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. 2004 Jan;186(2):543-55.
doi: 10.1128/JB.186.2.543-555.2004.

Pseudomonas syringae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter

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Free PMC article

Pseudomonas syringae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter

Lisa M Schechter et al. J Bacteriol. 2004 Jan.
Free PMC article

Abstract

Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis: The hrp-hrc-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphE(Pto) are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae.

Figures

FIG. 1.
FIG. 1.
Secretion of AvrPto-Cya hybrid proteins from P. syringae pv. tomato DC3000. DC3000 and CUCPB5114 (DC3000 ΔhrpK-hrpR::ΩCm) strains containing plasmids that express AvrPto(1-X)-Cya fusion proteins (where X is 164, 135, 100, 50, or 16) were grown in culture under conditions that induce hrp-mediated protein secretion, as described in Materials and Methods. These strains also contained pUFRO34, a plasmid that expresses the cytoplasmic NptII protein. Cultures were separated into cellular and supernatant fractions by centrifugation, and an immunoblot analysis was performed with protein samples electrophoresed on an SDS-12.5% PAGE gel. The supernatant samples loaded on the gel were 86-fold more concentrated than the cellular samples. The AvrPto(1-X)-Cya and NptII (29.1 kDa) proteins were detected by using antibodies to Cya and NptII, respectively. Lanes 1, 6, 11, and 16, AvrPto(1-164)-Cya (62.1 kDa); lanes 2, 7, 12, and 17, AvrPto(1-135)-Cya (58.6 kDa); lanes 3, 8, 13, and 18, AvrPto(1-100)-Cya (54.8 kDa); lanes 4, 9, 14, and 19, AvrPto(1-50)-Cya (49.0 kDa); lanes 5, 10, 15, and 20, AvrPto(1-16)-Cya (45.3 kDa). The positions of prestained protein standards on the gel are indicated on the right.
FIG. 2.
FIG. 2.
HRs elicited by AvrB-Cya fusion proteins in N. benthamiana after delivery by P. fluorescens 55 expressing the P. syringae pv. syringae 61 Hrp system. (A) Expression of AvrB-Cya hybrid proteins in P. fluorescens. P. fluorescens 55 strains containing plasmids that express the Hrp system from P. syringae pv. syringae 61 (pLN18) and AvrB(1-321)-Cya (pCPP3290) or AvrB(1-30)-Cya (pCPP3288) were grown in culture as described in Materials and Methods. Protein samples were separated on an SDS-12.5% PAGE gel, and an immunoblot analysis was performed by using antibodies to Cya. Lane 1, AvrB(1-321)-Cya (79.5 kDa); lane 2, AvrB(1-30)-Cya (46.8 kDa). The positions of prestained protein standards on the gel are indicated on the left. (B) AvrB(1-321)-Cya induces an HR in N. benthamiana. N. benthamiana leaves were infiltrated with suspensions (OD600, 0.4) of P. fluorescens 55 (Pf 55) strains expressing AvrB(1-321)-Cya, AvrB(1-30)-Cya, or AvrPto(1-100)-Cya and a wild-type Hrp system from pLN18 or a ΔhrcC mutant Hrp system from pCPP3297. Photographs were taken 48 h after inoculation.
FIG. 3.
FIG. 3.
Time course of cAMP accumulation in plants after inoculation with Pseudomonas or E. coli cells expressing AvrPto(1-164)-Cya. (A) Tomato (L. esculentum cv. Money Maker) and N. benthamiana plants were inoculated with DC3000 bacteria (OD600, 0.3) containing pCPP3214 [Cya(2-406)] (♦) or pCPP3221 [AvrPto(1-164)-Cya] (▪) or with CUCPB5114 (DC3000 ΔhrpK-hrpR::ΩCm) containing pCPP3221 (▴). Plants were also infiltrated with a cleared lysate prepared from a culture of E. coli containing pCPP3221 (×). (B) Tomato (L. esculentum cv. Money Maker) and N. benthamiana plants were inoculated with P. fluorescens 55 cells (OD600, 0.3) containing pLN18 and pCPP3214 (⋄), pLN18 and pCPP3221 (□), or pCPP3297 and pCPP3221 (▵). pLN18 and pCPP3297 contain the wild-type and ΔhrcC hrp system genes from P. syringae pv. syringae 61, respectively. Leaf samples were collected with a 0.8-cm-diameter cork borer 1, 3, 5, 7, and 9 h postinoculation. cAMP was quantified in triplicate for each sample, and the standard deviations are indicated by error bars. The graphs are based on data from one representative experiment. Repeated experiments on different days yielded similar results, although the cAMP levels varied by up to 50% for each strain.
FIG. 4.
FIG. 4.
Expression of DC3000 effector-Cya or effector candidate-Cya fusion proteins in P. fluorescens 55. P. fluorescens 55 strains containing plasmids that express the Hrp system from P. syringae pv. syringae 61 (pLN18) and the different Cya fusion proteins were grown in culture as described in Materials and Methods. Protein samples were separated on an SDS-7% PAGE gel, and an immunoblot analysis was performed by using antibodies to Cya. The estimated molecular masses of the hybrid proteins and the positions of prestained protein standards on the gel are indicated.
FIG. 5.
FIG. 5.
The hopPtoS4 and hopPtoAG genes in P. syringae pv. tomato DC3000 are interrupted by transposon insertions. Schematic diagrams of the hopPtoS4 (A) and hopPtoAG (B) regions of the DC3000 chromosome show the positions of coding sequences for effectors (solid arrows and boxes), transposases (open arrows), and a predicted chaperone (gray arrow). The hopPtoS4::tnpA and hopPtoAG::tnpA genes encode truncated products containing 118 and 152 amino acids, respectively. The bent arrows indicate the positions of promoters containing hrp boxes (Phrp).
FIG. 6.
FIG. 6.
HopPtoK-Cya and HopPtoQ-Cya induce HRs in N. benthamiana. N. benthamiana leaves were infiltrated with suspensions (OD600, 0.8) of P. fluorescens 55 strains expressing HopPtoK-Cya, HopPtoQ-Cya, or HopPtoT1-Cya and a wild-type P. syringae pv. syringae 61 Hrp system from pLN18 or a mutant P. syringae pv. syringae 61 Hrp system from pCPP3297. The photographs were taken 48 h after inoculation.

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