Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae downregulates the sphingolipid biosynthetic pathway leading to growth-arrest

Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors which suppress plant defense responses and promote bacterial growth followinginfection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf, S. cerevisiae library for mutants resistant toDspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1,Δskn1, Δcsg1, Δcsg2, Δorm1, Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases(LCBs) phytosphingosine and dihydrosphingosine also suppressed DspA/E-induced yeast growth defect. Expression of DspA/E in yeast downregulated LCBs biosynthesis and induced a rapid decrease in LCB levels,indicating that SPT, the first and rate limiting enzyme of the sphingolipid biosynthetic pathway was repressed. SPT downregulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation, affecting Cdc55-PP2A protein phosphatase activity, prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest

Mutational analysis of a predicted double β-propeller domain of the DspA/E effector of Erwinia amylovora

The bacterium Erwinia amylovora causes fire blight, an invasive disease that threatens apple trees, pear trees and other plants of the Rosaceae family. Erwinia amylovora pathogenicity relies on a type III secretion system and on a single effector DspA/E. This effector belongs to the widespread AvrE family of effectors whose biological function is unknown. In this manuscript, we performed a bioinformatic analysis of DspA/E- and AvrE-related effectors. Motif search identified nuclear localization signals, peroxisome targeting signals, endoplasmic reticulum membrane retention signals and leucine zipper motifs, but none of these motifs were present in all the AvrE-related effectors analysed. Protein threading analysis, however, predicted a conserved double β-propeller domain in the N-terminal part of all the analysed effector sequences. We then performed a random pentapeptide mutagenesis of DspA/E, which led to the characterization of 13 new altered proteins with a five amino acids insertion. Eight harboured the insertion inside the predicted β-propeller domain and six of these eight insertions impaired DspA/E stability or function. Conversely, the two remaining insertions generated proteins that were functional and abundantly secreted in the supernatant suggesting that these two insertions stabilized the protein

The HrpN Effector of Erwinia amylovora , Which Is Involved in Type III Translocation, Contributes Directly or Indirectly to Callose Elicitation on Apple Leaves

Erwinia amylovora is responsible for fire blight of apple and pear trees. Its pathogenicity depends on a type III secretion system (T3SS) mediating the translocation of effectors into the plant cell. The DspA/E effector suppresses callose deposition on apple leaves. We found that E. amylovora and Pseudomonas syringae DC3000 tts mutants or peptide flg22 do not trigger callose deposition as strongly as the dspA/E mutant on apple leaves. This suggests that, on apple leaves, callose deposition is poorly elicited by pathogen-associated molecular patterns (PAMPs) such as flg22 or other PAMPs harbored by tts mutants and is mainly elicited by injected effectors or by the T3SS itself. Callose elicitation partly depends on HrpW because an hrpW-dspA/E mutant elicits lower callose deposition than a dspA/E mutant. Furthermore, an hrpN-dspA/E mutant does not trigger callose deposition, indicating that HrpN is required to trigger this plant defense reaction. We showed that HrpN plays a general role in the translocation process. Thus, the HrpN requirement for callose deposition may be explained by its role in translocation: HrpN could be involved in the translocation of other effectors inducing callose deposition. Furthermore, HrpN may also directly contribute to the elicitation process because we showed that purified HrpN induces callose deposition

Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae downregulates the sphingolipid biosynthetic pathway leading to growth-arrest

Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors which suppress plant defense responses and promote bacterial growth followinginfection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf, S. cerevisiae library for mutants resistant toDspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1,Δskn1, Δcsg1, Δcsg2, Δorm1, Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases(LCBs) phytosphingosine and dihydrosphingosine also suppressed DspA/E-induced yeast growth defect. Expression of DspA/E in yeast downregulated LCBs biosynthesis and induced a rapid decrease in LCB levels,indicating that SPT, the first and rate limiting enzyme of the sphingolipid biosynthetic pathway was repressed. SPT downregulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation, affecting Cdc55-PP2A protein phosphatase activity, prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest

Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri

Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically composed of 12 components that form 2 major assemblies: the inner membrane complex embedded in the inner membrane and the core complex embedded in both the inner and outer membranes. Here we present the 3.3 Å-resolution cryo-electron microscopy model of the T4S system core complex from Xanthomonas citri, a phytopathogen that utilizes this system to kill bacterial competitors. An extensive mutational investigation was performed to probe the vast network of protein–protein interactions in this 1.13-MDa assembly. This structure expands our knowledge of the molecular details of T4S system organization, assembly and evolution