Pseudomonas syringae Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter

Lisa M. Schechter, Kathy A. Roberts, Yashitola Jamir, James R. Alfano, Alan Collmer

Research output: Contribution to journalArticle

152 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)543-555
Number of pages13
JournalJournal of bacteriology
Volume186
Issue number2
DOIs
StatePublished - Jan 1 2004

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Pseudomonas syringae
Humulus
Plant Cells
Proteins
Lycopersicon esculentum
Amino Acids
Sigma Factor
Type III Secretion Systems
Bacterial Proteins
Calmodulin
Multigene Family
Arabidopsis
Adenylyl Cyclases
Codon
Tobacco
Virulence
Fatty Acids

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Pseudomonas syringae Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter. / Schechter, Lisa M.; Roberts, Kathy A.; Jamir, Yashitola; Alfano, James R.; Collmer, Alan.

In: Journal of bacteriology, Vol. 186, No. 2, 01.01.2004, p. 543-555.

Research output: Contribution to journalArticle

Schechter, Lisa M. ; Roberts, Kathy A. ; Jamir, Yashitola ; Alfano, James R. ; Collmer, Alan. / Pseudomonas syringae Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter. In: Journal of bacteriology. 2004 ; Vol. 186, No. 2. pp. 543-555.
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