Degradation of class E MADS-domain transcription factors in Arabidopsis by a phytoplasmal effector, phyllogen

Abstract

Members of the SEPALLATA (SEP) gene sub-family encode class E floral homeotic MADS-domain transcription factors (MADS TFs) that specify the identity of floral organs. The Arabidopsis thaliana genome contains 4 ancestrally duplicated and functionally redundant SEP genes, SEP1–4. Recently, a gene family of unique effectors, phyllogens, was identified as an inducer of leaf-like floral organs in phytoplasmas (plant pathogenic bacteria). While it was shown that phyllogens target some MADS TFs, including SEP3 for degradation, it is unknown whether the other SEPs (SEP1, SEP2, and SEP4) of Arabidopsis are also degraded by them. In this study, we found that all 4 SEP proteins of Arabidopsis are degraded by a phyllogen using a transient co-expression assay in Nicotiana benthamiana. This finding indicates that phyllogens may broadly target class E MADS TFs of plants.

Keywords:

• Arabidopsis
• floral development
• MADS transcription factors
• phyllody
• phyllogen
• phytoplasma
• SEPALLATA

Many angiosperms have flowers composed of 4 whorls of floral organs: sepals, petals, stamens, and carpels. The identity of these floral organs is regulated by a specific combination of floral homeotic MADS-domain transcription factors (MADS TFs) constituting the ABCE model.^1-3 Among these MADS TF genes, class E genes of the SEPALLATA (SEP) gene sub-family are essential for the development of all 4 floral organs as well as for floral meristem determinacy^1,4 because they function as the “glue” for MADS TF tetrameric complex formation.^5,6 The Arabidopsis thaliana genome contains 4 functionally redundant SEP genes, SEP1–4. This is suggested by the findings that the triple mutant (sep1/2/3) and the quadruple mutant (sep1/2/3/4) exhibit abnormal flowers with green sepal- or leaf-like organs in all 4 whorls, and also exhibit a loss of floral meristem determinacy,^1,4 however, a mutant of one of SEP genes produces only subtle phenotypes, consistent with the possibility of functional redundancy. ¹

Phytoplasmas are obligate plant pathogenic bacteria with highly reduced genomes.^7,8 They inhabit both plant phloem sieve cells and insect cells altering their own gene expression,⁹ and cause drastic morphological changes in infected plants, including witches' broom (proliferation of small branches resulting in a characteristic bushy growth) and phyllody (a loss of floral meristem determinacy and the transformation of floral organs into leaves).^10,11 In recent publications, witches' broom and phyllody symptoms of plants were shown to be associated with aberrant expression patterns of auxin-related genes and the genes encoding MADS TFs, respectively.^12,13 Moreover, an inducer of witches' broom, designated TENGU, and a gene family of phyllody-inducing effectors, designated phyllogens, were identified from phytoplasmas.^12,14,15

Phyllogens interact with and degrade some MADS TFs, including SEP3 of Arabidopsis.^15,16 However, it is unknown whether the other 3 class E proteins (SEP1, SEP2, and SEP4) of Arabidopsis are also degraded by phyllogens, though their interactions with a phyllogen were shown in a yeast 2-hybrid assay.¹⁶ It is also interesting from the perspective of the phylogenetic differences between SEP genes: in an evolutionary analysis of SEP gene sub-family, SEP3 and the other 3 SEP genes of Arabidopsis were divided into 2 different clades.¹⁷

In this study, we examined whether a phyllogen degrades SEP1, SEP2, and SEP4 of Arabidopsis. We transiently expressed YFP-fused SEP1, SEP2, SEP3, SEP4, or bZIP63, a basic leucine zipper transcription factor involved in the glucose-abscisic acid interaction network of Arabidopsis,¹⁸ in Nicotiana benthamiana leaves by agroinfiltration in combination with either GUS protein (control) or a phyllogen (PHYL1) of OY-W phytoplasma,¹⁹ and monitored their accumulation and subcellular localization by confocal microscopy. As expected, all of the YFP-fused transcription factors localized to the nucleus when GUS was co-expressed (Fig. 1, upper panels). While co-expression with PHYL1 did not affect the accumulation or nuclear localization of YFP-fused bZIP63, it reduced the fluorescence derived from YFP-fused SEP1, SEP2, and SEP4 as well as SEP3-YFP (Fig. 1, lower panels), indicating that all 4 SEP proteins of Arabidopsis can be degraded in the presence of phyllogens in vivo. This result agrees well with the fact that transgenic Arabidopsis plants expressing phyllogens show floral homeotic phenotypes similar to those of triple or quadruple mutants of sep genes.^1,4,14,15 Moreover, SEP3- and SEP1/2/4-like genes are highly conserved among various angiosperms as genes of the SEP sub-family encoding class E MADS TFs.¹⁷ Therefore, it is likely that phyllogens broadly target class E proteins of angiosperms, including eudicots, monocots, and other taxa, for degradation. Future studies investigating the target specificity of phyllogens will shed further light on the relationships between phyllody caused by phytoplasmas and the function of class E genes in angiosperms.

Figure 1. PHYL1 induces the degradation of SEP1–4 proteins. Agrobacterium cultures (OD₆₀₀ = 1.0) for transient expression of YFP-fused proteins (bZIP63 and SEP1–4) and GUS or PHYL1 protein were mixed at a ratio of 1:10 and infiltrated into Nicotiana benthamiana leaves. The accumulation and subcellular localization of the transiently expressed YFP-fused proteins were observed 50 h after infiltration. Scale bar = 100 µm. The Agrobacterium strain and plasmids were the same as in the previous study, respectively.¹⁵ The composition of the infiltration buffer is as follows: 10 mM morpholinepropanesulfonic acid (MES, pH 5.6), 10 mM MgCl₂, and 150 µM acetosyringone. Nuclear localization of the transcription factors used in this study was confirmed by DAPI staining (data not shown).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (category “S” of Scientific Research Grant 25221201), by the Funding Program for Next Generation World-Leading Researchers (project: GS005) initiated by the Council for Science and Technology Policy (CSTP), and by the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN).