A phytoplasma effector acts as a ubiquitin-like mediator between floral MADS-box proteins and proteasome shuttle proteins

Abstract

Plant pathogenic bacteria have developed effectors to manipulate host cell functions to facilitate infection. A certain number of effectors use the conserved ubiquitin–proteasome system in eukaryotic to proteolyze targets. The proteasome utilization mechanism is mainly mediated by ubiquitin interaction with target proteins destined for degradation. Phyllogens are a family of protein effectors produced by pathogenic phytoplasmas that transform flowers into leaves in diverse plants. Here, we present a noncanonical mechanism for phyllogen action that involves the proteosome and is ubiquitin-independent. Phyllogens induce proteasomal degradation of floral MADS-box transcription factors (MTFs) in the presence of RAD23 shuttle proteins, which recruit ubiquitinated proteins to the proteasome. Intracellular localization analysis revealed that phyllogen induced colocalization of MTF with RAD23. The MTF/phyllogen/RAD23 ternary protein complex was detected not only in planta but also in vitro in the absence of ubiquitin, showing that phyllogen directly mediates interaction between MTF and RAD23. A Lys-less nonubiquitinated phyllogen mutant induced degradation of MTF or a Lys-less mutant of MTF. Furthermore, the method of sequential formation of the MTF/phyllogen/RAD23 protein complex was elucidated, first by MTF/phyllogen interaction and then RAD23 recruitment. Phyllogen recognized both the evolutionarily conserved tetramerization region of MTF and the ubiquitin-associated domain of RAD23. Our findings indicate that phyllogen functionally mimics ubiquitin as a mediator between MTF and RAD23.

Random mutagenesis-based screening of the interface of phyllogen, a bacterial phyllody-inducing effector, for interaction with plant MADS-box proteins

“…P. aurantifolia’ witches’ broom of lime (WBDL) strain. PHYL genes were previously described as PHYL1 but were renamed by Kitazawa et al. (2022) .…”

“…For in planta anti-FLAG immunoprecipitation, Agrobacterium cultures for the expression of 3 × myc-fused SEP3 and SVP (SEP3- and SVP-3myc, respectively), 3 × FLAG-fused phyllogens (3FLAG-PHYL SY , -PHYL OY , and -PHYL WBDL , or their mutants), and P19, were adjusted to an OD 600 of 1.0, mixed at a ratio of 1:1:0.1, and infiltrated into N. benthamiana leaves. 3 × FLAG-fused phyllogen was immunoprecipitated using EZview Red ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich, St. Louis, MO, USA) 36 h after infiltration, as described previously ( Kitazawa et al., 2022 ). The proteins were separated by SDS-PAGE and detected by immunoblotting, as described previously ( Maejima et al., 2014 ).…”

“…Phyllogen also interacts with RADIATION SENSITIVE23 (RAD23) ( MacLean et al., 2014 ), which delivers ubiquitinated proteins to the proteasome ( Dantuma et al., 2009 ). Our recent study indicated that phyllogen interacts with its target MTF, and then with RAD23 without ubiquitin, resulting in delivery to the proteasome and degradation of the MTF in a ubiquitin-independent manner ( Kitazawa et al., 2022 ).…”

“…Previous reports have identified that the K domain is recognized by phyllogen ( MacLean et al., 2014 ; Liao et al., 2019 ). Recently, it was shown that phyllogen interacts with the self-tetramerization-involved region of SEP3 K domain, not the dimerization-involved region ( Kitazawa et al., 2022 ). The interaction of phyllogen with SEP3 requires the five residues in the tetramerization-involved region of SEP3 K domain (methionine at position 150 and leucine at positions 154, 157, 164, and 171) ( Kitazawa et al., 2022 ), which are highly conserved among E-class MTFs and responsible for its self-tetramerization ( Puranik et al., 2014 ; Rümpler et al., 2018 ).…”

Transcriptional regulation of plant innate immunity

“…Transcription factor WRKY40 Capsicum annuum [23] Ralstonia solanacearum PopP2 Transcription factor WRKY Arabidopsis thaliana [126] Pseudomonas syringae AvrRps4 Transcription factor WRKY Arabidopsis thaliana [127] Verticillium dahliae VdSCP41 Transcription factor CBP60g, SARD1 Arabidopsis thaliana [128] Pseudomonas syringae HopBB1 Transcription factor TCP14 Arabidopsis thaliana [129] Phytoplasma Phyllogen Transcription factor MADS-box Arabidopsis thaliana, Oryza sativa [130] Hyaloperonospora arabidopsidis…”