Identification of RipAZ1 as an avirulence determinant of Ralstonia solanacearum in Solanum americanum

Abstract

Ralstonia solanacearum causes bacterial wilt disease in many plant species. Type III-secreted effectors (T3Es) play crucial roles in bacterial pathogenesis. However, some T3Es are recognized by corresponding disease resistance proteins and activate plant immunity. In this study, we identified the R. solanacearum T3E protein RipAZ1 (Ralstonia injected protein AZ1) as an avirulence determinant in the black nightshade species Solanum americanum. Based on the S. americanum accession-specific avirulence phenotype of R. solanacearum strain Pe_26, 12 candidate avirulence T3Es were selected for further analysis. Among these candidates, only RipAZ1 induced a cell death response when transiently expressed in a bacterial wilt-resistant S. americanum accession. Furthermore, loss of ripAZ1 in the avirulent R. solanacearum strain Pe_26 resulted in acquired virulence. Our analysis of the natural sequence and functional variation of RipAZ1 demonstrated that the naturally occurring C-terminal truncation results in loss of RipAZ1-triggered cell death. We also show that the 213 amino acid central region of RipAZ1 is sufficient to induce cell death in S. americanum. Finally, we show that RipAZ1 may activate defence in host cell cytoplasm. Taken together, our data indicate that the nucleocytoplasmic T3E RipAZ1 confers R. solanacearum avirulence in S. americanum. Few avirulence genes are known in vascular bacterial phytopathogens and ripAZ1 is the first one in R. solanacearum that is recognized in black nightshades. This work thus opens the way for the identification of disease resistance genes responsible for the specific recognition of RipAZ1, which can be a source of resistance against the devastating bacterial wilt disease.

Keywords: Ralstonia solanacearum; Solanum americanum; avirulence; bacterial wilt; effector; effector-triggered immunity.

FIGURE 1

Ralstonia solanacearum strain Pe_26 induces accession‐specific disease resistance in Solanum americanum. Bacterial wilt symptoms (a) and a survival graph (b) of the S. americanum accessions SP2273 and SP2275 infected with wild‐type R. solanacearum strains Pe_26 and To_1. (a) Photographs from representative plants were taken at 14 days postinoculation. Scale bar = 5 cm. (b) The percentage of surviving plants was recorded for 10 days. The data used for the survival graph were collected from three independent experiments. Log‐rank (Mantel–Cox) test p values are .0001 and .373 in SP2273 and SP2275, respectively

FIGURE 2

Overexpression of RipAZ1 induces cell death in Solanum americanum SP2273. (a) The candidate avirulence effectors were agroinfiltrated in SP2273 leaf epidermal cells. RipA1_GMI1000 and yellow fluorescent protein (YFP) were used as controls. Photographs were taken at 2 days postinfiltration. White circles indicate the infected leaf area showing cell death. (b) Conductivity measurement of RipAZ1_{Pe_26} in SP2273 and SP2275. Four‐week‐old S. americanum leaves transiently expressing RipAZ1_{Pe_26} or control proteins, RipA1_GMI1000 and YFP, using agroinfiltration were taken for conductivity measurements. HPI indicates hours postinfiltration. The data used were collected from three independent experiments. Coloured shapes and grey lines indicate means and standard deviations, respectively. Different letters indicate groups showing statistically significant differences at 48 HPI (p < .05; Student's t test). p values of circle–triangle, triangle–square, and square–circle are as follows: 0.343, 6.88 × 10⁻¹¹, and 2.33 × 10⁻¹¹ in SP2273; 2.20 × 10⁻¹⁶, 0.885, and 4.20 × 10⁻¹⁶ in SP2275

FIGURE 3

RipAZ1 triggers accession‐specific immune responses in Solanum americanum. Bacterial wilt symptoms (a) and a survival graph (b) of accessions SP2273 and SP2275 infected with wild‐type, ΔripAZ1, or ΔripAZ1 (ripAZ1) strain of Pe_26. (a) Photographs from representative plants were taken at 10 days postinfiltration. Scale bar = 5 cm. (b) The percentage of survival plants was recorded for 10–20 days. The data used for survival graphs were collected from three independent experiments. Log‐rank (Mantel–Cox) test p values are <.0001 and .128 in SP2273 and SP2275, respectively

FIGURE 4

A naturally occurring C‐terminally truncated RipAZ1 variant does not induce cell death in Solanum americanum. Amino acid sequence comparison (a), cell death responses in SP2273 (b), and western blot analysis in Nicotiana benthamiana (c) of the six representative RipAZ1 natural variants selected for functional analysis. (a) Nonsynonymous single nucleotide polymorphisms are indicated as black lines. A grey box indicates the region with no sequence identity due to a frameshift mutation caused by 1‐base pair insertion as indicated with an asterisk. An amino acid in‐frame‐insertion is indicated with black triangles. Phylotype is abbreviated as “Phy”. (b) The photograph was taken at 2 days postinfiltration. White circles indicate the infected leaf area showing cell death. (c) Empty vector (EV; pICH86988) and yellow fluorescent protein (YFP)‐FLAG were used as controls. Proteins were immunoblotted with anti‐FLAG antibody. Leaf tissue was harvested at 2 days postinfiltration. Ponceau staining shows equal loading of proteins

FIGURE 5

Analysis of truncated RipAZ1 defines the minimal region sufficient for cell death‐inducing activity. Schematics (a), cell death responses in SP2273 (b), and western blot analysis in Nicotiana benthamiana (c) of truncated variants of RipAZ1_{Pe_51}‐FLAG. (a) The N‐ or C‐terminally truncated variants of RipAZ1_{Pe_51} were based on the predicted secondary structure (Figure S10). Numbers indicate amino acids. Variants inducing cell death in SP2273 are indicated on the right panel with “+”. (b) The photograph was taken at 2 days postinfiltration. White circles indicate the infected leaf area showing cell death. (c) Empty vector (EV; pICH86988) and YFP‐FLAG were used as controls. Proteins were immunoblotted with anti‐FLAG antibody. Leaf tissue was harvested at 2 days postinfiltration. The protein bands with expected molecular weight are indicated with white asterisks. Ponceau staining shows equal loading of proteins

FIGURE 6

RipAZ1‐YFP localizes in the nucleus and cytoplasm when transiently expressed in Nicotiana benthamiana leaf epidermal cells. Subcellular localization in N. benthamiana (a) and cell death responses in SP2273 (b) of RipAZ1‐YFP variants when transiently expressed using agroinfiltration. (a) YFP signals were observed at 2 days postinfiltration. Scale bar = 50 μm. (b) The photograph was taken at 2 days postinfiltration