Spiralin is not essential for helicity, motility, or pathogenicity but is required for efficient transmission of Spiroplasma citri by its leafhopper vector Circulifer haematoceps.

Spiralin is the most abundant protein at the surface of the plant pathogenic mollicute Spiroplasma citri and hence might play a role in the interactions of the spiroplasma with its host plant and/or its insect vector. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. A spiralin-green fluorescent protein (GFP) translational fusion was engineered and introduced into S. citri by using an oriC-based targeting vector. According to the strategy used, integration of the plasmid by a single-crossover recombination at the spiralin gene resulted in the expression of the spiralin-GFP fusion protein. Two distinct mutants were isolated. Western and colony immunoblot analyses showed that one mutant (GII3-9a5) did produce the spiralin-GFP fusion protein, which was found not to fluoresce, whereas the other (GII3-9a2) produced neither the fusion protein nor the wild-type spiralin. Both mutants displayed helical morphology and motility, similarly to the wild-type strain GII-3. Genomic DNA analyses revealed that GII3-9a5 was unstable and that GII3-9a2 was probably derived from GII3-9a5 by a double-crossover recombination between plasmid sequences integrated into the GII3-9a5 chromosome and free plasmid. When injected into the leafhopper vector Circulifer haematoceps, the spiralinless mutant GII3-9a2 multiplied to high titers in the insects (1.1 x 10(6) to 2.8 x 10(6) CFU/insect) but was transmitted to the host plant 100 times less efficiently than the wild-type strain. As a result, not all plants were infected, and symptom production in these plants was delayed for 2 to 4 weeks compared to that in the wild-type strain. In the infected plants however, the mutant multiplied to high titers (1.2 x 10(6) to 1.4 x 10(7) CFU/g of midribs) and produced the typical symptoms of the disease. These results indicate that spiralin is not essential for pathogenicity but is required for efficient transmission of S. citri by its insect vector.

Glucose and trehalose PTS permeases of Spiroplasma citri probably share a single IIA domain, enabling the spiroplasma to adapt quickly to carbohydrate changes in its environment.

Spiroplasma citri is a plant-pathogenic mollicute phylogenetically related to Gram-positive bacteria. Spiroplasma cells are restricted to the phloem sieve tubes and are transmitted from plant to plant by the leafhopper vector Circulifer haematoceps. In the plant sieve tubes, S. citri grows on glucose and fructose, whereas in the leafhopper haemolymph the spiroplasma must grow on trehalose, the major sugar in insects. Previous studies in this laboratory have shown that fructose utilization was a key factor of spiroplasmal pathogenicity. To further study the implication of sugar metabolism in the interactions of S. citri with its plant host and its leafhopper vector, genes encoding permease enzymes II (EII(Glc) and EII(Tre)) of the S. citri phosphoenolpyruvate : glucose and phosphoenolpyruvate : trehalose phosphotransferase systems (PTS) were characterized. Mapping studies revealed that the EII(Glc) complex was split into two distinct polypeptides, IIA(Glc) and IICB(Glc), encoded by two separate genes, crr and ptsG, respectively. As expected, S. citri polypeptides IIA(Glc) and IICB(Glc) were more phylogenetically related to their counterparts from Gram-positive than to those from Gram-negative bacteria. The trehalose operon consisted of three genes treR, treP and treA, encoding a transcriptional regulator, the PTS permease (EII(Tre)) and the amylase, respectively. However, in contrast to the fructose-PTS permease, which is encoded as a single polypeptide (IIABC(Fru)) containing the three domains A, B and C, the trehalose-PTS permease (IIBC(Tre)) lacks its own IIA domain. No trehalose-specific IIA could be identified in the spiroplasmal genome, suggesting that the IIBC(Tre) permease probably functions with the IIA(Glc) domain. In agreement with this statement, yeast two-hybrid system experiments revealed that the IIA(Glc) domain interacted not only with IIB(Glc) but also with the IIB(Tre) domain. The results are discussed with respect to the ability of the spiroplasma to adapt from the phloem sap of the host plant to the haemolymph and salivary gland cells of the insect vector.

'Candidatus phytoplasma phoenicium' sp. nov., a novel phytoplasma associated with an emerging lethal disease of almond trees in Lebanon and Iran.

Almonds (Prunus amygdalus) represent an important crop in most Mediterranean countries. A new and devastating disease of almond trees in Lebanon was recently reported, characterized by the development of severe witches'-brooms on which no flowers or fruits developed, and leading to tree death within a few years. A phytoplasma was detected in diseased trees by PCR amplification of rRNA operon sequences, and RFLP patterns of amplified DNA indicated that the phytoplasma belonged to the pigeon pea witches'-broom (PPWB) group. In the present work, the presence of a phytoplasma in symptomatic plants was confirmed by electron microscopy; this phytoplasma was graft-transmissible to almond, plum and peach seedlings. The phytoplasma was characterized by sequence analysis of rRNA genes and was shown to be different from the phytoplasmas previously described in the PPWB group. A 16S rDNA phylogenetic tree identified the almond tree phytoplasma as a member of a distinct subclade of the class Mollicutes. Oligonucleotides have been defined for specific detection of the new phytoplasma. The almond phytoplasma from Lebanon was shown to be identical to a phytoplasma that induces a disease called 'almond brooming' in Iran, but different from another PPWB-group phytoplasma that infects herbaceous annual plants in Lebanon. Based on its unique properties, the name 'Candidatus Phytoplasma phoenicium' is proposed for the phytoplasma associated with almond witches'-broom in Lebanon and Iran.

Spiroplasma citri, a plant pathogenic molligute: relationships with its two hosts, the plant and the leafhopper vector.

Spiroplasma citri, the type species of the genus Spiroplasma (Spiroplasmataceae, Mollicutes), is restricted to the phloem sieve tubes and transmitted by phloem sap-feeding insects, as is characteristic of the phytopathogenic mollicutes. The spiroplasmas are the only mollicutes showing motility and helical morphology, apparently mediated by a contractile fibrillar cytoskeleton bound to the inner surface of the spiroplasmal membrane. MreB genes, which are involved in cell-shape determination, have been identified in S. citri. Identified genes of other functional groups are those involved in the transmission of S. citri by the leafhoppers and genes coding for lipoproteins, including spiralin, bound to the outer surface of the spiroplasma membrane. S. citri mutants that are unable to use fructose induce only mild and delayed symptoms. Fructose utilization by the sieve tube-restricted wild-type spiroplasmas is postulated to deprive the companion cells of fructose, thereby impairing sucrose loading into the sieve tubes.

"Candidatus Phlomobacter fragariae" Is the Prevalent Agent of Marginal Chlorosis of Strawberry in French Production Fields and Is Transmitted by the Planthopper Cixius wagneri (China).

ABSTRACT Marginal chlorosis has affected strawberry production in France for about 15 years. A phloem-restricted uncultured bacterium, "Candidatus Phlomobacter fragariae," is associated with the disease. A large-scale survey for marginal chlorosis in French strawberry production fields and nurseries by polymerase chain reaction amplification of "Ca. P. fragariae" 16S rDNA revealed that symptoms of marginal chlorosis were not always induced by "Ca. P. fragariae" and that the stolbur phytoplasma could induce identical symptoms. "Ca. P. fragariae" was found to be predominant in strawberry production fields, whereas the stolbur phytoplasma was predominantly detected in nurseries. Two transmission periods of the disease, one in spring and the other from late summer to early fall, were evident. Cixius wagneri planthoppers captured on infected strawberry plants were demonstrated to be efficient vectors of "Ca. P. fragariae." The involvement in natural disease spread of the whitefly Trialeurodes vaporariorum, previously shown to acquire and multiply "Ca. P. fragariae" under greenhouse conditions, remains uncertain.

New plasmid vectors for specific gene targeting in Spiroplasma citri.

In Spiroplasma citri gene inactivation through homologous recombination has been achieved by using the replicative, oriC plasmid pBOT1 as the disruption vector. However, plasmid recombination required extensive passaging of the transformants and, in most cases, recombination occurred at oriC rather than at the target gene. In the current study, we describe a new vector, in which the oriC fragment was reduced to the minimal sequences able to promote plasmid replication. Using this vector to inactivate the motility gene scm1 showed that size reduction of the oriC fragment did increase the frequency of recombination at the target gene. Furthermore, to avoid extensive passaging of the transformants, we developed a strategy in which the selective, tetracycline resistance phenotype can only be expressed once the plasmid has integrated into the chromosome by one single crossover recombination at the target gene. As an example, targeting of the spiralin gene is described.

Effect of polyclonal, monoclonal, and recombinant (single-chain variable fragment) antibodies on in vitro morphology, growth, and metabolism of the phytopathogenic mollicute Spiroplasma citri.

Antibodies are known to affect the morphology, growth, and metabolism of mollicutes and thus may serve as candidate molecules for a plantibody-based control strategy for plant-pathogenic spiroplasmas and phytoplasmas. Recombinant single-chain variable fragment (scFv) antibodies are easy to engineer and express in plants, but their inhibitory effects on mollicutes have never been evaluated and compared with those of polyclonal and monoclonal antibodies. We describe the morphology, growth, and glucose metabolism of Spiroplasma citri in the presence of polyclonal, monoclonal, and recombinant antibodies directed against the immunodominant membrane protein spiralin. We showed that the scFv antibodies had no effect on S. citri glucose metabolism but were as efficient as polyclonal antibodies in inhibiting S. citri growth in liquid medium. Inhibition of motility was also observed.

Fructose operon mutants of Spiroplasma citri.

Fructose-negative mutants of Spiroplasma citri wild-type strain GII-3 were selected by two methods. The first method is based on the selection of spontaneous xylitol-resistant mutants, xylitol being a toxic fructose analogue. Five such mutants were obtained, but only one, xyl3, was unable to use fructose and had no phosphoenolpuryvate:fructose phosphotransferase system (fructose-PTS) activity. Amplification and sequencing of the fructose permease gene of mutant xyl3 revealed the presence of an adenylic insertion leading to a truncated permease. The second method is based on inactivation of fruA and/or fruK by homologous recombination involving one crossing-over between the chromosomal genes and inactivated genes carried by replicative plasmids. Fructose-negative mutants were obtained at a frequency of about 10%. Fructose-PTS activity and 1-phosphofructokinase activity were not detected in four representative mutants that were characterized (H31, H45, E38 and E53). In strain H31, Southern blot analysis and PCR showed that the result of homologous recombination was, as expected, the presence in the chromosome of two mutated fruA-fruK copies with the plasmid sequence in between. Only the mutated copy, under control of the fructose operon promoter, was transcribed. This work describes for the first time the use of two methods to obtain fructose-auxotrophic mutants of S. citri. The method involving homologous recombination is a general procedure for gene disruption in S. citri.