The hrp genes of Pseudomonas cichorii are essential for pathogenicity on eggplant but not on lettuce

Pseudomonas cichorii causes necrotic lesions in eggplant and rot in lettuce. Through transposon insertion into P. cichorii strain SPC9018 we produced two mutants, 4-57 and 2-99, that lost virulence on eggplant but not lettuce. Analyses showed that a transposon was inserted into the hrpG gene in 4-57 and the hrcT gene in 2-99. Nucleotide sequences of the hrp genes of SPC9018 are homologous to those of Pseudomonas viridiflava BS group strains. The pathogenicity of 4-57 on eggplant was restored by transformation with an hrpF operon, originating from either SPC9018 or the BS group member P. viridiflava strain 9504 (Pv9504). These data suggested the involvement of hrp genes in the pathogenicity of SPC9018 on eggplant, and functional conservation of hrpF operons between SPC9018 and Pv9504. Both the hrpS mutant and the hrpL mutant were unable to cause necrotic lesions on eggplant leaves but retained their pathogenicity against lettuce. These results suggest that the pathogenicity of P. cichorii is hrp-dependent in eggplant, but not in lettuce

Amino Acid Substitutions in GyrA of Burkholderia glumae Are Implicated in Not Only Oxolinic Acid Resistance but Also Fitness on Rice Plants

Oxolinic acid (OA) resistance in field isolates of Burkholderia glumae, a causal agent of bacterial grain rot, is dependent on an amino acid substitution at position 83 in GyrA (GyrA83). In the present study, among spontaneous in vitro mutants from the OA-sensitive B. glumae strain Pg-10, we selected OA-resistant mutants that emerged at a rate of 5.7 × 10(−10). Nucleotide sequence analysis of the quinolone resistance-determining region in GyrA showed that Gly81Cys, Gly81Asp, Asp82Gly, Ser83Arg, Asp87Gly, and Asp87Asn are observed in these OA-resistant mutants. The introduction of each amino acid substitution into Pg-10 resulted in OA resistance, similar to what was observed for mutants with the responsible amino acid substitution. In vitro growth of recombinants with Asp82Gly was delayed significantly compared to that of Pg-10; however, that of the other recombinants did not differ significantly. The inoculation of each recombinant into rice spikelets did not result in disease. In inoculated rice spikelets, recombinants with Ser83Arg grew less than Pg-10 during flowering, and growth of the other recombinants was reduced significantly. On the other hand, the reduced growth of recombinants with Ser83Arg in spikelets was compensated for under OA treatment, resulting in disease. These results suggest that amino acid substitutions in GyrA of B. glumae are implicated in not only OA resistance but also fitness on rice plants. Therefore, GyrA83 substitution is thought to be responsible for OA resistance in B. glumae field isolates

バナジン酸によるエンドウ培養細胞におけるフィトアレキシン生産の誘導

We previously reported that the addition of orthovanadate suppressed the defense responses of plant differentiated tissues induced by a fungal elicitor.In this report,the effect of orthovanadate on the defense response of pea cultured cells was examined.The activities of ATPase and PI metabolism in plasma membrance fraction,which was prepared from suspension-cultured cells,were inhibited in vitro by orthovanadate as well as those in plasma membrances from pea epicotyl tissues. However,orthovanadate alone induced the accumulation of a phytoalexin, pisatin in suspention-clutured cells of pea in a manner similar to CuCl2.The viability of pea suspension-cultured cells was decreased by orthovanadate as well as by CuCl2.These results indicated that orthovanadate acts as an abiotic elicitor to pea suspension-cultured cell as observed in those of red bean,peanut and Perunia hybrida.バナジン酸は、エンドウ組織に褐紋病菌エリシターの処理で誘導される一連の防御応答を抑制することが報告されている。そこで、本研究ではバナジン酸のエンドウ培養細胞に対する影響を調べた。バナジン酸は培養細胞から分離した原形質膜画分のポリホスホイノシチド代謝系関連酵素やATPaseの活性を濃度依存的に阻害した。次に、褐紋病菌エリシター、塩化銅（非生物的エリシターの一種）、およびバナジン酸のin vivoでの影響を調べたところ、いずれの単独処理においてもエンドウ培養細胞のピサチン生産を誘導した。褐紋病菌エリシターはエンドウ培養細胞の細胞死（FDAの染色性喪失）を誘導しなかったが、塩化銅、バナジン酸は明らかな毒性を示した。以上の結果とこれまでの知見に基づいて、エンドウ培養細胞に対しては非生物的エリシターとして作用するバナジン酸の作用機構を考察した

Implications of Amino Acid Substitutions in GyrA at Position 83 in Terms of Oxolinic Acid Resistance in Field Isolates of Burkholderia glumae, a Causal Agent of Bacterial Seedling Rot and Grain Rot of Rice

Oxolinic acid (OA), a quinolone, inhibits the activity of DNA gyrase composed of GyrA and GyrB and shows antibacterial activity against Burkholderia glumae. Since B. glumae causes bacterial seedling rot and grain rot of rice, both of which are devastating diseases, the emergence of OA-resistant bacteria has important implications on rice cultivation in Japan. Based on the MIC of OA, 35 B. glumae field isolates isolated from rice seedlings grown from OA-treated seeds in Japan were divided into sensitive isolates (OSs; 0.5 μg/ml), moderately resistant isolates (MRs; 50 μg/ml), and highly resistant isolates (HRs; ≥100 μg/ml). Recombination with gyrA of an OS, Pg-10, led MRs and HRs to become OA susceptible, suggesting that gyrA mutations are involved in the OA resistance of field isolates. The amino acid at position 83 in the GyrA of all OSs was Ser, but in all MRs and HRs it was Arg and Ile, respectively. Ser83Arg and Ser83Ile substitutions in the GyrA of an OS, Pg-10, resulted in moderate and high OA resistance, respectively. Moreover, Arg83Ser and Ile83Ser substitutions in the GyrA of MRs and HRs, respectively, resulted in susceptibility to OA. These results suggest that Ser83Arg and Ser83Ile substitutions in GyrA are commonly responsible for resistance to OA in B. glumae field isolates

The Global Virulence Regulator PhcA Negatively Controls the Ralstonia solanacearum hrp Regulatory Cascade by Repressing Expression of the PrhIR Signaling Proteins▿ †

PhcA positively and negatively regulates many genes responsible for pathogenicity of Ralstonia solanacearum. The type III secretion system-encoding hrp regulon is one of the negatively controlled operons. PhcA bound to the promoter region of prhIR and repressed its expression, demonstrating that PhcA shuts down the most upstream component of a signal transfer system for hrpB activation

Contribution of Folate Biosynthesis to Ralstonia solanacearum Proliferation in Intercellular Spaces

The vigorous proliferation of Ralstonia solanacearum OE1-1 in host intercellular spaces after the invasion of host plants is necessary for the virulence of this bacterium. A folate auxotroph, RM, in which a mini-Tn5 transposon was inserted into pabB encoding para-aminobenzoate synthase component I, lost its ability to vigorously proliferate in intercellular spaces along with its systemic infectivity and virulence after inoculation into roots and infiltration into leaves of tobacco plants. Complementation of RM with the pabB gene allowed the mutant to multiply in intercellular spaces and to cause disease. In tobacco plants that were pretreated with folate, RM was able to vigorously proliferate in the intercellular spaces and cause disease. Interestingly, when it was inoculated through cut stems, the mutant multiplied in the plants and was virulent. Moreover, the mutant multiplied well in stem fluids but not in intercellular fluids, suggesting that the folate concentration within intercellular spaces may be a limiting factor for bacterial proliferation. Therefore, folate biosynthesis contributes to the vigorous proliferation of bacteria in intercellular spaces and leads to systemic infectivity resulting in virulence

バナジン酸によるエンドウ培養細胞におけるフィトアレキシン生産の誘導

We previously reported that the addition of orthovanadate suppressed the defense responses of plant differentiated tissues induced by a fungal elicitor.In this report,the effect of orthovanadate on the defense response of pea cultured cells was examined.The activities of ATPase and PI metabolism in plasma membrance fraction,which was prepared from suspension-cultured cells,were inhibited in vitro by orthovanadate as well as those in plasma membrances from pea epicotyl tissues. However,orthovanadate alone induced the accumulation of a phytoalexin, pisatin in suspention-clutured cells of pea in a manner similar to CuCl2.The viability of pea suspension-cultured cells was decreased by orthovanadate as well as by CuCl2.These results indicated that orthovanadate acts as an abiotic elicitor to pea suspension-cultured cell as observed in those of red bean,peanut and Perunia hybrida.バナジン酸は、エンドウ組織に褐紋病菌エリシターの処理で誘導される一連の防御応答を抑制することが報告されている。そこで、本研究ではバナジン酸のエンドウ培養細胞に対する影響を調べた。バナジン酸は培養細胞から分離した原形質膜画分のポリホスホイノシチド代謝系関連酵素やATPaseの活性を濃度依存的に阻害した。次に、褐紋病菌エリシター、塩化銅（非生物的エリシターの一種）、およびバナジン酸のin vivoでの影響を調べたところ、いずれの単独処理においてもエンドウ培養細胞のピサチン生産を誘導した。褐紋病菌エリシターはエンドウ培養細胞の細胞死（FDAの染色性喪失）を誘導しなかったが、塩化銅、バナジン酸は明らかな毒性を示した。以上の結果とこれまでの知見に基づいて、エンドウ培養細胞に対しては非生物的エリシターとして作用するバナジン酸の作用機構を考察した