Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum

Abstract

Ralstonia solanacearum is a Gram-negative soil-borne bacterium that causes bacterial wilt disease in more than 200 plant species, including economically important Solanaceae species. In R. solanacearum, the hypersensitive response and pathogenicity (Hrp) type III secretion system is required for both the ability to induce the hypersensitive response (HR) in nonhost plants and pathogenicity in host plants. Recently, 72 effector genes, called rip (Ralstonia protein injected into plant cells), have been identified in R. solanacearum RS1000. RS1002, a spontaneous nalixidic acid-resistant derivative of RS1000, induced strong HR in the nonhost wild eggplant Solanum torvum in an Hrp-dependent manner. An Agrobacterium-mediated transient expression system revealed that Rip36, a putative Zn-dependent protease effector of R. solanacearum, induced HR in S. torvum. A mutation in the putative Zn-binding motif (E149A) completely abolished the ability to induce HR. In agreement with this result, the RS1002-derived Δrip36 and rip36E149A mutants lost the ability to induce HR in S. torvum. An E149A mutation had no effect on the translocation of Rip36 into plant cells. These results indicate that Rip36 is an avirulent factor that induces HR in S. torvum and that a putative Zn-dependent protease motif is essential for this activity.

Figure 1

Hypersensitive responses (HRs) induced by inoculation of Ralstonia solanacearum wild‐type (WT) RS1002 and its derivatives in Solanum torvum  Torubamubiga (A, C) and tobacco (B) leaves. Leaves were infiltrated with bacterial suspensions (OD ₆₀₀ of 0.3) of R. solanacearum  RS1002 (WT), RS1662 (Δrip36), RS1668 (rip36E149A), RS1650 (avrA::Gm^r) or RS1273 (ΔhrpY), or the complemented strains RS1672 (Δrip36  Tnrip36 ⁺) or RS1673 (Δrip36  Tnrip36E149A), and incubated under a 16 h light/8 h dark cycle at 28 °C. Photographs show representative results of S. torvum at 1 day post‐inoculation (dpi) (A, C) or tobacco at 2 dpi (B) from three independent experiments, which gave similar results. The broken lines represent the infiltrated area. (D) Summary of the plant responses caused by various strains in S. torvum and tobacco leaves. NT, not tested.

Figure 2

Effects of Agrobacterium‐mediated transient expression of Rip36, Rip36E149A and AvrA in Solanum torvum  Torubamubiga and tobacco leaves. (A) Construction of a series of pEl2Ω‐MCS plasmids expressing the Rip effector. Leaves were infiltrated with Agrobacterium tumefaciens  GV3101 harbouring no plasmid, an empty plasmid, a plasmid expressing Rip36, Rip36E149A or AvrA at OD ₆₀₀ of 0.5, and incubated under a 16 h light/8 h dark cycle at 28 °C. Photographs show representative results of S. torvum at 2 days post‐inoculation (dpi) (B) or tobacco at 4 dpi (C) from three independent experiments, which gave similar results. The broken lines represent the infiltrated area. (D). Summary of plant responses. HR, hypersensitive response.

Figure 3

Translocation and stability of Rip36 and Rip36E149A. (A) The cyclic adenosine monophosphate (cAMP) levels of Solanum torvum leaves inoculated with Ralstonia solanacearum  RS1002 (hrp ⁺) or RS1273 (ΔhrpY) strains expressing the calmodulin‐dependent adenylate cyclase (Cya) fusion with Rip36 or Rip36E149A. The cAMP level is shown as an average of three replications with standard deviations in parentheses. (B) The stability of Rip36 and Rip36E149A in S. torvum leaves after infiltration. Leaves of S. torvum were infiltrated with RS1002 (lane 1) and the rip36E149A (lane 2) and Δrip36 (lane 3) mutants, and total proteins were prepared from the inoculated leaves at 15 h post‐inoculation. The Rip36 and Rip36E149A proteins were detected by an anti‐Rip36 peptide antibody after sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Photograph shows a representative result of Western blotting from two independent experiments. An arrow indicates the position of the 20‐kDa marker protein.

Figure 4

Effect of rip36 mutation on the growth of Ralstonia solanacearum  RS1002 on host eggplant (A) and nonhost Solanum torvum (B). Leaves were infiltrated with wild‐type (WT) RS1002 or Δrip36 and rip36E149A mutants at 5 × 10⁴ colony‐forming units (cfu)/mL. Bacterial growth was measured 2 and 5 days after inoculation. Error bars indicate the standard deviation measured from six biological replicates. Means of three independent experiments are presented. Asterisks indicate a significant difference from the growth of the RS1002 WT strain in a t‐test (***P < 0.001).