Sugar import and phytopathogenicity of Spiroplasma citri: glucose and fructose play distinct roles.

We have shown previously that the glucose PTS (phosphotransferase system) permease enzyme II of Spiroplasma citri is split into two distinct polypeptides, which are encoded by two separate genes, crr and ptsG. A S. citri mutant was obtained by disruption of ptsG through homologous recombination and was proved unable to import glucose. The ptsG mutant (GII3-glc1) was transmitted to periwinkle (Catharanthus roseus) plants through injection to the leaf-hopper vector. In contrast to the previously characterized fructose operon mutant GMT 553, which was found virtually nonpathogenic, the ptsG mutant GII3-glc1 induced severe symptoms similar to those induced by the wild-type strain GII-3. These results, indicating that fructose and glucose utilization were not equally involved in pathogenicity, were consistent with biochemical data showing that, in the presence of both sugars, S. citri used fructose preferentially. Proton nuclear magnetic resonance analyses of carbohydrates in plant extracts revealed the accumulation of soluble sugars, particularly glucose, in plants infected by S. citri GII-3 or GII3-glc1 but not in those infected by GMT 553. From these data, a hypothetical model was proposed to establish the relationship between fructose utilization by the spiroplasmas present in the phloem sieve tubes and glucose accumulation in the leaves of S. citri infected plants.

Identification of a novel cardenolide (2''-oxovoruscharin) from Calotropis procera and the hemisynthesis of novel derivatives displaying potent in vitro antitumor activities and high in vivo tolerance: structure-activity relationship analyses.

Analysis of the methanolic extract of Calotropis procera root barks enabled the identification of a novel cardenolide (2''-oxovoruscharin) to be made. Of the 27 compounds that we hemisynthesized, one (23) exhibited a very interesting profile with respect to its hemisynthetic chemical yield, its in vitro antitumor activity, its in vitro inhibitory influence on the Na+/K+-ATPase activity, and its in vivo tolerance. Compound 23 displayed in vitro antitumor activity on a panel of 57 human cancer cell lines similar to taxol, and higher than SN-38 (the active metabolite of irinotecan), two of the most potent drugs used in hospitals to combat cancer.

Identification of putative rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of bioinformatics approaches.

Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates.

Specific gene targeting in Spiroplasma citri: improved vectors and production of unmarked mutations using site-specific recombination.

In Spiroplasma citri, where homologous recombination is inefficient, specific gene targeting could only be achieved by using replicative, oriC plasmids. To improve the probability of selecting rare recombination events without fastidious, extensive passaging of the transformants, a new targeting vector was constructed, which was used to inactivate the crr gene encoding the IIA component of the glucose phosphotransferase system (PTS) permease. Selection of recombinants was based on a two-step strategy using two distinct selection markers, one of which could only be expressed once recombination had occurred through one single crossover at the target gene. According to this strategy, spiroplasmal transformants were screened and multiplied in the presence of gentamicin before the crr recombinants were selected for their resistance to tetracycline. In contrast to the wild-type strain GII-3, the crr-disrupted mutant GII3-gt1 used neither glucose nor trehalose, indicating that in S. citri the glucose and trehalose PTS permeases function with a single IIA component. In addition, the feasibility of using the transposon gammadelta TnpR/res recombination system to produce unmarked mutations in S. citri was demonstrated. In an arginine deiminase (arcA-disrupted) mutant, the tetM gene flanked by the res sequences was efficiently excised from the chromosome through expression of the TnpR resolvase from a replicative oriC plasmid. Due to oriC incompatibility, plasmid loss occurred spontaneously when selection pressure was removed. This approach will be helpful for constructing unmarked mutations and generating multiple mutants with the same selection marker in S. citri. It should also be relevant to other species of mollicutes.

Arabidopsis thaliana subcellular responses to compatible Erysiphe cichoracearum infections.

Subcellular events of Erysiphe cichoracearum infections of epidermal cells were visualized in living tissues of Arabidopsis plants carrying various green fluorescent protein (GFP)-tagged organelles via laser scanning confocal microscopy. Early in the infection sequence, cytoplasm and organelles moved towards penetration sites and accumulated near penetration pegs. Peroxisomes appeared to accumulate preferentially relative to the cytoplasm at penetration sites. Another early event, which preceded haustorium formation, was the aggregation of some GFP-tagged plasma membrane marker proteins into rings around penetration sites, which extended across cell-wall boundaries into neighboring cells. This feature localized to sites where papillae were deposited. The extrahaustorial membrane (EHM) encases the fungal feeding structure, the haustorium, separating it from the host cytoplasm. Eight plasma membrane markers were excluded from the EHM and remained in a collar-like formation around the haustorial neck. These observations support the suggestions that the EHM is a unique, specialized membrane and is different from the plasma membrane. Our results suggested two possibilities for the origin of the EHM: invagination of the plasma membrane coupled with membrane differentiation; or de novo synthesis of the EHM by targeted vesicle trafficking.

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.