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. 1999 Aug;181(16):4790-7.
doi: 10.1128/JB.181.16.4790-4797.1999.

The Avr (effector) proteins HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp (type III) protein secretion system in a temperature- and pH-sensitive manner

K van Dijk  1 , D E Fouts, A H Rehm, A R Hill, A Collmer, J R Alfano
Affiliations

The Avr (effector) proteins HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp (type III) protein secretion system in a temperature- and pH-sensitive manner

K van Dijk et al. J Bacteriol. 1999 Aug.

Abstract

We present here data showing that the Avr proteins HrmA and AvrPto are secreted in culture via the native Hrp pathways from Pseudomonas syringae pathovars that produce these proteins. Moreover, their secretion is strongly affected by the temperature and pH of the culture medium. Both HrmA and AvrPto were secreted at their highest amounts when the temperature was between 18 and 22 degrees C and when the culture medium was pH 6.0. In contrast, temperature did not affect the secretion of HrpZ. pH did affect HrpZ secretion, but not as strongly as it affected the secretion of HrmA. This finding suggests that there are at least two classes of proteins that travel the P. syringae pathway: putative secretion system accessory proteins, such as HrpZ, which are readily secreted in culture; and effector proteins, such as HrmA and AvrPto, which apparently are delivered inside plant cells and are detected in lower amounts in culture supernatants under the appropriate conditions. Because HrmA was shown to be a Hrp-secreted protein, we have changed the name of hrmA to hopPsyA to reflect that it encodes a Hrp outer protein from P. syringae pv. syringae. The functional P. syringae Hrp cluster encoded by cosmid pHIR11 conferred upon P. fluorescens but not Escherichia coli the ability to secrete HopPsyA in culture. The use of these optimized conditions should facilitate the identification of additional proteins traveling the Hrp pathway and the signals that regulate this protein traffic.

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Figures

FIG. 1
FIG. 1
Distribution of HrmA and β-lactamase in cultures of P. syringae pv. syringae 61(pCPP2318) or hrp mutant P. syringae pv. syringae 61-2089(pCPP2318). Bacterial cultures were grown at 25°C in hrp-derepressing medium and separated into cell-bound (C) and supernatant (S) fractions. The cell-bound fractions were concentrated 13.4-fold and the supernatant fractions were concentrated 100-fold relative to the initial culture volumes. The samples were subjected to SDS-PAGE and immunoblot analysis, and HrmA and β-lactamase were detected with either anti-HrmA or anti-β-lactamase antibodies followed by secondary antibodies conjugated to alkaline phosphatase as described in Materials and Methods. Pss wild-type, P. syringae pv. syringae 61(pCPP2318); Pss hrcC, P. syringae pv. syringae 61-2089(pCPP2318). The image of the immunoblot was captured using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
FIG. 2
FIG. 2
The secretion in culture of HrmA (HopPsyA) is affected by temperature, whereas the secretion of HrpZ is not. P. syringae pv. syringae 61(pCPP2318) cultures were grown in hrp-derepressing medium at the temperatures indicated. Bacterial cultures were separated into cell-bound (C) and supernatant (S) fractions by centrifugation, and the supernatant fractions were adjusted to be 7.5 times more concentrated than the cell-bound fractions. After the samples were separated by SDS-PAGE and transferred to PVDF membranes, HrmA, HrpZ, and β-lactamase were detected by immunoblotting using anti-HrmA, anti-HrpZ, and anti-β-lactamase antibodies, respectively, followed by secondary antibodies conjugated to alkaline phosphatase. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
FIG. 3
FIG. 3
The pH of the growth medium affects the secretion in culture of HrmA (HopPsyA) and HrpZ via the Hrp secretion system. P. syringae pv. syringae 61(pCPP2318) cultures were grown at 22°C in hrp-derepressing media differing only in the pH of the medium. Bacterial cultures were separated into cell-bound (C) and supernatant (S) fractions by centrifugation, and proteins were separated by SDS-PAGE. Immunoblot analysis was carried out as described in Materials and Methods, using anti-HrmA, anti-HrpZ, or anti-β-lactamase antibodies followed by secondary antibodies conjugated to alkaline phosphatase. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
FIG. 4
FIG. 4
AvrPto is secreted in culture from P. syringae pv. tomato DC3000 via the Hrp secretion system at 20°C but not at 30°C. P. syringae pv. tomato DC3000(pCPP2318) cultures were grown in hrp-derepressing medium at 20 and 30°C. The supernatant (S) and cell-bound (C) fractions were isolated as before, separated by SDS-PAGE, and analyzed by immunoblotting with anti-AvrPto or anti-β-lactamase antibodies. The preparations of the protein samples resulted in supernatant fractions that were concentrated 7.5 times more than the cell-bound fractions. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
FIG. 5
FIG. 5
AvrPto is secreted in culture from P. syringae pv. tomato DC3000 at pH 6.0 but not at pH 7.0. P. syringae pv. tomato DC3000(pCPP2318) cultures were grown at 20°C in hrp-derepressing medium adjusted to either pH 6.0 or pH 7.0. Isolated cell-bound (C) and supernatant (S) fractions were separated by SDS-PAGE and analyzed by immunoblotting using anti-AvrPto or anti-β-lactamase antibodies. Mature β-lactamase encoded by pCPP2318 was included as a control for cell lysis. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
FIG. 6
FIG. 6
Native AvrB cannot be detected in culture supernatants from P. syringae pv. glycinea race 0, even though heterologously expressed AvrPto is secreted. (A) P. syringae pv. glycinea (Psg) race 0(pCPP2318) was grown at 20°C in hrp-derepressing medium and separated into cell-bound (C) and supernatant (S) fractions. The fractions were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting using either anti-AvrPto or anti-β-lactamase antibodies. (B) P. syringae pv. glycinea race 0(pCPP2318, pCPP3026) was grown at 20°C in hrp-derepressing medium and separated into cell-bound (C) and supernatant (S) fractions. pCPP3026 encodes AvrPto from P. syringae pv. tomato DC3000. After separation of proteins by SDS-PAGE, immunoblot analysis was carried out with anti-AvrB, anti-AvrPto, or anti-β-lactamase antibodies. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
FIG. 7
FIG. 7
HrmA (HopPsyA) is secreted in culture from P. fluorescens 55(pHIR11) via the Hrp pathway but not from E. coli MC4100(pHIR11). (A) P. fluorescens (Pf) 55 carrying either a functional hrp cluster from P. syringae pv. syringae (pHIR11) or a defective P. syringae pv. syringae hrp cluster (pCPP2089) was grown in hrp-derepressing medium at 22°C. Cell-bound (C) and supernatant (S) fractions were separated by SDS-PAGE and analyzed on immunoblots with anti-HrmA antibodies. (B) E. coli (Ec) MC4100 carrying either pHIR11 or pCPP2089 was grown at 25°C in hrp-derepressing medium and separated into cell-bound (C) and supernatant (S) fractions. After separation of the fractions by SDS-PAGE, the presence of HrmA from each fraction was determined by immunoblot analysis with anti-HrmA antibodies. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
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References

    1. Alfano J R, Bauer D W, Milos T M, Collmer A. Analysis of the role of the Pseudomonas syringae pv. syringae HrpZ harpin in elicitation of the hypersensitive response in tobacco using functionally nonpolar hrpZ deletion mutants, truncated HrpZ fragments, and hrmA mutations. Mol Microbiol. 1996;19:715–728. - PubMed
    1. Alfano J R, Collmer A. Bacterial pathogens in plants: life up against the wall. Plant Cell. 1996;8:1683–1698. - PMC - PubMed
    1. Alfano J R, Collmer A. The type III (Hrp) secretion pathway of plant pathogenic bacteria: trafficking harpins, Avr proteins, and death. J Bacteriol. 1997;179:5655–5662. - PMC - PubMed
    1. Alfano J R, Kim H-S, Delaney T P, Collmer A. Evidence that the Pseudomonas syringae pv. syringae hrp-linked hrmA gene encodes an Avr-like protein that acts in an hrp-dependent manner within tobacco cells. Mol Plant-Microbe Interact. 1997;10:580–588. - PubMed
    1. Bergman T, Håkansson S, Forsberg A, Norlander L, Macellaro A, Backman T, Bolin I, Wolf-Watz H. Analysis of the V antigen lcrGVH-yopBD operon of Yersinia pseudotuberculosis: evidence for a regulatory role for LcrH and LcrV. J Bacteriol. 1991;173:1607–1616. - PMC - PubMed

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