Phytobacterial type III effectors HopX1, HopAB1 and HopF2 enhance sense-post-transcriptional gene silencing independently of plant R gene-effector recognition

Abstract

Plant- and animal-pathogenic bacteria deploy a variable arsenal of type III effector proteins (T3EP) to manipulate host defense. Specific biochemical functions and molecular or subcellular targets have been demonstrated or proposed for a growing number of T3EP but remain unknown for the majority of them. Here, we show that transient expression of genes coding certain bacterial T3EP (HopAB1, HopX1, and HopF2), which did not elicit hypersensitive response (HR) in transgenic green fluorescent protein (GFP) Nicotiana benthamiana 16C line, enhanced the sense post-transcriptional gene silencing (S-PTGS) triggered by agrodelivery of a GFP-expressing cassette and the silencing enhancement could be blocked by two well-known viral silencing suppressors. Further analysis using genetic truncations and site-directed mutations showed that the receptor recognition domains of HopAB1 and HopX1 are not involved in enhancing silencing. Our studies provide new evidence that phytobacterial pathogen T3EP manipulate the plant small interfering RNA pathways by enhancing silencing efficiency in the absence of effector-triggered immunity signaling and suggest that phytopathogenic bacterial effectors affect host RNA silencing in yet other ways than previously described.

Fig. 1

Effect of transient expression of hopX1, hopAB1, and hopX1m on green fluorescent protein (GFP) silencing in Nicotiana benthamiana 16C plants. A, GFP fluorescence assay. Mixtures (1:1) of agrobacteria carrying the expression cassettes or control plasmids (empty vector [EV]) indicated above the images were infiltrated in N. benthamiana 16C leaves photographed at 6 days postinoculation (dpi) under UV (A-I) and visible (A-II). Agroinfiltration was carried out as described (Hamilton et al. 2002). GFP fluorescence was monitored 4 and 6 dpi with a hand-held long-wavelength UV lamp. Plants were photographed with a NIKON COOLPIX 990 digital camera, under UV (A-I) and normal light (A-II) and pictures were processed using Adobe Photoshop CS2. B, Densitometric quantification of GFP fluorescence in infiltrated leaf areas, using the ImageJ program (developed at the United States National Institutes of Health). Fluorescence intensity values were determined after subtracting background fluorescence of noninfiltrated areas on the same leaf. Experiments were repeated at least 10 times on separate plants.

Fig. 2

Transient expression of hopX1, hopAB1 enhances the RNA-mediated green fluorescent protein (GFP) silencing, in contrast to the nonfunctional hopX1 allele (hopX1m) from Pseudomonas syringae pv. phaseolicola NPS3121. Mixtures of agrobacteria (1:1) carrying the effector or viral protein expression cassettes oir empty vector (EV) control plasmids indicated above the lanes were infiltrated in Nicotiana benthamiana 16C leaves. I-A, GFP mRNA and small interfering (si)RNA levels at 6 days postinoculation (dpi) in agroinfiltrated tissues are indicated above the images. A strong decrease of GFP mRNA and an increase of siRNA accumulation relative to the control are observed in the presence of hopX1 at 6 dpi, indicating enhancement of the GFP silencing by HopX1. Expression of viral suppressors P19 and P38 alone relieves silencing and hopX1 does not counteract this effect; GFP mRNA and siRNA levels in agroinfiltrated tissues are indicated above the images. I-B, Quantification (using the ImageJ program) of siRNA levels. In the presence of hopX1, the GFP siRNA level is increased approximately threefold compared with the empty vector control. II-A, GFP mRNA and siRNA levels at 6 dpi in agroinfiltrated tissues, as are indicated above the images. At 6 dpi, a decrease of GFP mRNA (below the control) and an increase of siRNA accumulation (above the control) was observed with hopAB1, indicating enhancement of GFP silencing. Expression of viral suppressors P19 and P38 alone or together with hopAB1 strongly increased GFP mRNA while strongly decreasing GFP siRNA levels. II-B, Quantification (using the ImageJ program) of siRNA levels. In the presence of hopAB1, the GFP siRNA level is increased approximately twofold compared with the empty vector control. III-A, GFP mRNA and siRNA levels in agroinfil-trated tissues as indicated below the images. GFP siRNAs appear at 5 to 6 dpi. Strong GFP siRNA accumulation is seen in leaves treated with hopX1 but not in those treated with hopX1m. III-B, Quantification of siRNA levels in hopX1- and hopX1m-treated leaves.

Fig. 3

Overlap infiltration assay (Chakravarthy et al. 2009; Oh and Collmer 2005) with agrobacteria carrying expression cassettes for hopX1 or hopAB1 and for viral silencing suppressor P19 or P38. Mixtures (1:1) of agrobacteria carrying separate expression cassettes for green fluorescent protein (GFP), type III effector proteins (HopX1 or HopAB1), and the viral silencing suppressors P19 or P38, each under the Cauliflower mosaic virus 35S promoter, were infiltrated in Nicotiana benthamiana 16C leaves in combinations indicated above the images and photographed 6 days postinoculation. The type III effector silencing enhancer phenotype on green fluorescent protein (GFP) accumulation is fully reversed (strong fluorescence in the overleaping co-infiltration area) by both viral suppressors. A, N. benthamiana 16C leaves infiltrated with 1:1 mixtures of agrobacteria containing 35S::GFP/35S::hopX1, and 35S::GFP/35S:: P19 or 35S::GFP/35S::P38. B, Leaves infiltrated with 1:1 mixtures of agrobacteria containing 35S::GFP/35S::hopAB1 and 35S::GFP/35S::P19 or 35S::GFP/35S::P38. Experiments were repeated at least eight times on separate N. benthamiana 16C plants with similar results.

Fig. 4

Effect of transiently expressed hopAB1 wild type (w.t.) and hopAB1-CTD (HopAB1_376-539) on green fluorescent protein (GFP) silencing in Nicotiana benthamiana 16C plants. hopAB1 w.t. and the hopAB1-CTD have been cloned under the transcription regulation of the 35S Cauliflower mosaic virus promoter in the pBin-Hyg-Tx vector. A, GFP fluorescence assay. Mixtures (1:1) of agrobacteria carrying the expression constructs indicated above the images were infiltrated in N. benthamiana 16C leaves photographed at 5 days postinoculation (dpi) under UV (A-I) and visible (A-II) light. Agroinfiltration was carried out as described (Hamilton et al. 2002). GFP fluorescence was monitored 4, 5, and 6 dpi with a handheld long-wavelength UV lamp. B, Densitometric quantification of GFP fluorescence in infiltrated areas of leaves. Experiments were repeated at least 10 times on separate plants with similar results.

Fig. 5

Effect of transiently expressed hopX1 wild type (w.t.), hopX1 mutant 1 (N-terminal domain), and hopX1 mutant 2 (catalytic triad) alleles on green fluorescent protein (GFP) silencing in Nicotiana benthamiana 16C plants. hopX1 w.t. and the two mutants have been cloned under the transcription regulation of a dexamethasone (DEX)-inducible promoter in the pTA7002 vector. A, GFP fluorescence assay. Mixtures (1:1) of agrobacteria carrying the expression constructs indicated above the images were infiltrated in N. benthamiana 16C leaves photographed at 6 days postinoculation (dpi) under UV (A-I) and visible (A-II) light. Agroinfiltration was carried out as described (Hamilton et al. 2002). GFP fluorescence was monitored 4 and 6 dpi with a handheld long-wavelength UV lamp. B, Densitometric quantification of GFP fluorescence in infiltrated areas of leaves. Experiments were repeated at least 10 times on separate plants with similar results.

Fig. 6

Effect of transient expression of cassettes for hopAB1, HopAB1-C terminal domain (residues 376-539), HopX1, and HopX1-A domain and catalytic triad mutants and HopAB1-C terminal domain (residues 376 to 539) on the lsiRNA and nat-siRNA accumulation in agroinfiltrated efr-1 Arabidopsis plants. For these experiments, Pseudomonas syringae pv. tomato DC3000 carrying the avrRpt2 gene was used as a positive technical control for comparison.

Fig. 7

Inhibition of crown gall tumor development in petioles of Nicotiana benthamiana. Petioles of wild-type (w.t.) N. benthamiana leaves were photographed 28 days after inoculation with: A, Agrobacterium tumefaciens A281 (A. tumefaciens C58C1 [pTiBo542], induces crown gall tumors); B, A. tumefaciens A281 carrying the 35S::hopX1 cassette; and C, A. tumefaciens A281 carrying pART27 (empty vector [EV]) used as negative control. The relevant elements of the T-DNAs are schematically drawn below the photographs. 35S refers to the Cauliflower mosaic virus 35S promoter and OCS refers to the octopine synthase gene transcription terminator region. Four petioles were inoculated for each treatment and all experiments were repeated at least four times. Crown gall tumors were observed in all A, w.t. Ti and C, EV treatments. In contrast, no gall formation was observed in 80% of the petioles inoculated with A. tumefaciens A281 currying the 35S::hopX1 expression cassette.