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.

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.

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.

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.

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.

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.

Involvement of a minimal actin-binding region of Spiroplasma citri phosphoglycerate kinase in spiroplasma transmission by its leafhopper vector.

BACKGROUND: Spiroplasma citri is a wall-less bacterium that colonizes phloem vessels of a large number of host plants. Leafhopper vectors transmit S. citri in a propagative and circulative manner, involving colonization and multiplication of bacteria in various insect organs. Previously we reported that phosphoglycerate kinase (PGK), the well-known glycolytic enzyme, bound to leafhopper actin and was unexpectedly implicated in the internalization process of S. citri into Circulifer haematoceps cells. METHODOLOGY/PRINCIPAL FINDINGS: In an attempt to identify the actin-interacting regions of PGK, several overlapping PGK truncations were generated. Binding assays, using the truncations as probes on insect protein blots, revealed that the actin-binding region of PGK was located on the truncated peptide designated PGK-FL5 containing amino acids 49-154. To investigate the role of PGK-FL5-actin interaction, competitive spiroplasma attachment and internalization assays, in which His(6)-tagged PGK-FL5 was added to Ciha-1 cells prior to infection with S. citri, were performed. No effect on the efficiency of attachment of S. citri to leafhopper cells was observed while internalization was drastically reduced. The in vivo effect of PGK-FL5 was confirmed by competitive experimental transmission assays as injection of PGK-FL5 into S. citri infected leafhoppers significantly affected spiroplasmal transmission. CONCLUSION: These results suggest that S. citri transmission by its insect vector is correlated to PGK ability to bind actin.

I-SceI-mediated plasmid deletion and intra-molecular recombination in Spiroplasma citri.

S. citri wild-type strain GII3 carries six plasmids (pSci1 to -6) that are thought to encode determinants involved in the transmission of the spiroplasma by its leafhopper vector. In this study we report the use of meganuclease I-SceI for plasmid deletion in S. citri. Plasmids pSci1NT-I and pSci6PT-I, pSci1 and pSci6 derivatives that contain the tetM selection marker and a unique I-SceI recognition site were first introduced into S. citri strains 44 (having no plasmid) and GII3 (carrying pSci1-6), respectively. Due to incompatibility of homologous replication regions, propagation of the S. citri GII3 transformant in selective medium resulted in the replacement of the natural pSci6 by pSci6PT-I. The spiroplasmal transformants were further transformed by an oriC plasmid carrying the I-SceI gene under the control of the spiralin gene promoter. In the S. citri 44 transformant, expression of I-SceI resulted in rapid loss of pSciNT-I showing that expression of I-SceI can be used as a counter-selection tool in spiroplasmas. In the case of the S. citri GII3 transformant carrying pSci6PT-I, expression of I-SceI resulted in the deletion of plasmid fragments comprising the I-SceI site and the tetM marker. Delineating the I-SceI generated deletions proved they had occurred though recombination between homologous sequences. To our knowledge this is the first report of I-SceI mediated intra-molecular recombination in mollicutes.

Partial chromosome sequence of Spiroplasma citri reveals extensive viral invasion and important gene decay.

The assembly of 20,000 sequencing reads obtained from shotgun and chromosome-specific libraries of the Spiroplasma citri genome yielded 77 chromosomal contigs totaling 1,674 kbp (92%) of the 1,820-kbp chromosome. The largest chromosomal contigs were positioned on the physical and genetic maps constructed from pulsed-field gel electrophoresis and Southern blot hybridizations. Thirty-eight contigs were annotated, resulting in 1,908 predicted coding sequences (CDS) representing an overall coding density of only 74%. Cellular processes, cell metabolism, and structural-element CDS account for 29% of the coding capacity, CDS of external origin such as viruses and mobile elements account for 24% of the coding capacity, and CDS of unknown function account for 47% of the coding capacity. Among these, 21% of the CDS group into 63 paralog families. The organization of these paralogs into conserved blocks suggests that they represent potential mobile units. Phage-related sequences were particularly abundant and include plectrovirus SpV1 and SVGII3 and lambda-like SpV2 sequences. Sixty-nine copies of transposases belonging to four insertion sequence (IS) families (IS30, IS481, IS3, and ISNCY) were detected. Similarity analyses showed that 21% of chromosomal CDS were truncated compared to their bacterial orthologs. Transmembrane domains, including signal peptides, were predicted for 599 CDS, of which 58 were putative lipoproteins. S. citri has a Sec-dependent protein export pathway. Eighty-four CDS were assigned to transport, such as phosphoenolpyruvate phosphotransferase systems (PTS), the ATP binding cassette (ABC), and other transporters. Besides glycolytic and ATP synthesis pathways, it is noteworthy that S. citri possesses a nearly complete pathway for the biosynthesis of a terpenoid.

Sequences essential for transmission of Spiroplasma citri by its leafhopper vector, Circulifer haematoceps, revealed by plasmid curing and replacement based on incompatibility.

Spiroplasma citri GII3 contains highly related low-copy-number plasmids pSci1 to -6. Despite the strong similarities between their replication regions, these plasmids coexist in the spiroplasma cells, indicating that they are mutually compatible. The pSci1 to -6 plasmids encode the membrane proteins known as S. citri adhesion-related proteins (ScARPs) (pSci1 to -5) and the hydrophilic protein P32 (pSci6), which had been tentatively associated with insect transmission, as they were not detected in non-insect-transmissible strains. With the aim of further investigating the role of plasmid-encoded determinants in insect transmission, we have constructed S. citri mutant strains that differ in their plasmid contents by developing a plasmid curing/replacement strategy based on the incompatibility of plasmids having identical replication regions. Experimental transmission of these S. citri plasmid mutants through injection into the leafhopper vector Circulifer haematoceps revealed that pSci6, more precisely, the pSci6_06 coding sequence, encoding a protein of unknown function, was essential for transmission. In contrast, ScARPs and P32 were dispensable for both acquisition and transmission of the spiroplasmas by the leafhopper vector, even though S. citri mutants lacking pSci1 to -5 (encoding ScARPs) were acquired and transmitted at lower efficiencies than the wild-type strain GII3.

First report of a tetracycline-inducible gene expression system for mollicutes.

Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO(2) from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO(2) tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO(2) promoter. Adding tetracycline (>50 ng ml(-1)) to the medium induced high-level expression of spiralin. Interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO(2) system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.

Transmission of different isolates of Spiroplasma citri to carrot and citrus by Circulifer tenellus (Hemiptera: Cicadellidae).

Carrot purple leaf disease was first reported in 2006 in the state of Washington and was associated with Spiroplasma citri. The disease also was reported in California in 2008. The objectives of this work were to fulfill Koch's postulates and to determine 1) whether the beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), transmits carrot [Daucus carota L. subsp. Sativus (Hoffm.) Arcang] isolates of S. citri; and 2) whether carrot and citrus [Citrus sinensis (L.) Osb.]-derived spiroplasmas are pathogenic to both plant species. C. tenellus adults received a 24-h acquisition access period to a diet containing carrot-derived S. citri. After 30 d, insects were transferred to healthy carrot seedlings (five per plant). Negative controls were carrot and periwinkle [Catharanthus roseus (L.) G. Don] plants exposed to diet-only-fed insects, and positive controls were periwinkle plants exposed to insects fed on spiroplasma-supplemented diet. Purple carrot leaves and small, chlorotic periwinkle leaves were evident 10-45 d after exposure. Spiroplasmas were reisolated only from symptomatic plants, and polymerase chain reaction (PCR) confirmed their identity as S. citri. No symptoms occurred, and no spiroplasma-specific PCR amplifications or spiroplasma cultures were obtained from plants exposed to diet only-fed insects. Carrot-derived S. citri was transmitted to 15 and 50% of carrot and periwinkle plants exposed, respectively. Insects exposed to S. citri isolates from carrot or citrus transmitted the pathogen to both their host of origin and to the other plant host (carrot or citrus), showing no isolate-host specificity. Our findings confirm that carrot is a host of S. citri. Although carrot is not a preferred host of C. tenellus, it is likely that inoculative leafhoppers feed on carrot during seasonal migration.

Genetic diversity of Spiroplasma citri strains from different regions, hosts, and isolation dates.

Spiroplasma citri, a phloem-limited pathogen, causes citrus stubborn disease (CSD). Losses due to CSD in California orchards have grown over the past decade. To investigate the possibility of introduction or emergence of a new strain, a study of genetic diversity among S. citri strains from various locations was conducted using random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR) of 35 strains cultured from 1980 to 1993, and of 35 strains cultured from 2005 to 2006. Analysis using 20 primer pairs revealed considerable diversity among strains. However, no unique genetic signatures were associated with recently collected strains compared with those collected 15 to 28 years ago, and no geographically associated pattern was distinguishable. S. citri strains from carrot and daikon radish contain some unique DNA fragments, suggesting some host plant influence. Multiple strains from single trees also showed genetic diversity. Sequencing of five RAPD bands that differed among strains showed that diversity-related gene sequences include virus fragments, and fragments potentially encoding a membrane lipoprotein, a DNA modification enzyme, and a mobilization element. No differences in colony morphology were observed among the strains. The lack of correlation between PCR patterns and isolation date or collection site is inconsistent with the hypothesis that recent infections are due to the introduction or emergence of novel pathogen strains.