR regulatory genes, pagI and pagR, were characterized in strain P. agglomerans pv. gypsophilae Pag824-1 and shown to be convergently transcribed and separated by 14 bp. The deduced PagI (23.8 kDa) and PagR (26.9 kDa) show high similarity with SmaI (41% identity) and SmaR (43% identity), respectively, of Serratia sp. American Type Culture Collection 39006. PagR possesses characteristic autoinducer binding and a helix-turn-helix DNA-binding domain. Gall formation by P. agglomerans pv. gypsophilae depends on a plasmid-borne hrp/hrc gene cluster, type III effectors, and phytohormones. Disruption of pagI, pagR, or both genes simultaneously in Pag824-1 reduced gall size in gypsophila cuttings by 50 to 55% when plants were inoculated with 106 CFU/ml. Higher reductions in gall size (70 to 90%) were achieved by overexpression of pagI or addition of exogenous C4-HSL. Expression of the hrp/hrc regulatory gene hrpL and the type III effector pthG in the pagI mutant, as measured with quantitative reverse-transcriptase polymerase chain reaction, was reduced by 5.8 and 6.6, respectively, compared with the wild type, suggesting an effect of the QS system on the Hrp regulon.

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Elucidating the Functions of Type III Effectors from Necrogenic and Tumorigenic Bacterial Pathogens

10.32747/2010.7592638.bard ◽

2010 ◽

Author(s):

James Alfano ◽

Isaac Barash ◽

Thomas Clemente ◽

Paul E. Staswick ◽

Guido Sessa ◽

...

Keyword(s):

Innate Immunity ◽

Pseudomonas Syringae ◽

Bacterial Pathogens ◽

Functional Redundancy ◽

Pantoea Agglomerans ◽

Gall Formation ◽

Type Iii Effector ◽

Type Iii Effectors ◽

Hormone Levels ◽

Type Iii

Many phytopathogenic bacteria use a type III protein secretion system (T3SS) to inject type III effectors into plant cells. In the experiments supported by this one-year feasibility study we investigated type III effector function in plants by using two contrasting bacterial pathogens: Pseudomonas syringae pv. tomato, a necrotrophic pathogen and Pantoea agglomerans, a tumorigenic pathogen. The objectives are listed below along with our major conclusions, achievements, and implications for science and agriculture. Objective 1: Compare Pseudomonas syringae and Pantoea agglomerans type III effectors in established assays to test the extent that they can suppress innate immunity and incite tumorigenesis. We tested P. agglomerans type III effectors in several innate immunity suppression assays and in several instances these effectors were capable of suppressing plant immunity, outputs that are suppressed by P. syringae effectors. Interestingly, several P. syringae effectors were able to complement gall production to a P. agglomerans pthGmutant. These results suggest that even though the disease symptoms of these pathogens are dramatically different, their type III effectors may function similarly. Objective 2: Construct P. syringae mutants in different combinations of type III-related DNA clusters to reduce type III effector redundancy. To determine their involvement in pathogenicity we constructed mutants that lack individual and multiple type III-related DNA clusters using a Flprecombinase-mediated mutagenesis strategy. The majority of single effector mutants in DC3000 have weak pathogenicity phenotypes most likely due to functional redundancy of effectors. Supporting this idea, Poly-DNAcluster deletion mutants were more significantly reduced in their ability to cause disease. Because these mutants have less functional redundancy of type III effectors, they should help identify P. syringae and P. agglomerans effectors that contribute more significantly to virulence. Objective 3: Determine the extent that P. syringae and P. agglomerans type III effectors alter hormone levels in plants. Inhibition of auxin polar transport by 2,3,5-triiodobenzoic acid (TIBA) completely prevented gall formation by P. agglomerans pv. gypsophilae in gypsophila cuttings. This result supported the hypothesis that auxin and presumably cytokinins of plant origin, rather than the IAA and cytokinins secreted by the pathogen, are mandatory for gall formation. Transgenic tobacco with pthGshowed various phenotypic traits that suggest manipulation of auxin metabolism. Moreover, the auxin levels in pthGtransgenic tobacco lines was 2-4 times higher than the control plants. External addition of auxin or cytokinins could modify the gall size in gypsophila cuttings inoculated with pthGmutant (PagMx27), but not with other type III effectors. We are currently determining hormone levels in transgenic plants expressing different type III effectors. Objective 4: Determine whether the P. agglomerans effectors HsvG/B act as transcriptional activators in plants. The P. agglomerans type III effectors HsvG and HsvB localize to the nucleus of host and nonhost plants and act as transcription activators in yeast. Three sites of adjacent arginine and lysine in HsvG and HsvB were suspected to act as Nuclear localization signals (NLS) domains. A nuclear import assay indicated two of the three putative NLS domains were functional NLSs in yeast. These were shown to be active in plants by fusing HsvG and HsvB to YFP. localization to the nucleus was dependent on these NLS domains. These achievements indicate that our research plan is feasible and suggest that type III effectors suppress innate immunity and modulate plant hormones. This information has the potential to be exploited to improve disease resistance in agricultural crops.

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The type III effectors HsvG and HsvB of gall-forming Pantoea agglomerans determine host specificity and function as transcriptional activators

Molecular Microbiology ◽

10.1111/j.1365-2958.2006.05301.x ◽

2006 ◽

Vol 61 (5) ◽

pp. 1118-1131 ◽

Cited By ~ 40

Author(s):

Gal Nissan ◽

Shulamit Manulis-Sasson ◽

Dan Weinthal ◽

Henia Mor ◽

Guido Sessa ◽

...

Keyword(s):

Host Specificity ◽

Pantoea Agglomerans ◽

Transcriptional Activators ◽

Type Iii Effectors ◽

Type Iii ◽

And Function

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Tracking the Secretion of Fluorescently Labeled Type III Effectors from Single Bacteria in Real Time

Methods in Molecular Biology - Protein Secretion ◽

10.1007/978-1-60327-412-8_14 ◽

2010 ◽

pp. 241-256 ◽

Cited By ~ 3

Author(s):

Nandi Simpson ◽

Laurent Audry ◽

Jost Enninga

Keyword(s):

Real Time ◽

Type Iii Effectors ◽

Type Iii

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Identification of type III effectors modulating the symbiotic properties of Bradyrhizobium vignae strain ORS3257 with various Vigna species

Scientific Reports ◽

10.1038/s41598-021-84205-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pongpan Songwattana ◽

Clémence Chaintreuil ◽

Jenjira Wongdee ◽

Albin Teulet ◽

Mamadou Mbaye ◽

...

Keyword(s):

Positive Impact ◽

Symbiotic Efficiency ◽

Type Iii Effectors ◽

Type Iii ◽

Vigna Species ◽

Symbiotic Properties

AbstractThe Bradyrhizobium vignae strain ORS3257 is an elite strain recommended for cowpea inoculation in Senegal. This strain was recently shown to establish symbioses on some Aeschynomene species using a cocktail of Type III effectors (T3Es) secreted by the T3SS machinery. In this study, using a collection of mutants in different T3Es genes, we sought to identify the effectors that modulate the symbiotic properties of ORS3257 in three Vigna species (V. unguiculata, V. radiata and V. mungo). While the T3SS had a positive impact on the symbiotic efficiency of the strain in V. unguiculata and V. mungo, it blocked symbiosis with V. radiata. The combination of effectors promoting nodulation in V. unguiculata and V. mungo differed, in both cases, NopT and NopAB were involved, suggesting they are key determinants for nodulation, and to a lesser extent, NopM1 and NopP1, which are additionally required for optimal symbiosis with V. mungo. In contrast, only one effector, NopP2, was identified as the cause of the incompatibility between ORS3257 and V. radiata. The identification of key effectors which promote symbiotic efficiency or render the interaction incompatible is important for the development of inoculation strategies to improve the growth of Vigna species cultivated in Africa and Asia.

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The Proteasome Acts as a Hub for Plant Immunity and Is Targeted by Pseudomonas Type III Effectors

PLANT PHYSIOLOGY ◽

10.1104/pp.16.00808 ◽

2016 ◽

Vol 172 (3) ◽

pp. 1941-1958 ◽

Cited By ~ 37

Author(s):

Suayib Üstün ◽

Arsheed Sheikh ◽

Selena Gimenez-Ibanez ◽

Alexandra Jones ◽

Vardis Ntoukakis ◽

...

Keyword(s):

Plant Immunity ◽

Type Iii Effectors ◽

Type Iii

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Genome context influences evolutionary flexibility of nearly identical type III effectors in two phytopathogenic Pseudomonads

10.1101/2021.11.22.469601 ◽

2021 ◽

Author(s):

David A Baltrus ◽

Qian Feng ◽

Brian H Kvitko

Keyword(s):

Pseudomonas Syringae ◽

Evolutionary Dynamics ◽

Effector Proteins ◽

In Planta ◽

Genomic Context ◽

Type Iii Effectors ◽

Type Iii ◽

Genome Context ◽

The Impact ◽

Multiple Type

Integrative Conjugative Elements (ICEs) are replicons that can insert and excise from chromosomal locations in a site specific manner, can conjugate across strains, and which often carry a variety of genes useful for bacterial growth and survival under specific conditions. Although ICEs have been identified and vetted within certain clades of the agricultural pathogen Pseudomonas syringae, the impact of ICE carriage and transfer across the entire P. syringae species complex remains underexplored. Here we identify and vet an ICE (PmaICE-DQ) from P. syringae pv. maculicola ES4326, a strain commonly used for laboratory virulence experiments, demonstrate that this element can excise and conjugate across strains, and contains loci encoding multiple type III effector proteins. Moreover, genome context suggests that another ICE (PmaICE-AOAB) is highly similar in comparison with and found immediately adjacent to PmaICE-DQ within the chromosome of strain ES4326, and also contains multiple type III effectors. Lastly, we present passage data from in planta experiments that suggests that genomic plasticity associated with ICEs may enable strains to more rapidly lose type III effectors that trigger R-gene mediated resistance in comparison to strains where nearly isogenic effectors are not present in ICEs. Taken together, our study sheds light on a set of ICE elements from P. syringae pv. maculicola ES4326 and highlights how genomic context may lead to different evolutionary dynamics for shared virulence genes between strains.

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Genome-Assisted Development of a Diagnostic Protocol for Distinguishing High Virulence Pseudomonas syringae pv. tomato Strains

Plant Disease ◽

10.1094/pdis-08-14-0833-re ◽

2015 ◽

Vol 99 (4) ◽

pp. 527-534 ◽

Cited By ~ 5

Author(s):

Lisa A. Jones ◽

Surya Saha ◽

Alan Collmer ◽

Christine D. Smart ◽

Magdalen Lindeberg

Keyword(s):

New York ◽

Pseudomonas Syringae ◽

Genome Comparison ◽

Type Iii Effectors ◽

Type Iii ◽

Host Interaction ◽

Group I ◽

Virulent Strains ◽

High Virulence ◽

Relationship Of

A severe outbreak of bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, occurred in central New York in 2009. Isolate 09150, collected from this outbreak and subsequently named NYS-T1, was found to be highly virulent on tomato. To better understand the relationship of 09150 to other P. syringae strains and develop a diagnostic assay for aggressive strains of this pathogen, the 09150 genome was sequenced. Genome comparison revealed it to be highly similar to a previously sequenced isolate, T1. Genetic factors linked to host interaction including type III effectors, toxin biosynthetic genes, and elicitors of host innate immunity were identified. Type III effector repertoires were compared with other strains in the high virulence T1-like subgroup and lower virulence DC3000/P. syringae pv. maculicola subgroup within P. syringae phylogenetic Group I. Primers for conventional PCR were developed using sequences for avrA, hopW, conserved in the former subgroup and hopN, present in the latter. These were tested on isolates in the two subgroups, other pseudomonads, and other bacterial pathogens of tomato. Primers developed for avaA and hopW were diagnostic for more virulent strains of P. syringae pv. tomato while primers for hopN were diagnostic for P. syringae pv. tomato DC3000 and related P. syringe pv. maculicola strains. Primers designed against hopR distinguished both of these P. syringae subgroups from other P. syringae strains.

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