A unique virulence factor for proliferation and dwarfism in plants identified from a phytopathogenic bacterium

Abstract

One of the most important themes in agricultural science is the identification of virulence factors involved in plant disease. Here, we show that a single virulence factor, tengu-su inducer (TENGU), induces witches' broom and dwarfism and is a small secreted protein of the plant-pathogenic bacterium, phytoplasma. When tengu was expressed in Nicotiana benthamiana plants, these plants showed symptoms of witches' broom and dwarfism, which are typical of phytoplasma infection. Transgenic Arabidopsis thaliana lines expressing tengu exhibited similar symptoms, confirming the effects of tengu expression on plants. Although the localization of phytoplasma was restricted to the phloem, TENGU protein was detected in apical buds by immunohistochemical analysis, suggesting that TENGU was transported from the phloem to other cells. Microarray analyses showed that auxin-responsive genes were significantly down-regulated in the tengu-transgenic plants compared with GUS-transgenic control plants. These results suggest that TENGU inhibits auxin-related pathways, thereby affecting plant development.

Fig. 1.

Identification of a virulence factor inducing phytoplasma disease symptoms. (A) N. benthamiana plants inoculated with A. tumefaciens harboring empty vector (pCAMV) (Left), pCAMV-PAM765 (tengu) (Center), or pCAMV-PAM486 (Right). (Lower) Stems are highlighted with red lines; leaf petioles are highlighted with black lines. The center plant showed witches' broom disease symptoms (a dramatically increased shoot system). (B) The number of leaves per plant following inoculation with the viral vector. The error bars indicate the SD. An asterisk indicates a significant difference (P < 0.05). 1, pCAMV; 2, pCAMV-PAM765 (tengu).

Fig. 2.

Comparison of 35S∷PAM765 (tengu) transgenic plants and phytoplasma (OY strain)-infected plants. (A) Phenotypes of the OY-infected plants. (Left) uninfected plant (control). (Center and Right) OY-infected plants. The center and right plants show severe dwarfism and witches' broom symptoms, respectively. (B) Phenotypes of the 35S∷PAM765 transgenic A. thaliana lines. (Left) 35S∷GUS transgenic line (control). (Center and Right) 35S∷PAM765 transgenic lines. The center and right transgenic lines show severe dwarfism with short internodes and witches' broom symptoms, respectively.

Fig. 3.

Analysis of branching in 35S∷PAM765 (tengu) transgenic plants and phytoplasma-infected plants. (A) A 35S∷GUS transgenic plant (control). (B–E) Phenotypes of the 35S∷PAM765 transgenic A. thaliana lines. (B–D) The 35S∷PAM765 transgenic plants exhibited defects in phyllotaxis (2 or more flowers growing from a single point on the stem). (E) A 35S∷PAM765 transgenic plant with sterile flowers. (F) An uninfected plant (control). (G–I) Phenotypes of the OY-infected plants. (G and H) The OY-infected plants exhibited defects in phyllotaxis, similar to (B–D). (I) An OY-infected plant with sterile flowers, as in (E). (Scale bars, 50 mm.) (J) The transcription of tengu was examined by quantitative real-time RT-PCR and the results were normalized against the expression of tufB. The error bars indicate the SD. An asterisk indicates a significant difference (P < 0.05). 1, Phytoplasma-infected plants (C. coronarium). 2, Phytoplasma-infected insects (M. striifrons).

Fig. 4.

Immunohistochemical detection of TENGU and Amp proteins in apical meristem tissue. (A and B) Apical meristem tissue sections from OY-infected plants were reacted with the anti-TENGU antibody (A) or the anti-Amp antibody (B). Bars, 1 mm. (C) An apical meristem tissue section from healthy plant was reacted with the anti-TENGU antibody. (Scale bar, 1 mm.) (D, F, and H) Enlarged section of (A). (E, G, and I) Enlarged section of (B). (D and E) Branching region of axillary buds. (Scale bar, 200 μm.) (F and G) Tip region of stem. Bar, 200 μm. (H and I) Apical meristem. (Scale bar, 100 μm.) ph; phloem, pa; parenchyma. (J) Analysis of the subcellular localization of TENGU. Chimeric constructs (35S∷GFP [left], 35S∷GFP-tengu [center], and 35S∷tengu-GFP [right]) were transiently expressed in onion epidermal cells.