Specialized 16SrX phytoplasmas induce diverse morphological and physiological changes in their respective fruit crops.

The host-pathogen combinations-Malus domestica (apple)/`Candidatus Phytoplasma mali´, Prunus persica (peach)/`Ca. P. prunorum´ and Pyrus communis (pear)/`Ca. P. pyri´ show different courses of diseases although the phytoplasma strains belong to the same 16SrX group. While infected apple trees can survive for decades, peach and pear trees die within weeks to few years. To this date, neither morphological nor physiological differences caused by phytoplasmas have been studied in these host plants. In this study, phytoplasma-induced morphological changes of the vascular system as well as physiological changes of the phloem sap and leaf phytohormones were analysed and compared with non-infected plants. Unlike peach and pear, infected apple trees showed substantial reductions in leaf and vascular area, affecting phloem mass flow. In contrast, in infected pear mass flow and physicochemical characteristics of phloem sap increased. Additionally, an increased callose deposition was detected in pear and peach leaves but not in apple trees in response to phytoplasma infection. The phytohormone levels in pear were not affected by an infection, while in apple and peach trees concentrations of defence- and stress-related phytohormones were increased. Compared with peach and pear trees, data from apple suggest that the long-lasting morphological adaptations in the vascular system, which likely cause reduced sap flow, triggers the ability of apple trees to survive phytoplasma infection. Some phytohormone-mediated defences might support the tolerance.

Two Phytoplasmas Elicit Different Responses in the Insect Vector Euscelidius variegatus Kirschbaum.

Phytoplasmas are plant-pathogenic bacteria transmitted by hemipteran insects. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of flavescence dorée phytoplasma (FDp). The two phytoplasmas induce different effects on this species: CYp slightly improves whereas FDp negatively affects insect fitness. To investigate the molecular bases of these different responses, transcriptome sequencing (RNA-seq) analysis of E. variegatus infected with either CYp or FDp was performed. The sequencing provided the first de novo transcriptome assembly for a phytoplasma vector and a starting point for further analyses on differentially regulated genes, mainly related to immune system and energy metabolism. Insect phenoloxidase activity, immunocompetence, and body pigmentation were measured to investigate the immune response, while respiration and movement rates were quantified to confirm the effects on energy metabolism. The activation of the insect immune response upon infection with FDp, which is not naturally transmitted by E. variegatus, confirmed that this bacterium is mostly perceived as a potential pathogen. Conversely, the acquisition of CYp, which is naturally transmitted by E. variegatus, seems to increase the insect fitness by inducing a prompt response to stress. This long-term relationship is likely to improve survival and dispersal of the infected insect, thus enhancing the opportunity of phytoplasma transmission.

An immunodominant membrane protein (Imp) of 'Candidatus Phytoplasma mali' binds to plant actin.

The phytopathogenic, cell-wall-less phytoplasmas exhibit a dual life cycle: they multiply in the phloem of their host plant and in the body of their insect vector. Their membrane proteins are in direct contact with both hosts and are supposed to play a crucial role in the phytoplasma spread within the plant as well as by the insect vector. Three types of nonhomologous but highly abundant and immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: Amp, IdpA, and Imp. Although recent results indicate that Amp is involved in vector specificity interacting with insect proteins such as actin, little is known about the interaction of IDP with the plant. We could demonstrate that transiently expressed Imp of 'Candidatus Phytoplasma mali' as well as the Imp without transmembrane domain (Imp▴Tm) bind with plant actins in vivo. Moreover, in vitro co-sediment and binding assays showed that Escherichia coli-expressed recombinant Imp▴Tm-His binds to both G- and F-actins isolated from rabbit muscle. Transgenic plants expressing Imp- or Imp▴Tm-green fluorescent protein did not exhibit any remarkable change of phenotype compared with the wild-type plant. These results indicate that Imp specifically binds to plant actin and a role of Imp-actin binding in phytoplasma motility is hypothesized.

Toward immunomodulation of witches broom disease of lime (WBDL) by targeting immunodominant membrane protein (IMP) of candidatus Phytoplasma aurantifolia.

The witches' broom disease of lime (WBDL) caused by Candidatus Phytoplasma aurantifolia is the most devastating disease of acidian lime in southern part of Iran as it destroy thousands of trees yearly throughout these regions. Traditional methods such as eradication of infected trees and insect vector control have shown limited effect on this case. Therefore, alternative approaches such as plantibody-mediated resistance, have been considered. Throughout present study we prepared sufficient amount of antigen that is required for generation of specific monoclonal recombinant antibodies against Immunodominant membrane protein (IMP) which will be exploited for plantibody-mediated resistance approach. The gene encoding IMP protein was obtained by PCR amplification using specific primers and DNA extracted from the infected plants. Amplified fragment was then inserted into T/A cloning vector. Intact clones containing the right sequence was selected after digestion, PCR amplification and subsequent sequencing analysis. IMP encoding region having the right sequence was sub-cloned into pET28a bacterial expression vector. Large scale expression of His tagged recombinant protein was performed in the BL21-de3 strain of E. coli and purification under native conditions was carried out through immobilized metal ion affinity chromatography (IMAC) in a column containing Ni-NTA agarose beads. Successful expression and purification steps were confirmed by SDS-PAGE and western blotting analyses. The results obtained indicated the successful production of about 18 mg purified recombinant IMP protein with a low level of contamination in one liter cultured medium. Finally the purified protein was dialyzed in phosphate saline buffer and applied for immunization of mice.

Immunological detection of the Weligama coconut leaf wilt disease associated phytoplasma: Development and validation of a polyclonal antibody based indirect ELISA.

Weligama coconut leaf wilt disease (WCLWD) causes heavy losses in the coconut cultivations of southern Sri Lanka. The in-house developed and validated indirect ELISA was based on specific polyclonal antibodies raised in female New Zealand White rabbits, against partially purified WCLWD associated phytoplasma. This ELISA has the potential to distinguish secA PCR confirmed, WCLWD associated phytoplasma positive palms from phytoplasma free palms at high accuracy (93%) and sensitivity (92.7%), but with marginal specificity (79%). The calculated ELISA cross reactivity index (CRI) values were low for sugarcane white leaf (7%) and sugarcane grassy shoot (8%) infected leaves, but with marked highCRIfor both Bermuda grass white leaf (69%) and areca nut yellow leaf (70%) infected leaves. SecA gene based phylogenetic relationships of the WCLWD associated phytoplasma with these other locally prevalent phytoplasma strains elucidated this immunological cross reactivity, which was further reiterated by virtual restriction fragment length polymorphism analysis. Based on scanning electron microscopy, this study provides additional visual evidence, for the presence of phytoplasmas in WCLWD infected tissues.

The development of monoclonal antibodies to the secA protein of Cape St. Paul wilt disease phytoplasma and their evaluation as a diagnostic tool.

Partial recombinant secA proteins were produced from six different phytoplasma isolates representing five 16Sr groups and the expressed, purified recombinant (partial secA) protein from Cape St. Paul wilt disease phytoplasma (CSPWD, 16SrXXII) was used to immunise mice. Monoclonal antibodies (mAbs) were selected by screening hybridoma supernatants for binding to the recombinant proteins. To characterise the binding to proteins from different phytoplasmas, the antibodies were screened by ELISA and western blotting, and epitope mapping was undertaken. Eight different mAbs with varying degrees of specificity against recombinant proteins from different phytoplasma groups were selected. Western blotting revealed that the mAbs bind to proteins in infected plant material, two of which were specific for phytoplasmas. ELISA testing of infected material, however, gave negative results suggesting that either secA was not expressed at sufficiently high levels, or conformational changes of the reagents adversely affected detection. This work has shown that the phytoplasma secA gene is not a suitable antibody target for routine detection, but has illustrated proof of principle for the methodology.

Generation and expression in plants of a single-chain variable fragment antibody against the immunodominant membrane protein of Candidatus phytoplasma aurantifolia.

Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

Cloning, expression analysis, and sequence diversity of genes encoding two different immunodominant membrane proteins in poinsettia branch-inducing phytoplasma (PoiBI).

Poinsettia branch-inducing phytoplasma (PoiBI) is a phytopathogenic bacterium that infects poinsettia, and is associated with the free-branching morphotype (characterized by many axillary shoots and flowers) of many commercially grown poinsettias. The major membrane proteins of phytoplasmas are classified into three general types, that is, immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp). These membrane proteins are often used as targets for the production of antibodies used in phytoplasma detection. Herein, we cloned and sequenced the imp and idpA genes of PoiBI strains from 26 commercial poinsettia cultivars. Although the amino acid sequences of the encoded IdpA proteins were invariant, those of the encoded Imp varied among the PoiBI isolates, with no synonymous nucleotide substitution. Western blotting and immunohistochemical analyses revealed that the amount of Imp expressed exceeded that of IdpA, in contrast to the case of a related phytoplasma-disease, western X-disease, for which the major membrane protein appears to be IdpA, not Imp. These results suggest that even phylogenetically close phytoplasmas express different types of major membrane proteins.

The major antigenic membrane protein of "Candidatus Phytoplasma asteris" selectively interacts with ATP synthase and actin of leafhopper vectors.

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and ß subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

Cloning of immunodominant membrane protein genes of phytoplasmas and their in planta expression.

Phytoplasmas are plant pathogenic bacteria that cause devastating yield losses in diverse crops worldwide. Although the understanding of the pathogen biology is important in agriculture, the inability to culture phytoplasmas has hindered their full characterization. Previous studies demonstrated that immunodominant membrane proteins could be classified into three types, immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp), and they are nonhomologous to each other. Here, cloning and sequencing of imp-containing genomic fragments were performed for several groups of phytoplasma including the aster yellows and rice yellow dwarf groups, for which an imp sequence has not previously been reported. Sequence comparison analysis revealed that Imps are highly variable among phytoplasmas, and clear positive selection was observed in several Imps, suggesting that Imp has important roles in host-phytoplasma interactions. As onion yellows (OY) phytoplasma was known to have Amp as the immunodominant membrane protein, the protein accumulation level of Imp in planta was measured compared with that of Amp. The resulting accumulation of Imp was calculated as approximately one-tenth that of Amp, being consistent with the immunodominant property of Amp in OY. It is suggested that an ancestral type of immunodominant membrane protein could be Imp, and subsequently the expression level of Amp or IdpA is increased in several phytoplasma groups.

'Candidatus Phytoplasma graminis' and 'Candidatus Phytoplasma caricae', two novel phytoplasmas associated with diseases of sugarcane, weeds and papaya in Cuba.

During 2003, surveys of sugarcane yellow leaf disease and papaya bunchy top-like disease were carried out on plantations in Havana province, Cuba, to determine the roles of weeds and Auchenorrhyncha insects in the epidemiology of these diseases. More than 250 plant and insect samples were collected and indexed by using a nested PCR for phytoplasma 16S rDNA with the generic primer pairs P1/P7 and R16F2n/R16R2. The PCR products were further characterized by restriction fragment length polymorphism using HaeIII, AluI, Sau3AI, Tru9I, HhaI, HpaII and TaqI endonucleases, giving patterns that distinguished them from those of the other reference phytoplasmas analysed. Phylogenetic analysis of 16S rRNA gene sequences identified the phytoplasmas present in sugarcane (Saccharum officinarum L.), Cynodon dactylon L., Conyza canadensis L. Cronq., Sorghum halepense L. Pers., Macroptilium lathyroides L. Urb., Saccharosydne saccharivora (Westwood) and Cedusa spp., and those in papaya (Carica papaya L.) and Empoasca papayae, as two novel provisional phytoplasma species. We propose that these phytoplasmas should be given Candidatus status, as 'Candidatus Phytoplasma graminis' and 'Candidatus Phytoplasma caricae', respectively.

'Candidatus Phytoplasma cynodontis', the phytoplasma associated with Bermuda grass white leaf disease.

Bermuda grass white leaf (BGWL) is a destructive, phytoplasmal disease of Bermuda grass (Cynodon dactylon). The causal pathogen, the BGWL agent, differs from other phytoplasmas that cluster in the same major branch of the phytoplasma phylogenetic clade in <2.5% of 16S rDNA nucleotide positions, the threshold for assigning species rank to phytoplasmas under the provisional status 'Candidatus'. Thus, the objective of this work was to examine homogeneity of BGWL isolates and to determine whether there are, in addition to 16S rDNA, other markers that support delineation of the BGWL agent at the putative species level. Phylogenetic analyses revealed that the 16S rDNA sequences of BGWL strains were identical or nearly identical. Clear differences that support separation of the BGWL agent from related phytoplasmas were observed within the 16S-23S rDNA spacer sequence, by serological comparisons, in vector transmission and in host-range specificity. From these results, it can be concluded that the BGWL phytoplasma is a discrete taxon at the putative species level, for which the name 'Candidatus Phytoplasma cynodontis' is proposed. Strain BGWL-C1 was selected as the reference strain. Phytoplasmas that are associated with brachiaria white leaf, carpet grass white leaf and diseases of date palms showed 16S rDNA and/or 16S-23S rDNA spacer sequences that were identical or nearly identical to those of the BGWL phytoplasmas. However, the data available do not seem to be sufficient for a proper taxonomic assignment of these phytoplasmas.

Secretion of immunodominant membrane protein from onion yellows phytoplasma through the Sec protein-translocation system in Escherichia coli.

A gene that encodes a putative SecE protein, which is a component of the Sec protein-translocation system, was cloned from the onion yellows phytoplasma (OY). The identification of this gene and the previously reported genes encoding SecA and SecY provides evidence that the Sec system exists in phytoplasma. In addition, a gene encoding an antigenic membrane protein (Amp) (a type of immunodominant membrane protein) of OY was cloned and sequenced. The OY amp gene consisted of 702 nt encoding a protein of 233 aa which was highly similar to Amp of aster yellows phytoplasma (AY). Part of OY Amp was overexpressed in Escherichia coli, purified, and used to raise an anti-Amp polyclonal antibody. The anti-Amp antibody reacted specifically with an OY-infected plant extract in Western blot analysis and was therefore useful for the detection of OY as well as Amp. Amp has a conserved protein motif that is known to be exported by the Sec system of E. coli. A partial OY Amp protein expressed in E. coli was localized in the periplasm as a shorter, putatively processed form of the protein. It had probably been exported from the cytoplasm to the periplasm through the Sec system. Moreover, OY Amp protein expressed in OY and detected in OY-infected plants was apparently also processed. Because phytoplasmas cannot be cultured or transformed, little information is available regarding their protein secretion systems. This study suggests that the Sec system operates in this phytoplasma to export OY Amp.