Ralstonia solanacearum Type III Effectors with Predicted Nuclear Localization Signal Localize to Various Cell Compartments and Modulate Immune Responses in Nicotiana spp

Abstract

Ralstonia solanacearum (Rso) is a causal agent of bacterial wilt in Solanaceae crops worldwide including Republic of Korea. Rso virulence predominantly relies on type III secreted effectors (T3Es). However, only a handful of Rso T3Es have been characterized. In this study, we investigated subcellular localization of and manipulation of plant immunity by 8 Rso T3Es predicted to harbor a nuclear localization signal (NLS). While 2 of these T3Es elicited cell death in both Nicotiana benthamiana and N. tabacum, only one was dependent on suppressor of G2 allele of skp1 (SGT1), a molecular chaperone of nucleotide-binding and leucine-rich repeat immune receptors. We also identified T3Es that differentially regulate flg22-induced reactive oxygen species production and gene expression. Interestingly, several of the NLS-containing T3Es translationally fused with yellow fluorescent protein accumulated in subcellular compartments other than the cell nucleus. Our findings bring new clues to decipher Rso T3E function in planta.

Keywords: Nicotiana spp.; Ralstonia solanacearum; bacterial wilt; innate immunity; type III effectors.

Fig. 1

RipA1 and RipE1 induce cell death in Nicotiana spp. N. benthamiana (N.b) and N. tabacum (N.t) leaves were infiltrated with Agrobacterium tumefaciens AGL1 strain carrying C-terminally 3×FLAG-tagged T3E, BAX, or PopP2. Photographs were taken 4 days post-infiltration. The number of patches showing cell death out of total infiltrated patches for that treatment in more than three independent experiments is indicated under each photograph panel.

Fig. 2

NbSGT1 is required for cell death triggered by RipE1. (A) Effector-triggered cell death in silenced plants. Nicotiana benthamiana plants silenced for EV or NbSGT1 were infiltrated with Agrobacterium tumefaciens AGL1 strain carrying RipA1, RipE1, BAX, or GFP (as in Fig. 1). Photographs were taken 7 days post-infiltration. The number of patches showing cell death out of total infiltrated patches for that treatment in three independent experiments is indicated under each photograph panel. (B) Analysis of NbSGT1 gene expression in silenced plants. NbSGT1 and NbActin gene-specific fragments were amplified by reverse transcription polymerase chain reaction..

Fig. 3

RipAD, RipAF1, and RipD impair flg22-induced reactive oxygen species (ROS) production. ROS production upon treatment with 100 nM flg22 was continuously recorded for 75 min for Nicotiana benthamiana leaf tissue expressing T3E, GFP, or AvrPto. (A) ROS production curve over duration of the experiment, and (B) total ROS production for the experiment. Data presented in (A) are mean ± standard error of mean (SEM; n = 16) of relative light units (RLU) from one representative experiment. Total ROS production (B) is presented as mean percentage relative to total ROS production in GFP from one representative experiment. A statistically significant difference compared to GFP-expressing leaf is indicated by asterisks (Student’s t-test, ***P < 0.001).

Fig. 4

RipAD, RipAO, and RipD impair flg22-induced defense gene expression. Induction of defense marker gene expression in Nicotiana benthamiana leaf tissue expressing T3E or GFP was monitored 60 min after 100 nM flg22 treatment. Mock treatment (water) in GFP-expressing leaf is included as a negative control. Gene expression was normalized by NbEF1α expression and is shown as the ratio of expression level (%) compared to flg22-treated GFP sample. Data presented are the mean of three independent experiments ± standard error of mean (n = 3). Statistical significance compared to GFP-expressing leaf is indicated by asterisks (Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 5

Subcellular localization of the predicted nuclear localization signal-containing T3Es. Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens AGL1 strain carrying YFP-tagged T3E and different organelle markers fused with mCherry (-mC) as indicated. Fluorescence was observed 2 days post-infiltration by confocal microscopy. Pictures are representative of three independent experiments. Scale bars = 25 μm (RipAD, RipAB, RipAO, and RipD), 50 μm (RipL), 10 μm (RipAF1).