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Introduction: The adhesion of flavescence dorée phytoplasma to the midgut epithelium cells of their insect vectors is partially mediated by the variable membrane protein A (VmpA), an adhesin which shows lectin properties. In order to identify the insect receptor for VmpA, we identified Euscelidius variegatus cell proteins interacting with recombinant VmpA-His 6 ., Methods: The E. variegatus proteins were identified by mass spectrometry analysis of VmpA- E. variegatus protein complexes formed upon in vitro interaction assays. To assess their impact in VmpA binding, we reduced the expression of the candidate genes on E. variegatus cells in culture by dsRNA-mediated RNAi. The effect of candidate gene knockdown on VmpA binding was measured by the capacity of E. variegatus cells to bind VmpA-coated fluorescent beads., Results and Discussion: There were 13 candidate proteins possessing potential N-glycosylation sites and predicted transmembrane domains selected. The decrease of expression of an unknown transmembrane protein with leucine-rich repeat domains (uk1_LRR) was correlated with the decreased adhesion of VmpA beads to E. variegatus cells. The uk1_LRR was more expressed in digestive tubes than salivary glands of E. variegatus . The protein uk1_LRR could be implicated in the binding with VmpA in the early stages of insect infection following phytoplasmas ingestion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Canuto, Duret, Dubrana, Claverol, Malembic-Maher, Foissac and Arricau-Bouvery.)

Phytoplasmas are insect-borne bacterial pathogens capable of secreting effectors into host cells and interfering with host plant defense response processes. Previous studies have found that the Candidatus Phytoplasma tritici effector SWP12 binds to and destabilizes the wheat transcription factor TaWRKY74, increasing wheat susceptibility to phytoplasmas. Here, we used a Nicotiana benthamiana transient expression system to identify two key functional sites of SWP12 and screened a series of truncated mutants and amino acid substitution mutants to determine whether they inhibit Bax-induced cell death. Using a subcellular localization assay and online structure analysis websites, we found that structure rather than intracellular localization probably affects the function of SWP12. D33A and P85H are two inactive substitution mutants, neither of which interacts with TaWRKY74, and P85H does not inhibit Bax-induced cell death, suppress flg22-triggered reactive oxygen species (ROS) bursts, degrade TaWRKY74, or promote phytoplasma accumulation. D33A can weakly suppress Bax-induced cell death and flg22-triggered ROS bursts and degrade a portion of TaWRKY74 and weakly promote phytoplasma accumulation. S53L, CPP, and EPWB are three SWP12 homolog proteins from other phytoplasmas. Sequence analysis revealed that D33 was conserved in these proteins, and they exhibited the same polarity at P85. Transient expression in N. benthamiana showed that these proteins could inhibit Bax-induced cell death and suppress ROS bursts. Our findings clarified that P85 and D33 of SWP12 play critical and minor roles, respectively, in suppressing the plant defense response and that they play a preliminary role in determining the functions of homologous proteins., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Phytoplasmas are uncultivable, phloem-limited, phytopathogenic bacteria that represent a major threat to agriculture worldwide. Phytoplasma membrane proteins are in direct contact with hosts and presumably play a crucial role in phytoplasma spread within the plant as well as by the insect vector. Three highly abundant types of immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp). Although recent results indicate that Amp is involved in host specificity by interacting with host proteins such as actin, little is known about the pathogenicity of IDP in plants. In this study, we identified an antigenic membrane protein (Amp) of rice orange leaf phytoplasma (ROLP), which interacts with the actin of its vector. In addition, we generated Amp-transgenic lines of rice and expressed Amp in tobacco leaves by the potato virus X (PVX) expression system. Our results showed that the Amp of ROLP can induce the accumulation of ROLP and PVX in rice and tobacco plants, respectively. Although several studies have reported interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins, this example demonstrates that Amp protein can not only interact with the actin protein of its insect vector but can also directly inhibit host defense responses to promote the infection. The function of ROLP Amp provides new insights into the phytoplasma-host interaction.

"Candidatus Phytoplasma australiense" is associated with floral malformations in sesame but the interaction remains largely unexplored. A label-free quantitative shotgun proteomics approach through liquid chromatography-mass spectrometry quadruple time-of-flight was used to analyze changes in the proteome of asymptomatic (control) and symptomatic (phytoplasma-infected) sesame plants to identify proteins differentially expressed during phytoplasma infection at early stages of flower development. A total of 3457 and 1704 proteins were identified from asymptomatic and symptomatic samples respectively through proteome profiling with three runs per sample. Several differentially abundant proteins (DAPs) were identified which might be involved in sesame-phytoplasma interaction. The DAPs identified were related to transcription, cell division, chromosome partitioning, defense mechanisms, negative regulation of flower development, amino acid transport and metabolism, signal transduction and RNA processing, and its modifications. Of these proteins, 21 were downregulated while 212 were significantly upregulated in symptomatic sesame plants compared to the control plants. The floral development-related proteins like UBP16 and DCAF1 were found to be downregulated while negative regulators/repressors of floral development genes, HUA2, PIE1, and ICU2, were upregulated in symptomatic samples indicating phytoplasma's role in altering the expression of these genes. Validation of these genes through quantitative retro-transcripted PCR suggested that the DAPs observed in symptomatic sesame might be induced by phytoplasma presence to suppress flowering via negative regulation of flower development., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Main Conclusion: Loss of CALS7 appears to confer increased susceptibility to phytoplasma infection in Arabidopsis, altering expression of genes involved in sugar metabolism and membrane transport. Callose deposition around sieve pores, under control of callose synthase 7 (CALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. Wild-type and Atcals7ko mutants were, therefore, employed to unveil the mode of involvement of CALS7 in the plant's response to phytoplasma infection. The fresh weights of healthy and CY-(Chrysanthemum Yellows) phytoplasma-infected Arabidopsis wild type and mutant plants indicated two superimposed effects of the absence of CALS7: a partial impairment of photo-assimilate transport and a stimulated phytoplasma proliferation as illustrated by a significantly increased phytoplasma titre in Atcal7ko mutants. Further studies solely dealt with the effects of CALS7 absence on phytoplasma growth. Phytoplasma infection affected sieve-element substructure to a larger extent in mutants than in wild-type plants, which was also true for the levels of some free carbohydrates. Moreover, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) and transmembrane transport (AtSWEET11) in mutants and wild-type plants, but an increased gene expression of carbohydrate transmembrane transporters (AtSWEET12, AtSTP13, AtSUC3) in infected mutants only. It remains still unclear how the absence of AtCALS7 leads to gene upregulation and how an increased intercellular mobility of carbohydrates and possibly effectors contributes to a higher susceptibility. It is also unclear if modified sieve-pore structures in mutants allow a better spread of phytoplasmas giving rise to higher titre., (© 2022. The Author(s).)

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., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Phytoplasmas are sieve-elements restricted wall-less, pleomorphic pathogenic microorganisms causing devastating damage to over 700 plant species worldwide. The invasion of sieve elements by phytoplasmas has several consequences on nutrient transport and metabolism, anyway studies about changes of the mineral-nutrient profile following phytoplasma infections are scarce and offer contrasting results. Here, we examined changes in macro- and micronutrient concentration in tomato plant upon 'Candidatus Phytoplasma solani' infection. To investigate possible effects of 'Ca. P. solani' infection on mineral element allocation, the mineral elements were separately analysed in leaf midrib, leaf lamina and root. Moreover, we focused our analysis on the transcriptional regulation of genes encoding trans-membrane transporters of mineral nutrients. To this aim, a manually curated inventory of differentially expressed genes encoding transporters in tomato leaf midribs was mined from the transcriptional profile of healthy and infected tomato leaf midribs. Results highlighted changes in ion homeostasis in the host plant, and significant modulations at transcriptional level of genes encoding ion transporters and channels., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Witches'-broom (WB, excessive initiation, and outgrowth of axillary buds) is one of the remarkable symptoms in plants caused by phytoplasmas, minute wall-less intracellular bacteria. In healthy plants, axillary bud initiation and outgrowth are regulated by an intricate interplay of nutrients (such as sugars), hormones, and environmental factors. However, how these factors are involved in the induction of WB by phytoplasma is poorly understood. We postulated that the WB symptom is a manifestation of the pathologically induced redistribution of sugar and phytohormones. Employing potato purple top phytoplasma and its alternative host tomato ( Solanum lycopersicum ), sugar metabolism and transportation, and the spatiotemporal distribution of phytohormones were investigated. A transmission electron microscopy (TEM) analysis revealed that starch breakdown was inhibited, resulting in the degradation of damaged chloroplasts, and in turn, premature leaf senescence. In the infected source leaves, two marker genes encoding asparagine synthetase ( Sl-ASN ) and trehalose-6-phosphate synthase ( Sl-TPS ) that induce early leaf senescence were significantly up-regulated. However, the key gibberellin biosynthesis gene that encodes ent-kaurene synthase ( Sl-KS ) was suppressed. The assessment of sugar content in various infected tissues (mature leaves, stems, roots, and leaf axils) indicated that sucrose transportation through phloem was impeded, leading to sucrose reallocation into the leaf axils. Excessive callose deposition and the resulting reduction in sieve pore size revealed by aniline blue staining and TEM provided additional evidence to support impaired sugar transport. In addition, a spatiotemporal distribution study of cytokinin and auxin using reporter lines detected a cytokinin signal in leaf axils where the axillary buds initiated. However, the auxin responsive signal was rarely present in such leaf axils, but at the tips of the newly elongated buds. These results suggested that redistributed sucrose as well as cytokinin in leaf axils triggered the axillary bud initiation, and auxin played a role in the bud elongation. The expression profiles of genes encoding squamosa promoter-binding proteins ( Sl-SBP1 ), and BRANCHED1 ( Sl-BRC1a and Sl-BRC1b ) that control axillary bud release, as determined by quantitative reverse transcription (qRT)-PCR, indicated their roles in WB induction. However, their interactions with sugars and cytokinins require further study. Our findings provide a comprehensive insight into the mechanisms by which phytoplasmas induce WB along with leaf chlorosis, little leaf, and stunted growth.

Phytoplasmas enhance transmission by manipulating plant architecture. Recently, Huang et al. report that SAP05, an effector from a phytoplasma that causes witches' broom, targets host transcription factors for proteasomal degradation by binding host ubiquitin receptor RPN10. These findings provide opportunities for engineering phytoplasma-resistant plants and developing protein therapeutics., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Like many other pathogens, the obligate parasitic bacteria phytoplasmas reply on secreted effectors to cause diseases in their plant hosts. Huang et al. revealed that a phytoplasma effector degrades plant proteins independent of ubiquitination. Bypassing this degradation step makes Arabidopsis thaliana plants resistant to this parasite effector., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Vector-borne plant diseases have significant ecological and economic impacts, affecting farm profitability and forest composition throughout the world. Bacterial vector-borne pathogens have evolved sophisticated strategies to interact with their hemipteran insect vectors and plant hosts. These pathogens reside in plant vascular tissue, and their study represents an excellent opportunity to uncover novel biological mechanisms regulating intracellular pathogenesis and to contribute to the control of some of the world's most invasive emerging diseases. In this perspective, we highlight recent advances and major unanswered questions in the realm of bacterial vector-borne disease, focusing on liberibacters, phytoplasmas, spiroplasmas, and Xylella fastidiosa., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Background: Among several TF families unique to eukaryotes, the basic leucine zipper (bZIP) family is one of the most important. Chinese jujube (Ziziphus jujuba Mill.) is a popular fruit tree species in Asia, and its fruits are rich in sugar, vitamin C and so on. Analysis of the bZIP gene family of jujube has not yet been reported. In this study, ZjbZIPs were identified firstly, their expression patterns were further studied in different tissues and in response to various abiotic and phytoplasma stresses, and their protein-protein interactions were also analyzed., Results: At the whole genome level, 45 ZjbZIPs were identified and classified into 14 classes. The members of each class of bZIP subfamily contain a specific conserved domain in addition to the core bZIP conserved domain, which may be related to its biological function. Relative Synonymous Codon Usage (RSCU) analysis displayed low values of NTA and NCG codons in ZjbZIPs, which would be beneficial to increase the protein production and also indicated that ZjbZIPs were at a relative high methylation level. The paralogous and orthologous events occurred during the evolutionary process of ZjbZIPs. Thirty-four ZjbZIPs were mapped to but not evenly distributed among 10 pseudo- chromosomes. 30 of ZjbZIP genes showed diverse tissue-specific expression in jujube and wild jujube trees, indicating that these genes may have multiple functions. Some ZjbZIP genes were specifically analyzed and found to play important roles in the early stage of fruit development. Moreover, some ZjbZIPs that respond to phytoplasma invasion and abiotic stress environmental conditions, such as salt and low temperature, were found. Based on homology comparisons, prediction analysis and yeast two-hybrid, a protein interaction network including 42 ZjbZIPs was constructed., Conclusions: The bioinformatics analyses of 45 ZjbZIPs were implemented systematically, and their expression profiles in jujube and wild jujube showed that many genes might play crucial roles during fruit ripening and in the response to phytoplasma and abiotic stresses. The protein interaction networks among ZjbZIPs could provide useful information for further functional studies.

Flavescence dorée (FD) is a severe epidemic disease of grapevines caused by FD phytoplasma (FDP) transmitted by the leafhopper vector Scaphoideus titanus . The recent sequencing of the 647-kbp FDP genome highlighted an unusual number of genes encoding ATP-dependent zinc proteases FtsH, which have been linked to variations in the virulence of " Candidatus Phytoplasma mali" strains. The aims of the present study were to predict the FtsH repertoire of FDP, to predict the functional domains and topologies of the encoded proteins in the phytoplasma membrane and to measure the expression profiles in different hosts. Eight complete ftsH genes have been identified in the FDP genome. In addition to ftsH 6, which appeared to be the original bacterial ortholog, the other seven gene copies were clustered on a common distinct phylogenetic branch, suggesting intra-genome duplication of ftsH . The expression of these proteins, quantified in plants and insect vectors in natural and experimental pathosystems, appeared to be modulated in a host-dependent manner. Two of the eight FtsH C-tails were predicted by Phobius software to be extracellular and, therefore, in direct contact with the host cellular content. As phytoplasmas cannot synthesize amino acids, our data raised questions regarding the involvement of FtsH in the adaptation to hosts via potentially enhanced recycling of phytoplasma cellular proteins and host protein degradation.

Phytoplasmas reside exclusively in sieve tubes, tubular arrays of sieve element-companion cell complexes. Hence, the cell biology of sieve elements may reveal (ultra)structural and functional conditions that are of significance for survival, propagation, colonization, and effector spread of phytoplasmas. Electron microscopic images suggest that sieve elements offer facilities for mobile and stationary stages in phytoplasma movement. Stationary stages may enable phytoplasmas to interact closely with diverse sieve element compartments. The unique, reduced sieve element outfit requires permanent support by companion cells. This notion implies a future focus on the molecular biology of companion cells to understand the sieve element-phytoplasma inter-relationship. Supply of macromolecules by companion cells is channelled via specialized symplasmic connections. Ca2+-mediated gating of symplasmic corridors is decisive for the communication within and beyond the sieve element-companion cell complex and for the dissemination of phytoplasma effectors. Thus, Ca2+ homeostasis, which affects sieve element Ca2+ signatures and induces a range of modifications, is a key issue during phytoplasma infection. The exceptional physical and chemical environment in sieve elements seems an essential, though not the only factor for phytoplasma survival., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Elucidating the molecular mechanisms underlying plant disease development has become an important aspect of phytoplasma research in the last years. Especially unraveling the function of phytoplasma effector proteins has gained interesting insights into phytoplasma-host interaction at the molecular level. Here, we describe how to analyze and visualize the interaction of a phytoplasma effector with its proteinaceous host partner using bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana mesophyll protoplasts. The protocol comprises a description of how to isolate protoplasts from leaves and how to transform these protoplasts with BiFC expression vectors containing the phytoplasma effector and the host interaction partner, respectively. If an interaction occurs, a fluorescent YFP-complex is reconstituted in the protoplast, which can be visualized using fluorescence microscopy.

Considering the crude methods used to control phytoplasma diseases, a deeper knowledge on the defence mechanisms recruited by the plant to face phytoplasma invasion is required. Recently, we demonstrated that Arabidopsis mutants lacking AtSEOR1 gene showed a low phytoplasma titre. In wild type plants AtSEOR1 and AtSEOR2 are tied in filamentous proteins. Knockout of the AtSEOR1 gene may pave the way for an involvement of free AtSEOR2 proteins in defence mechanisms. Among the proteins conferring resistance against pathogenic bacteria, AtRPM1-interacting protein has been found to interact with AtSEOR2 in a high-quality, matrix-based yeast-two hybrid assay. For this reason, we investigated the expression levels of Arabidopsis AtRIN4, and the associated AtRPM1 and AtRPS2 genes in healthy and Chrysanthemum yellows-infected wild-type and Atseor1ko lines.

Phytoplasmas are plant-pathogenic, phloem-colonizing, cell wall-less microorganisms that are primarily dependent on insect transmission for their spread and survival. The life cycle of phytoplasmas involves replication in insects and host plants. Until recently, phytoplasmas have resisted all attempts at cultivation in cell-free media, making these pathogens poorly characterized on a physiological and biochemical basis. However, host-pathogen relationships can be studied by investigating immunodominant membrane proteins (IDPs), which are located on the exterior surfaces of phytoplasma cells and are the most abundant proteins of the cell membrane. These membrane proteins come in direct contact with both insect and plant hosts and are thought to play a crucial role in phytoplasma spread both within the plant and by insect vectors. Therefore, there is great interest in studying this class of proteins. We summarize and discuss important investigations about these membrane proteins, which have already provided a better understanding of the host-phytoplasma relationship.

Phytoplasmas are bacterial phytopathogens that release virulence effectors into sieve cells and act systemically to affect the physiological and morphological state of host plants to promote successful pathogenesis. We show here that transgenic Nicotiana benthamiana lines expressing the secreted effector SAP11 from Candidatus Phytoplasma mali exhibit an altered aroma phenotype. This phenomenon is correlated with defects in the development of glandular trichomes and the biosynthesis of 3-isobutyl-2-methoxypyrazine (IBMP). IBMP is a volatile organic compound (VOC) synthesized by an O-methyltransferase, via a methylation step, from a non-volatile precursor, 3-isobutyl-2-hydroxypyrazine (IBHP). Based on comparative and functional genomics analyses, NbOMT1, which encodes an O-methyltransferase, was found to be highly suppressed in SAP11-transgenic plants. We further silenced NbOMT1 through virus-induced gene silencing and demonstrated that this enzyme influenced the accumulation of IBMP in N. benthamiana In vitro biochemical analyses also showed that NbOMT1 can catalyse IBHP O-methylation in the presence of S-adenosyl-L-methionine. Our study suggests that the phytoplasma effector SAP11 has the ability to modulate host VOC emissions. In addition, we also demonstrated that SAP11 destabilized TCP transcription factors and suppressed jasmonic acid responses in N. benthamiana These findings provide valuable insights into understanding how phytoplasma effectors influence plant volatiles., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

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.

PHYL1 and SAP54 are orthologs of pathogenic effectors of Aster yellow witches'-broom (AYWB) phytoplasma and Peanut witches'-broom (PnWB) phytoplasma, respectively. These effectors cause virescence and phyllody symptoms (hereafter leafy flower) in phytoplasma-infected plants. T0 lines of transgenic Arabidopsis expressing the PHYL1 or SAP54 genes (PHYL1 or SAP54 plants) show a leafy flower phenotype and result in seedless, suggesting that PHYL1 and SAP54 interfere with reproduction stage that restrict gain-of-function studies in the next generation of transgenic plants. Turnip mosaic virus (TuMV) mild strain (TuGK) has an Arg182Lys mutation in the helper-component proteinase (HC-ProR182K) that blocks suppression of the miRNA pathway and prevents symptom development in TuGK-infected plants. We exploited TuGK as a viral vector for gain-of-function studies of PHYL1 and SAP54 in Arabidopsis plants. TuGK-PHYL1- and TuGK-SAP54-infected Arabidopsis plants produced identical leafy flower phenotypes and similar gene expression profiles as PHYL1 and SAP54 plants. In addition, the leafy flower formation rate was enhanced in TuGK-PHYL1- or TuGK-SAP54-infected Arabidopsis plants that compared with the T0 lines of PHYL1 plants. These results provide more evidence and novel directions for further studying the mechanism of PHYL1/SAP54-mediated leafy flower development. In addition, the TuGK vector is a good alternative in transgenic plant approaches for rapid gene expression in gain-of-function studies.

Background: Flavescence dorée (FD) of grapevine is a phloem bacterial disease that threatens European vineyards. The disease is associated with a non-cultivable mollicute, a phytoplasma that is transmitted by the grapevine leafhopper Scaphoideus titanus in a persistent, propagative manner. The specificity of insect transmission is presumably mediated through interactions between the host tissues and phytoplasma surface proteins comprising the so-called variable membrane proteins (Vmps). Plant spiroplasmas and phytoplasmas share the same ecological niches, the phloem sieve elements of host plants and the hemocoel of insect vectors. Unlike phytoplasmas, however, spiroplasmas, and Spiroplasma citri in particular, can be grown in cell-free media and genetically engineered. As a new approach for studying phytoplasmas-insect cell interactions, we sought to mimic phytoplasmas through the construction of recombinant spiroplasmas exhibiting FD phytoplasma Vmps at the cell surface., Results: Here, we report the expression of the FD phytoplasma VmpA in S. citri. Transformation of S. citri with plasmid vectors in which the vmpA coding sequence was under the control of the S. citri tuf gene promoter resulted in higher accumulation of VmpA than with the native promoter. Expression of VmpA at the spiroplasma surface was achieved by fusing the vmpA coding sequence to the signal peptide sequence of the S. citri adhesin ScARP3d, as revealed by direct colony immunoblotting and immunogold labelling electron microscopy. Anchoring of VmpA to the spiroplasma membrane was further demonstrated by Triton X-114 protein partitioning and Western immunoblotting. Using the same strategy, the secretion of free, functionally active β-lactamase (used as a model protein) into the culture medium by recombinant spiroplasmas was achieved., Conclusions: Construction of recombinant spiroplasmas harbouring the FD phytoplasma variable membrane protein VmpA at their surface was achieved, which provides a new biological approach for studying interactions of phytoplasma surface proteins with host cells. Likewise, the secretion of functional β-lactamase by recombinant spiroplasmas established the considerable promise of the S. citri expression system for delivering phytoplasma effector proteins into host cells.

Phytoplasmas are a diverse but phylogenetically coherent group of cell-wall-less bacteria affiliated with the class Mollicutes. Due to difficulties in establishing axenic culture, phytoplasmas were assigned to a provisional genus, 'Candidatus Phytoplasma', and the genus was embraced within the order Acholeplasmatales. However, phytoplasmas differ significantly from species of the genus Acholeplasma in their habitat specificities, modes of life, metabolic capabilities, genomic architectures, and phylogenetic positions. This communication describes the unique ecological, nutritional, biochemical, genomic and phylogenetic properties that distinguish phytoplasmas from species of the genus Acholeplasma and all other taxa in the class Mollicutes. Since such distinguishing properties of the phytoplasmas are not referable to the descriptions of the order Acholeplasmatales and of all other existing orders, namely Mycoplasmatales, Entomoplasmatales and Anaeroplasmatales, this communication raises the question of whether 'Candidatus Phytoplasma' should be retained in the order Acholeplasmatales or whether a novel provisional order and family should be created to accommodate the genus 'Ca. Phytoplasma'., (© 2015.)

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.

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.

Phytoplasmas ('Candidatus Phytoplasma') are insect-vectored plant pathogens. The genomes of these bacteria are small with limited metabolic capacities making them dependent on their plant and insect hosts for survival. In contrast to mycoplasmas and other relatives in the class Mollicutes, phytoplasmas encode genes for malate transporters and malic enzyme (ME) for conversion of malate into pyruvate. It was hypothesized that malate is probably a major energy source for phytoplasmas as these bacteria are limited in the uptake and processing of carbohydrates. In this study, we investigated the metabolic capabilities of 'Candidatus (Ca.) phytoplasma' aster yellows witches'-broom (AYWB) malic enzyme (ME). We found that AYWB-ME has malate oxidative decarboxylation activity, being able to convert malate to pyruvate and CO2 with the reduction of either NAD or NADP, and displays distinctive kinetic mechanisms depending on the relative concentration of the substrates. AYWB-ME activity was strictly modulated by the ATP/ADP ratio, a feature which has not been found in other ME isoforms characterized to date. In addition, we found that the 'Ca. Phytoplasma' AYWB PduL-like enzyme (AYWB-PduL) harbours phosphotransacetylase activity, being able to convert acetyl-CoA to acetyl phosphate downstream of pyruvate. ATP also inhibited AYWB-PduL activity, as with AYWB-ME, and the product of the reaction catalysed by AYWB-PduL, acetyl phosphate, stimulated AYWB-ME activity. Overall, our data indicate that AYWB-ME and AYWB-PduL activities are finely coordinated by common metabolic signals, like ATP/ADP ratios and acetyl phosphate, which support their participation in energy (ATP) and reducing power [NAD(P)H] generation from malate in phytoplasmas., (© 2014 The Authors.)

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'., (© 2014 The Authors.The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

'Candidatus Phytoplasma mali' is a phytopathogenic bacterium of the family Acholeplasmataceae assigned to the class Mollicutes. This causative agent of the apple proliferation colonizes in Malus domestica the sieve tubes of the plant phloem resulting in a range of symptoms such as witches'--broom formation, reduced vigor and affecting size and quality of the crop. The disease is responsible for strong economical losses in Europe. Although the genome sequence of the pathogen is available, there is only limited information on expression of selected genes and metabolic key features that have not been examined on the transcriptomic or proteomic level so far. This situation is similar to many other phytoplasmas. In the work presented here, RNA-Seq and mass spectrometry shotgun techniques were applied on tissue samples from Nicotiana occidentalis infected by 'Ca. P. mali' strain AT providing insights into transcriptome and proteome of the pathogen. Data analysis highlights expression of 208 genes including 14 proteins located in the terminal inverted repeats of the linear chromosome. Beside a high portion of house keeping genes, the recently discussed chaperone GroES/GroEL is expressed. Furthermore, gene expression involved in formation of a type IVB and of the Sec-dependent secretion system was identified as well as the highly expressed putative pathogenicity-related SAP11-like effector protein. Metabolism of phytoplasmas depends on the uptake of spermidine/putescine, amino acids, co-factors, carbohydrates and in particular malate/citrate. The expression of these transporters was confirmed and the analysis of the carbohydrate cycle supports the suggested alternative energy-providing pathway for phytoplasmas releasing acetate and providing ATP. The phylogenetic analyses of malate dehydrogenase and acetate kinase in phytoplasmas show a closer relatedness to the Firmicutes in comparison to Mycoplasma species indicating an early divergence of the Acholeplasmataceae from the Mollicutes.

Phytoplasmas have the smallest genome among bacteria and lack many essential genes required for biosynthetic and metabolic functions, making them unculturable, phloem-limited plant pathogens. In this study, we observed that transgenic Arabidopsis (Arabidopsis thaliana) expressing the secreted Aster Yellows phytoplasma strain Witches' Broom protein11 shows an altered root architecture, similarly to the disease symptoms of phytoplasma-infected plants, by forming hairy roots. This morphological change is paralleled by an accumulation of cellular phosphate (Pi) and an increase in the expression levels of Pi starvation-induced genes and microRNAs. In addition to the Pi starvation responses, we found that secreted Aster Yellows phytoplasma strain Witches' Broom protein11 suppresses salicylic acid-mediated defense responses and enhances the growth of a bacterial pathogen. These results contribute to an improved understanding of the role of phytoplasma effector SAP11 and provide new insights for understanding the molecular basis of plant-pathogen interactions.

Recent advances in the automation of metabolic model reconstruction have led to the availability of draft-quality metabolic models (predicted reaction complements) for multiple bacterial species. These reaction complements can be considered as trait representations and can be used for ancestral state reconstruction to infer the most likely metabolic complements of common ancestors of all bacteria with generated metabolic models. We present here an ancestral state reconstruction for 141 extant bacteria and analyze the reaction gains and losses for these bacteria with respect to their lifestyles and pathogenic nature. A simulated annealing approach is used to look at coordinated metabolic gains and losses in two bacteria. The main losses of Onion yellows phytoplasma OY-M, an obligate intracellular pathogen, are shown (as expected) to be in cell wall biosynthesis. The metabolic gains made by Clostridium difficile CD196 in adapting to its current habitat in the human colon is also analyzed. Our analysis shows that the capability to utilize N-Acetyl-neuraminic acid as a carbon source has been gained, rather than having been present in the Clostridium ancestor, as has the capability to synthesize phthiocerol dimycocerosate, which could potentially aid the evasion of the host immune response. We have shown that the availability of large numbers of metabolic models, along with conventional approaches, has enabled a systematic method to analyze metabolic evolution in the bacterial domain.

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.

Phytoplasmas are a group of bacteria that are associated with hundreds of plant diseases. Due to their economical importance and the difficulties involved in the experimental study of these obligate pathogens, genome sequencing and comparative analysis have been utilized as powerful tools to understand phytoplasma biology. To date four complete phytoplasma genome sequences have been published. However, these four strains represent limited phylogenetic diversity. In this study, we report the shotgun sequencing and evolutionary analysis of a peanut witches'-broom (PnWB) phytoplasma genome. The availability of this genome provides the first representative of the 16SrII group and substantially improves the taxon sampling to investigate genome evolution. The draft genome assembly contains 13 chromosomal contigs with a total size of 562,473 bp, covering ∼90% of the chromosome. Additionally, a complete plasmid sequence is included. Comparisons among the five available phytoplasma genomes reveal the differentiations in gene content and metabolic capacity. Notably, phylogenetic inferences of the potential mobile units (PMUs) in these genomes indicate that horizontal transfer may have occurred between divergent phytoplasma lineages. Because many effectors are associated with PMUs, the horizontal transfer of these transposon-like elements can contribute to the adaptation and diversification of these pathogens. In summary, the findings from this study highlight the importance of improving taxon sampling when investigating genome evolution. Moreover, the currently available sequences are inadequate to fully characterize the pan-genome of phytoplasmas. Future genome sequencing efforts to expand phylogenetic diversity are essential in improving our understanding of phytoplasma evolution.

Infection of Mexican lime trees (Citrus aurantifolia L.) with the specialized bacterium "CandidatusPhytoplasma aurantifolia" causes witches' broom disease. Witches' broom disease has the potential to cause significant economic losses throughout western Asia and North Africa. We used label-free quantitative shotgun proteomics to study changes in the proteome of Mexican lime trees in response to infection by "Ca. Phytoplasma aurantifolia". Of 990 proteins present in five replicates of healthy and infected plants, the abundances of 448 proteins changed significantly in response to phytoplasma infection. Of these, 274 proteins were less abundant in infected plants than in healthy plants, and 174 proteins were more abundant in infected plants than in healthy plants. These 448 proteins were involved in stress response, metabolism, growth and development, signal transduction, photosynthesis, cell cycle, and cell wall organization. Our results suggest that proteomic changes in response to infection by phytoplasmas might support phytoplasma nutrition by promoting alterations in the host's sugar metabolism, cell wall biosynthesis, and expression of defense-related proteins. Regulation of defense-related pathways suggests that defense compounds are induced in interactions with susceptible as well as resistant hosts, with the main differences between the two interactions being the speed and intensity of the response.

Proteome analysis is becoming a powerful tool in the functional characterization of organisms, and takes a broad, comprehensive, systematic approach to understanding biology. Following the sequencing of the phytoplasma genomes, the next step is to characterize the expressed proteome of phytoplasmas to acquire the verification and functional annotation of all predicted genes and their protein products. Here, we describe the protocol of mulberry dwarf phytoplasma purification, phytoplasma protein extraction and separation by SDS-PAGE, in-gel tryptic digestion of the proteins, separation of the digested peptides by liquid chromatography, and identification of the peptides by mass spectrometry. The protocol described here is also applicable to the analysis of other phytoplasma proteomes.

The phytopathogenic, cell-wall-less phytoplasmas exhibit a dual life cycle: they multiply in the phloem of their host plant and in the body of their insect vector. Their membrane proteins are in direct contact with both hosts and are supposed to play a crucial role in the phytoplasma spread within the plant as well as by the insect vector. Three types of nonhomologous but highly abundant and immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: Amp, IdpA, and Imp. Although recent results indicate that Amp is involved in vector specificity interacting with insect proteins such as actin, little is known about the interaction of IDP with the plant. We could demonstrate that transiently expressed Imp of 'Candidatus Phytoplasma mali' as well as the Imp without transmembrane domain (Imp▴Tm) bind with plant actins in vivo. Moreover, in vitro co-sediment and binding assays showed that Escherichia coli-expressed recombinant Imp▴Tm-His binds to both G- and F-actins isolated from rabbit muscle. Transgenic plants expressing Imp- or Imp▴Tm-green fluorescent protein did not exhibit any remarkable change of phenotype compared with the wild-type plant. These results indicate that Imp specifically binds to plant actin and a role of Imp-actin binding in phytoplasma motility is hypothesized.

Phytoplasmas are specialised bacteria that are obligate parasites of plant phloem tissue and insects. These bacteria have resisted all attempts of cell-free cultivation. Genome research is of particular importance to analyse the genetic endowment of such bacteria. Here we review the gene content of the four completely sequenced 'Candidatus Phytoplasma' genomes that include those of 'Ca. P. asteris' strains OY-M and AY-WB, 'Ca. P. australiense,' and 'Ca. P. mali'. These genomes are characterized by chromosome condensation resulting in sizes below 900 kb and a G + C content of less than 28%. Evolutionary adaption of the phytoplasmas to nutrient-rich environments resulted in losses of genetic modules and increased host dependency highlighted by the transport systems and limited metabolic repertoire. On the other hand, duplication and integration events enlarged the chromosomes and contribute to genome instability. Present differences in the content of membrane and secreted proteins reflect the host adaptation in the phytoplasma strains. General differences are obvious between different phylogenetic subgroups. 'Ca. P. mali' is separated from the other strains by its deviating chromosome organization, the genetic repertoire for recombination and excision repair of nucleotides or the loss of the complete energy-yielding part of the glycolysis. Apart from these differences, comparative analysis exemplified that all four phytoplasmas are likely to encode an alternative pathway to generate pyruvate and ATP.

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches' Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.

Surface proteins play an important role in phytoplasma life cycle. The antigenic membrane protein (AMP) of 'Candidatus Phytoplasma asteris' has been shown to interact with the insect microfilaments. Due to the synteny of the groL-amp-nadE genetic locus between phytoplasma genomes, the gene stamp that encodes the antigenic membrane protein of stolbur phytoplasma has been cloned and characterized. It encodes a 157 amino acid-long protein with a predicted signal peptide and a C-terminal hydrophobic alpha-helix. STAMP was 26-40% identical to AMP of 'Candidatus Phytoplasma asteris' strains and 40% identical to AMP of 'Ca. P. japonicum'. The expression of STAMP in Escherichia. coli produced a 16 kDa peptide recognized by an anti-stolbur monoclonal antibody. Stamp was more variable than the house-keeping gene secY and the ratio between non-synonymous over synonymous mutations (dN/dS ) was 2.78 for stamp as compared to 0.64 for secY. This indicates that stamp is submitted to a positive diversifying selection pressure., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Phytoplasma research begins to bloom (75). Indeed, this review shows that substantial progress has been made with the identification of phytoplasma effectors that alter flower development, induce witches' broom, affect leaf shape, and modify plant-insect interactions. Phytoplasmas have a unique life cycle among pathogens, as they invade organisms of two distinct kingdoms, namely plants (Plantae) and insects (Animalia), and replicate intracellularly in both. Phytoplasmas release effectors into host cells of plants and insects to target host molecules, and in plants these effectors unload from the phloem to access distal tissues and alter basic developmental processes. The effectors provide phytoplasmas with a fitness advantage by modulating their plant and insect hosts. We expect that further research on the functional characterization of phytoplasma effectors will generate new knowledge that is relevant to fundamental aspects of plant sciences and entomology, and for agriculture by improving yields of crops affected by phytoplasma diseases., (Copyright © 2011 by Annual Reviews. All rights reserved.)

Phytoplasmas are bacterial plant pathogens that have devastating effects on the yields of crops and plants worldwide. They are intracellular parasites of both plants and insects, and are spread among plants by insects. How phytoplasmas can adapt to two diverse environments is of considerable interest; however, the mechanisms enabling the "host switching" between plant and insect hosts are poorly understood. Here, we report that phytoplasmas dramatically alter their gene expression in response to "host switching" between plant and insect. We performed a detailed characterization of the dramatic change that occurs in the gene expression profile of Candidatus Phytoplasma asteris OY-M strain (approximately 33% of the genes change) upon host switching between plant and insect. The phytoplasma may use transporters, secreted proteins, and metabolic enzymes in a host-specific manner. As phytoplasmas reside within the host cell, the proteins secreted from phytoplasmas are thought to play crucial roles in the interplay between phytoplasmas and host cells. Our microarray analysis revealed that the expression of the gene encoding the secreted protein PAM486 was highly upregulated in the plant host, which is also observed by immunohistochemical analysis, suggesting that this protein functions mainly when the phytoplasma grows in the plant host. Additionally, phytoplasma growth in planta was partially suppressed by an inhibitor of the MscL osmotic channel that is highly expressed in the plant host, suggesting that the osmotic channel might play an important role in survival in the plant host. These results also suggest that the elucidation of "host switching" mechanism may contribute to the development of novel pest controls.

Forty-eight apple trees infected by 'Candidatus Phytoplasma mali' were examined using single-strand conformation polymorphism (SSCP) and sequence analysis of a variable hflB gene fragment and the immunodominant membrane protein-encoding imp gene. SSCP analysis of polymerase chain reaction-amplified hflB gene fragments revealed diverse profiles, differing in number and position of the bands. The 'Ca. P. mali' content of a single infected apple tree was termed an accession. Cloning of fragments from accessions that yielded fewer bands resulted in clone populations showing uniform or moderately polymorphic SSCP patterns and largely homogenous nucleotide sequences. In contrast, inserts from accessions yielding more bands were heterogeneous and formed two to four distinct groups of profiles. DNA fragments from such accessions were diverse and clustered distantly when subjected to phylogenetic analysis, mostly as two homogenous groups plus one or a few other sequences. Similar results were obtained upon imp gene examination. The collective data indicate that accessions exhibiting more complex patterns were composed of two or three distinct 'Ca. P. mali' strains. There is evidence that multiple infections are of pathological relevance due to strain interactions leading to shifts in the populations. In triply infected trees of one accession, no specific symptoms were induced by the presence of two of the strains. The rare appearance of pronounced symptoms was associated with a separate strain that possessed a unique SSCP profile and a unique hflB sequence. The two mild strains from this apple accession also induced only mild symptoms on periwinkle and tobacco and occurred specifically in one of these plants.

Phytoplasmas replicate intracellularly in plants and insects and are dependent on both hosts for dissemination in nature. Phytoplasmas have small genomes lacking genes for major metabolic pathways. Nevertheless, their genomes harbour multicopy gene clusters that were named potential mobile units (PMUs). PMU1 is the largest most complete repeat among the PMUs in the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB). PMU1 is c. 20 kb in size and contains 21 genes encoding DNA replication and predicted membrane-targeted proteins. Here we show that AY-WB has a chromosomal linear PMU1 (L-PMU1) and an extrachromosomal circular PMU1 (C-PMU1). The C-PMU1 copy number was consistently higher by in average approximately fivefold in insects compared with plants and PMU1 gene expression levels were also considerably higher in insects indicating that C-PMU1 synthesis and expression are regulated. We found that the majority of AY-WB virulence genes lie on chromosomal PMU regions that have similar gene content and organization as PMU1 providing evidence that PMUs contribute to phytoplasma host adaptation and have integrated into the AY-WB chromosome., (© 2010 Blackwell Publishing Ltd.)

'Candidatus Phytoplasma asteris', onion yellows strain (OY), a mildly pathogenic line (OY-M), is a phytopathogenic bacterium transmitted by Macrosteles striifrons leafhoppers. OY-M contains two types of plasmids (EcOYM and pOYM), each of which possesses a gene encoding the putative transmembrane protein, ORF3. A non-insect-transmissible line of this phytoplasma (OY-NIM) has the corresponding plasmids (EcOYNIM and pOYNIM), but pOYNIM lacks orf3. Here we show that in OY-M, orf3 is transcribed from two putative promoters and that on EcOYNIM, one of the promoter sequences is mutated and the other deleted. We also show by immunohistochemical analysis that ORF3 is not expressed in OY-NIM-infected plants. Moreover, ORF3 protein seems to be preferentially expressed in OY-M-infected insects rather than in plants. We speculate that ORF3 may play a role in the interactions of OY with its insect host.

One of the most important themes in agricultural science is the identification of virulence factors involved in plant disease. Here, we show that a single virulence factor, tengu-su inducer (TENGU), induces witches' broom and dwarfism and is a small secreted protein of the plant-pathogenic bacterium, phytoplasma. When tengu was expressed in Nicotiana benthamiana plants, these plants showed symptoms of witches' broom and dwarfism, which are typical of phytoplasma infection. Transgenic Arabidopsis thaliana lines expressing tengu exhibited similar symptoms, confirming the effects of tengu expression on plants. Although the localization of phytoplasma was restricted to the phloem, TENGU protein was detected in apical buds by immunohistochemical analysis, suggesting that TENGU was transported from the phloem to other cells. Microarray analyses showed that auxin-responsive genes were significantly down-regulated in the tengu-transgenic plants compared with GUS-transgenic control plants. These results suggest that TENGU inhibits auxin-related pathways, thereby affecting plant development.

Phytoplasmas are plant pathogenic bacteria that cause devastating yield losses in diverse crops worldwide. Although the understanding of the pathogen biology is important in agriculture, the inability to culture phytoplasmas has hindered their full characterization. Previous studies demonstrated that immunodominant membrane proteins could be classified into three types, immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp), and they are nonhomologous to each other. Here, cloning and sequencing of imp-containing genomic fragments were performed for several groups of phytoplasma including the aster yellows and rice yellow dwarf groups, for which an imp sequence has not previously been reported. Sequence comparison analysis revealed that Imps are highly variable among phytoplasmas, and clear positive selection was observed in several Imps, suggesting that Imp has important roles in host-phytoplasma interactions. As onion yellows (OY) phytoplasma was known to have Amp as the immunodominant membrane protein, the protein accumulation level of Imp in planta was measured compared with that of Amp. The resulting accumulation of Imp was calculated as approximately one-tenth that of Amp, being consistent with the immunodominant property of Amp in OY. It is suggested that an ancestral type of immunodominant membrane protein could be Imp, and subsequently the expression level of Amp or IdpA is increased in several phytoplasma groups.

The fully sequenced genome of aster yellows phytoplasma strain witches' broom (AY-WB; Candidatus Phytoplasma asteris) was mined for the presence of genes encoding secreted proteins based on the presence of N-terminal signal peptides (SP). We identified 56 secreted AY-WB proteins (SAP). These SAP are candidate effector proteins potentially involved in interaction with plant and insect cell components. One of these SAP, SAP11, contains an N-terminal SP sequence and a eukaryotic bipartite nuclear localization signal (NLS). Transcripts for SAP11 were detected in AY-WB-infected plants. Yellow fluorescence protein (YFP)-tagged SAP11 accumulated in Nicotiana benthamiana cell nuclei, whereas the nuclear targeting of YFP-tagged SAP11 mutants with disrupted NLS was inhibited. The nuclear transport of YFP-SAP11 was also inhibited in N. benthamiana plants in which the expression of importin alpha was knocked down using virus-induced gene silencing (VIGS). Furthermore, SAP11 was detected by immunocytology in nuclei of young sink tissues of China aster plants infected with AY-WB. In summary, this work shows that AY-WB phytoplasma produces a protein that targets the nuclei of plant host cells; this protein is a potential phytoplasma effector that may alter plant cell physiology.

A Japanese hydrangea phyllody (JHP) disease found throughout Japan causes economic damage to the horticultural industry. JHP phytoplasma-infected Japanese hydrangea plants show several disease symptoms involved in floral malformations, such as virescence, phyllody and proliferation. Here, we cloned and characterized the antigenic membrane protein (Amp) gene homolog from the JHP phytoplasma (JHP-amp), expressed the JHP-Amp protein in Escherichia coli cells, and then obtained an antibody against JHP-Amp. The antibody against JHP-Amp had no cross-reactions with the antibody against the Amp protein from a closely related onion yellows phytoplasma. This serologic specificity is probably due to the high diversity of the hydrophilic domains in the Amp proteins. The in situ detection of the JHP-Amp protein revealed that the JHP phytoplasma was localized to the phloem tissues in the malformed flower. This study shows that the JHP-Amp protein is indeed a membrane protein, which is expressed at detectable level in the JHP phytoplasma-infected hydrangea.

Background: Phytoplasmas are insect-transmitted, uncultivable bacterial plant pathogens that cause diseases in hundreds of economically important plants. They represent a monophyletic group within the class Mollicutes (trivial name mycoplasmas) and are characterized by a small genome with a low GC content, and the lack of a firm cell wall. All mycoplasmas, including strains of 'Candidatus (Ca.) Phytoplasma asteris' and 'Ca. P. australiense', examined so far have circular chromosomes, as is the case for almost all walled bacteria., Results: Our work has shown that 'Ca. Phytoplasma mali', the causative agent of apple proliferation disease, has a linear chromosome. Linear chromosomes were also identified in the closely related provisional species 'Ca. P. pyri' and 'Ca. P. prunorum'. The chromosome of 'Ca. P. mali' strain AT is 601,943 bp in size and has a GC content of 21.4%. The chromosome is further characterized by large terminal inverted repeats and covalently closed hairpin ends. Analysis of the protein-coding genes revealed that glycolysis, the major energy-yielding pathway supposed for 'Ca. P. asteris', is incomplete in 'Ca. P. mali'. Due to the apparent lack of other metabolic pathways present in mycoplasmas, it is proposed that maltose and malate are utilized as carbon and energy sources. However, complete ATP-yielding pathways were not identified. 'Ca. P. mali' also differs from 'Ca. P. asteris' by a smaller genome, a lower GC content, a lower number of paralogous genes, fewer insertions of potential mobile DNA elements, and a strongly reduced number of ABC transporters for amino acids. In contrast, 'Ca. P. mali' has an extended set of genes for homologous recombination, excision repair and SOS response than 'Ca. P. asteris'., Conclusion: The small linear chromosome with large terminal inverted repeats and covalently closed hairpin ends, the extremely low GC content and the limited metabolic capabilities reflect unique features of 'Ca. P. mali', not only within phytoplasmas, but all mycoplasmas. It is expected that the genome information obtained here will contribute to a better understanding of the reduced metabolism of phytoplasmas, their fastidious nutrition requirements that prevented axenic cultivation, and the mechanisms involved in pathogenicity.

The chromosome sequence of "Candidatus Phytoplasma australiense" (subgroup tuf-Australia I; rp-A), associated with dieback in papaya, Australian grapevine yellows in grapevine, and several other important plant diseases, was determined. The circular chromosome is represented by 879,324 nucleotides, a GC content of 27%, and 839 protein-coding genes. Five hundred two of these protein-coding genes were functionally assigned, while 337 genes were hypothetical proteins with unknown function. Potential mobile units (PMUs) containing clusters of DNA repeats comprised 12.1% of the genome. These PMUs encoded genes involved in DNA replication, repair, and recombination; nucleotide transport and metabolism; translation; and ribosomal structure. Elements with similarities to phage integrases found in these mobile units were difficult to classify, as they were similar to both insertion sequences and bacteriophages. Comparative analysis of "Ca. Phytoplasma australiense" with "Ca. Phytoplasma asteris" strains OY-M and AY-WB showed that the gene order was more conserved between the closely related "Ca. Phytoplasma asteris" strains than to "Ca. Phytoplasma australiense." Differences observed between "Ca. Phytoplasma australiense" and "Ca. Phytoplasma asteris" strains included the chromosome size (18,693 bp larger than OY-M), a larger number of genes with assigned function, and hypothetical proteins with unknown function.

To characterize potentially important surface-exposed proteins of the phytoplasma causing chrysanthemum yellows (CY), new primers were designed based on the conserved regions of 3 membrane protein genes of the completely sequenced onion yellows and aster yellows witches' broom phytoplasmas and were used to amplify CY DNA. The CY genes secY, amp, and artI, encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively, were cloned and completely sequenced. Alignment of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16S rDNA-based classification, while amp sequences were highly variable within the 'Candidatus Phytoplasma asteris'. Five CY partial sequences were cloned into the pRSetC expression vector, and 3 of the encoded protein fragments (Amp 64/651, Amp 64/224, ArtI 131/512) were expressed as fusion antigens for the production of CY-specific polyclonal antibodies (A416 against Amp 64/224; A407 against ArtI 131/512). A416 recognized, in Western blots, the full-length Amp from CY-infected plants (periwinkle, daisy) and insect vectors (Euscelidius variegatus, Macrosteles quadripunctulatus). A416 also reacted to European aster yellows, to primula yellows phytoplasmas, to northern Italian strains of 'Ca. Phytoplasma asteris' from lettuce and gladiolus, but it did not react to American aster yellows phytoplasma.