Multiple sequence alignment of the SLT domain from HrpH and other selected proteins. The proteins are as follows: Slt70, E. coli K-12 (POAGC3); IagB, S. enterica (NP_457271); IpgF, S. flexneri (NP_085298); Hpa2, X. oryzae pv. oryzae (ABJ80895); VirB1, A. tumefaciens (NP_536285); and HopP1, MltD, and HrpH from P. syringae pv. tomato DC3000 (AAO56180, AAO57184, and NP_791205, respectively). Boxes indicate three conserved sequence motifs typical for the goose egg white lysozyme-like domain (LT_GEWL). Overlines mark the conserved two α-helices (I and III) and the beta sheet (II) of the LT-GEWL family. The asterisk indicates the catalytic glutamate.

Evidence that HrpH and HopP1, but not HopAJ1, travel the P. syringae T3SS. (A) Translocation tests using P. syringae pv. tomato DC3000. N. benthamiana leaves were infiltrated via a blunt syringe with DC3000 at 3 × 10⁸ CFU/ml carrying plasmids expressing the indicated protein with a C-terminal Cya reporter fusion. The three vectors used different promoters: pCPP3234 (tac promoter), pCPP5295 (native promoters), and pCPP5371 (avrPto1 promoter). Regardless of the promoter, none of the test proteins was translocated by a T3SS-deficient DC3000 ΔhrcC mutant, as indicated by cAMP levels below 2.0 pmol/μg of total bacterial protein (data not shown). (B) Assays using P. fluorescens heterologously expressing a P. syringae T3SS to test for translocation of HrpH and derivatives HrpH_17-495, HrpH(E148A), and HrpH_1-241. N. benthamiana leaves were infiltrated with P. fluorescens at 3 × 10⁸ CFU/ml carrying pLN18 or ΔhrcC derivative pCPP3297 and pCPP3234 derivatives expressing the indicated test proteins as Cya fusions. In both panels, samples were collected from N. benthamiana leaves 6 h after inoculation, and data represent the means and standard deviations of populations measured from two leaf disks from each of two different leaves.

Effect of deleting the three P. syringae pv. tomato DC3000 HrpL-regulated LT genes on elicitation of the HR in tobacco leaves. (A) HR threshold assays for LT mutants. The tested strains were DC3000 wild type (WT), three mutants with single deletions (ΔhrpH, ΔhopP1, and ΔhopAJ1, respectively), two double mutants (ΔhrpH hopP1 and ΔhrpH hopAJ1), one triple mutant (ΔhrpH hopP1 hopAJ1), ΔhrpH_242-496 producing a HrpH derivative lacking the C-terminal region, and a T3SS-deficient mutant (ΔhrcC) as a negative control. Strains were inoculated with a blunt syringae at 6 × 10⁶ and 3 × 10⁶ CFU/ml, as indicated, and leaves were photographed 48 h later. (B) HR threshold assays for complemented LT mutants. Two of the single-deletion mutants, ΔhopAJ1 and ΔhrpH, were complemented by expressing HopAJ1 and HrpH, respectively, from plasmids using a vector pCPP5372 avrPto1 promoter. For the plants used in this assay, the threshold bacterial population for HR elicitation was 6 × 10⁶ CFU/ml.

Effect of ΔhrpH mutation and ΔhrpH hopP1 hopAJ1 triple mutation on the virulence of P. syringae pv. tomato DC3000 in tomato and N. benthamiana. (A) Bacterial speck symptoms in tomato leaves. Plants were inoculated by dipping in inoculum containing the indicated strains at 3 × 10⁶ CFU/ml and photographed 10 days later. (B) Bacterial growth in tomato leaves. Inoculum at 3 × 10⁴ CFU/ml was infiltrated with a blunt syringe into leaves and populations were measured at 0, 2, and 4 days after inoculation. The data show the means and standard deviations of populations measured from three leaf disks from each of three different leaves. (C) Effect on virulence in N. benthamiana of deleting hrpH, hopAJ1, and hopP1 from a DC3000 ΔhopQ1-1 mutant. The indicated strains were infiltrated at 3 × 10⁴ CFU/ml into leaf panels with a blunt syringe, and the leaf was photographed 7 days later. Circles mark the approximate inoculation area. To aid visualization of necrotic lesions, leaves were distained with Carnoy's fluid.

Predicted domain architecture of the three P. syringae pv. tomato DC3000 HrpL-regulated LTs and mutant derivatives and comparison with other representative bacterial LTs. (A) Architecture of HrpH and mutant derivatives used in the present study. The Pfam domains are MltD_N (PF06471, the N-terminal domain of membrane-bound LT), SLT (PF01464, transglycosylase), and a Pfam class B domain shared with PSPTO3714. The location of the E148A mutation is indicated with a triangle. (B) Domain architecture of other relevant LT proteins: MltD, P. syringae pv. tomato DC3000 PSPTO3714; IagB, Salmonella enterica (NP_457271); IpgF, Shigella flexneri (NP_085298); Hpa2, Xanthomonas oryzae pv. oryzae (ABJ80895); and VirB1, Agrobacterium tumefaciens (NP_536285). The LysM domains in MltD are Pfam PF01476 (lysin motif). (C) Domain architecture of the other two HrpL-regulated LTs of DC3000. The HopP1 HrpW domain, determined by alignment with HrpW, includes the N-terminal T3SS targeting region. HopAJ1 has an MLTB domain (COG2951, membrane-bound lytic murein transglycosylase B) and a PGB domain (PF01471, peptidoglycan binding domain 1).

Inhibition of E. coli growth in culture by induced production of HrpH. The data show growth in LB medium of E. coli BL21(DE3) cells harboring pET-DEST42 expressing hrpH or hrpH(E148A) from the vector T7lac promoter. Expression of the indicated LT genes was induced by the addition of 0.5 mM IPTG at time zero.

Contribution of HrpH and functionally equivalent LTs to the ability of T3SS-proficient P. fluorescens(pCPP5316) to translocate HopA1-Cya and elicit a HopA1-dependent HR in tobacco. (A) The P. syringae pv. syringae 61 DNA insert in cosmid pHIR11 contains a complete hrp-hrc gene cluster, including genes encoding positive regulators (diagonal lines) and T3SS substrates (shaded). pCPP5316 is pHIR11 with a hopA1-cya translational fusion. (B) Translocation of HopA1-Cya by P. fluorescens carrying pCPP5316 and pCPP5316 ΔhrpH, with or without test LT genes expressed in trans, as indicated by HR elicitation in tobacco leaves and by Cya reporter assays. Leaf tissue was photographed 24 h after inoculation with 6 × 10⁸ CFU/ml; a positive response (+) is indicated by confluent collapse and bleaching of the tissue. For Cya translocation reporter tests, samples were collected 6 h after syringe infiltration of tobacco leaf tissue with 6 × 10⁸ CFU/ml and assayed for cAMP levels as described in the text. The data show the means and standard deviations for two leaf disks from each of two different leaves. The LT genes were cloned with a stop codon and expressed by inducing the vector pCPP3234 tac promoter with 0.1 mM IPTG. The HR assay panels are aligned with the corresponding translocation bar graph results and labels below.

Ability of P. syringae pv. tomato DC3000 wild-type, ΔhrpH mutant, and ΔhrpH hopP1 hopAJ1 triple mutant to translocate representative T3SS substrates into host tomato cells and secrete them in culture. (A) Translocation assay. Tomato leaves were infiltrated with DC3000 (WT), ΔhrpH mutant (ΔH), or ΔhrpH hopP1 hopAJ1 triple mutant (ΔH,P,AJ) cells that also carried pCPP5371 expressing the indicated T3SS substrates with a C-terminal Cya fusion. Bacteria were infiltrated via a blunt syringe at 6 × 10⁶ CFU/ml, and samples were collected from leaves 6 h later. The results present the apparent translocation (based on cAMP concentration) by the mutants relative to the WT for each test substrate, with the WT levels for each substrate normalized to 100. The data show the means and standard deviations for two leaf disks from each of two different leaves. The actual mean values of the wild type (pmol of cAMP/μg of total protein) were 127 for HopA1, 93 for AvrPto1, and 30 for HrpZ1 for the experiment shown. Note that the use of a moderate level of inoculum and the avrPto1 promoter in vector pCPP5371 for all three test substrates results in a relatively low level of translocation. None of the test proteins was translocated by a T3SS-deficient DC3000 ΔhrcC mutant, as indicated by cAMP levels below 2.0 pmol/μg of protein (data not shown). The experiment was repeated three times with similar results. (B) Assay for secretion of AvrPto1 and HrpZ1 produced from their native genes in the DC3000 chromosome under Hrp-inducing conditions in culture. The indicated DC3000 wild-type (WT) and mutant strains were grown in Hrp-inducing minimal broth to an OD₆₀₀ of 0.5 and separated by centrifugation into cell-bound “C” and supernatant “S” fractions. Proteins in each fraction were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by immunoblotting with polyclonal antibodies to AvrPto1 and HrpZ1.

Dominant-negative effect of HrpH(E148A) and HrpH_1-241 on the ability of wild-type P. syringae pv. tomato DC3000 to elicit the HR in nonhost tobacco and produce disease lesions and bacterial growth in host tomato. (A) HR elicitation in tobacco leaf. Bacteria were infiltrated with a blunt syringe at the indicated levels into a tobacco leaf, and the leaf was photographed 48 h later. The indicated plasmid-produced HrpH derivatives were expressed from the avrPto1 promoter in pCPP5372. (B) Lesion formation on tomato plants. Tomato plants were inoculated by dipping in the indicated strains at 3 × 10⁶ CFU/ml and photographed 10 days later. (C) Bacterial growth in tomato leaves. Inoculum at 3 × 10⁴ CFU/ml was infiltrated with a blunt syringe into different leaves, and populations were measured at 0, 2, and 4 days after inoculation. The data show the means and standard deviations of populations measured from three leaf disks from each of three different leaves.