Frédéric Laigret

Automated SNP detection in expressed sequence tags: statistical considerations and application to maritime pine sequences.

We developed an automated pipeline for the detection of single nucleotide polymorphisms (SNPs) in expressed sequence tag (EST) data sets, by combining three DNA sequence analysis programs:Phred, Phrap and PolyBayes. This application requires access to the individual electrophoregram traces. First, a reference set of 65 SNPs was obtained from the sequencing of 30 gametes in 13 maritime pine (Pinus pinaster Ait.) gene fragments (6671 bp), resulting in a frequency of 1 SNP every 102.6 bp. Second, parameters of the three programs were optimized in order to retrieve as many true SNPs, while keeping the rate of false positive as low as possible. Overall, the efficiency of detection of true SNPs was 83.1%. However, this rate varied largely as a function of the rare SNP allele frequency: down to 41% for rare SNP alleles (frequency ` 10%), up to 98% for allele frequencies above 10%. Third, the detection method was applied to the 18498 assembled maritime pine (Pinus pinaster Ait.) ESTs, allowing to identify a total of 1400 candidate SNPs, in contigs containing between 4 and 20 sequence reads. These genetic resources, described for the first time in a forest tree species, were made available at http://www.pierroton.inra/genetics/Pinesnps. We also derived an analytical expression for the SNP detection probability as a function of the SNP allele frequency, the number of haploid genomes used to generate the EST sequence database, and the sample size of the contigs considered for SNP detection. The frequency of the SNP allele was shown to be the main factor influencing the probability of SNP detection.

Heterogeneity of Genome Sizes within the Genus Spiroplasma

Organisms belonging to the genus Spiroplasma are currently classified into 23 groups, 17 of which have been assigned species epithets. We determined the genome sizes of representatives of 20 groups by using pulsed-field gel electrophoresis. Each genome size was deduced from the mobility of linear nonrestricted DNA, as well as from the sum of the sizes of restriction fragments obtained after digestion with NotI, a restriction endonuclease with a limited number of restriction sites in spiroplasma DNA. The values which we obtained indicated that the genome sizes of members of the genus Spiroplasma range from 940 to 2,220 kbp.

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.

Mutagenesis by Insertion of Tn4001 into the Genome of Spiroplasma citri: Characterization of Mutants Affected in Plant Pathogenicity and Transmission to the Plant by the Leafhopper Vector Circulifer haematoceps

Two hundred and fifty-seven transposon Tn4001 mutants of Spiroplasma citri strain GII3 were used for transmission assays by the leafhopper vector Circulifer haematoceps into periwinkle (Catharanthus roseus) plants. Multiplication of the mutants in the two hosts, the leafhopper and the plant, as well as the symptom expression in the plant were studied. Two mutants, GMT 470 and GMT 553, caused no symptoms on plants. Tn4001 is inserted as a single copy in the genome of these mutants. Mutant GMT 470 did not multiply, or multiplied only poorly, in the leaf-hopper and was not transmitted by the insect to the plant, nor to culture medium through Parafilm membrane. The growth rate of GMT 470 in SP4 medium was twice as slow as that of wild-type strain GII3. Mutant GMT 553 multiplied in the leafhopper as well as the wild-type spiro-plasma, and was transmitted by the leafhoppers into the plants, where it reached the same titers as the wild-type strain but in approximately twice as much time. The plants containing hi...

Partial Chromosome Sequence of Spiroplasma citri Reveals Extensive Viral Invasion and Important Gene Decay

ABSTRACT 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.

Mycoplasmas, plants, insect vectors: a matrimonial triangle.

Plant pathogenic mycoplasmas were discovered by electron microscopy, in 1967, long after the discovery and culture in 1898 of the first pathogenic mycoplasma of animal origin, Mycoplasma mycoides. Mycoplasmas are Eubacteria of the class Mollicutes, a group of organisms phylogenetically related to Gram-positive bacteria. Their more characteristic features reside in the small size of their genomes, the low guanine (G) plus cytosine (C) content of their genomic DNA and the lack of a cell wall. Plant pathogenic mycoplasmas are responsible for several hundred diseases and belong to two groups: the phytoplasmas and the spiroplasmas. The phytoplasmas (previously called MLOs, for mycoplasma like organisms) were discovered first; they are pleiomorphic, and have so far resisted in vitro cultivation. Phytoplasmas represent the largest group of plant pathogenic Mollicutes. Only three plant pathogenic spiroplasmas are known today. Spiroplasma citri, the agent of citrus stubborn was discovered and cultured in 1970 and shown to be helical and motile. S. kunkelii is the causal agent of corn stunt. S. phoeniceum, responsible for periwinkle yellows, was discovered in Syria. There are many other spiroplasmas associated with insects and ticks. Plant pathogenic mycoplasmas are restricted to the phloem sieve tubes in which circulates the photosynthetically-enriched sap, the food for many phloem-feeding insects (aphids, leafhoppers, psyllids, etc.). Interestingly, phytopathogenic mycoplasmas are very specifically transmitted by leafhoppers or psyllid species. In this paper, the most recent knowledge on phytopathogenic mycoplasmas in relation with their insect and plant habitats is presented as well as the experiments carried out to control plant mycoplasma diseases, by expression of mycoplasma-directed-antibodies in plants (plantibodies).

Fructose utilization and pathogenicity of Spiroplasma citri: characterization of the fructose operon.

Transposon Tn4001 mutagenesis of Spiroplasma citri wild-type (wt) strain GII-3 led to the isolation and characterization of non-phytopathogenic mutant GMT 553. In this mutant, transposon Tn4001 is inserted within the first gene of the fructose operon. This operon comprises three genes. The first gene (fruR) codes for a putative transcriptional regulator protein belonging to the deoxyribonucleoside repressor (DeoR) family. Sequence similarities and functional complementation of mutant GMT 553 with different combinations of the wt genes of the fructose operon showed that the second gene (fruA) codes for the permease of the phosphoenolpyruvate:fructose phosphotransferase system (fructose PTS), and the third, fruK, for the 1-phosphofructokinase (1-PFK). Transcription of the fructose operon in wt strain GII-3 resulted in two messenger RNAs, one of 2.8kb and one of 3.8kb. Insertion of Tn4001 in the genome of mutant GMT 553 abolished transcription of the fructose operon, and resulted in the inability of this mutant to use fructose. Functional complementation experiments demonstrated that fructose utilization was restored with fruR-fruA-fruK, fruA-fruK or fruA only, but not with fruR or fruR-fruA. This is the first time that an operon for sugar utilization has been functionally characterized in the mollicutes.

Identification of a Plant-Derived Mollicute as a Strain of an Avian Pathogen, Mycoplasma iowae, and Its Implications for Mollicute Taxonomy

Strain PPAV, a filamentous but nonhelical mollicute, was isolated from aborted apple seeds in France in late 1979. This organism grew well in SP-4 broth, fermented glucose, and required sterol for growth, and most of its properties suggested that it belonged to the genus Mycoplasma. However, it was serologically distinct; in addition, unlike other Mycoplasma species, genome measurements consistently yielded values of about 1,000 MDa (ca. 1,500 kbp), and the organism had a growth temperature optimum of 43 degrees C. A comparison of strain PPAV 16S rRNA sequences with those of other mollicutes revealed a high degree of sequence similarity to a strain of Mycoplasma iowae, which is commonly encountered in poultry. This relationship was confirmed by performing a restriction endonuclease pattern analysis and DNA-DNA hybridization tests. The genome size of type strain 695 of M. iowae was determined to be about 1,000 MDa (1,500 kbp) by renaturation kinetics, a value which is much higher than any other value known in the genus. Additional measurements by pulsed-field gel electrophoresis yielded values of 1,300 kbp for both strain PPAV and M. iowae. Subsequent phenotypic comparisons supported this relationship. Serologic tests with strain PPAV and other strains of M. iowae confirmed the findings of other investigators that this species is serologically heterogeneous. The high optimum temperature for growth of strain PPAV was also shared by a number of M. iowae isolates. Genome size is an inappropriate character for taxonomic assignment to the family Mycoplasmataceae because strain PPAV and other established species in this family are now known to have genomes ranging in size from 1,000 to 1,400 kbp.

Cloning and sequence analysis of the eburicol 14α-demethylase gene of the obligate biotrophic grape powdery mildew fungus

In order to obtain molecular data concerning field resistance of Uncinula necator, the causal agent of grape powdery mildew, to sterol demethylation inhibitors, a major group of fungicides, the gene encoding the target of these compounds (eburicol 14alpha-demethylase) was cloned and sequenced from this obligately biotrophic phytopathogenic fungus. This single-copy gene encodes a 524 amino acid protein which displays high similarity to other known sterol 14alpha-demethylases (CYP51s). The coding sequence is interrupted by two short introns at positions identical to introns in Penicillium italicum CYP51, which is the only other known CYP51 gene in which introns have been identified. Intron excision was verified by cDNA sequencing.

Chromosomal Heterogeneity among Various Strains of Spiroplasma citri

The genomes of various Spiroplasma citri strains were digested with several restriction enzymes, and the fragments were analyzed by pulsed-field gel electrophoresis. Polymorphism of the restriction patterns was observed. The genome sizes of the strains obtained when we added the restriction fragment sizes ranged from 1,650 to 1,910 kbp. Physical and genetic maps of 12 strains were constructed by using several DNA probes as genetic markers. The relative positions of mapped loci were conserved in most of the strains; the main differences were differences in the order and number of restriction sites and differences in the sizes of certain fragments. The distribution of viral sequences, which occur at multiple sites in the 5. citri genome and are homologous to the sequences of S. citri virus SpV1 strains R8A2B and S102, was also studied.