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. 2013 May 14;14(7):678–692. doi: 10.1111/mpp.12039

Figure 5.

figure

Detection of HrpE3 secretion via the type III secretion (T3S) system. (A) Amino acid sequence alignment of Xanthomonas oryzae pv. oryzicola (Xoc) HrpE (ABH07404), X. campestris pv. vesicatoria (Xcv) HpaE (YP_362146) and the fused chimeric protein nE3‐cE; the different amino acids among HrpE3, HpaE and nE3‐cE are shown in grey and black letters, and the same in red letters. (B) Schematic maps showing HrpE3 constructs fused to AvrXa10. The 50 amino acids at the N‐terminus of HrpE3 contain a higher percentage of serine (S) and proline (P) residues, and may comprise a T3S signal. The 50 N‐terminal amino acids of HrpE3 were fused to a truncated version of AvrXa10Δ in which the first 28 amino acids were deleted, generating a fusion designated E3avrXa10Δ. The transcription of this fusion was driven by three fragments containing 397, 201 and 101 bp of hrpE3 upstream DNA; these were designated pP1E3avrXa10Δ, pP2E3avrXa10Δ and pP3E3avrXa10Δ, respectively. The black rectangle indicates the location of the putative plant‐inducible promoter (PIP) box in the promoter region of hrpE3. (C) The X. oryzae pv. oryzae (Xoo)–rice pathosystem was used to evaluate secretion and translocation of the HrpE3‐AvrXa10 chimeric proteins. For comparative purposes, an HpaE‐AvrXa10 chimeric protein was included. This construct was designated pP2EavrXa10Δ and contained the 50 N‐terminal amino acids from HpaE fused to AvrXa10Δ; the promoter driving transcription was the 201‐bp region upstream of hrpE3. Xoo strains containing the fusions were inoculated into rice cultivar IRBB10 or IR24 (see Experimental procedures). The native avrXa10 was used as a positive control (construct pavrXa10) and the N‐terminal truncated avrXa10Δ under control of the 201‐bp hrpE3 promoter region (pP2avrXa10Δ) served as a negative control. PXO99A and PΔhrcU containing the empty vector pURF034 were also included. Phenotypes were photographed 3 days after inoculation in three independent experiments. (D) Detection of the fused genes E3avrXa10Δ and EavrXa10Δ in rice IRBB10 leaves infiltrated with Xoo strains and analysed by reverse transcription‐polymerase chain reaction (RT‐PCR) at 12 h post‐inoculation (hpi) (detailed in Experimental procedures). The 16S rRNA gene of Xoo was used as an internal control.