Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Feb 15;108(7):2975-80.
doi: 10.1073/pnas.1013031108. Epub 2011 Jan 31.

Genetic disassembly and combinatorial reassembly identify a minimal functional repertoire of type III effectors in Pseudomonas syringae

Sébastien Cunnac  1 , Suma Chakravarthy, Brian H Kvitko, Alistair B Russell, Gregory B Martin, Alan Collmer
Affiliations

Genetic disassembly and combinatorial reassembly identify a minimal functional repertoire of type III effectors in Pseudomonas syringae

Sébastien Cunnac et al. Proc Natl Acad Sci U S A. .

Abstract

The virulence of Pseudomonas syringae and many other proteobacterial pathogens is dependent on complex repertoires of effector proteins injected into host cells by type III secretion systems. The 28 well-expressed effector genes in the repertoire of the model pathogen P. syringae pv. tomato DC3000 were deleted to produce polymutant DC3000D28E. Growth of DC3000D28E in Nicotiana benthamiana was symptomless and 4 logs lower than that of DC3000ΔhopQ1-1, which causes disease in this model plant. DC3000D28E seemed functionally effectorless but otherwise WT in diagnostic phenotypes relevant to plant interactions (for example, ability to inject the AvrPto-Cya reporter into N. benthamiana). Various effector genes were integrated by homologous recombination into native loci or by a programmable or random in vivo assembly shuttle (PRIVAS) system into the exchangeable effector locus in the Hrp pathogenicity island of DC3000D28E. The latter method exploited dual adapters and recombination in yeast for efficient assembly of PCR products into programmed or random combinations of multiple effector genes. Native and PRIVAS-mediated integrations were combined to identify a minimal functional repertoire of eight effector genes that restored much of the virulence of DC3000ΔhopQ1-1 in N. benthamiana, revealing a hierarchy in effector function: AvrPtoB acts with priority in suppressing immunity, enabling other effectors to promote further growth (HopM1 and HopE1), chlorosis (HopG1), lesion formation (HopAM1-1), and near full growth and symptom production (AvrE, HopAA1-1, and/or HopN1 functioning synergistically with the previous effectors). DC3000D28E, the PRIVAS method, and minimal functional repertoires provide new resources for probing the plant immune system.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Deletion of 10 T3E genes in CUCPB5500 yields DC3000D28E (CUCPB5585), which is strongly reduced in growth in N. benthamiana. Bacteria were inoculated with a blunt syringe at 3 × 104 cfu/mL in MgCl2 buffer, and populations in three 0.8-cm leaf disks were determined at 6 dpi. The arithmetic mean of log-transformed values of four replicate infiltrations from independent plants is displayed ± SD. Strains with the same letter are not statistically different based on a Tukey's honestly significant difference (HSD) test (α = 0.05). The cells in the genotype grid are shadowed in dark gray if the corresponding strains carry a deletion of the matching genomic region or are left white if the locus is WT. The Ω for hopY1 indicates that the gene was interrupted by insertion of a spectinomycin resistance cassette flanked by FRT sites. This experiment was repeated three times with similar results.
Fig. 2.
Fig. 2.
DC3000D28E (CUCPB5585) is functionally effectorless but otherwise WT in diagnostic assays. (A) Equivalent growth of DC3000D28E and DC3000ΔhopQ1-1 in liquid mannitol glutamate minimal medium supplemented with 50 μM iron citrate (means ± SD of the absorbance of triplicate cultures). (B) Equivalent translocation of an AvrPto-Cya fusion by DC3000D28E and DC3000. Plasmid pCPP5702 encoding the reporter gene under transcriptional control of the avrPto promoter was introduced into DC3000D28E, DC3000, and the ΔhrcQ-U T3SS− mutant. The resulting strains were infiltrated into N. benthamiana leaves at two different densities (107 and 108 cfu/mL) to control for assay saturation. cAMP concentrations were determined from tissues sampled from three independent leaves per treatment 7 h postinoculation. Means ± SD from one of two replicate experiments are shown. (C) Reduced ability of DC3000D28E to elicit ETI-like rapid plant cell death in N. benthamiana and N. tabacum. DC3000D28E and controls DC3000 (incompatible on both Nicotiana spp.), ΔhopQ1-1 (incompatible on N. tabacum), and ΔhrcQ-U cell suspensions in MgCl2 buffer, adjusted to three densities covering the dynamic range of the assay, were infiltrated into leaves, and the plant response was photographed 48 h later. Cell death response: +, positive; −, null; ±, partial. Each experiment was repeated at least three times with similar results. (D) Ability of DC3000D28E to be transcomplemented in mixed infections with virulent DC3000ΔhopQ1-1. Equal volumes of DC3000D28E and ΔhopQ1-1 strains (Left) or ΔhrcQ-U and ΔhopQ1-1 strains (Right) standardized at 3 × 106 cfu/mL (an inoculum level high enough for DC3000ΔhopQ1-1 to produce conditions favoring bacterial growth throughout the inoculated tissue) were mixed and infiltrated into leaves of N. benthamiana. Values (means ± SD of three samples) for the query strains and DC3000ΔhopQ1-1 were calculated by subtracting colony-forming unit counts on nonselective media (all strains) from colony-forming units on selective media (spectinomycin-resistant query strains). The experiment was repeated three times with similar results.
Fig. 3.
Fig. 3.
Restoration to native loci in DC3000D28E of genes encoding the AvrPto and AvrE REGs suggests that AvrPto and AvrPtoB interfere at an early phase of the host immune response. White fill in the genotype grid indicates that the locus was restored (note that the CEL includes hopM1). Strains harboring combinations of multiple WT loci were constructed by sequential integrations. Growth assays were performed at 6 dpi as in Fig. 1 with means ± SD of the bacterial populations calculated using values from three replicate leaves per strain. Means with the same letters are not statistically different based on a Tukey's HSD test (α = 0.05). This experiment was repeated three times with similar results.
Fig. 4.
Fig. 4.
The programmable or random in vivo assembly shuttle (PRIVAS) system exploits dual adapter recombination for facile integration of combinatorial gene sets into the DC3000 exchangeable effector locus (EEL). (A) Structure of a typical T3E genetic unit (GU) for PRIVAS. Primary PCR reactions with gene-specific oligonucleotide primers harboring 20-bp 3′ extensions amplify GUs flanked on each side by universal adaptor (UA) regions 1 or 2. (B) Secondary PCR reactions use these primary products as templates and flexible adapter (FA) primers composed of UA-specific segments at their 3′ end and one of a set of ~35-bp FA-homology regions at their 5′ end to yield UA-FA dual adapter-flanked GUs that are used as the elementary building blocks for in vivo assembly in yeast. The configuration of the gene sets, including gene orientation, can be fully programmed during construction by designing FA-flanked GUs so that a unique combination of recombination events between FAs leads to the closure of a circular DNA molecule containing the sequences of the shuttle vector as depicted in B for 3 GUs. The shuttle vector's backbone provides the origins of replication and selection markers for yeast and E. coli as well as an origin of transfer for conjugation into P. syringae. After transformation with a suitable pool of GUs and the linearized shuttle vector, plasmid DNA is extracted from yeast cells surviving selection and transferred into E. coli for subsequent conjugation into a recipient P. syringae strain and single cross-over integration into the EEL. (C) PRIVAS also can be used in random mode for the creation of complex combinatorial libraries of gene sets of variable configuration but of fixed size (equal to three in C). If several distinct GUs sharing the same pair of external FAs specifying a given position within the gene sets are included in the assembly reaction, identical FAs compete for recombination and hence, incorporation in growing DNA molecules. The final circular products contain polymorphic sets composed of GUs drawn from distinct bins of GUs at desired positions as illustrated in C.
Fig. 5.
Fig. 5.
Successive PRIVAS-mediated integration of eight T3Es into DC3000D28E reveals a hierarchy contributing to chlorosis, lesion formation, and near WT growth in N. benthamiana. PRIVAS was used in programmed mode to create various combinations of hopE1, hopAM1-1, and hopG1. The resulting gene sets were integrated at the EEL of DC3000D28E derivatives CUCPB6017 or CUCPB6019, which had avrPtoB and hopM1, respectively, or the entire CEL natively restored, as indicated by white-filled cells in the genotype grid. (A) Symptoms in N. benthamiana leaves. Leaves were infiltrated with two levels of inoculum, and the plants were kept in a chamber with 70–80% relative humidity. The fraction of plants showing symptoms and the nature of symptoms scored is shown; plus sign indicates chlorosis, and asterisk indicates cell death. (B) Bacterial growth in N. benthamiana. Bacteria were inoculated at 3 × 104 cfu/mL, and populations measured 6 dpi. The least-squares means ± SD of log (cfu/cm2) are shown. Means with the same letter are not significantly different using the Tukey–Kramer multiple comparisons method (α = 0.05). Eight independent experiments were performed with different subsets of the nine strains shown, with a minimum of three plants per strain in every experiment and a total of 213 data points. Randomized block design was used for data analysis using the statistical analysis program SAS.
See this image and copyright information in PMC

References

    1. Kenny B, Valdivia R. Host-microbe interactions: Bacteria. Curr Opin Microbiol. 2009;12:1–3. - PubMed
    1. Dean P, Kenny B. The effector repertoire of enteropathogenic E. coli: Ganging up on the host cell. Curr Opin Microbiol. 2009;12:101–109. - PMC - PubMed
    1. Buell CR, et al. The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000. Proc Natl Acad Sci USA. 2003;100:10181–10186. - PMC - PubMed
    1. Chang JH, et al. A high-throughput, near saturating screen for type III effector genes from Pseudomonas syringae. Proc Natl Acad Sci USA. 2005;102:2549–2554. - PMC - PubMed
    1. Vinatzer BA, Jelenska J, Greenberg JT. Bioinformatics correctly identifies many type III secretion substrates in the plant pathogen Pseudomonas syringae and the biocontrol isolate P. fluorescens SBW25. Mol Plant Microbe Interact. 2005;18:877–888. - PubMed

Publication types

MeSH terms

LinkOut - more resources