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

Abstract

Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis. The hrp-hrcencoded 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.Pseudomonas syringae is a host-specific bacterial pathogen of plants whose various pathovars cause necrotic lesions on leaves or fruits of susceptible plants and elicit the hypersensitive response (HR), a defense-associated localized programmed cell death, in resistant plants. P. syringae interactions with plants depend on the hrp (for "hypersensitive response and pathogenicity") gene cluster, which encodes a type III secretion system (TTSS) or Hrp system that injects bacterial proteins into plant cells (1). Similar TTSSs exist in a variety of other gram-negative plant and animal pathogens (13, 28). The P. syringae proteins injected into host cells by the Hrp system are primarily known as effectors or Avr (avirulence) or Hop (Hrp-dependent outer protein) proteins. Hop proteins may contribute to P. syringae virulence by suppressing plant immunity. For example, AvrB, AvrRpm1...

Elucidation of a pH-folding switch in the Pseudomonas syringae effector protein AvrPto

“…These results suggest that translocation of AvrPto via the T3SS should be highly sensitive to temperature, with more efficient translocation at higher temperatures corresponding to higher unfolded AvrPto populations. Translocation assays performed in plants equilibrated to 24°C show definitive delivery of AvrPto to the host-cell cytoplasm (26). However, in culture medium at pH 6, P. syringae efficiently secretes AvrPto at 20°C but not at 30°C (17).…”

RPG1-B-Derived Resistance to AvrB-Expressing Pseudomonas syringae Requires RIN4-Like Proteins in Soybean

“…Positive interactions between coinfiltrated proteins resulted in reconstitution of EYFP and thereby fluorescence detectable by confocal microscopy. A reduced inoculum of AvrB-expressing cells was used for infiltration to minimize autofluorescence from AvrB-induced cell death (Schechter et al, 2004;Supplemental Fig. S1B).…”

Comparative Large-Scale Analysis of Interactions between Several Crop Species and the Effector Repertoires from Multiple Pathovars of Pseudomonas and Ralstonia

“…We used previously published data and our own sequence searches to identify genes encoding confirmed and putative effectors representing nearly the entire secretomes of four pathovars of Ps and one strain of Ralstonia solanacearum (Rs; Supplemental Table S1). Specifically, we cloned 42 of 54 genes encoding effectors and related proteins from Ps pv tomato DC3000 (Pto DC3000), 24 of 36 from Ps pv phaseolicola 1448A (Pph 1448A), and 22 of 27 from Ps pv syringae B728a (Psy B728a; Table I; Guttman et al, 2002;Greenberg and Vinatzer, 2003;Chang et al, 2004;Schechter et al, 2004Schechter et al, , 2006Joardar et al, 2005;Vinatzer et al, 2005Vinatzer et al, , 2006Lindeberg et al, 2006; http://pseudomonassyringae.org/). The sequence of the Ps pv maculicola strain ES4326 (Pma ES4326) genome was not available to us when we initiated these studies; therefore, we could not determine the exact number of effector genes in this strain.…”

“…First, BLASTX analysis was used to identify homologs of previously identified effectors. Second, all open reading frames located within 300 bp downstream of sequences resembling the conserved hrp-promoter element were translated and analyzed for characteristics common to known effectors, including a high Ser or Pro bias in the first 50 amino acids, an aliphatic amino acid at the third or fourth position, and the lack of negatively charged residues in the first 12 positions (Guttman et al, 2002;Petnicki-Ocwieja et al, 2002;Schechter et al, 2004). Finally, open reading frames immediately downstream of putative effector genes identified above were also analyzed because genes encoding effectors are often clustered in the genome and can exist in operons.…”