Plant pathogens alter the course of plant developmental processes, resulting in abnormal morphology in infected host plants. Phytoplasmas are unique plant-pathogenic bacteria that transform plant floral organs into leaf-like structures and cause the emergence of secondary flowers. These distinctive symptoms have attracted considerable interest for many years. Here, we revealed the molecular mechanisms of the floral symptoms by focusing on a phytoplasma-secreted protein, PHYL1, which induces morphological changes in flowers that are similar to those seen in phytoplasma-infected plants. PHYL1 is a homolog of the phytoplasmal effector SAP54 that also alters floral development. Using yeast two-hybrid and in planta transient co-expression assays, we found that PHYL1 interacts with and degrades the floral homeotic MADS domain proteins SEPALLATA3 (SEP3), APETALA1 (AP1) and CAULIFLOWER (CAL). This degradation of MADS domain proteins was dependent on the ubiquitin–proteasome pathway. The expression of floral development genes downstream of SEP3 and AP1 was disrupted in 35S::PHYL1 transgenic plants. PHYL1 was genetically and functionally conserved among other phytoplasma strains and species. We designate PHYL1, SAP54 and their homologs as members of the phyllody-inducing gene family of ‘phyllogens’.

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.

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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.

Fig mosaic virus (FMV), a negative-strand RNA virus, is recognized as a causal agent of fig mosaic disease. We performed RT-PCR for 14 FMV isolates collected from symptomatic fig plants in Japan and Serbia using primers corresponding to the conserved 13 nt stretches found at the termini of FMV genomic segments. The resulting simultaneous amplification of all FMV genomic segments yielded four previously identified segments of FMV and two novel segments. These novel FMV genomic RNA segments were found in each of the 14 FMV isolates analysed. In Northern blot studies, both the sense and antisense strands of these novel RNA molecules accumulated in FMV-infected fig leaves but not in uninfected fig leaves, confirming that they replicate as FMV genomic segments. Sequence analysis showed that the novel RNA segments are similar, in their structural organization and molecular evolutionary patterns, to those of known FMV genomic RNA segments. Our findings thus indicate that these newly discovered RNA segments are previously unidentified FMV genomic segments, which we have designated RNA5 and RNA6.

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.

Plants exhibit a wide variety of disease symptoms in response to pathogen attack. In general, most plant symptoms are recognized as harmful effects on host plants, and little is known about positive aspects of symptoms for infected plants. Herein, we report the beneficial role of purple top symptoms, which are characteristic of phytoplasma-infected plants. First, by using plant mutants defective in anthocyanin biosynthesis, we demonstrated that anthocyanin accumulation is directly responsible for the purple top symptoms, and is associated with reduction of leaf cell death caused by phytoplasma infection. Furthermore, we revealed that phytoplasma infection led to significant activation of the anthocyanin biosynthetic pathway and dramatic accumulation of sucrose by about 1000-fold, which can activate the anthocyanin biosynthetic pathway. This is the first study to demonstrate the role and mechanism of the purple top symptoms in plant–phytoplasma interactions.

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