New Assays for Rapid Detection of Beet Leafhopper-Associated Plant Pathogens, 'Candidatus Phytoplasma trifolii', Beet Curly Top Virus, and Spiroplasma citri.

The beet leafhopper Circulifer tenellus is an important pest of agricultural crops in the United States, where it transmits beet curly top virus, beet leafhopper-transmitted virescence agent phytoplasma, and Spiroplasma citri to numerous crops, affecting yield and quality. Each of these pathogens have been linked to serious disease outbreaks within Washington State in the past century. To mitigate the risk of disease, growers target the beet leafhopper in their insect pest management programs. Knowledge of pathogen prevalence in beet leafhopper populations could help growers make better management decisions, but timely diagnostics is required. Four new assays were developed for the rapid detection of the beet leafhopper-associated pathogens. These include two assays that detect Beet leafhopper transmitted virescence agent (a PCR and a real-time PCR SYBR green assay), a duplex PCR assay that simultaneously detects beet curly top virus and Spiroplasma citri, and a multiplex real-time PCR assay for the simultaneous detection of all three pathogens. The screening of dilution series generated from plant total nucleic acid extracts with these new assays typically led to detection at levels 10- to 100-fold more sensitive than the conventional PCR assays currently used. These new tools will allow the rapid detection of beet leafhopper-associated pathogens in both plant and insect specimens and will have the potential to be used in diagnostic laboratories seeking to disseminate fast and accurate results to growers for implementation in their insect pest monitoring programs.

Metabolite Signature and Differential Expression of Genes in Washington Navel Oranges (Citrus sinensis) Infected by Spiroplasma citri.

Spiroplasma citri is the pathogen that causes citrus stubborn disease (CSD). Infection of citrus with S. citri has been shown to cause leaf mottling, reduce fruit yield, and stunt tree growth. Fruit from trees exhibiting symptoms of CSD are misshapen and discolored. The symptoms of CSD are easily confused with nutrient deficiencies or symptoms of citrus greening disease. In this study, young Washington navel oranges (Citrus sinensis) were graft-inoculated with budwood originating from trees confirmed to be infected with S. citri. Leaf samples were collected monthly for 10 months for metabolomics and differential gene expression analyses. Significant differences in the concentration of metabolites and expressed genes were observed between control and S. citri-infected trees throughout the experiment. Metabolites and genes associated with important defense and stress pathways, including jasmonic acid signaling, cell wall modification, amino acid biosynthesis, and the production of antioxidant and antimicrobial secondary metabolites, were impacted by S. citri throughout the study, and even prior to symptom development. This work fills a current gap in knowledge surrounding the pathogenicity of S. citri and provides an updated mechanistic explanation for the development of CSD symptoms in S. citri-infected plants.

Filament organization of the bacterial actin MreB is dependent on the nucleotide state.

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. Despite belonging to the actin family, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we provide insights into the effect of nucleotide state on membrane binding of Spiroplasma citri MreB5 (ScMreB5). Filaments of ScMreB5WT and an ATPase-deficient mutant, ScMreB5E134A, assemble independently of the nucleotide state. However, capture of the filament dynamics revealed that efficient filament formation and organization through lateral interactions are affected in ScMreB5E134A. Hence, the catalytic glutamate functions as a switch, (a) by sensing the ATP-bound state for filament assembly and (b) by assisting hydrolysis, thereby potentially triggering disassembly, as observed in other actins. Glu134 mutation and the bound nucleotide exhibit an allosteric effect on membrane binding, as observed from the differential liposome binding. We suggest that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis among actins has been repurposed in MreBs for modulating filament organization on the membrane.

Genome analysis of Spiroplasma citri strains from different host plants and its leafhopper vectors.

BACKGROUND: Spiroplasma citri comprises a bacterial complex that cause diseases in citrus, horseradish, carrot, sesame, and also infects a wide array of ornamental and weed species. S. citri is transmitted in a persistent propagative manner by the beet leafhopper, Neoaliturus tenellus in North America and Circulifer haematoceps in the Mediterranean region. Leafhopper transmission and the pathogen's wide host range serve as drivers of genetic diversity. This diversity was examined in silico by comparing the genome sequences of seven S. citri strains from the United States (BR12, CC-2, C5, C189, LB 319, BLH-13, and BLH-MB) collected from different hosts and times with other publicly available spiroplasmas. RESULTS: Phylogenetic analysis using 16S rRNA sequences from 39 spiroplasmas obtained from NCBI database showed that S. citri strains, along with S. kunkelii and S. phoeniceum, two other plant pathogenic spiroplasmas, formed a monophyletic group. To refine genetic relationships among S. citri strains, phylogenetic analyses with 863 core orthologous sequences were performed. Strains that clustered together were: CC-2 and C5; C189 and R8-A2; BR12, BLH-MB, BLH-13 and LB 319. Strain GII3-3X remained in a separate branch. Sequence rearrangements were observed among S. citri strains, predominantly in the center of the chromosome. One to nine plasmids were identified in the seven S. citri strains analyzed in this study. Plasmids were most abundant in strains isolated from the beet leafhopper, followed by strains from carrot, Chinese cabbage, horseradish, and citrus, respectively. All these S. citri strains contained one plasmid with high similarity to plasmid pSci6 from S. citri strain GII3-3X which is known to confer insect transmissibility. Additionally, 17 to 25 prophage-like elements were identified in these genomes, which may promote rearrangements and contribute to repetitive regions. CONCLUSIONS: The genome of seven S. citri strains were found to contain a single circularized chromosome, ranging from 1.58 Mbp to 1.74 Mbp and 1597-2232 protein-coding genes. These strains possessed a plasmid similar to pSci6 from the GII3-3X strain associated with leafhopper transmission. Prophage sequences found in the S. citri genomes may contribute to the extension of its host range. These findings increase our understanding of S. citri genetic diversity.

Multiplex detection of "Candidatus Liberibacter asiaticus" and Spiroplasma citri by qPCR and droplet digital PCR.

"Candidatus Liberibacter asiaticus" (CLas) and Spiroplasma citri are phloem-limited bacteria that infect citrus and are transmitted by insect vectors. S. citri causes citrus stubborn disease (CSD) and is vectored by the beet leafhopper in California. CLas is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid. CLas is a regulatory pathogen spreading in citrus on residential properties in southern California and is an imminent threat to spread to commercial citrus plantings. CSD is endemic in California and has symptoms in citrus that can be easily confused with HLB. Therefore, the objective of this study was to develop a multiplex qPCR and duplex droplet digital PCR (ddPCR) assay for simultaneous detection of CLas and S. citri to be used where both pathogens can co-exist. The multiplex qPCR assay was designed to detect multicopy genes of CLas-RNR (5 copies) and S. citri-SPV1 ORF1 (13 copies), respectively, and citrus cytochrome oxidase (COX) as internal positive control. Absolute quantitation of these pathogens was achieved by duplex ddPCR as a supplement for marginal qPCR results. Duplex ddPCR allowed higher sensitivity than qPCR for detection of CLas and S. citri. ddPCR showed higher tolerance to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced cost and can serve to augment the official regulatory protocol for CLas detection in California. Moreover, the ddPCR provided unambiguous absolute detection of CLas and S. citri at very low concentrations without any standards for pathogen titer.

MreB5 Is a Determinant of Rod-to-Helical Transition in the Cell-Wall-less Bacterium Spiroplasma.

In most rod-shaped bacteria, the spatial coordination of cell wall synthesis machinery by MreBs is the main theme for shape determination and maintenance in cell-walled bacteria [1-9]. However, how rod or spiral shapes are achieved and maintained in cell-wall-less bacteria is currently unknown. Spiroplasma, a helical Mollicute that lacks cell wall synthesis genes, encodes five MreB paralogs and a unique cytoskeletal protein fibril [10, 11]. Here, we show that MreB5, one of the five MreB paralogs, contributes to cell elongation and is essential for the transition from rod-to-helical shape in Spiroplasma. Comparative genomic and proteomic characterization of a helical and motile wild-type Spiroplasma strain and a non-helical, non-motile natural variant helped delineate the specific roles of MreB5. Moreover, complementation of the non-helical strain with MreB5 restored its helical shape and motility by a kink-based mechanism described for Spiroplasma [12]. Earlier studies had proposed that length changes in fibril filaments are responsible for the change in handedness of the helical cell and kink propagation during motility [13]. Through structural and biochemical characterization, we identify that MreB5 exists as antiparallel double protofilaments that interact with fibril and the membrane, and thus potentially assists in kink propagation. In summary, our study provides direct experimental evidence for MreB in maintaining cell length, helical shape, and motility-revealing the role of MreB in sculpting the cell in the absence of a cell wall.

Whole genome sequence of five strains of Spiroplasma citri isolated from different host plants and its leafhopper vector.

OBJECTIVES: Spiroplasma citri is a bacterium with a wide host range and is the causal agent of citrus stubborn and brittle root diseases of citrus and horseradish, respectively. S. citri is transmitted in a circulative, persistent manner by the beet leafhopper, Neoaliturus (Circulifer) tenellus (Baker), in North America. Five strains of S. citri were cultured from citrus, horseradish, and N. tenellus from different habitats and times. DNA from cultures were sequenced and genome assembled to expand the database to improve detection assays and better understand its genetics and evolution. DATA DESCRIPTION: The whole genome sequence of five strains of S. citri are described herein. The S. citri chromosome was circularized for all five strains and ranged from 1,576,550 to 1,742,208 bp with a G + C content of 25.4-25.6%. Characterization of extrachromosomal DNAs resulted in identification of one or two plasmids, with a G + C content of 23.3 to 27.6%, from plant hosts; and eight or nine plasmids, with a G + C content of 21.65 to 29.19%, from N. tenellus. Total genome size ranged from 1,611,714 to 1,832,173 bp from plants and 1,968,976 to 2,155,613 bp from the leafhopper. All sequence data has been deposited in DDBJ/ENA/GenBank under the accession numbers CP046368-CP046373 and CP047426-CP047446.

Genome Sequence Resource for Spiroplasma citri, Strain CC-2, Associated with Citrus Stubborn Disease in California.

Spiroplasma citri is a bacterium that causes stubborn disease of citrus and infects other crops, ornamentals, and weeds. It is transmitted by leafhoppers in a circulative manner. Due to limited sequence data on S. citri, the bacterium was isolated from naturally infected Chinese cabbage grown on a farm in Fresno County, CA. DNA from S. citri CC-2 was extracted from a pure culture in LD8 and subjected to PacBio sequencing. Four contigs were obtained with a single circular chromosome of 1,709,192 bp and three plasmids of 40,210, 39,313, and 2,921 bp in size. The genome developed herein extends the sequence database of S. citri and is the first whole-genome sequence record of S. citri from California.

Large variability in the motility of spiroplasmas in media of different viscosities.

Spiroplasmas are bacteria that do not possess flagella and their motility is linked to kink propagation coupled to changes in the cell body helicity. While the motility of bacteria with flagellar motion has been studied extensively, less work has been devoted to the motility of spiroplasmas. We first show that the motility of such bacteria has large variability from individual to individual as well as large fluctuations in time. The Brownian motion of such bacteria both in orientation and translation is also highlighted. We propose a simple model to disentangle the different components of this motility by examining trajectories of single bacteria in different viscosity solvents. The mean velocity of the bacteria turns out to depend on the viscosity of the medium as it increases with viscosity. Further, the temporal fluctuations of the bacteria motility turn out to be very strong with a direct link to tumbling events particular to this bacteria.

Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR.

Droplet digital polymerase chain reaction (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.

Proteolytic Post-Translational Processing of Adhesins in a Pathogenic Bacterium.

Mollicutes, including mycoplasmas and spiroplasmas, have been considered as good representatives of the « minimal cell ¼ concept: these wall-less bacteria are small in size and possess a minimal genome and restricted metabolic capacities. However, the recent discovery of the presence of post-translational modifications unknown so far, such as the targeted processing of membrane proteins of mycoplasma pathogens for human and swine, revealed a part of the hidden complexity of these microorganisms. In this study, we show that in the phytopathogen, insect-vectored Spiroplasma citri GII-3 adhesion-related protein (ScARP) adhesins are post-translationally processed through an ATP-dependent targeted cleavage. The cleavage efficiency could be enhanced in vitro when decreasing the extracellular pH or upon the addition of polyclonal antibodies directed against ScARP repeated units, suggesting that modification of the surface charge and/or ScARP conformational changes could initiate the cleavage. The two major sites for primary cleavage are localized within predicted disordered regions and do not fit any previously reported cleavage motif; in addition, the inhibition profile and the metal ion requirements indicate that this post-translational modification involves at least one non-conventional protease. Such a proteolytic process may play a role in S. citri colonization of cells of the host insect. Furthermore, our work indicates that post-translational cleavage of adhesins represents a common feature to mollicutes colonizing distinct hosts and that processing of surface antigens could represent a way to make the most out of a minimal genome.

Stubborn Disease in Iran: Diversity of Spiroplasma citri Strains in Circulifer haematoceps Leafhoppers Collected in Sesame Fields in Fars Province.

Spiroplasma citri is a bacterial pathogen responsible for the economically important citrus stubborn disease. Sesame and citrus seeds serve as hosts for both S. citri and its leafhopper vector Circulifer haematoceps. To evaluate whether sesame could act as a reservoir for citrus-infecting strains or not, the genetic diversity among S. citri strains found in leafhoppers collected in citrus and citrus-free sesame fields was investigated. Among 26 periwinkle plants exposed to the collected C. haematoceps leafhoppers, 12 plants developed typical stubborn symptoms. All symptomatic periwinkles were polymerase chain reaction positive using S. citri-specific primer pairs targeting the spiralin and P89 genes. Phylogenetic trees based on spiralin gene sequence analysis indicated that the novel field-collected strains clustered with those belonging to two formerly defined S. citri groups (groups 6 and 1). In addition, our results strongly suggest that group 1 strains could be transmitted from sesame-infected plants to citrus trees by C. haematoceps, while group 6 strains may not infect citrus trees.

Differential expression of Spiroplasma citri surface protein genes in the plant and insect hosts.

BACKGROUND: Spiroplasma citri is a cell wall-less, plant pathogenic bacteria that colonizes two distinct hosts, the leafhopper vector and the host plant. Given the absence of a cell wall, surface proteins including lipoproteins and transmembrane polypeptides are expected to play key roles in spiroplasma/host interactions. Important functions in spiroplasma/insect interactions have been shown for a few surface proteins such as the major lipoprotein spiralin, the transmembrane S. citri adhesion-related proteins (ScARPs) and the sugar transporter subunit Sc76. S. citri efficient transmission from the insect to the plant is expected to rely on its ability to adapt to the different environments and more specifically to regulate the expression of genes encoding surface-exposed proteins. RESULTS: Genes encoding S. citri lipoproteins and ScARPs were investigated for their expression level in axenic medium, in the leafhopper vector Circulifer haematoceps and in the host plant (periwinkle Catharanthus roseus) either insect-infected or graft-inoculated. The vast majority of the lipoprotein genes tested (25/28) differentially responded to the various host environments. Considering their relative expression levels in the different environments, the possible involvement of the targeted genes in spiroplasma host adaptation was discussed. In addition, two S. citri strains differing notably in their ability to express adhesin ScARP2b and pyruvate dehydrogenase E1 component differed in their capacity to multiply in the two hosts, the plant and the leafhopper vector. CONCLUSIONS: This study provided us with a list of genes differentially expressed in the different hosts, leading to the identification of factors that are thought to be involved in the process of S. citri host adaptation. The identification of such factors is a key step for further understanding of S. citri pathogenesis. Moreover the present work highlights the high capacity of S. citri in tightly regulating the expression level of a large set of surface protein genes, despite the small size of its genome.

Immune response and survival of Circulifer haematoceps to Spiroplasma citri infection requires expression of the gene hexamerin.

Spiroplasma citri is a cell wall-less bacterium that infects plants. It is transmitted by the leafhopper Circulifer haematoceps, which hosts this bacterium in the haemocel and insect tissues. Bacterial factors involved in spiroplasma colonization of the insect host have been identified, but the immune response of the leafhopper to S. citri infection remains unknown. In this study, we showed that C. haematoceps activates both humoral and cellular immune responses when challenged with bacteria. When infected by S. citri, C. haematoceps displayed a specific immune response, evidenced by activation of phagocytosis and upregulation of a gene encoding the protein hexamerin. S. citri infection also resulted in decreased phenoloxidase-like activity. Inhibition of hexamerin by RNA interference resulted in a significant reduction in phenoloxidase-like activity and increased mortality of infected leafhoppers. Therefore, the gene hexamerin is involved in S. citri control by interfering with insect phenoloxidase activity.

Heterologous expression and processing of the flavescence dorée phytoplasma variable membrane protein VmpA in Spiroplasma citri.

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 ß-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 ß-lactamase by recombinant spiroplasmas established the considerable promise of the S. citri expression system for delivering phytoplasma effector proteins into host cells.

Invasion of insect cells by Spiroplasma citri involves spiralin relocalization and lectin/glycoconjugate-type interactions.

Spiroplamas are helical, cell wall-less bacteria belonging to the Class Mollicutes, a group of microorganisms phylogenetically related to low G+C, Gram-positive bacteria. Spiroplasma species are all found associated with arthropods and a few, including Spiroplasma citri are pathogenic to plant. Thus S. citri has the ability to colonize cells of two very distinct hosts, the plant and the insect vector. While spiroplasmal factors involved in transmission by the leafhopper Circulifer haematoceps have been identified, their specific contribution to invasion of insect cells is poorly understood. In this study we provide evidence that the lipoprotein spiralin plays a major role in the very early step of cell invasion. Confocal laser scanning immunomicroscopy revealed a relocalization of spiralin at the contact zone of adhering spiroplasmas. The implication of a role for spiralin in adhesion to insect cells was further supported by adhesion assays showing that a spiralin-less mutant was impaired in adhesion and that recombinant spiralin triggered adhesion of latex beads. We also showed that cytochalasin D induced changes in the surface-exposed glycoconjugates, as inferred from the lectin binding patterns, and specifically improved adhesion of S. citri wild-type but not of the spiralin-less mutant. These results indicate that cytochalasin D exposes insect cell receptors of spiralin that are masked in untreated cells. In addition, competitive adhesion assays with lectins strongly suggest spiralin to exhibit glycoconjugate binding properties similar to that of the Vicia villosa agglutinin (VVA) lectin.

Spiralin diversity within Iranian strains of Spiroplasma citri.

The first-cultured and most-studied spiroplasma is Spiroplasma citri, the causal agent of citrus stubborn disease, one of the three plant-pathogenic, sieve-tube-restricted, and leafhopper vector-transmitted mollicutes. In Iranian Fars province, S. citri cultures were obtained from stubborn affected citrus trees, sesame and safflower plants, and from the leafhopper vector Circulifer haematoceps. Spiralin gene sequences from different S. citri isolates were amplified by PCR, cloned, and sequenced. Phylogenetic trees based on spiralin gene sequence showed diversity and indicated the presence of three clusters among the S. citri strains. Comparison of the amino acid sequences of eleven spiralins from Iranian strains and those from the reference S. citri strain GII-3 (241 aa), Palmyre strain (242 aa), Spiroplasma kunkelii (240 aa), and Spiroplasma phoeniceum (237 aa) confirmed the conservation of general features of the protein. However, the spiralin of an S. citri isolate named Shiraz I comprised 346 amino acids and showed a large duplication of the region comprised between two short repeats previously identified in S. citri spiralins. We report in this paper the spiralin diversity in Spiroplasma strains from southern Iran and for the first time a partial internal duplication of the spiralin gene.

The repetitive domain of ScARP3d triggers entry of Spiroplasma citri into cultured cells of the vector Circulifer haematoceps.

Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.

Expression patterns of genes involved in the defense and stress response of Spiroplasma citri infected Madagascar Periwinkle Catharanthus roseus.

Madagascar periwinkle is an ornamental and a medicinal plant, and is also an indicator plant that is highly susceptible to phytoplasma and spiroplasma infections from different crops. Periwinkle lethal yellows, caused by Spiroplasma citri, is one of the most devastating diseases of periwinkle. The response of plants to S. citri infection is very little known at the transcriptome level. In this study, quantitative real-time PCR (RT-qPCR) was used to investigate the expression levels of four selected genes involved in defense and stress responses in naturally and experimentally Spiroplasma citri infected periwinkles. Strictosidine ß-glucosidase involved in terpenoid indole alkaloids (TIAs) biosynthesis pathway showed significant upregulation in experimentally and naturally infected periwinkles. The transcript level of extensin increased in leaves of periwinkles experimentally infected by S. citri in comparison to healthy ones. A similar level of heat shock protein 90 and metallothionein expression was observed in healthy, naturally and experimentally spiroplasma-diseased periwinkles. Overexpression of Strictosidine ß-glucosidase demonstrates the potential utility of this gene as a host biomarker to increase the fidelity of S. citri detection and can also be used in breeding programs to develop stable disease-resistance varieties.

Selection and characterization of Spiroplasma citri mutants by random transposome mutagenesis.

Phytopathogenic spiroplasmas can multiply in vascular plants and insects. A deeper understanding of this dual-host life could be furthered through the identification by random mutagenesis of spiroplasma genes required. The ability of the EZ::TN™ Tnp transposome™ system to create random insertional mutations in the genome of Spiroplasma citri was evaluated. The efficiency of electroporation-mediated transformation of S. citri BR3-3X averaged 28.8 CFUs/ng transposome for 10(9) spiroplasma cells. Many transformants appearing on the selection plates were growth impaired when transferred to broth. Altering broth composition in various ways did not improve their growth. However, placing colonies into a small broth volume resulted in robust growth and successful subsequent passages of a subset of transformants. PCR using primers for the dihydrofolate reductase gene confirmed the transposon's presence in the genomes of selected transformants. Southern blot hybridization and nucleotide sequencing suggested that insertion was random within the chromosome and usually at single sites. The insertions were stable. Growth rates of all transformants were lower than that of the wild-type S. citri, but none lost the ability to adhere to a Circulifer tenellus (CT-1) cell line. The EZ::TN™ Tnp transposome™ system represents an additional tool for genetic manipulation of the fastidious spiroplasmas.