Publications

3558

Huanglongbing (HLB) is a global devastating citrus disease that is mainly caused by “Candidatus Liberibacter asiaticus” (CLas). It is mostly transmitted by the insect Asian citrus psyllid (ACP, Diaphorina citri) in a persistent and proliferative manner. CLas traverses multiple barriers to complete an infection cycle and is likely involved in multiple interactions with D. citri. However, the protein–protein interactions between CLas and D. citri are largely unknown. Here, we report on a vitellogenin-like protein (Vg_VWD) in D. citri that interacts with a CLas flagellum (flaA) protein. We found that Vg_VWD was upregulated in CLas-infected D. citri. Silencing of Vg_VWD in D. citri via RNAi silencing significantly increased the CLas titer, suggesting that Vg_VWD plays an important role in the CLas–D. citri interaction. Agrobacterium-mediated transient expression assays indicated that Vg_VWD inhibits BAX- and INF1-triggered necrosis and suppresses the callose deposition induced by flaA in Nicotiana benthamiana. These findings provide new insights into the molecular interaction between CLas and D. citri.

AbstractThe effect of population bottlenecks and genome reduction on enzyme function is poorly understood. ‘CandidatusLiberibacter solanacearum’ is a bacterium with a reduced genome that is transmitted vertically to the egg of an infected psyllid—a population bottleneck that imposes genetic drift and is predicted to affect protein structure and function. Here, we define the effects of genome reduction and genetic drift on the function ofCa. L. solanacearum dihydrodipicolinate synthase (CLsoDHDPS), which catalyses the committed branchpoint reaction in diaminopimelate and lysine biosynthesis. We demonstrate thatCLsoDHDPS is expressed inCa. L. solanacearum and expression is increased ∼2-fold in the insect host compared toin planta.CLsoDHDPS has increased aggregation propensity, implying mutations have destabilised the enzyme but are compensated for through elevated chaperone expression and a stabilised oligomeric state.CLsoDHDPS uses a ternary-complex kinetic mechanism, which is unique among DHDPS enzymes, has unusually low catalytic ability, but an unusually high substrate affinity. Structural studies demonstrate that the active site is more open, and the structure ofCLsoDHDPS with both pyruvate and the substrate analogue succinic-semialdehyde reveals that the product is both structurally and energetically different and therefore evolution has in this case fashioned a new enzyme. Our study reveals the effects of genome reduction and genetic drift on the function of essential enzymes and provides insights on bacteria-host co-evolutionary association. We suggest that bacteria with endosymbiotic lifestyles present a rich vein of interesting enzymes useful for understanding enzyme function and/or informing protein engineering efforts.

Although no loss of crown shape or unusual growth were evident on two mature Chinaberry trees (Melia azedarach L.) located near the citadel in central Hué city, Vietnam, leaves on both trees displayed distinctive interveinal yellowing during September 2003. This symptom was reminiscent in appearance to foliar discoloration previously observed on mature Chinaberry trees in El Torno, Santa Cruz, Bolivia that was subsequently attributed to phytoplasma infection of these trees (2). Eight samples of yellowed leaves were collected from affected trees and preserved by pressing and drying for later analysis. Total nucleic acids were extracted from 0.5 g of each leaf sample and assayed for phytoplasma DNA using a polymerase chain reaction (PCR) assay with phytoplasma universal rRNA primer pair P1 and P7 (4). No visible product was generated from any Chinaberry sample while a product of expected size (1.8 kb) was obtained from DNA of a periwinkle plant (Catharanthus roseus (L.) G. Don) infected with “Candidatus Phytoplasma asteris”-related strain eastern aster yellows (EAY) and included as a known positive control in the assay. After P1/P7-primed products were reamplified by PCR with nested phytoplasma universal 16S rRNA primer pair R16mF2/R16mR1 (1), a 1.4-kb product of predicted size was obtained from the eight samples and EAY positive control, whereas no product was obtained from DNA of seed-grown healthy periwinkle included as a negative control. Digests of nested PCR products (1.4 kb) with HaeIII or MseI endonuclease, and electrophoresis of digests through 8% polyacrylamide gels, revealed no apparent differences in restriction fragment patterns among products from Chinaberry samples. However, HaeIII and MseI patterns differed from those obtained by digestion of nested PCR products from EAY, a known 16SrI-A subgroup phytoplasma (3), with these enzymes. Chinaberry phytoplasmas were definitively identified as group 16SrI strains after reevaluation of samples by a PCR incorporating ribosomal protein (rp) gene primer pair rpF1/rpR1 and reamplification of resulting products with nested 16SrI group-specific primer pair rp(I)F1A/rp(I)R1A (3). A 1.2-kb product of expected size was obtained from all Chinaberry samples and EAY positive control only. Restriction fragment length polymorphism patterns produced by DraI or SspI endonuclease digests of nested PCR products (1.2 kb) revealed no differences among Chinaberry samples, although patterns associated with each enzyme differed from those observed for the EAY positive control. Sequence comparison and phylogenetic analysis of Chinaberry yellows phytoplasma (CbY-V) 16Sr DNA (GenBank Accession No. AY863003) determined this strain to be most closely related (99.65%) to Epilobium phyllody phytoplasma, a 16SrI-B subgroup strain (3). However, based on analysis of rp gene sequences (GenBank Accession No. DQ321823), strain CbY-V was judged most similar (99.59%) to cabbage proliferation, a well characterized 16SrI-B subgroup, rpI-B subgroup phytoplasma (3). To our knowledge, this is the first record of phytoplasma infection of Chinaberry, a common urban shade tree in Vietnam. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) N. A. Harrison et al. Plant Pathol. 52:147, 2003. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004 (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

Significance TM7 is one of the most enigmatic bacterial phyla among the uncultivated candidate phyla referred to as “microbial dark matter,” and it has potential pathogenic associations. We revealed molecular insights into its uncultivability and pathogenicity, as well its unique epibiotic and parasitic lifestyle phases. These novel discoveries shed significant light on the biological, ecological, and medical importance of TM7, as well as providing useful information for culturing other TM7 and currently uncultivable bacteria that may evade standard cultivation approaches.

TXW-1, a Borrelia strain isolated in March 1998 from an adult male Dermacentor variabilis tick feeding on a coyote from Webb county, Texas, USA, was characterized by using randomly amplified polymorphic DNA (RAPD) analysis, RFLP and sequence analysis of flaB and rrs (16S rRNA gene), DNA–DNA hybridization analysis, SDS-PAGE and Western blotting with mAbs. It shows different banding patterns in RFLP analysis of flaB and forms distinct branches in phylogenetic analysis derived from flaB and rrs genes. It differs from other borreliae based on the banding patterns obtained by RAPD analysis. This strain contains a small, 38-kDa endoflagellar protein. DNA–DNA hybridization experiments revealed that the levels of DNA reassociation between TXW-1 and previously described relapsing fever borreliae were 38·64 % (Borrelia turicatae), 38·40 % (Borrelia parkeri), 7·39 % (Borrelia hermsii) and 18·30 % (Borrelia coriaceae). However, the level of DNA relatedness between B. parkeri and B. turicatae was 78·78 %. Sequence analyses of flaB and rrs genes indicate that the similarities of nucleotide sequences among TXW-1 and B. turicatae or B. parkeri are less than that between B. turicatae and B. parkeri, and that the genetic distances among TXW-1 and B. turicatae or B. parkeri are greater than that between B. turicatae and B. parkeri. TXW-1 lacks an ospC gene. Electron microscope observations showed that this spirochaete had different morphological structures compared to previously described relapsing fever borreliae. All the results obtained from the above-mentioned analyses indicate that TXW-1 is different from other described Borrelia species and that it represents a novel species of Borrelia. We have been unable to revive frozen cultures and so can not meet the requirements of the Bacteriological Code to deposit viable type material at two different culture collections. Therefore we use the Candidatus designation; based on these results, the species ‘Candidatus Borrelia texasensis' is proposed.