Spatiotemporal dynamics and quantitative analysis of phytoplasmas in insect vectors.
ABSTRACT: Phytoplasmas are transmitted by insect vectors in a persistent propagative manner; however, detailed movements and multiplication patterns of phytoplasmas within vectors remain elusive. In this study, spatiotemporal dynamics of onion yellows (OY) phytoplasma in its vector Macrosteles striifrons were investigated by immunohistochemistry-based 3D imaging, whole-mount fluorescence staining, and real-time quantitative PCR. The results indicated that OY phytoplasmas entered the anterior midgut epithelium by seven days after acquisition start (daas), then moved to visceral muscles surrounding the midgut and to the hemocoel at 14-21 daas; finally, OY phytoplasmas entered into type III cells of salivary glands at 21-28 daas. The anterior midgut of the alimentary canal and type III cells of salivary glands were identified as the major sites of OY phytoplasma infection. Fluorescence staining further revealed that OY phytoplasmas spread along the actin-based muscle fibers of visceral muscles and accumulated on the surfaces of salivary gland cells. This accumulation would be important for phytoplasma invasion into salivary glands, and thus for successful insect transmission. This study demonstrates the spatiotemporal dynamics of phytoplasmas in insect vectors. The findings from this study will aid in understanding of the underlying mechanism of insect-borne plant pathogen transmission.
Project description:Phytoplasmas are uncultivated plant pathogens and cell wall-less bacteria and are transmitted from plant to plant by hemipteran insects. The phytoplasma's circulative propagative cycle in insects requires the crossing of the midgut and salivary glands, and primary adhesion to cells is an initial step toward the invasion process. The flavescence dorée (FD) phytoplasma possesses a set of variable membrane proteins (Vmps) exposed on its surface, and this pathogen is suspected to interact with insect cells. The results showed that VmpA is expressed by the flavescence dorée phytoplasma present in the midgut and salivary glands. Phytoplasmas cannot be cultivated at present, and no mutant can be produced to investigate the putative role of Vmps in the adhesion of phytoplasma to insect cells. To overcome this difficulty, we engineered the Spiroplasma citri mutant G/6, which lacks the ScARP adhesins, for VmpA expression and used VmpA-coated fluorescent beads to determine if VmpA acts as an adhesin in ex vivo adhesion assays and in vivo ingestion assays. VmpA specifically interacted with Euscelidius variegatus insect cells in culture and promoted the retention of VmpA-coated beads to the midgut of E. variegatus In this latest case, VmpA-coated fluorescent beads were localized and embedded in the perimicrovillar membrane of the insect midgut. Thus, VmpA functions as an adhesin that could be essential in the colonization of the insect by the FD phytoplasmas.IMPORTANCE Phytoplasmas infect a wide variety of plants, ranging from wild plants to cultivated species, and are transmitted by different leafhoppers, planthoppers, and psyllids. The specificity of the phytoplasma-insect vector interaction has a major impact on the phytoplasma plant host range. As entry into insect cells is an obligate process for phytoplasma transmission, the bacterial adhesion to insect cells is a key step. Thus, studying surface-exposed proteins of phytoplasma will help to identify the adhesins implicated in the specific recognition of insect vectors. In this study, it is shown that the membrane protein VmpA of the flavescence dorée (FD) phytoplasma acts as an adhesin that is able to interact with cells of Euscelidius variegatus, the experimental vector of the FD phytoplasma.
Project description: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 ? and ? 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.
Project description:Phytoplasmas (class, Mollicutes) are insect-transmissible and plant-pathogenic bacteria that multiply intracellularly in both plants and insects through host switching. Our previous study revealed that phytoplasmal sigma factor rpoD of OY-M strain (rpoDOY) could be a key regulator of host switching, because the expression level of rpoDOY was higher in insect hosts than in plant hosts. In this study, we developed an in vitro transcription assay system to identify RpoDOY-dependent genes and the consensus promoter elements. The assay revealed that RpoDOY regulated some housekeeping, virulence, and host-phytoplasma interaction genes of OY-M strain. The upstream region of the transcription start sites of these genes contained conserved -35 and -10 promoter sequences, which were similar to the typical bacterial RpoD-dependent promoter elements, while the -35 promoter elements were variable. In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools. The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns. These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.
Project description:North American Grapevine Yellows (NAGY) is a destructive disease of grapevines caused by phytoplasmas, wall-less bacteria that are insect-transmitted and found in plant phloem tissues. Although the disease was recognized in vineyards in the eastern United States since the 1980s, the identities of vectors remain unknown. The objectives of this study were to survey potential phytoplasma vector insects inhabiting Virginia vineyards that expressed NAGY symptoms and to evaluate their ability to transmit phytoplasmas associated with NAGY. Phytoplasmas were identified as 'Candidatus Phytoplasma pruni'-related NAGYIIIβ strains and 'Ca. Phytoplasma asteris'-related NAGYI-B strains. To determine the identities of the potential vectors, artificial feeding solution was used to evaluate the ability of leafhopper species to release phytoplasmas during feeding and phytoplasma strains were identified using molecular tools. Out of 49 insect species screened, Jikradia olitoria was the only insect that released phytoplasmas into the feeding solutions; all phytoplasmas, thus, detected were identified as NAGYIIIβ strains by nucleotide sequencing of three different genomic regions. No NAGYI-B strain was detected. To our knowledge, this is the first evidence of a potential insect vector of a specific phytoplasma associated with NAGY disease, and it is the first report of J. olitoria being a putative vector of a plant pathogenic phytoplasma.
Project description:Phytoplasmas ("Candidatus Phytoplasma," class Mollicutes) cause disease in hundreds of economically important plants and are obligately transmitted by sap-feeding insects of the order Hemiptera, mainly leafhoppers and psyllids. The 706,569-bp chromosome and four plasmids of aster yellows phytoplasma strain witches' broom (AY-WB) were sequenced and compared to the onion yellows phytoplasma strain M (OY-M) genome. The phytoplasmas have small repeat-rich genomes. This comparative analysis revealed that the repeated DNAs are organized into large clusters of potential mobile units (PMUs), which contain tra5 insertion sequences (ISs) and genes for specialized sigma factors and membrane proteins. So far, these PMUs appear to be unique to phytoplasmas. Compared to mycoplasmas, phytoplasmas lack several recombination and DNA modification functions, and therefore, phytoplasmas may use different mechanisms of recombination, likely involving PMUs, for the creation of variability, allowing phytoplasmas to adjust to the diverse environments of plants and insects. The irregular GC skews and the presence of ISs and large repeated sequences in the AY-WB and OY-M genomes are indicative of high genomic plasticity. Nevertheless, segments of approximately 250 kb located between the lplA and glnQ genes are syntenic between the two phytoplasmas and contain the majority of the metabolic genes and no ISs. AY-WB appears to be further along in the reductive evolution process than OY-M. The AY-WB genome is approximately 154 kb smaller than the OY-M genome, primarily as a result of fewer multicopy sequences, including PMUs. Furthermore, AY-WB lacks genes that are truncated and are part of incomplete pathways in OY-M.
Project description:Phytoplasmas are bacterial plant pathogens that have devastating effects on the yields of crops and plants worldwide. They are intracellular parasites of both plants and insects, and are spread among plants by insects. How phytoplasmas can adapt to two diverse environments is of considerable interest; however, the mechanisms enabling the "host switching" between plant and insect hosts are poorly understood. Here, we report that phytoplasmas dramatically alter their gene expression in response to "host switching" between plant and insect. We performed a detailed characterization of the dramatic change that occurs in the gene expression profile of Candidatus Phytoplasma asteris OY-M strain (approximately 33% of the genes change) upon host switching between plant and insect. The phytoplasma may use transporters, secreted proteins, and metabolic enzymes in a host-specific manner. As phytoplasmas reside within the host cell, the proteins secreted from phytoplasmas are thought to play crucial roles in the interplay between phytoplasmas and host cells. Our microarray analysis revealed that the expression of the gene encoding the secreted protein PAM486 was highly upregulated in the plant host, which is also observed by immunohistochemical analysis, suggesting that this protein functions mainly when the phytoplasma grows in the plant host. Additionally, phytoplasma growth in planta was partially suppressed by an inhibitor of the MscL osmotic channel that is highly expressed in the plant host, suggesting that the osmotic channel might play an important role in survival in the plant host. These results also suggest that the elucidation of "host switching" mechanism may contribute to the development of novel pest controls.
Project description:Many insect-transmissible pathogens are transmitted by specific insect species and not by others, even if they are closely related. The molecular mechanisms underlying such strict pathogen-insect specificity are poorly understood. Candidatus Phytoplasma asteris, OY strain, line W (OY), is a phytopathogenic bacterium transmitted from plant to plant by sap-feeding insect vectors (leafhoppers). Our study focused on an abundant cell-surface membrane protein of the phytoplasma named antigenic membrane protein (Amp), which is not homologous with any reported functional protein. Immunofluorescence microscopy of the phytoplasma-infected insect showed that OY phytoplasma was localized to the microfilaments of the visceral smooth muscle surrounding the insect's intestinal tract. The affinity column assay showed that Amp forms a complex with three insect proteins: actin, myosin heavy chain, and myosin light chain. Amp-microfilament complexes were detected in all OY-transmitting leafhopper species, but not in the non-OY-transmitting leafhoppers, suggesting that the formation of the Amp-microfilament complex is correlated with the phytoplasma-transmitting capability of leafhoppers. Although several studies have reported interactions between pathogens and mammalian microfilaments, this is an example of host-specific interactions between a bacterial surface protein and a host microfilament in insect cells. Our data also suggest that the utilization of a host microfilament may be a universal system for pathogenic bacteria infecting mammals or insects.
Project description:TAXONOMY:Superkingdom Prokaryota; Kingdom Monera; Domain Bacteria; Phylum Firmicutes (low-G+C, Gram-positive eubacteria); Class Mollicutes; Candidatus (Ca.) genus Phytoplasma. HOST RANGE:Ca. Phytoplasma comprises approximately 30 distinct clades based on 16S rRNA gene sequence analyses of approximately 200 phytoplasmas. Phytoplasmas are mostly dependent on insect transmission for their spread and survival. The phytoplasma life cycle involves replication in insects and plants. They infect the insect but are phloem-limited in plants. Members of Ca. Phytoplasma asteris (16SrI group phytoplasmas) are found in 80 monocot and dicot plant species in most parts of the world. Experimentally, they can be transmitted by approximately 30, frequently polyphagous insect species, to 200 diverse plant species. DISEASE SYMPTOMS:In plants, phytoplasmas induce symptoms that suggest interference with plant development. Typical symptoms include: witches' broom (clustering of branches) of developing tissues; phyllody (retrograde metamorphosis of the floral organs to the condition of leaves); virescence (green coloration of non-green flower parts); bolting (growth of elongated stalks); formation of bunchy fibrous secondary roots; reddening of leaves and stems; generalized yellowing, decline and stunting of plants; and phloem necrosis. Phytoplasmas can be pathogenic to some insect hosts, but generally do not negatively affect the fitness of their major insect vector(s). In fact, phytoplasmas can increase fecundity and survival of insect vectors, and may influence flight behaviour and plant host preference of their insect hosts. DISEASE CONTROL:The most common practices are the spraying of various insecticides to control insect vectors, and removal of symptomatic plants. Phytoplasma-resistant cultivars are not available for the vast majority of affected crops.
Project description:Background:Phytoplasmas are phloem-limited plant pathogenic bacteria in the class Mollicutes transmitted by sap-feeding insect vectors of the Order Hemiptera. Vectors still have not yet been identified for about half of the 33 known phytoplasma groups and this has greatly hindered efforts to control the spread of diseases affecting important crops. Extensive gaps of knowledge on actual phytoplasma vectors and on the plant disease epidemiology prevent our understanding of the basic underlying biological mechanisms that facilitate interactions between insects, phytoplasmas and their host plants. New information:This paper presents a complete online database of Hemiptera-Phytoplasma-Plant (HPP) biological interactions worldwide, searchable via an online interface. The raw data are available through Zenodo at https://doi.org/10.5281/zenodo.2532738. The online database search interface was created using the 3I software (Dmitriev 2006) which enhances data usability by providing a customised web interface (http://trivellone.speciesfile.org/) that provides an overview of the recorded biological interactions and ability to discover particular interactions by searching for one or more phytoplasma, insect or plant taxa. The database will facilitate synthesis of all available and relevant data on the observed associations between phytoplasmas and their insect and plant hosts and will provide useful data to generate and test ecological and evolutionary hypotheses.
Project description:Flavescence dorée (FD) of grapevine is a phloem bacterial disease that threatens European vineyards. The disease is associated with a non-cultivable mollicute, a phytoplasma that is transmitted by the grapevine leafhopper Scaphoideus titanus in a persistent, propagative manner. The specificity of insect transmission is presumably mediated through interactions between the host tissues and phytoplasma surface proteins comprising the so-called variable membrane proteins (Vmps). Plant spiroplasmas and phytoplasmas share the same ecological niches, the phloem sieve elements of host plants and the hemocoel of insect vectors. Unlike phytoplasmas, however, spiroplasmas, and Spiroplasma citri in particular, can be grown in cell-free media and genetically engineered. As a new approach for studying phytoplasmas-insect cell interactions, we sought to mimic phytoplasmas through the construction of recombinant spiroplasmas exhibiting FD phytoplasma Vmps at the cell surface.Here, we report the expression of the FD phytoplasma VmpA in S. citri. Transformation of S. citri with plasmid vectors in which the vmpA coding sequence was under the control of the S. citri tuf gene promoter resulted in higher accumulation of VmpA than with the native promoter. Expression of VmpA at the spiroplasma surface was achieved by fusing the vmpA coding sequence to the signal peptide sequence of the S. citri adhesin ScARP3d, as revealed by direct colony immunoblotting and immunogold labelling electron microscopy. Anchoring of VmpA to the spiroplasma membrane was further demonstrated by Triton X-114 protein partitioning and Western immunoblotting. Using the same strategy, the secretion of free, functionally active β-lactamase (used as a model protein) into the culture medium by recombinant spiroplasmas was achieved.Construction of recombinant spiroplasmas harbouring the FD phytoplasma variable membrane protein VmpA at their surface was achieved, which provides a new biological approach for studying interactions of phytoplasma surface proteins with host cells. Likewise, the secretion of functional β-lactamase by recombinant spiroplasmas established the considerable promise of the S. citri expression system for delivering phytoplasma effector proteins into host cells.