Draft Genome Sequence of "Candidatus Phytoplasma pruni" Strain CX, a Plant-Pathogenic Bacterium.

"Candidatus Phytoplasma pruni" strain CX, belonging to subgroup 16SrIII-A, is a plant-pathogenic bacterium causing economically important diseases in many fruit crops. Here, we report the draft genome sequence, which consists of 598,508 bases, with a G+C content of 27.21 mol%.

Larval stage Lymantria dispar microRNAs differentially expressed in response to parasitization by Glyptapanteles flavicoxis parasitoid.

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression by targeting messenger RNAs and causing cleavage or translation blockage. miRNAs induced after parasitization of the lepidopteran host Lymantria dispar by the parasitoid wasp Glyptapanteles flavicoxis, which introduces a polydnavirus and other parasitoid factors, were examined to identify induced miRNAs that might regulate host genes and contribute to host immunosuppression and other effects. miRNA profiling of parasitized larval hemocytes versus non-parasitized ones by microarray hybridization to mature insect and virus miRNAs identified 27 differentially expressed miRNAs after parasitization. This was confirmed by real-time relative qPCR for insect miRNAs (dme-mir-1, -8, -14, -184, -276, -277, -279, -289, -let-7) using miRNA-specific TaqMan assays. Certain cellular miRNAs were differentially expressed in larval tissues, such as the potentially developmentally linked mir-277, signifying a need for functional studies.

Differential expression of the CrV1 haemocyte inactivation-associated polydnavirus gene in the African maize stem borer Busseola fusca (Fuller) parasitized by two biotypes of the endoparasitoid Cotesia sesamiae (Cameron).

Polydnaviruses are rarely studied for their natural variation in immune suppressive abilities. The polydnavirus harboring braconid Cotesia sesamiae, a widespread endoparasitoid of Busseola fusca and Sesamia calamistis in sub-Saharan Africa exists as two biotypes. In Kenya, the western biotype completes development in B. fusca larvae. However, eggs of the coastal C. sesamiae are encapsulated in this host and ultimately, no parasitoids emerge from parasitized B. fusca larvae. Both biotypes develop successfully in S. calamistis larvae. Encapsulation activity by B. fusca larvae towards eggs of the avirulent C. sesamiae was detectable six hours post-parasitization. The differences in encapsulation of virulent and avirulent strains were associated with differences in nucleotide sequences and expression of a CrV1 polydnavirus (PDV) gene, which is associated with haemocyte inactivation in the Cotesia rubecula/Pieris rapae system. CrV1 expression was faint or absent in fat body and haemolymph samples from B. fusca parasitized by the avirulent C. sesamiae, which exhibited encapsulation of eggs. Expression was high in fat body and haemolymph samples from both B. fusca and S. calamistis larvae parasitized by the virulent C. sesamiae, encapsulation in the former peaking at the same time points as CrV1 expression in the latter. Non synonymous difference in CrV1 gene sequences between virulent and avirulent wasp suggests that variations in B. fusca parasitism by C. sesamiae may be due to qualitative differences in CrV1-haemocyte interactions.

Characterization and transcriptional analysis of protein tyrosine phosphatase genes and an ankyrin repeat gene of the parasitoid Glyptapanteles indiensis polydnavirus in the parasitized host.

Glyptapanteles indiensis (Braconidae, Hymenoptera) is an endoparasitoid of Lymantria dispar, the gypsy moth. Expression of G. indiensis polydnavirus (GiBV)-encoded genes within the pest host results in inhibition of immune response and development and alteration of physiology, enabling successful development of the parasitoid. Here, GiBV genome segment F (segF), an 18.6 kb segment shown to encode nine protein tyrosine phosphatase (PTP) genes and a single ankyrin repeat gene (ank), is analysed. PTPs have presumed function as regulators of signal transduction, while ankyrin repeat genes are hypothesized to function in inhibition of NF-kappaB signalling in the parasitized host. In this study, transcription of each gene was mapped by 5'- and 3'-RACE (rapid amplification of cDNA ends) and temporal and tissue-specific expression was examined in the parasitized host. For polydnavirus gene prediction in the parasitized host, no available gene prediction parameters were entirely precise. The mRNAs for each GiBV segF gene initiated between 30 and 112 bp upstream of the translation initiation codon. All were encoded in single open reading frames (ORFs), with the exception of PTP9, which was transcribed as a bicistronic message with the adjacent ank gene. RT-PCR indicated that all GiBV segF PTPs were expressed early in parasitization and, for most, expression was sustained over the course of at least 7 days after parasitization, suggesting importance in both early and sustained virus-induced immunosuppression and alteration of physiology. Tissue-specific patterns of PTP expression of GiBV segF genes were variable, suggesting differing roles in facilitating parasitism.

Baculovirus-based expression of an insect viral protein in 12 different insect cell lines.

The ability of 12 unique lepidopteran insect cell lines from Anticarsia gemmatalis, Heliothis virescens, Lymantria dispar (two lines), Mamestra brassica, Plutella xylostella, Spodoptera frugiperda (two lines), and Trichoplusia ni (three lines) to support production of a recombinant polydnavirus (PDV) protein (GiPDV 1.1) expressed using the Bac-to-Bac baculovirus expression system was examined. Polydnavirus gene GiPDV 1.1 was cloned into the pFastBac baculovirus vector under the control of the polyhedron promoter, followed by generation of recombinant bacmid-GiPDV 1.1 by site-specific transposition. The ability of each insect cell line to support recombinant PDV gene expression was estimated using reverse transcriptase-polymerase chain reaction and Western blot. Each insect cell line infected with recombinant bacmid-GiPDV 1.1 and tested in this study was capable of supporting and producing recombinant protein. Time course expression analysis showed that 72-96 h after transfection to be the optimal time for harvest of recombinant protein for each insect cell line.

'Candidatus Phytoplasma asteris', a novel phytoplasma taxon associated with aster yellows and related diseases.

Aster yellows (AY) group (16SrI) phytoplasmas are associated with over 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Strains that belong to the AY group form a phylogenetically discrete subclade within the phytoplasma clade and are related most closely to the stolbur phytoplasma subclade, based on analysis of 16S rRNA gene sequences. AY subclade strains are related more closely to their culturable relatives, Acholeplasma spp., than any other phytoplasmas known. Within the AY subclade, six distinct phylogenetic lineages were revealed. Congruent phylogenies obtained by analyses of tuf gene and ribosomal protein (rp) operon gene sequences further resolved the diversity among AY group phytoplasmas. Distinct phylogenetic lineages were identified by RFLP analysis of 16S rRNA, tuf or rp gene sequences. Ten subgroups were differentiated, based on analysis of rp gene sequences. It is proposed that AY group phytoplasmas represent at least one novel taxon. Strain OAY, which is a member of subgroups 16SrI-B, rpI-B and tufI-B and is associated with evening primrose (Oenothera hookeri) virescence in Michigan, USA, was selected as the reference strain for the novel taxon 'Candidatus Phytoplasma asteris'. A comprehensive database of diverse AY phytoplasma strains and their geographical distribution is presented.

Quantitation of a Glyptapanteles indiensis polydnavirus gene expressed in parasitized host, Lymantria dispar, by real-time quantitative RT-PCR.

Glyptapanteles indiensis is a polydnavirus-carrying wasp that parasitizes early instar gypsy moth larvae. During oviposition, the wasp injects calyx fluid containing polydnavirus along with its eggs into the host. Within the host, expression of polydnavirus genes triggers a set of changes in host physiology, which are of critical importance for the survival of the wasp. In the present study, a G. indiensis polydnavirus (GiPDV) gene, represented by cDNA clone GiPDV 1.1, was selected for expression analysis in the parasitized host. The GiPDV 1.1 gene transcript was detected in host hemolymph 30 min post-parasitization (pp) and continued to be detected for six days. The level of GiPDV 1.1 expression varied in different host tissues and expression in the brain was lower than in the hemolymph. The findings suggest that GiPDV 1.1 could be involved in early protection of parasitoid eggs from host cellular encapsulation. The temporal and spatial variations in PDV gene expression in different host tissues post-parasitization affirm their specific host regulation mechanism.

Morphological and genomic characterization of the polydnavirus associated with the parasitoid wasp Glyptapanteles indiensis (Hymenoptera: Braconidae).

Glyptapanteles indiensis polydnavirus (GiPDV) is essential for successful parasitization of the larval stage of the lepidopteran Lymantria dispar (gypsy moth) by the endoparasitic wasp Glyptapanteles indiensis. This virus has not been characterized previously. Ultrastructural studies of GiPDV showed that virions had a rod-like or rectangular form and each contained as many as ten nucleocapsids enclosed by a single unit membrane envelope. Field inversion gel electrophoresis (FIGE) analysis of the virus genomic DNA revealed that GiPDV had a segmented genome composed of 13 dsDNA segments, ranging in size from approximately 11 kb to more than 30 kb. Four genomic segments were present in higher molar concentration than the others. Further characterization of the GiPDV genome yielded several cDNA clones which derived from GiPDV-specific mRNAs, and Northern blot analysis confirmed expression of isolated cDNA clones in the parasitized host. Each was present on more than one GiPDV genomic DNA segment, suggesting the existence of related sequences among DNA segments. It has been proposed previously that in polydnavirus systems, genome segmentation, hypermolar ratio segments and segment nesting may function to increase the copy number of essential genes and to increase the levels of gene expression in the absence of virus replication. The present data support this notion and suggest that GiPDV morphology and genomic organization may be intrinsically linked to the function and evolutionary strategies of the virus.

Quantitative expression analysis of a Glyptapanteles indiensis polydnavirus protein tyrosine phosphatase gene in its natural lepidopteran host, Lymantria dispar.

In the present study, expression of a newly identified Glyptapanteles indiensis polydnavirus (GiPDV) gene encoding a putative protein tyrosine phosphatase (PDVPTP) was monitored in vivo in the parasitized host, L. dispar, using one step RT-PCR. Expression levels of the PDVPTP transcript were also evaluated in various host tissues at different times post parasitization (pp) using RT quantitative competitive PCR (RT-qcPCR). Expression levels varied, with the most abundant transcript detected in host haemolymph 2 h pp. The high expression level in host haemolymph at an early stage of parasitization suggested a potential role for viral PDVPTP in disruption of the host immune system and protection of the endoparasitoid egg from encapsulation. Additionally, the PDVPTP gene or its homolog(s) mapped to more than one GiPDV genomic DNA segment, which may account for its increased level of expression in the absence of virus replication.

Polydnavirus integration in lepidopteran host cells in vitro.

The long-term persistence of polydnavirus (PDV) DNA in infected lepidopteran cell cultures has suggested that at least some of the virus sequences become integrated permanently into the cell genome. In the current study, we provide supportive evidence of this event. Cloned libraries were prepared from two different Lymantria dispar (gypsy moth) cell lines that had been maintained in continuous culture for more than five years after infection with Glyptapanteles indiensis PDV (GiPDV). Junction clones containing both insect chromosomal and polydnaviral sequences were isolated. Precise integration junction sites were identified by sequence comparison of linear (integrated) and circular forms of the GiPDV genome segment F, from which viral sequences originated. Host chromosomal sequences at the site of integration varied between the two L. dispar cell lines but virus sequence junctions were identical and contained a 4-base pair CATG palindromic repeat. The GiPDV segment F does not encode any self-replication or self-insertion proteins, suggesting a host-derived mechanism is responsible for its in vitro integration. The chromosomal site of one junction clone contained sequences indicative of a new L. dispar retrotransposon, including a putative reverse transcriptase and integrase located upstream of the site of viral integration. A potential mechanism is proposed for the integration of PDV DNA in vitro. It remains to be seen if integration of the virus also occurs in the lepidopteran host in vivo.

Molecular identification of a new phytoplasma associated with alfalfa witches'-broom in oman.

ABSTRACT Alfalfa (Medicago sativa) plants showing witches'-broom symptoms typical of phytoplasmas were observed from Al-Batinah, Al-Sharqiya, Al-Bureimi, and interior regions of the Sultanate of Oman. Phytoplasmas were detected from all symptomatic samples by the specific amplification of their 16S-23S rRNA gene. Polymerase chain reaction (PCR), utilizing phytoplasma-specific universal primer pairs, consistently amplified a product of expected lengths when DNA extract from symptomatic samples was used as template. Asymptomatic plant samples and the negative control yielded no amplification. Restriction fragment length polymorphism profiles of PCR-amplified 16S-23S rDNA of alfalfa using the P1/P7 primer pair identified phytoplasmas belonging to peanut witches'-broom group (16SrII or faba bean phyllody). Restriction enzyme profiles showed that the phytoplasmas detected in all 300 samples belonged to the same ribosomal group. Extensive comparative analyses on P1/P7 amplimers of 20 phytoplasmas with Tru9I, Tsp509I, HpaII, TaqI, and RsaI clearly indicated that this phytoplasma is different from all the other phytoplasmas employed belonging to subgroup 16SrII, except tomato big bud phytoplasma from Australia, and could be therefore classified in subgroup 16SrII-D. The alfalfa witches'-broom (AlfWB) phytoplasma P1/P7 PCR product was sequenced directly after cloning and yielded a 1,690-bp product. The homology search showed 99% similarity (1,667 of 1,690 base identity) with papaya yellow crinkle (PapayaYC) phytoplasma from New Zealand. A phylogenetic tree based on 16S plus spacer regions sequences of 35 phytoplasmas, mainly from the Southern Hemisphere, showed that AlfWB is a new phytoplasma species, with closest relationships to PapayaYC phytoplasmas from New Zealand and Chinese pigeon pea witches'-broom phytoplasmas from Taiwan but distinguishable from them considering the different associated plant hosts and the extreme geographical isolation.

Phytoplasma: phytopathogenic mollicutes.

During the past decade, research has yielded new knowledge about the plant and insect host ranges, geographical distribution, and phylogenetic relationships of phytoplasmas, and a taxonomic system has emerged in which distinct phytoplasmas are named as separate "Candidatus phytoplasma species." In large part, this progress has resulted from the development and use of molecular methods to detect, identify, and classify phytoplasmas. While these advances continue, research has recently begun on the phytoplasma genome, how phytoplasmas cause disease, the role of mixed phytoplasmal infections in plant diseases, and molecular/genetic phenomena that underlie symptom development in plants. These and other recent advances are laying the foundation for future progress in understanding the mechanisms of phytoplasma pathogenicity, organization of the phytoplasma genome, evolution of new phytoplasma strains and emergence of new diseases, bases of insect transmissibility and specificity of transmission, and plant gene expression in response to phytoplasmal infection, as well as the design of novel approaches to achieve effective control of phytoplasmal diseases.

Both point mutation and RNA recombination contribute to the sequence diversity of citrus viroid III.

Field-grown citrus trees often harbor complex mixtures of 4-5 different viroid species, and the presence of citrus viroid III (CVd-III) has been shown to reduce the rate of tree growth without inducing disease. To more fully define the structure of its quasi-species, we have examined nine citrus viroid complexes for the presence of previously undescribed sequence variants of CVd-III. Analysis of 86 full-length cDNAs generated from these nine viroid complexes by RT-PCR revealed the presence of 20 new CVd-III variants. Chain lengths ranged from 293-297 nucleotides, and sequence changes were confined largely to the lower portions of the central conserved region and variable domain. The previously described variants CVd-IIIa (297 nt) and CVd-IIIb (294 nt) were clearly predominant, but phylogenetic analysis indicated that certain isolates may contain representatives of two additional fitness peaks. At least one group of CVd-III variants appears to have arisen as a result of RNA recombination. Populations recovered from diseased/declining trees were the most diverse, but even dwarfing isolates originating from old line Shamouti trees showed considerable variability.

Evidence for integration of Glyptapanteles indiensis polydnavirus DNA into the chromosome of Lymantria dispar in vitro.

Polydnaviruses replicate within calyx cells of the female ovaries of certain species of parasitic wasps and are required for the successful parasitization of lepidopteran hosts. These viruses, which have unusual double-stranded circular DNA segmented genomes, are integrated as proviruses into the genomes of their associated wasp hosts and are believed to be transmitted vertically through germline tissue. Here, by combined Southern hybridization, polymerase chain reaction (PCR) assays and viral sequence analyses we provide evidence that DNA originating from two distinct double-stranded circular segments of the polydnavirus genome from the braconid Glyptapanteles indiensis (GiPDV) integrates in vitro into the genome of cells derived from the natural host, Lymantria dispar. The G. indiensis polydnavirus DNA, as a result of its unique ability to be integrated in part into the chromosome of cells derived from its lepidopteran host, has potential to be developed as an in vitro cell transformation system.

Transfection of Lymantria dispar insect cell lines.

Lepidopteran cell lines derived from the gypsy moth, Lymantria dispar, have not been widely used in protein expression studies or systems because they are weakly adherent, have specific growth requirements and characteristics, and are generally difficult to transfect. Using lipid-mediated transfection of a reporter plasmid, we modify the standard method for transfection of L. dispar-derived embryonic cell lines IPLB-LdEp and -LdEIta, obtaining transfection efficiencies of 34% and 30%, respectively, as determined by image analysis assays. Using the standard lipid-mediated method, we obtain transfection efficiencies for L. dispar-derived cell line IPLB-Ld652Y of at least 40% with high mean expression levels, indicating the IPLB-Ld652Y cell line may be a superior choice for expression studies or systems requiring L. dispar-derived cells.

Transformation of lepidopteran and coleopteran insect cell lines by Glyptapanteles indiensis polydnavirus DNA.

Recently investigators showed that polydnavirus DNA from the parasitic wasp Glyptapanteles indiensis could transform gypsy moth L. dispar cell lines in vitro (McKelvey et al., 1996). Here we show GiPDV DNA is capable of transforming in vitro to varying degrees lepidopteran (IPLB-TN-R2, IPLB-SF-21, IAL-PID2, IPLB-HvT1) and coleopteran (IPLB-DU182E) insect cell lines derived from various somatic tissue types. An insect cell line derived from dipteran Aedes albopictus (C7/10) could not be transformed with G. indiensis polydnavirus.

Phytoplasma: ecology and genomic diversity.

ABSTRACT The recent development of molecular-based probes such as mono- and polyclonal antibodies, cloned phytoplasma DNA fragments, and phytoplasma-specific primers for polymerase chain reaction (PCR) has allowed for advances in detection and identification of uncultured phytoplasmas (formerly called mycoplasma-like organisms). Comprehensive phylogenetic studies based on analysis of 16S ribosomal RNA (rRNA) or both 16S rRNA and ribosomal protein gene operon sequences established the phylogenetic position of phytoplasmas as members of the class Mollicutes, and the revealed phylogenetic interrelationships among phytoplasmas formed a basis for their classification. Based on restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rRNA gene sequences, phytoplasmas are currently classified into 14 groups and 38 subgroups that are consistent with groups delineated based on phylogenetic analysis using parsimony of 16S rRNA gene sequences. In the past decades, numerous phyto-plasma strains associated with plants and insect vectors have been identified using molecular-based tools. Genomic diversity of phytoplasma groups appears to be correlated with their sharing common insect vectors, host plants, or both in nature. The level of exchange of genetic information among phytoplasma strains in a given group is determined by three-way, vector-phytoplasma-plant interactions. A putative mechanism for the creation of new ecological niches and the evolution of new ecospecies is proposed.

Phylogeny and classification of bacteria in the genera Clavibacter and Rathayibacter on the basis of 16s rRNA gene sequence analyses.

A phylogenetic analysis by parsimony of 16S rRNA gene sequences (16S rDNA) revealed that species and subspecies of Clavibacter and Rathayibacter form a discrete monophyletic clade, paraphyletic to Corynebacterium species. Within the Clavibacter-Rathayibacter clade, four major phylogenetic groups (subclades) with a total of 10 distinct taxa were recognized: (I) species C. michiganensis; (II) species C. xyli; (III) species R. iranicus and R. tritici; and (IV) species R. rathayi. The first three groups form a monophyletic cluster, paraphyletic to R. rathayi. On the basis of the phylogeny inferred, reclassification of members of Clavibacter-Rathayibacter group is proposed. A system for classification of taxa in Clavibacter and Rathayibacter was developed based on restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA sequences. The groups delineated on the basis of RFLP patterns of 16S rDNA coincided well with the subclades delineated on the basis of phylogeny. In contrast to previous classification systems, which are based primarily on phenotypic properties and are laborious, the RFLP analyses allow for rapid differentiation among species and subspecies in the two genera.

Phytoplasma induced free-branching in commercial poinsettia cultivars.

Free-branching poinsettia cultivars that produce numerous axillary shoots are essential for propagating desirable multi-flowered poinsettias (Euphorbia pulcherrima Wild. Klotz). For more than a decade, a biological agent has been suspected to cause free-branching in poinsettias. Attempts to identify the branching agent have failed. Isolation of the pathogen was accomplished using a living host and it was concluded that an unculturable phytoplasma is the cause of free-branching in poinsettias. This is the first reported example of a pathogenic phytoplasma as the causal agent of a desirable and economically important trait.

Transformation of gypsy moth (Lymantria dispar) cell lines by infection with Glyptapanteles indiensis polydnavirus.

Glyptapanteles indiensis, a species of braconid parasitic wasp, infects its host Lymantria dispar (gypsy moth) with a polydnavirus (GiPDV) to suppress the host immune system during parasitization. Here it is shown that GiPDV can infect L. dispar cell lines and that a portion of the GiPDV genome is stably maintained in infected cells. Results of Southern hybridization analyses suggested that this portion of the GiPDV genome is integrated into the L. dispar cellular genome. This is the first report of an insect viral DNA molecule that can apparently integrate into lepidopteran insect cells.