Phytoplasma-conserved phyllogen proteins induce phyllody across the Plantae by degrading floral MADS domain proteins

Abstract

ABCE-class MADS domain transcription factors (MTFs) are key regulators of floral organ development in angiosperms. Aberrant expression of these genes can result in abnormal floral traits such as phyllody. Phyllogen is a virulence factor conserved in phytoplasmas, plant pathogenic bacteria of the class Mollicutes. It triggers phyllody in Arabidopsis thaliana by inducing degradation of A- and E-class MTFs. However, it is still unknown whether phyllogen can induce phyllody in plants other than A. thaliana, although phytoplasma-associated phyllody symptoms are observed in a broad range of angiosperms. In this study, phyllogen was shown to cause phyllody phenotypes in several eudicot species belonging to three different families. Moreover, phyllogen can interact with MTFs of not only angiosperm species including eudicots and monocots but also gymnosperms and a fern, and induce their degradation. These results suggest that phyllogen induces phyllody in angiosperms and inhibits MTF function in diverse plant species.

Keywords: ABCE model; MADS domain transcription factor; floral development; phyllody; phyllogen; phytoplasma.

Fig. 1.

Characteristics of phyllogen-induced phyllody in petunia flowers. Whole flower (A) and each of the floral organs (B–E) of ALSV-empty-infected plants: (B) sepals, (C) petals, (D) stamens, and (E) a pistil. No malformation was observed in any floral organ. Whole flower (F) and each of the floral organs (G–J) of ALSV-PHYL1_OY-infected plants. Flower malformations were observed in each floral organ. (G) Sepals showing leaf-like round shapes. (H) Petals becoming small and green at the tips and edges of the limbs. (I) Stamens with no pollen, but with small leaf-like structures on top of their anthers. Two left stamens did not show malformation, but pollen was already shed. An enlarged view of an abnormal anther (dotted square) is shown in the upper right of the picture. (J) Pistil replaced by leaf-like structures with trichomes. Arrowheads indicate newly developed floral buds. (K) Flower of ALSV-PHYL1_PnWB-infected petunia. The observed phyllody was very similar to that of ALSV-PHYL1_OY-infected petunia plants. Bars indicate 1 cm.

Fig. 2.

Characteristics of PHYL1_PnWB-induced phyllody on sunflower and sesame flowers. (A–D) Flowers of ALSV-empty-infected sunflower. A disc floret and ray floret are shown in (C) and (D), respectively. No flower malformation was observed in any floral organ. (E–I) Flowers of ALSV-PHYL1_PnWB-infected sunflower. Flower malformations were observed on both disk and ray florets. (G) Disk floret showing mild malformations. Bract and sepals became green and elongated. (H) Disk floret showing severe malformations. Petals became green and a pistil changed into a leaf-like structure. (I) Ray floret showing malformations. Corolla became small, and its color changed slightly to green. (J–S) Characteristics of phyllogen-induced phyllody in sesame flowers. Whole flower (J) and each of the floral organs (K–N) of ALSV-empty-infected sesame plants: (K) sepals, (L) separated petals, (M) stamens, and (N) a pistil. No malformation was observed in any floral organ. Whole flower (O) and each of the floral organs (P–S) of ALSV-PHYL1_PnWB-infected plants. Flower malformations were observed in each floral organ. (P) Sepals showing large leaf-like structures. (Q) Petals becoming green from the tips. (R) Stamens with no pollen, but with small leaf-like structures on top of their anthers. (S) Pistils replaced by leaf-like structures. Bars indicate 1 cm. B, bracts; S, sepals; Pe, petals; Pi, pistils.

Fig. 3.

PHYL1_OY interacts with A- and E-class MADS domain transcription factors (MTFs) of angiosperms in yeast cells. The MTFs listed in Table 1 were fused to the GAL4 activation domain (AD). These AD-fused MTFs were expressed in the yeast strain AH109, with the GAL4 DNA-binding domain (BD) or BD-fused PHYL1_OY. The AD-fused SEP3 is a positive control, which interacts with PHYL1_OY (Maejima et al., 2014a). Yeast cells harboring the appropriate AD and BD vectors were adjusted to an OD₆₀₀ of 0.1. Aliquots (10 µl) of these cells were spotted on synthetically defined medium lacking leucine/tryptophan (–LW), lacking leucine/tryptophan/histidine (–LWH), lacking leucine/tryptophan/histidine and containing 5 mM 3-amino-1,2,4-triazole (–LWH+3AT), or lacking tryptophan/leucine/adenine/histidine (–LWAH). The plates were incubated for 4 d at 30 °C.

Fig. 4.

PHYL1_OY induces degradation of A- and E-class MADS domain transcription factors (MTFs) of angiosperms in Nicotiana benthamiana epidermal cells. Each of the MTFs was fused to the YFP protein and co-expressed with either GUS or PHYL1_OY. We used bZIP63–YFP and SEP3–YFP as negative and positive controls, respectively. Agrobacterium cultures (OD₆₀₀=1.0) carrying each of the YFP-fused proteins and those carrying either GUS or PHYL1_OY were mixed at a ratio of 1:10, and infiltrated into N. benthamiana leaves. YFP fluorescence was observed 36 h after infiltration. Graphs show the number of the YFP signals quantified in a relative manner. The average YFP signal of each MTF expressed with GUS was set as 1.0. Each bar represents the average of YFP signals observed in four leaf areas of 2.4 mm². The bar indicates 100 µm. Asterisks indicate statistically significant differences compared with GUS (**P<0.01 by the one-tailed Student’s t-test). The experiment was performed independently three times. (This figure is available in colour at JXB online.)

Fig. 5.

qRT-PCR analyses of MADS domain transcription factor (MTF) genes in floral buds of ALSV-empty- and ALSV-PHYL1_OY-infected plants. Each bar represents the average of nine plants. The expression levels of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were used for normalization. The average expression levels in the control were set as 1.0. Asterisks indicate statistically significant differences compared with the control (*P<0.05 by the two-tailed Student’s t-test).

Fig. 6.

PHYL1_OY targets the MTFs of gymnosperms and a fern. MADS domain transcription factors (MTFs) were chosen from two gymnosperms (CjMADS14 from Cryptomeria japonica and DAL1 from Picea abies) and a fern (CRM6 from Ceratopteris pteridoides). (A) A yeast two-hybrid assay showed interaction between PHYL1_OY and the MTFs in yeast cells. Each of the AD-fused MTFs was expressed in the yeast strain AH109, with BD or BD-fused PHYL1_OY. Experimental details are described in the legend to Fig. 3. (B) The YFP reporter assay showed PHYL1_OY-dependent degradation of the MTFs in Nicotiana benthamiana leaves. Accumulation of each MTF was evaluated by immunoblotting using an anti-GFP antibody.