UC Riverside International Organization of Citrus Virologists Conference Proceedings ( 1957-2010 ) Title Insect-Transmitted

Spiroplasma citri , the causal agent of citrus stubborn disease, was the first mollicute of plant origin to have been cultured as early as 1970. Even today, none of the many phytoplasmas (formerly MLOs) have been obtained in culture. S. citri was also the first representative of a new genus in the Mollicutes, the genus Spiroplasma with unique properties: motility and helical morphology. Being available in culture, many cellular and molecular properties of S. citri could be studied in the ensuing 20 yr: cell growth and division, cell polarity, spiroplasma proteins, extrachromosomal DNA (viruses and plasmids), DNA and RNA polymerases, insensitivity to rifampin, cloning and sequencing of protein genes (such as spiralin, fibril, etc.), heterologous expression of spiralin in Escherichia coli , sequences of viral genomes and identification of UGA as a tryptophane codon, sequences of UGA and UGG tryptophane tRNAs, physical and genomic map of the S. citri genome, 16S rDNA based taxonomy and phylogeny, cloning and sequencing of the replication origin ( oriC ) of the spiroplasmal chromosome. As interesting as the above studies might have been, they threw no light on the most characteristic features of the spiroplasma: multiplication in, and transmission by, leafhopper vectors, mechanisms of phytopathogenicity, helical morphology and motility. To approach these problems, molecular genetics had to be used, and the required tools had first to be developed: transformation (by electroporation or PEG-mediated) or transfection (with virus SpV1 replicative form), development of S. citri oriC artificial plasmids such as shuttle plasmid pBOT1 used, for instance, to complement S. citri mutants with wild type genes, transposon Tn 4001 mutagenesis, and production of mutants by gene disruption through homologous recombination. These tools have made it possible to identify genes involved in the mechanism of phytopathogenicity (fructose operon), motility, morphology, and helicity (gene scm 1, fibril gen, gene mre B1), leafhopper transmission (gene sc76 ), and the regulation of spiroplasma operons (genes fruR and treR ). In fact, the preceding studies are part of what is called “functional genomics” (what are the genes involved in a function? ). They were undertaken before the era of “genomics” or the sequencing of full genomes. Sequencing and annotation of the S. citri genome is however underway. This will greatly accelerate functional genomics, and render possible comparisons with sequenced genomes of other phytopathogenic bacteria of the apoplast, the phloem or the xylem. The S. citri genome should be available in 2003. Many more exciting years are ahead of us. The first 15 yr of research on Spiroplasma citri , the causal agent of citrus stubborn disease, were reviewed at the 10th congress of the IOCV covering references up to 1988 (3). The major points covered in the 1986 review will be summarized and updated in this introduction. The essential part of the present review will be devoted to functional genomics, a field developed more recently. Sequencing of the S. citri genome is also underway, and will be briefly mentioned. The first plant mollicutes, now called phytoplasmas, were discovered in 1967 (11), but have not yet been obtained in culture. S. citri was seen in 1970 in the sieve tubes of stubborn affected sweet orange leaves, and could be cultured as early as 1971. Hence, S. citri was the first mollicute cultured from plants. The organism had an optimal growth temperature of 32°C. All plant mollicutes, i.e. phytoplasmas and spiroplasmas, are restricted to the phloem sieve tubes. The stubborn agent was characterized and described as a new mollicute with motility and helical morphology in 1973, but the mecanisms involved in these features begin to be understood only today, as will be seen later. The organism was *Invited presentation. Fifteenth IOCV Conference, 2002—Insect-Transmitted Procaryotes 279 shown to be the causal agent of stubborn disease in 1974. The leafhopper Circulifer tenellus was found to be the major vector of S. citri in the U.S.A. in 1976. C. haematoceps , absent from the U.S.A. , transmits the spiroplasma in the Old World as found in 1986, but C. tenellus is also present. Growth and division of this unique, helical mollicute in liquid medium was studied in the early 1980s. The smallest viable cell of S. citri was found to be a two-turn helix (elementary helix). The elementary helices grow into longer parental cells, which then divide by constriction to yield elementary helices. The helical morphology is conserved during this process. The discovery of S. citri led to the isolation of many other spiroplasmas, mainly from insects and ticks. There were 30 different species in 1986, almost 50 in 2001. In addition to S. citri , there are only two other spiroplasmas of plant origin: S. kunkelii , a corn pathogen, and S. phoeniceum , isolated from naturally infected periwinkle ( Catharanthus roseus ) plants in Syria (3). The molecular and cellular biology of spiroplasmas was reviewed in 1989 (5). Spiroplasmas as infectious agents of plants, arthropods and vertebrates were reviewed in 1997 (4). S. citri is serologically related to, and shares DNA homology with the two other plant pathogenic spiroplasmas, as well as with S. melliferum , a honeybee pathogen. By 1987, three S. citri viruses (SpV1, SpV2, and SpV3), and one S. melliferum virus (SpV4) had been discovered. SpV1 is a filamentous non-lytic Plectrovirus within the Inoviridae , while SpV4 is an icosahedral lytic Spiromicrovirus within the Microviridae (9, 28). The single-stranded, circular DNA genomes of SpV4 and SpV1 have been sequenced in 1987 and 1990, respectively (28). Much data on molecular biology and gene organization have been acquired over the years. Some will be mentioned here. The mollicutes are phylogenetically related to Gram positive bacteria with low G+C DNA (33). This explains many of the properties of S. citri. The G+C content of S. citri DNA is 26%. The genome size varies from 1,650 to 1,910 kbp according to the S. citri strain considered. (36). The physical and genomic map of the S. citri genome was established in 1992 (37). Viral SpV1 DNA sequences are inserted at several sites within the genome (1). Features for DNA transcription (RNA polymerase, promoter and terminator) are similar to those of Gram positive bacteria. However, contrary to bacteria, S. citri and all other mollicutes, are insensitive to rifampin. It was shown in 1996 that this property is due to the fact that amino acid 526 in the β subunit of RNA polymerase is asparagin (18). In spiroplasmas and other mollicutes, UGA is not a stop codon, but codes for tryptophan. Spiroplasmas have thus two tryptophan codons: UGG (regular codon) and UGA. The UGG-tRNA and the UGA-tRNA have been isolated and sequenced (10). Spiralin is the major membrane protein of S. citri, and the spiralin gene was the first mollicute gene to be expressed in Escherichia coli . Many other mollicute genes could not be expressed in this way. When the spiralin gene was eventually sequenced (7 , 25 ) , it was found that there were no tryptophan codons, and especially no UGA codons, in the gene, in agreement with the absence of tryptophan in the protein, and hence the E. coli ribosomes were able to translate the whole gene. However, with mollicute genes containing UGAtryptophan codons, the bacterial ribosomes stop at the first UGA codon encountered, as UGA means “stop” to them, and no complete, functional protein is obtained.