Flower malformation represented by phyllody is a common symptom of phytoplasma infection induced by a novel family of phytoplasma effectors called phyllogens. Despite the accumulation of functional and structural phyllogen information, the molecular mechanisms of phyllody have not yet been integrated with their evolutionary aspects due to the limited data on their homologs across diverse phytoplasma lineages. Here, we developed a novel universal PCR‐based approach to identify 25 phytoplasma phyllogens related to nine “Candidatus Phytoplasma” species, including four species whose phyllogens have not yet been identified. Phylogenetic analyses showed that the phyllogen family consists of four groups (phyl‐A, ‐B, ‐C, and ‐D) and that the evolutionary relationships of phyllogens were significantly distinct from those of phytoplasmas, suggesting that phyllogens were transferred horizontally among phytoplasma strains and species. Although phyllogens belonging to the phyl‐A, ‐C, and ‐D groups induced phyllody, the phyl‐B group lacked the ability to induce phyllody. Comparative functional analyses of phyllogens revealed that a single amino acid polymorphism in phyl‐B group phyllogens prevented interactions between phyllogens and A‐ and E‐class MADS domain transcription factors (MTFs), resulting in the inability to degrade several MTFs and induce phyllody. Our finding of natural variation in the function of phytoplasma effectors provides new insights into molecular mechanisms underlying the aetiology of phytoplasma diseases.
Phyllogen, a bacterial virulence factor, induced phyllody in various eudicot species, and had broad-spectrum degradation activity on MADS domain transcription factors of plants, suggesting phyllogen universally functions in plants.
SUMMARYOne of the plant host resistance machineries to viruses is attributed to recessive alleles of genes encoding critical host factors for virus infection. This type of resistance, also referred to as recessive resistance, is useful for revealing plant-virus interactions and for breeding antivirus resistance in crop plants. Therefore, it is important to identify a novel host factor responsible for robust recessive resistance to plant viruses. Here, we identified a mutant from an ethylmethane sulfonate (EMS)-mutagenized Arabidopsis population which confers resistance to plantago asiatica mosaic virus (PlAMV, genus Potexvirus). Based on map-based cloning and single nucleotide polymorphism analysis, we identified a premature termination codon in a functionally unknown gene containing a GYF domain, which binds to proline-rich sequences in eukaryotes. Complementation analyses and robust resistance to PlAMV in a T-DNA mutant demonstrated that this gene, named Essential for poteXvirus Accumulation 1 (EXA1), is indispensable for PlAMV infection. EXA1 contains a GYF domain and a conserved motif for interaction with eukaryotic translation initiation factor 4E (eIF4E), and is highly conserved among monocot and dicot species. Analysis using qRT-PCR and immunoblotting revealed that EXA1 was expressed in all tissues, and was not transcriptionally responsive to PlAMV infection in Arabidopsis plants. Moreover, accumulation of PlAMV and a PlAMV-derived replicon was drastically diminished in the initially infected cells by the EXA1 deficiency. Accumulation of two other potexviruses also decreased in exa1-1 mutant plants. Our results provided a functional annotation to GYF domain-containing proteins by revealing the function of the highly conserved EXA1 gene in plant-virus interactions.
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.
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