Ketoglutarate transport protein KgtP is secreted through the type III secretion system and contributes to virulence in Xanthomonas oryzae pv. oryzae

Abstract

The phytopathogenic prokaryote Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight (BB) of rice and utilizes a type III secretion system (T3SS) to deliver T3SS effectors into rice cells. In this report, we show that the ketoglutarate transport protein (KgtP) is secreted in an HpaB-independent manner through the T3SS of X. oryzae pv. oryzae PXO99(A) and localizes to the host cell membrane for α-ketoglutaric acid export. kgtP contained an imperfect PIP box (plant-inducible promoter) in the promoter region and was positively regulated by HrpX and HrpG. A kgtP deletion mutant was impaired in bacterial virulence and growth in planta; furthermore, the mutant showed reduced growth in minimal media containing α-ketoglutaric acid or sodium succinate as the sole carbon source. The reduced virulence and the deficiency in α-ketoglutaric acid utilization by the kgtP mutant were restored to wild-type levels by the presence of kgtP in trans. The expression of OsIDH, which is responsible for the synthesis of α-ketoglutaric acid in rice, was enhanced when KgtP was present in the pathogen. To our knowledge, this is the first report demonstrating that KgtP, which is regulated by HrpG and HrpX and secreted by the T3SS in Xanthomonas oryzae pv. oryzae, transports α-ketoglutaric acid when the pathogen infects rice.

Fig 1

kgtP is required for full virulence and growth of X. oryzae pv. oryzae in planta. (A) Symptoms induced by different X. oryzae pv. oryzae strains inoculated to leaves of 2-month-old rice (IR24, a susceptible cultivar) by the leaf-clipping procedure. Photographs were taken 14 dpi. (B) Bacterial blight lesion lengths in rice. Values represent the means ± standard deviations from three replicates, each containing five leaves. (C) Bacterial growth in inoculated leaves. Inoculated leaves were removed each day for 4 dpi and homogenized in sterile water. The homogenates were diluted and plated on NA plates with appropriate antibiotics. Bacterial CFU were counted after incubation at 28°C for 3 days. Data are the means ± standard deviations from three replicates.

Fig 2

Selected carbohydrates impact growth of the kgtP mutant. Growth of X. oryzae pv. oryzae strains in NCM minimal medium containing α-ketoglutaric acid (A) or sodium succinate (B) as the sole carbon source is depicted. The initial concentration of the strains was adjusted to an OD₆₀₀ of 0.05 in NCM supplemented with α-ketoglutaric acid or sodium succinate. Aliquots were taken in triplicate at 12-h intervals for 108 h. Bacteria were incubated at 28°C, and growth was determined by measuring the OD₆₀₀. Values given are the means ± standard deviations of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments. (C) Expression analysis of kgtP in X. oryzae pv. oryzae by RT-PCR. RNAs were isolated from cultures of wild-type PXO99^A, PΔkgtP, CPΔkgtP, PΔhrpX, and PΔhrpG strains grown in NB, MMX, rice suspension cells, and NCM supplemented with 0.5% α-ketoglutaric acid for 16 h. The quantity of kgtP mRNA in the tested strains was determined by semiquantitative RT-PCR and the primer kgtP-F1/R1 (see Table S1 in the supplemental material). PCR products were electrophoretically separated on a 1.0% agarose gel. The 16S rRNA PCR product of the pathogen was used as an internal control.

Fig 3

Regulation and secretion of the kgtP gene product. (A) Schematic map showing the kgtP promoter containing the PIP box motifs fused with GFP (top row, in construct pkgtPPGFP), the kgtP promoter, and the N-terminal 50 amino acids of KgtP fused with Δ28AvrXa10 (middle row, in construct pNKgtPΔAvrXa10), and the residue proline (P) and serine (S) consititution of the N-terminal 50 amino acids of KgtP (bottom row). (B) Interaction of X. oryzae pv. oryzae strains with rice suspension cells after a 16-h cocultivation period. Images were acquired by light (LM) or fluorescence (FM) microscopy. X. oryzae pv. oryzae bacterial cells appeared as black dots at rice cell surfaces or gray dots surrounding rice cells under LM and green spindly dots surrounding or near rice cells under FM, while rice cells turned red under FM. The white bar indicates 50 μm. 1, PXO99^A(pkgtPPgfp); 2, PΔhrpX(pkgtPPgfp). (C) X. oryzae pv. oryzae strains (3 × 10⁸ CFU/ml) were infiltrated into 2-week-old seedling leaves of IRBB10 rice with needleless syringes. Symptoms were photographed 3 dpi in three independent experiments. The top panels showed symptoms caused by PXO99^A(pUFR034), PXO99^A(pΔ28AvrX10), PXO99^A(pAvrX10), and PXO99^A(pNKgtPΔ28AvrXa10), and the bottom panels displayed no symptoms triggered by PΔhrcU(pUFR034), PΔhrcU(pΔ28AvrX10), PΔhrcU(pAvrX10), or PΔhrcU(pNKgtPΔ28AvrXa10). (D) Detection of KgtP secretion by immunoblot analysis. X. oryzae pv. oryzae wild-type PXO99^A, PΔhrcU, PΔhrpF, PΔhpaB, PΔhpaP, and PΔhrpE expressing pKgtP-c-Myc were incubated in XOM3 medium. Total protein extracts (TE) and culture supernatants (SN) were analyzed by immunoblotting using anti-c-Myc antibodies.

Fig 4

Subcellular localization of KgtP. (A) Transmembrane distribution and schematic map of deletion constructs of KgtP with GFP. The black boxes indicate the membrane-spanning domains in KgtP protein of X. oryzae pv. oryzae based on the homologous KgtP of E. coli (45, 46). KgtPΔ6 and KgtPΔ12 displayed 6 and 12 membrane-spanning domain deletions, respectively, in the KgtP protein of X. oryzae pv. oryzae. (B) Subcellular localization of KgtP-GFP in tobacco epidermal cells. Expression was driven by the CaMV 35S promoter. For confocal laser scanning microscopy, samples were taken 24 h postinoculation. Images were acquired by light microscopy (LM) or fluorescence microscopy (FM).

Fig 5

KgtP is required for wild-type levels of OsIDH expression in rice. RNAs were isolated from rice leaves that were infiltrated with cultures of X. oryzae pv. oryzae PXO99^A, PΔkgtP, and PΔhrcU strains for 8 h. (A) Semiquantitative RT-PCR analysis. The OsIDH mRNA level of tested strains was determined with the primer OsIDH-F/R (see Table S1 in the supplemental material). PCR products were then electrophoretically separated on a 1% agarose gel. The EF1-a gene of rice was used as an internal standard control. (B) Real-time quantitative RT-PCR analysis. The relative mRNA level of the OsIDH genes in rice inoculated with PΔkgtP was calculated with respect to the level of the corresponding transcripts in rice inoculated with wild-type PXO99^A. Values given are the means ± standard deviations of triplicate measurements from a representative experiment. The experiment was repeated at least three times, and similar results were obtained.

Fig 6

Working model of KgtP in X. oryzae pv. oryzae-rice pathosystem. kgtP, encoding the ketoglutarate transport protein of X. oryzae pv. oryzae PXO99^A, is positively regulated by HrpX and HrpG. The protein is presumably secreted in an HpaB-independent manner through the T3SS and localizes to the host cell membrane for α-ketoglutaric acid import. The model speculates that KgtP transports α-ketoglutaric acid to X. oryzae pv. oryzae from external substrates for growth survival and from rice cells for parasitism. PEP, phosphoenolpyruvate; CoA, coenzyme A.