Monique Garnier

Phloem-and xylem-restricted plant pathogenic bacteria

1. Introduction: an overview This review concerns plant pathogenic bacteria, which are strictly restricted either to the sieve tubes in the phloem or to the vessels in the xylem. These bacteria are endogenous as opposed to exogenous bacteria such as species of Erwinia , Pseudomonas , Ralstonia or Xanthomonas , which colonize the apoplast (intercellular spaces) of plant tissues, even though some of the exogenous bacteria, such as Xylophilus ampelina , can also induce vascular infections. Due to their vascular habitat, the endogenous bacteria have a systemic distribution throughout the plant, they are transmitted from plant to plant by graft inoculation, and most of them are vectored by insects which feed in the phloem (leafhoppers, psyllids), or the xylem (sharpshooters). Because of these virus-like properties, the diseases caused by endogenous bacteria have long been taken for virus diseases. The principal conducting cells of the phloem are the sieve tube elements [37]. These elements are joined end to end into sieve tubes, and are associated with parenchymatic, nucleated cells, the companion cells, an important role of which is to load sucrose into the sieve tubes. The sieve tube elements are living cells, which become enucleated at maturity. The sieve plates are lateral wall areas between two adjacent sieve elements. The sieve plates are clustered with pores, resembling giant plasmodesmata and interconnecting two adjacent sieve elements through their cytoplasms. The diameter of a pore ranges from a fraction of a micron (m )t o 15m and more, and is large enough to allow passage of sieve tube-restricted bacteria. The principal conducting elements of the xylem are tracheids and vessel members [37]. Both are dead cells, contain no cytoplasm, and have lignified secondary walls. The vessel elements are joined end to end into vessels, and the adjoining ends have open perforation plates. These openings allow relatively unimpeded longitudinal spread of the xylem-restricted bacteria within

Mycoplasmas of Plants and Insects

Plants and insects are habitats of several mollicute genera including Acholeplasma, Entomoplasma, Mesoplasma, Spiroplasma, and the provisionally classified phytoplasmas. Plant- and insect-associated acholeplasmas, entomoplasmas and mesoplasmas occur as saprophytes on plant surfaces including flowers, and in insects probably as commensals or symbionts (90, 106). They are pleomorphic, nonhelical in shape and culturable in vitro and consist of relatively few species. There is no indication that they are of economic importance, and only their phylogenetic positions will be discussed in this chapter. For more information, the reader is referred to references 89, 107, 109.

Catharanthus roseus Genes Regulated Differentially by Mollicute Infections

A differential display of mRNAs was used to isolate periwinkle cDNAs differentially expressed following infection with one of three mollicutes: Spiroplasma citri, Candidatus Phytoplasma aurantifolia, and stolbur phytoplasma. Twenty-four differentially expressed cDNAs were characterized by Northern blots and sequence analysis. Eight of them had homologies with genes in databanks coding for proteins involved in photosynthesis, sugar transport, response to stress, or pathways of phytosterol synthesis. The regulation of these genes in periwinkle plants infected by additional phloem-restricted bacteria showed that they were not specific to a given mollicute, but correlations with particular symptoms could be established. Expression of transketolase was down regulated following infection with a pathogenic strain of S. citri. No down regulation was observed for the nonphytopathogenic mutant GMT553, which is deficient for fructose utilization.

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.

Spiralin Is Not Essential for Helicity, Motility, or Pathogenicity but Is Required for Efficient Transmission of Spiroplasma citri by Its Leafhopper Vector Circulifer haematoceps

ABSTRACT 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 × 106 to 2.8 × 106 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 × 106 to 1.4 × 107 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.

Stable Transformation of the Xylella fastidiosa Citrus Variegated Chlorosis Strain with oriC Plasmids

ABSTRACT Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain ofX. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kanr). In plasmid p16KdAori, the oriCfragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori theoriC fragment was restricted to thednaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kanr gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.

Spiroplasma phoeniceum sp. nov., a new plant-pathogenic species from Syria.

Sixteen spiroplasma isolates, recovered over a 2-year period from symptomatic periwinkle plants (Catharanthus roseus) collected in eight different locations in Syria, were compared with other established Spiroplasma species or serogroups. Serological analysis of selected representatives of the new isolates revealed sharing of some antigenic components with several spiroplasmas currently classified within subgroups of group I of the genus. Strain P40Twas selected as the type strain and examined, meeting the criteria proposed by the International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes. The organism was shown to belong to the class Mollicutes by its morphology, ultrastructure of its limiting membrane, colony characteristics, and filtration patterns. The helicity and motility of the cells indicated its placement within the family Spiroplasmataceae. Although some serological cross-reactions could be observed with representatives of group I subgroups, strain P40Tcould be readily distinguished from other plant or insect pathogenic spiroplasmas in subgroup I-1 (Spiroplasma citri), subgroup I-2 (S. melliferum), or subgroup I-3 (S. kunkelii) and from spiroplasmas assigned to subgroups I-4 through I-7 and groups II through XI. Cholesterol was required for growth. Glucose was fermented, and arginine was hydrolyzed. The base composition (guanine plus cytosine) of the deoxyribonucleic acid of strain P40Twas found to be 26 mol%. Deoxyribonucleic acid-deoxyribonucleic acid hybridization comparisons between strain P40Tand other subgroup I representatives revealed approximately 60% relatedness to S. citri and S. kunkelii and 50% relatedness to S. melliferum. Experimental transmission of two of the new isolates (P40Tand P354) occurred through inoculation of spiroplasma broth cultures into leafhoppers (Macrosteles fascifrons), multiplication of the organism in the insects, and subsequent transmission of the organism by insect feeding on aster or periwinkle plants. The organism was also successfully recovered from broth cultures of symptomatic tissues of experimentally infected periwinkle plants, thus fulfilling Kochs postulates. We propose that such strains be named Spiroplasma phoeniceum. Strain P40Thas been deposited in the American Type Culture Collection (= ATCC 43115T)

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.

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.