RESUMO
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.
Assuntos
Phytoplasma , Complexo de Endopeptidases do Proteassoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Phytoplasma/genética , Proteínas de Bactérias/metabolismo , Fatores de Transcrição/metabolismo , Plantas/metabolismoRESUMO
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.
RESUMO
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.
RESUMO
The genus 'Candidatus Phytoplasma' was proposed to accommodate cell wall-less bacteria that are molecularly and biochemically incompletely characterized, and colonize plant phloem and insect vector tissues. This provisional classification is highly relevant due to its application in epidemiological and ecological studies, mainly aimed at keeping the severe phytoplasma plant diseases under control worldwide. Given the increasing discovery of molecular diversity within the genus 'Ca. Phytoplasma', the proposed guidelines were revised and clarified to accommodate those 'Ca. Phytoplasma' species strains sharing >98.65â% sequence identity of their full or nearly full 16S rRNA gene sequences, obtained with at least twofold coverage of the sequence, compared with those of the reference strain of such species. Strains sharing <98.65â% sequence identity with the reference strain but >98.65â% with other strain(s) within the same 'Ca. Phytoplasma' species should be considered related strains to that 'Ca. Phytoplasma' species. The guidelines herein, keep the original published reference strains. However, to improve 'Ca. Phytoplasma' species assignment, complementary strains are suggested as an alternative to the reference strains. This will be implemented when only a partial 16S rRNA gene and/or a few other genes have been sequenced, or the strain is no longer available for further molecular characterization. Lists of 'Ca. Phytoplasma' species and alternative reference strains described are reported. For new 'Ca. Phytoplasma' species that will be assigned with identity ≥98.65â% of their 16S rRNA gene sequences, a threshold of 95â% genome-wide average nucleotide identity is suggested. When the whole genome sequences are unavailable, two among conserved housekeeping genes could be used. There are 49 officially published 'Candidatus Phytoplasma' species, including 'Ca. P. cocostanzaniae' and 'Ca. P. palmae' described in this manuscript.
Assuntos
Phytoplasma , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Filogenia , Phytoplasma/genética , Doenças das Plantas/microbiologia , Plantas , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Regardless of the general model of translation in eukaryotic cells, a number of studies suggested that many mRNAs encode multiple proteins. Leaky scanning, which supplies ribosomes to downstream open reading frames (ORFs) by readthrough of upstream ORFs, has great potential to translate polycistronic mRNAs. However, the mRNA elements controlling leaky scanning and their biological relevance have rarely been elucidated, with exceptions such as the Kozak sequence. Here, we have analyzed the strategy of a plant RNA virus to translate three movement proteins from a single RNA molecule through leaky scanning. The in planta and in vitro results indicate thatthe significantly shorter 5' untranslated region (UTR) of the most upstream ORF promotes leaky scanning, potentially fine-tuning the translation efficiency of the three proteins in a single RNA molecule to optimize viral propagation. Our results suggest that the remarkably short length of the leader sequence, like the Kozak sequence, is a translational regulatory element with a biologically important role, as previous studies have shown biochemically. IMPORTANCEPotexvirus, a group of plant viruses, infect a variety of crops, including cultivated crops. It has been thought that the three transition proteins that are essential for the cell-to-cell transfer of potexviruses are translated from two subgenomic RNAs, sgRNA1 and sgRNA2. However, sgRNA2 has not been clearly detected. In this study, we have shown that sgRNA1, but not sgRNA2, is the major translation template for the three movement proteins. In addition, we determined the transcription start site of sgRNA1 in flexiviruses and found that the efficiency of leaky scanning caused by the short 5' UTR of sgRNA1, a widely conserved feature, regulates the translation of the three movement proteins. When we tested the infection of viruses with mutations introduced into the length of the 5' UTR, we found that the movement efficiency of the virus was affected. Our results provide important additional information on the protein translation strategy of flexiviruses, including Potexvirus, and provide a basis for research on their control as well as the need to reevaluate the short 5' UTR as a translational regulatory element with an important role in vivo.
Assuntos
Vírus de Plantas , Biossíntese de Proteínas , Vírus de RNA , Regiões 5' não Traduzidas/genética , Fases de Leitura Aberta , Vírus de Plantas/genética , Biossíntese de Proteínas/genética , Vírus de RNA/genética , RNA Mensageiro/genética , RNA Viral/genética , RNA Viral/metabolismoRESUMO
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.
Assuntos
Phytoplasma , Proteínas de Saccharomyces cerevisiae , Flores/metabolismo , Phytoplasma/metabolismo , Plantas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismoRESUMO
Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.
Assuntos
Potexvirus/genética , Potexvirus/metabolismo , Proteínas do Complexo da Replicase Viral/genética , Sequência de Aminoácidos/genética , Proteínas de Membrana/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Doenças das Plantas/virologia , Prolina/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Replicon/genética , Nicotiana/virologia , Proteínas Virais/metabolismo , Proteínas do Complexo da Replicase Viral/metabolismo , Replicação Viral/genéticaRESUMO
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.
Assuntos
Proteínas de Bactérias , Phytoplasma , Aminoácidos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Flores/crescimento & desenvolvimento , Flores/microbiologia , Regulação Bacteriana da Expressão Gênica , Transferência Genética Horizontal , Genes Bacterianos , Proteínas de Domínio MADS/metabolismo , Filogenia , Phytoplasma/genética , Phytoplasma/metabolismo , Phytoplasma/patogenicidade , Doenças das Plantas/etiologia , Doenças das Plantas/microbiologia , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Phytoplasma/química , Cristalografia por Raios X , Estrutura Molecular , Doenças das Plantas/microbiologia , Conformação Proteica em alfa-HéliceRESUMO
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.
Assuntos
Phytoplasma/genética , Animais , Sequência de Bases , Sequência Conservada , Biblioteca Gênica , Genoma Bacteriano , Insetos/microbiologia , Phytoplasma/patogenicidade , Plantas/microbiologia , Regiões Promotoras Genéticas , RNA Bacteriano/genética , RNA não Traduzido/genética , Análise de Sequência de RNA , Sítio de Iniciação de TranscriçãoRESUMO
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.
Assuntos
Toxinas Bacterianas , Proteínas de Domínio MADS/metabolismo , Phytoplasma/patogenicidade , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plantas/microbiologia , Fatores de Virulência/fisiologia , Toxinas Bacterianas/genética , Cycadopsida/genética , Cycadopsida/microbiologia , Gleiquênias/genética , Gleiquênias/microbiologia , Flores/microbiologia , Regulação da Expressão Gênica de Plantas , Magnoliopsida/genética , Magnoliopsida/microbiologia , Phytoplasma/fisiologia , Proteólise , Fatores de Virulência/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Proteínas de Bactérias , Flores/crescimento & desenvolvimento , Proteínas de Domínio MADS/metabolismo , Phytoplasma/metabolismo , Doenças das Plantas/microbiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/farmacologia , Regulação da Expressão Gênica de Plantas , Família Multigênica , Folhas de Planta/crescimento & desenvolvimento , Proteólise/efeitos dos fármacos , Nicotiana/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Phytoplasmas (class, Mollicutes) are insect-transmissible and plant-pathogenic bacteria that multiply intracellularly in both plants and insects through host switching. Our previous study revealed that phytoplasmal sigma factor rpoD of OY-M strain (rpoDOY) could be a key regulator of host switching, because the expression level of rpoDOY was higher in insect hosts than in plant hosts. In this study, we developed an in vitro transcription assay system to identify RpoDOY-dependent genes and the consensus promoter elements. The assay revealed that RpoDOY regulated some housekeeping, virulence, and host-phytoplasma interaction genes of OY-M strain. The upstream region of the transcription start sites of these genes contained conserved -35 and -10 promoter sequences, which were similar to the typical bacterial RpoD-dependent promoter elements, while the -35 promoter elements were variable. In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools. The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns. These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.
Assuntos
Proteínas de Bactérias/metabolismo , Phytoplasma/metabolismo , Fator sigma/metabolismo , Animais , Proteínas de Bactérias/genética , Sequência de Bases , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Bacteriano , Insetos/microbiologia , Dados de Sequência Molecular , Phytoplasma/genética , Doenças das Plantas/microbiologia , Plantas/microbiologia , Regiões Promotoras Genéticas , Fator sigma/genética , Transcrição Gênica , Virulência/genéticaRESUMO
Despite plants infected by pathogens are often unable to produce offspring, it remains unclear how sterility is induced in host plants. In this study, we demonstrate that TENGU, a phytoplasmal virulence peptide known as a dwarfism inducer, acts as an inducer of sterility. Transgenic expression of TENGU induced both male and female sterility in Arabidopsis thaliana flowers similar to those observed in double knockout mutants of auxin response factor 6 (ARF6) and ARF8, which are known to regulate floral development in a jasmonic acid (JA)-dependent manner. Transcripts of ARF6 and ARF8 were significantly decreased in both tengu-transgenic and phytoplasma-infected plants. Furthermore, JA and auxin levels were actually decreased in tengu-transgenic buds, suggesting that TENGU reduces the endogenous levels of phytohormones by repressing ARF6 and ARF8, resulting in impaired flower maturation. TENGU is the first virulence factor with the effects on plant reproduction by perturbation of phytohormone signaling.
Assuntos
Arabidopsis , Proteínas de Bactérias , Ciclopentanos/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Ácidos Indolacéticos/metabolismo , Oxilipinas/metabolismo , Peptídeos , Phytoplasma , Infertilidade das Plantas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Flores/genética , Flores/metabolismo , Flores/microbiologia , Peptídeos/genética , Peptídeos/metabolismo , Phytoplasma/genética , Phytoplasma/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Adhesins are microbial surface proteins that mediate the adherence of microbial pathogens to host cell surfaces. In Mollicutes, several adhesins have been reported in mycoplasmas and spiroplasmas. Adhesins P40 of Mycoplasma agalactiae and P89 of Spiroplasma citri contain a conserved amino acid sequence known as the Mollicutes adhesin motif (MAM), whose function in the host cell adhesion remains unclear. Here, we show that phytoplasmas, which are plant-pathogenic mollicutes transmitted by insect vectors, possess an adhesion-containing MAM that was identified in a putative membrane protein, PAM289 (P38), of the 'Candidatus Phytoplasma asteris,' OY strain. P38 homologs and their MAMs were highly conserved in related phytoplasma strains. While P38 protein was expressed in OY-infected insect and plant hosts, binding assays showed that P38 interacts with insect extract, and weakly with plant extract. Interestingly, the interaction of P38 with the insect extract depended on MAM. These results suggest that P38 is a phytoplasma adhesin that interacts with the hosts. In addition, the MAM of adhesins is important for the interaction between P38 protein and hosts.
Assuntos
Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Cebolas/microbiologia , Phytoplasma/fisiologia , Doenças das Plantas/microbiologia , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dados de Sequência Molecular , Phytoplasma/química , Phytoplasma/genética , Alinhamento de SequênciaRESUMO
RNA silencing plays an important antiviral role in plants and invertebrates. To counteract antiviral RNA silencing, most plant viruses have evolved viral suppressors of RNA silencing (VSRs). TRIPLE GENE BLOCK PROTEIN1 (TGBp1) of potexviruses is a well-characterized VSR, but the detailed mechanism by which it suppresses RNA silencing remains unclear. We demonstrate that transgenic expression of TGBp1 of plantago asiatica mosaic virus (PlAMV) induced developmental abnormalities in Arabidopsis thaliana similar to those observed in mutants of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) required for the trans-acting small interfering RNA synthesis pathway. PlAMV-TGBp1 inhibits SGS3/RDR6-dependent double-stranded RNA synthesis in the trans-acting small interfering RNA pathway. TGBp1 interacts with SGS3 and RDR6 and coaggregates with SGS3/RDR6 bodies, which are normally dispersed in the cytoplasm. In addition, TGBp1 forms homooligomers, whose formation coincides with TGBp1 aggregation with SGS3/RDR6 bodies. These results reveal the detailed molecular function of TGBp1 as a VSR and shed new light on the SGS3/RDR6-dependent double-stranded RNA synthesis pathway as another general target of VSRs.
RESUMO
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'.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Flores/metabolismo , Proteínas de Domínio MADS/metabolismo , Phytoplasma/metabolismo , Folhas de Planta/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/genética , Sequência de Bases , Flores/genética , Flores/ultraestrutura , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Interações Hospedeiro-Patógeno , Immunoblotting , Proteínas de Domínio MADS/genética , Microscopia Confocal , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Phytoplasma/genética , Folhas de Planta/genética , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Ligação Proteica , Proteólise , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Arabidopsis/metabolismo , Petunia/metabolismo , Phytoplasma/fisiologia , Antocianinas/biossíntese , Arabidopsis/genética , Morte Celular , DNA Bacteriano/análise , Interações Hospedeiro-Patógeno , Monossacarídeos/metabolismo , Petunia/genética , Phytoplasma/genética , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Reação em Cadeia da PolimeraseRESUMO
Parasitic bacteria that infect eukaryotes, such as animals and plants, often have reduced genomes, having lost important metabolic genes as a result of their host-dependent life cycles. Genomic sequencing of these bacteria has revealed their survival strategies and adaptations to parasitism. Phytoplasmas (class Mollicutes, genus 'Candidatus Phytoplasma') are intracellular bacterial pathogens of plants and insects and cause devastating yield losses in diverse low- and high-value crops worldwide. The complete genomic sequences of four Candidatus Phytoplasma species have been reported. The genomes encode even fewer metabolic functions than other bacterial genomes do, which may be the result of reductive evolution as a consequence of their life as an intracellular parasite. This review summarizes current knowledge of the diversity and common features of phytoplasma genomes, including the factors responsible for pathogenicity.
RESUMO
Phytoplasmas are insect-borne plant pathogenic bacteria that alter host morphology. TENGU, a small peptide of 38 residues, is a virulence factor secreted by phytoplasmas that induces dwarfism and witches' broom in the host plant. In this study, we demonstrate that plants process TENGU in order to generate small functional peptides. First, virus vector-mediated transient expression demonstrated that the amino-terminal 11 amino acids of TENGU are capable of causing symptom development in Nicotiana benthamiana plants. The deletion of the 11th residue significantly diminished the symptom-inducing activity of TENGU, suggesting that these 11 amino acids constitute a functional domain. Second, we found that TENGU undergoes proteolytic processing in vitro, generating peptides of 19 and 21 residues including the functional domain. Third, we observed similar processing of TENGU in planta, and an alanine substitution mutant of TENGU, for which processing was compromised, showed reduced symptom induction activity. All TENGU homologs from several phytoplasma strains possessed similar symptom induction activity and went through processing, which suggests that the processing of TENGU might be related to its function.