Corn Stunt Spiroplasma in Dicotyledonous Plants

Corn stunt spiroplasma (CSS) was transmitted by the leafhopper vector Euscelidius variegatus (Kirschbaum) and produced symptoms on four dicotyledonous plant species, Sinapis alba L. (mustard), Pisum sativitm L. (pea), Raphanus sativus L. (radish) and Spinacia oleracea L. (spinach). The vectors became infective by microinjection with a broth culture of CSS. This insect also acquired CSS from infected mustard plants and transmitted it to healthy ones.     

Corn Stunt Spiroplasma in the Leafhopper Euscelidius variegatus

Corn stunt spiroplasma (CSS) multiplied in all leafhoppers Euscelidius variegatus injected with a culture of CSS, reaching titres of over 1x10⁶ colony forming units (cfu) per insect and 2x10⁴ cfu per salivary gland of each insect. CSS could be isolated from the haemolymph and the salivary glands at any time after injection. The growth of CSS in culture was inhibited by insect extract at concentrations greater than the equivalent of 0.1 insect/ml. Transmission of CSS to sterile feeding solution and to broad beans were compared using 24 h feeding periods. A porportion of 1.7 % of injected leafhoppers began to transmit to sterile feeding solution through membranes by the 4th day after injection, and reached a maximum of 30 % by day 14. Similar insects started transmitting to broad bean plants on day 12 (2 %), reaching a maximum of 7.5 % by day 14. The number of spiroplasmas transmitted by each insect to sterile feeding solution increased from 3, cfu on day 4 to a maximum of 80 cfu by day 14. Helices were seen in the haemolymph at any time after injection. However, partially deformed cells were not present until the 1st week and clumps of 3–4 cells and small aggregates until the 3rd week after injection. The salivary glands of injected insects contained membrane-bound “pockets” or “colonies” packed with pleomorphic, filamentous (helical and non-helical) cells and aggregates. Intracellular colonies were always at the periphery of the acini and were easily detectable by fluorescence microscopy after staining with a DNA-binding fluorescent stain. Pleomorphic and filamentous cells were also seen intercellularly in the salivary glands.     

Isolation and Culture of Corn Stunt Spiroplasma in Serum-Free Medium

A serum-free medium (medium A₆), supports the primary isolation and cultivation in vitro of corn stunt spiroplasma (CSS). The growth characteristics of CSS in A₆ medium are similar to those in the conventional serum dependent, ketoglutarate sorbitol medium (SMK).     

Integrative and free Spiroplasma citri oriC plasmids: expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri.

The replication region (oriC) of the Spiroplasma citri chromosome has been recently sequenced, and a 2-kbp DNA fragment was characterized as an autonomously replicating sequence (F. Ye, J. Renaudin, J. M. Bové, and F. Laigret, Curr. Microbiol. 29:23-29, 1994). In the present studies, we have combined this DNA fragment, containing the dnaA gene and the flanking dnaA boxes, with a ColE1-derived Escherichia coli replicon and the Tet M determinant, which confers resistance to tetracycline. The recombinant plasmid, named pBOT1, was introduced into S. citri cells, in which it replicated. Plasmid pBOT1 was shuttled from E. coli to S. citri and back to E. coli. In S. citri, replication of pBOT1 did not require the presence of a functional dnaA gene on the plasmid. However, the dnaA box region downstream of the dnaA gene was essential. Upon passaging of the S. citri transformants, the plasmid integrated into the spiroplasmal host chromosome by recombination at the replication origin. The integration process led to duplication of the oriC sequences. In contrast to the integrative pBOT1, plasmid pOT1, which does not contain the E. coli replicon, was stably maintained as a free extrachromosomal element. Plasmid pOT1 was used as a vector to introduce into S. citri the G fragment of the cytadhesin P1 gene of Mycoplasma pneumoniae and the spiralin gene of Spiroplasma phoeniceum. The recombinant plasmids, pOTPG with the G fragment and pOTPS with the spiralin gene, were stably maintained in spiroplasmal transformants. Expression of the heterologous S. phoeniceum spiralin in S. citri was demonstrated by Western immunoblotting.     

Antigenic relatedness between the spiralins of Spiroplasma citri and Spiroplasma melliferum.

Four spiralins were compared by rocket immunoelectrophoresis, quantitative immunoblotting techniques, and the spiroplasma deformation test with the use of antispiralin (polyclonal) monospecific antibodies. This investigation revealed that the spiralins of Spiroplasma citri and S. melliferum are antigenically related and that probably no more than two epitopes simultaneously saturable with antibodies are shared by the two proteins. One at least of these epitopes is accessible to antibodies on the spiroplasma cell surface.     

Characterization of the recA gene regions of Spiroplasma citri and Spiroplasma melliferum.

In previous studies (A. Marais, J. M. Bove, and J. Renaudin, J. Bacteriol. 178:862-870, 1996), we have shown that the recA gene of Spiroplasma citri R8A2 was restricted to the first 390 nucleotides of the N-terminal part. PCR amplification and sequencing studies of five additional strains of S. citri have revealed that these strains had the same organization at the recA region as the R8A2 strain. In contrast to S. citri, Spiroplasma melliferum was found to contain a full-length recA gene. However, in all five S. melliferum strains tested, a TAA stop codon was found within the N-terminal region of the recA reading frame. Our results suggest that S. melliferum, as well as S. citri, is RecA deficient. In agreement with the recA mutant genotype of S. citri and S. melliferum, we have shown that these organisms are highly sensitive to UV irradiation.     

Comparison of the membrane composition of Spiroplasma citri and the corn stunt Spiroplasma.

Components of membranes isolated from Spiroplasma citri and corn stunt spiroplasma grown at 28 degrees C were analyzed. On a protein basis, lipid phosphorus was lower and cholesterol was higher in S. citri. Only minor differences between the two species were found in fatty acid composition, reduced nicotinamide adenine dinucleotide diaphorase, and adenosine triphosphatase.     

Fructose utilization and phytopathogenicity of Spiroplasma citri

Spiroplasma citri is a plant-pathogenic mollicute. Recently, the so-called nonphytopathogenic S. citri mutant GMT 553 was obtained by insertion of transposon Tn4001 into the first gene of the fructose operon. Additional fructose operon mutants were produced either by gene disruption or selection of spontaneous xylitol-resistant strains. The behavior of these spiroplasma mutants in the periwinkle plants has been studied. Plants infected via leafhoppers with the wild-type strain GII-3 began to show symptoms during the first week following the insect-transmission period, and the symptoms rapidly became severe. With the fructose operon mutants, symptoms appeared only during the fourth week and remained mild, except when reversion to a fructose+ phenotype occurred. In this case, the fructose+ revertants quickly overtook the fructose- mutants and the symptoms soon became severe. When mutant GMT 553 was complemented with the fructose operon genes that restore fructose utilization, severe pathogenicity, similar to that of the wild-type strain, was also restored. Finally, plants infected with the wild-type strain and grown at 23 degrees C instead of 30 degrees C showed late symptoms, but these rapidly became severe. These results are discussed in light of the role of fructose in plants. Fructose utilization by the spiroplasmas could impair sucrose loading into the sieve tubes by the companion cells and result in accumulation of carbohydrates in source leaves and depletion of carbon sources in sink tissues.     

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.     

Multiplication and morphology of Spiroplasma citri in the leafhopper Euscelis plebejus

Spiroplasma citri multiplied in all Euscelis plebejus leaf hoppers injected and sometimes reached titres of over 1 × 10⁷ colony forming units per insect. Spiroplasmas could be isolated from the haemolymph at all times although helices were only apparent for a few days after injection. The salivary glands of injected insects contained membrane bound pockets densely packed with mycoplasma-like bodies. These bodies were frequently infected with virus-like particles similar to those found in cultures of S. citri. Spiroplasmas had little effect on the longevity of the leafhoppers.     

Involvement of a Spiroplasma citri plasmid in the erythromycin-resistance transfer

An erythromycin-resistant strain (M4 Er-1) was selected from Spiroplasma citri M4+. The transfer by transformation of the erythromycin-resistance character to the erythromycin-sensitive S. citri strain R8A2+ was studied. Transfer became effective and reproducible when cells were treated with alkali cations plus polyethylene glycol. Comparison of the efficiency of transformation of the erythromycin-sensitive strain S. citri R8A2+ by total and extrachromosomal DNA purified from the erythromycin-resistant strain M4 Er-1 showed that the plasmid pM42 was able to transfer the erythromycin-resistance. pM42 was mapped with restriction endonucleases and found to be related to the pMH1 plasmid previously isolated from S. citri MH. Hybridization analysis of DNA from sensitive and resistant strains has shown that a sequence from pM42, analogous to a sequence from pMH1, was integrated at a specific locus in the chromosome of the erythromycin-resistant cells, i.e., of the transformed R8A2 cells and of the spontaneous mutant M4 Er-1 strain.     

Composition and enzyme activities of Spiroplasma citri membranes.

Spiroplasma citri was cultured in three different media that supplied cholesterol and fatty acids from: (i) horse serum, (ii) pleuropneumonia-like organism (PPLO) serum fraction, or (iii) bovine serum albumin-fatty acid-cholesterol. The ability of PPLO serum fraction to support growth varied by lot number. Neither PPLO serum fraction nor the bovine serum albumin medium supported growth as well as the horse serum medium. Analysis of cholesterol, lipid phosphorus, and membrane protein showed the horse serum- and PPLO-grown cells to be indistinguishable, but the bovine serum albumin-grown cells were deficient in lipid phosphorus. The three cultures did not show markedly different fatty acid compositions, but, in all cases, the cultures preferentially incorporated palmitic acid and discriminated against linoleic acid. Cultures grown for different times from logarithmic growth through a degenerative phase showed relatively constant ratios of cholesterol/protein and lipid phosphorus/protein. Fatty acid composition was also relatively constant at the different stages. Adenosine triphosphatase and p-nitrophenyl phosphatase were mainly associated with the membrane, whereas reduced nicotinamide adenine dinucleotide oxidase was either readily removed or not associated with the membrane. The reduced nicotinamide adenine dinucleotide oxidase was inactivated at temperatures above 35 degrees C.     

Lipid composition and lipid metabolism of Spiroplasma citri.

In a horse serum-based medium containing a full complement of fatty acids, cells of Spiroplasma citri were seen to preferentially incorporate palmitic acid. In the same medium, which had a steryl ester-to-sterol ratio of 3.64, a steryl ester-to-sterol ratio of 0.23 was seen in the cells, cholesterol being preferentially incorporated over cholesteryl ester. Like most other mycoplasmas, S. citri was shown to be unable to synthesize fatty acids or esterify cholesterol. The neutral lipids of S. citri grown in a medium containing horse serum consisted of free cholesterol, cholesteryl ester, free fatty acids, triglycerides and diglycerides. All polar lipids were phospholipids, with no glycolipids detected. These phospholipids, which are characteristic of many mycoplasmas, are phosphatidyl glycerol, diphosphatidyl glycerol, and their lyso derivatives. Sphingomyelin was also incorporated when cells were grown on horse serum. A sterol requirement for the growth of S. citri was confirmed using a serum-free medium supplemented with bovine serum albumin, palmitic acid, and various concentrations of sterols dissolved in Tween 80. The addition of palmitic acid stimulated growth but was not essential for growth. S citri was shown to grow best on cholesterol and beta-sitosterol and was able to grow on stigmasterol and ergosterol to a lesser degree. No growth was obtained using mevalonate, deoxycholate, or taurodeoxycholate as an alternative to sterol. S. citri was also able to grow when palmitic acid was replaced with oleic acid, linoleic acid, or linolenic acid. Alterations in the lipid composition of the growth medium and hence in the lipid composition of S. citri induced changes in the characteristic helical morphology of the cells, concurrent with loss of cell viability. Culture, age, and pH were also factors in determining cell morphology and viability.     

Spiralins of Spiroplasma citri and Spiroplasma melliferum: amino acid sequences and putative organization in the cell membrane

Spiralin is the major membrane protein of the helical mollicute Spiroplasma citri. A similar protein occurs in the membrane of Spiroplasma melliferum, an organism related to S. citri. The gene encoding spiralin has been sequenced. A restriction fragment of the spiralin gene has been used as a probe to detect the gene encoding S. melliferum spiralin. A 4.6-kilobase-pair ClaI DNA fragment from S. melliferum strongly hybridized with the probe. This fragment was inserted in pBR322 and cloned in Escherichia coli. It was further subcloned in the replicative forms of M13mp18 and M13mp19, and its nucleotide sequence was determined (GenBank accession number M33991). An open reading frame showing 88.6% base sequence homology with the S. citri spiralin gene could be identified and was assumed to be the gene encoding S. melliferum spiralin. The deduced amino acid sequence of the protein had 75% homology with the spiralin sequence. In particular, the two proteins possess a stretch of 20 amino acids which can form an alpha-helix, in which all polar amino acids occupy approximately one-third of the axial projection down the helix. On the basis of these data and published data, we propose a topological model for the structural organization of the spiralin in the cell membrane of spiroplasmas.     

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.     

A physical and genetic map of the Spiroplasma citri genome.

A physical and genetic map of the Spiroplasma citri genome has been constructed using several restriction enzymes and pulsed field gel electrophoresis. A number of genes were subsequently localized on the map by the use of appropriate probes. The genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest of all Mollicutes studied so far. It contains multisite insertions of Spiroplasma virus 1 (SpV1) sequences. The physical and genetic map of the S. citri genome shares several features with that of other Mollicutes, especially those in the Mycoplasma mycoides cluster. This supports the finding that S. citri and these Mycoplasma spp. are phylogenetically related.     

Filter Paper Dot-Immunobinding Assay for Detection of Spiroplasma citri

A rapid filter paper dot-immunobinding assay was adapted to detect the wall-less mollicute Spiroplasma citri in medium, plants, or insects. Filter paper spotted with sample was incubated first in dilute antiserum, then in protein A-peroxidase, and finally in a substrate of 4-chloro-1-naphthol plus hydrogen peroxide. The detection limit averaged 2.3 × 10¹⁰ CFU/ml in cultures, and S. citri was detected in single infected leafhoppers. This assay was less sensitive but more rapid and economical than an enzyme-linked immunosorbent assay.