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

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 proteasome and is ubiquitin-independent. Phyllogens induce proteasomal degradation of floral MADS-box transcription factors (MTFs) in the presence of RADIATION-SENSITIVE23 (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.

Crystal structure of phyllogen, a phyllody-inducing effector protein of phytoplasma.

Phytoplasmas are plant pathogenic bacteria that often induce unique phyllody symptoms in which the floral organs are transformed into leaf-like structures. Recently, a novel family of bacterial effector genes, called phyllody-inducing genes (phyllogens), was identified as being involved in the induction of phyllody by degrading floral MADS-domain transcription factors (MTFs). However, the structural characteristics of phyllogens are unknown. In this study, we elucidated the crystal structure of PHYL1OY, a phyllogen of 'Candidatus Phytoplasma asteris' onion yellows strain, at a resolution of 2.4 Å. The structure of PHYL1 consisted of two α-helices connected by a random loop in a coiled-coil manner. In both α-helices, the distributions of hydrophobic residues were conserved among phyllogens. Amino acid insertion mutations into either α-helix resulted in the loss of phyllody-inducing activity and the ability of the phyllogen to degrade floral MTF. In contrast, the same insertion in the loop region did not affect either activity, indicating that both conserved α-helices are important for the function of phyllogens. This is the first report on the crystal structure of an effector protein of phytoplasmas.

Comprehensive screening of antimicrobials to control phytoplasma diseases using an in vitro plant-phytoplasma co-culture system.

Phytoplasmas are plant-pathogenic bacteria that infect many important crops and cause serious economic losses worldwide. However, owing to an inability to culture phytoplasmas, screening of antimicrobials on media is difficult. The only antimicrobials being used to control phytoplasmas are tetracycline-class antibiotics. In this study, we developed an accurate and efficient screening method to evaluate the effects of antimicrobials using an in vitro plant-phytoplasma co-culture system. We tested 40 antimicrobials, in addition to tetracycline, and four of these (doxycycline, chloramphenicol, thiamphenicol and rifampicin) decreased the accumulation of 'Candidatus (Ca.) Phytoplasma asteris'. The phytoplasma was eliminated from infected plants by the application of both tetracycline and rifampicin. We also compared nucleotide sequences of rRNAs and amino acid sequences of proteins targeted by antimicrobials between phytoplasmas and other bacteria. Since antimicrobial target sequences were conserved among various phytoplasma species, the antimicrobials that decreased accumulation of 'Ca. P. asteris' may also have been effective against other phytoplasma species. These approaches will provide new strategies for phytoplasma disease management.

'Candidatus Phytoplasma noviguineense', a novel taxon associated with Bogia coconut syndrome and banana wilt disease on the island of New Guinea.

Bogia coconut syndrome (BCS) is one of the lethal yellowing (LY)-type diseases associated with phytoplasma presence that are seriously threatening coconut cultivation worldwide. It has recently emerged, and is rapidly spreading in northern parts of the island of New Guinea. BCS-associated phytoplasmas collected in different regions were compared in terms of 16S rRNA gene sequences, revealing high identity among them represented by strain BCS-BoR. Comparative analysis of the 16S rRNA gene sequences revealed that BCS-BoR shared less than a 97.5 % similarity with other species of 'Candidatus Phytoplasma', with a maximum value of 96.08 % (with strain LY; GenBank accession no. U18747). This result indicates the necessity and propriety of a novel taxon for BCS phytoplasmas according to the recommendations of the IRPCM. Phylogenetic analysis was also conducted on 16S rRNA gene sequences, resulting in a monophyletic cluster composed of BCS-BoR and other LY-associated phytoplasmas. Other phytoplasmas on the island of New Guinea associated with banana wilt and arecanut yellow leaf diseases showed high similarities to BCS-BoR and were closely related to BCS phytoplasmas. Based on the uniqueness of their 16S rRNA gene sequences, a novel taxon 'Ca.Phytoplasma noviguineense' is proposed for these phytoplasmas found on the island of New Guinea, with strain BCS-BoR (GenBank accession no. LC228755) as the reference strain. The novel taxon is described in detail, including information on the symptoms of associated diseases and additional genetic features of the secY gene and rp operon.

EXA1, a GYF domain protein, is responsible for loss-of-susceptibility to plantago asiatica mosaic virus in Arabidopsis thaliana.

One 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.

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

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.

Target degradation specificity of phytoplasma effector phyllogen is regulated by the recruitment of host proteasome shuttle protein.

Phytoplasmas infect a wide variety of plants and can cause distinctive symptoms including the conversion of floral organs into leaf-like organs, known as phyllody. Phyllody is induced by an effector protein family called phyllogens, which interact with floral MADS-box transcription factors (MTFs) responsible for determining the identity of floral organs. The MTF/phyllogen complex then interacts with the proteasomal shuttle protein RADIATION SENSITIVE23 (RAD23), which facilitates delivery of the MTF/phyllogen complex to the host proteasome for MTF degradation. Previous studies have indicated that the MTF degradation specificity of phyllogens is determined by their ability to bind to MTFs. However, in the present study, we discovered a novel mechanism determining the degradation specificity through detailed functional analyses of a phyllogen homologue of rice yellow dwarf phytoplasma (PHYLRYD ). PHYLRYD degraded a narrower range of floral MTFs than other phyllody-inducing phyllogens, resulting in compromised phyllody phenotypes in plants. Interestingly, PHYLRYD was able to bind to some floral MTFs that PHYLRYD was unable to efficiently degrade. However, the complex of PHYLRYD and the non-degradable MTF could not interact with RAD23. These results indicate that the MTF degradation specificity of PHYLRYD is correlated with the ability to form the MTF/PHYLRYD /RAD23 ternary complex, rather than the ability to bind to MTF. This study elucidated that phyllogen target specificity is regulated by both the MTF-binding ability and RAD23 recruitment ability of the MTF/phyllogen complex.

Use of the 23S rRNA gene as a target template in the universal loop-mediated isothermal amplification (LAMP) of genomic DNA from phytoplasmas.

Plant-pathogenic bacteria cause numerous diseases in host plants and can result in serious damage. Timely and accurate diagnostic techniques are, therefore, crucial. While advances in molecular techniques have led to diagnostic systems able to distinguish known plant pathogens at the species or strain level, systems covering larger categories are mostly lacking. In this study, a specific and universal LAMP-based diagnostic system was developed for phytoplasmas, a large group of insect-borne plant-pathogenic bacteria that cause significant agricultural losses worldwide. Targeting the 23S rRNA gene of phytoplasma, the newly designed primer set CaPU23S-4 detected 31 'Candidatus Phytoplasma' tested within 30 min. This primer set also showed high specificity, without false-positive results for other bacteria (including close relatives of phytoplasmas) or healthy plants. The detection sensitivity was ~10,000 times higher than that of PCR methods for phytoplasma detection. A simple, rapid method of DNA extraction, by boiling phytoplasma-infected tissues, was developed as well. When used together with the universal LAMP assay, it enabled the prompt and accurate detection of phytoplasmas from plants and insects. The results demonstrate the potential of the 23S rRNA gene as a versatile target for the LAMP-based universal detection of bacteria at the genus level and provide a novel avenue for exploring this gene as molecular marker for phytoplasma presence detection.IMPORTANCEPhytoplasmas are associated with economically important diseases in crops worldwide, including lethal yellowing of coconut palm, "flavescence dorée" and "bois noir" of grapevine, X-disease in stone fruits, and white leaf and grassy shoot in sugarcane. Numerous LAMP-based diagnostic assays, mostly targeting the 16S rRNA gene, have been reported for phytoplasmas. However, these assays can only detect a limited number of 'Candidatus Phytoplasma' species, whereas the genus includes at least 50 of these species. In this study, a universal, specific, and rapid diagnostic system was developed that can detect all provisionally classified phytoplasmas within 1 h by combining the LAMP technique targeting the 23S rRNA gene with a simple method for DNA extraction. This diagnostic system will facilitate the on-site detection of phytoplasmas and may aid in the discovery of new phytoplasma-associated diseases and putative insect vectors, irrespective of the availability of infrastructure and experimental resources.

Potential mobile units drive the horizontal transfer of phytoplasma effector phyllogen genes.

Phytoplasmas are obligate intracellular plant pathogenic bacteria that can induce phyllody, which is a type of abnormal floral organ development. Phytoplasmas possess phyllogens, which are effector proteins that cause phyllody in plants. Phylogenetic comparisons of phyllogen and 16S rRNA genes have suggested that phyllogen genes undergo horizontal transfer between phytoplasma species and strains. However, the mechanisms and evolutionary implications of this horizontal gene transfer are unclear. Here, we analyzed synteny in phyllogen flanking genomic regions from 17 phytoplasma strains that were related to six 'Candidatus' species, including three strains newly sequenced in this study. Many of the phyllogens were flanked by multicopy genes within potential mobile units (PMUs), which are putative transposable elements found in phytoplasmas. The multicopy genes exhibited two distinct patterns of synteny that correlated with the linked phyllogens. The low level of sequence identities and partial truncations found among these phyllogen flanking genes indicate that the PMU sequences are deteriorating, whereas the highly conserved sequences and functions (e.g., inducing phyllody) of the phyllogens suggest that the latter are important for phytoplasma fitness. Furthermore, although their phyllogens were similar, PMUs in strains related to 'Ca. P. asteris' were often located in different regions of the genome. These findings strongly indicate that PMUs drive the horizontal transfer of phyllogens among phytoplasma species and strains. These insights improve our understanding of how symptom-determinant genes have been shared among phytoplasmas.

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

To understand protein function deeply, it is important to identify how it interacts physically with its target. Phyllogen is a phyllody-inducing effector that interacts with the K domain of plant MADS-box transcription factors (MTFs), which is followed by proteasome-mediated degradation of the MTF. Although several amino acid residues of phyllogen have been identified as being responsible for the interaction, the exact interface of the interaction has not been elucidated. In this study, we comprehensively explored interface residues based on random mutagenesis using error-prone PCR. Two novel residues, at which mutations enhanced the affinity of phyllogen to MTF, were identified. These residues, and all other known interaction-involved residues, are clustered together at the surface of the protein structure of phyllogen, indicating that they constitute the interface of the interaction. Moreover, in silico structural prediction of the protein complex using ColabFold suggested that phyllogen interacts with the K domain of MTF via the putative interface. Our study facilitates an understanding of the interaction mechanisms between phyllogen and MTF.

Complete Genome Sequence of Tea Plant Necrotic Ring Blotch Virus Detected from a Tea Plant in Japan.

We report the complete genome sequence of a Japanese isolate of Tea plant necrotic ring blotch virus (TPNRBV-J). The predicted TPNRBV-J genes have the same organization as those of a Chinese isolate, and the 5' termini of the segments have conserved nucleotide sequences.

Complete Genome Sequence of Clover Yellow Mosaic Virus Isolated from White Clover in Japan.

Clover yellow mosaic virus (ClYMV) infecting white clover was isolated in Japan, and the complete genome sequence was determined.

Complete Genome Sequence of Mirabilis Crinkle Mosaic Virus Isolated from Pokeweed in Japan.

The complete genome sequence of a pokeweed (Phytolacca americana L.) isolate of mirabilis crinkle mosaic virus (MiCMV) in Japan was determined.

Functional variation in phyllogen, a phyllody-inducing phytoplasma effector family, attributable to a single amino acid polymorphism.

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.

Complete Genome Sequence of a Carrot Torradovirus 1 Isolate, Obtained from Angelica keiskei in Japan.

The complete genome sequence of the first Japanese isolate of carrot torradovirus 1 (CaTV1-J), which infects Angelica keiskei, was determined. This is the first report of a CaTV1 isolate obtained from A. keiskei.

Genome-Wide Analysis of the Transcription Start Sites and Promoter Motifs of Phytoplasmas.

Phytoplasmas are obligate intracellular parasitic bacteria that infect both plants and insects. We previously identified the sigma factor RpoD-dependent consensus promoter sequence of phytoplasma. However, the genome-wide landscape of RNA transcripts, including non-coding RNAs (ncRNAs) and RpoD-independent promoter elements, was still unknown. In this study, we performed an improved RNA sequencing analysis for genome-wide identification of the transcription start sites (TSSs) and the consensus promoter sequences. We constructed cDNA libraries using a random adenine/thymine hexamer primer, in addition to a conventional random hexamer primer, for efficient sequencing of 5'-termini of AT-rich phytoplasma RNAs. We identified 231 TSSs, which were classified into four categories: mRNA TSSs, internal sense TSSs, antisense TSSs (asTSSs), and orphan TSSs (oTSSs). The presence of asTSSs and oTSSs indicated the genome-wide transcription of ncRNAs, which might act as regulatory ncRNAs in phytoplasmas. This is the first description of genome-wide phytoplasma ncRNAs. Using a de novo motif discovery program, we identified two consensus motif sequences located upstream of the TSSs. While one was almost identical to the RpoD-dependent consensus promoter sequence, the other was an unidentified novel motif, which might be recognized by another transcription initiation factor. These findings are valuable for understanding the regulatory mechanism of phytoplasma gene expression.

Deficiency of the eIF4E isoform nCBP limits the cell-to-cell movement of a plant virus encoding triple-gene-block proteins in Arabidopsis thaliana.

One of the important antiviral genetic strategies used in crop breeding is recessive resistance. Two eukaryotic translation initiation factor 4E family genes, eIF4E and eIFiso4E, are the most common recessive resistance genes whose absence inhibits infection by plant viruses in Potyviridae, Carmovirus, and Cucumovirus. Here, we show that another eIF4E family gene, nCBP, acts as a novel recessive resistance gene in Arabidopsis thaliana toward plant viruses in Alpha- and Betaflexiviridae. We found that infection by Plantago asiatica mosaic virus (PlAMV), a potexvirus, was delayed in ncbp mutants of A. thaliana. Virus replication efficiency did not differ between an ncbp mutant and a wild type plant in single cells, but viral cell-to-cell movement was significantly delayed in the ncbp mutant. Furthermore, the accumulation of triple-gene-block protein 2 (TGB2) and TGB3, the movement proteins of potexviruses, decreased in the ncbp mutant. Inoculation experiments with several viruses showed that the accumulation of viruses encoding TGBs in their genomes decreased in the ncbp mutant. These results indicate that nCBP is a novel member of the eIF4E family recessive resistance genes whose loss impairs viral cell-to-cell movement by inhibiting the efficient accumulation of TGB2 and TGB3.

First Complete Genome Sequence of Cherry virus A.

The 5'-terminal genomic sequence of Cherry virus A (CVA) has long been unknown. We determined the first complete genome sequence of an apricot isolate of CVA (7,434 nucleotides [nt]). The 5'-untranslated region was 107 nt in length, which was 53 nt longer than those of known CVA sequences.

Complete Genome Sequence of Alternanthera mosaic virus, Isolated from Achyranthes bidentata in Asia.

Alternanthera mosaic virus (AltMV) infecting Achyranthes bidentata was first detected in Asia, and the complete genome sequence (6,604 nucleotides) was determined. Sequence identity analysis and phylogenetic analysis confirmed that this isolate is the most phylogenetically distant AltMV isolate worldwide.

Complete Genome Sequences of Two Hydrangea Ringspot Virus Isolates from Japan.

Hydrangea ringspot virus (HdRSV) is a plant RNA virus, naturally infectingHydrangea macrophylla Here, we report the first genomic sequences of two HdRSV isolates from hydrangea plants in Japan. The overall nucleotide sequences of these Japanese isolates were 96.0 to 96.3% identical to those of known European isolates.