Limits...
Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species.

Rashidi M, Galetto L, Bosco D, Bulgarelli A, Vallino M, Veratti F, Marzachì C - BMC Microbiol. (2015)

Bottom Line: Pre-feeding of E. variegatus and M. quadripunctulatus on anti-Amp antibody resulted in a significant decrease of acquisition efficiencies in both species.Inoculation efficiency of microinjected E. variegatus with CYP suspension and anti-Amp antibody was significantly reduced compared to that of the control with phytoplasma suspension only.The possibility that this was due to reduced infection efficiency or antibody-mediated inhibition of phytoplasma multiplication was ruled out.

View Article: PubMed Central - PubMed

Affiliation: Istituto per la Protezione Sostenibile delle Piante, CNR, Torino, Italy. rashidii.mahnaz@gmail.com.

ABSTRACT

Background: Phytoplasmas are bacterial plant pathogens (class Mollicutes), transmitted by phloem feeding leafhoppers, planthoppers and psyllids in a persistent/propagative manner. Transmission of phytoplasmas is under the control of behavioral, environmental and geographical factors, but molecular interactions between membrane proteins of phytoplasma and vectors may also be involved. The aim of the work was to provide experimental evidence that in vivo interaction between phytoplasma antigenic membrane protein (Amp) and vector proteins has a role in the transmission process. In doing so, we also investigated the topology of the interaction at the gut epithelium and at the salivary glands, the two barriers encountered by the phytoplasma during vector colonization.

Methods: Experiments were performed on the 'Candidatus Phytoplasma asteris' chrysanthemum yellows strain (CYP), and the two leafhopper vectors Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum. To specifically address the interaction of CYP Amp at the gut epithelium barrier, insects were artificially fed with media containing either the recombinant phytoplasma protein Amp, or the antibody (A416) or both, and transmission, acquisition and inoculation efficiencies were measured. An abdominal microinjection protocol was employed to specifically address the interaction of CYP Amp at the salivary gland barrier. Phytoplasma suspension was added with Amp or A416 or both, injected into healthy E. variegatus adults and then infection and inoculation efficiencies were measured. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with A416 antibody. The organs were then either observed in confocal microscopy or subjected to DNA extraction and phytoplasma quantification by qPCR, to visualize and quantify possible differences among treatments in localization/presence/number of CYP cells.

Results: Artificial feeding and abdominal microinjection protocols were developed to address the two barriers separately. The in vivo interactions between Amp of 'Candidatus Phytoplasma asteris' Chrysanthemum yellows strain (CYP) and vector proteins were studied by evaluating their effects on phytoplasma transmission by Euscelidius variegatus and Macrosteles quadripunctulatus leafhoppers. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with anti-Amp antibody. To visualize possible differences among treatments in localization/presence of CYP cells, the organs were observed in confocal microscopy. Pre-feeding of E. variegatus and M. quadripunctulatus on anti-Amp antibody resulted in a significant decrease of acquisition efficiencies in both species. Inoculation efficiency of microinjected E. variegatus with CYP suspension and anti-Amp antibody was significantly reduced compared to that of the control with phytoplasma suspension only. The possibility that this was due to reduced infection efficiency or antibody-mediated inhibition of phytoplasma multiplication was ruled out. These results provided the first indirect proof of the role of Amp in the transmission process.

Conclusion: Protocols were developed to assess the in vivo role of the phytoplasma native major antigenic membrane protein in two phases of the vector transmission process: movement through the midgut epithelium and colonization of the salivary glands. These methods will be useful also to characterize other phytoplasma-vector combinations. Results indicated for the first time that native CYP Amp is involved in vivo in specific crossing of the gut epithelium and salivary gland colonization during early phases of vector infection.

Show MeSH

Related in: MedlinePlus

Putative mechanism of phytoplasma adhesion to and internalization in the epithelium of insect vector midgut (a, b) and salivary glands (c, d). Following infectious nutrition (a, b) or abdominal microinjection (c, d), phytoplasma cells reach the microvilli of the brush border membrane of vector midgut epithelium (a) or the salivary gland epithelium (c) of the vector, where native antigenic membrane protein (Amp) molecules within the phytoplasma cell membrane may specifically interact with putative vector receptors, and start vesicle-mediated colonization of host salivary glands (c). Masking of native Amp by its antibody A416 (b, d) impedes the interaction with putative vector receptors, therefore blocking midgut crossing and decreasing acquisition efficiency (b) or affecting salivary gland colonization and decreasing inoculation efficiency (d)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4589916&req=5

Fig3: Putative mechanism of phytoplasma adhesion to and internalization in the epithelium of insect vector midgut (a, b) and salivary glands (c, d). Following infectious nutrition (a, b) or abdominal microinjection (c, d), phytoplasma cells reach the microvilli of the brush border membrane of vector midgut epithelium (a) or the salivary gland epithelium (c) of the vector, where native antigenic membrane protein (Amp) molecules within the phytoplasma cell membrane may specifically interact with putative vector receptors, and start vesicle-mediated colonization of host salivary glands (c). Masking of native Amp by its antibody A416 (b, d) impedes the interaction with putative vector receptors, therefore blocking midgut crossing and decreasing acquisition efficiency (b) or affecting salivary gland colonization and decreasing inoculation efficiency (d)

Mentions: The reduced transmission efficiency of E. variegatus, microinjected with CYP suspension plus A416, is the long-lasting effect of an interaction that presumably occurs at early stages after microinjection. To address this hypothesis, an internalization assay was developed (modified according to [33]), consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with A416 antibody. The organs were then either observed in confocal microscopy or subjected to DNA extraction and phytoplasma quantification by qPCR, to visualize and quantify possible differences among treatments in localization/presence/concentration of CYP cells. This approach was selected to investigate under standardized conditions (organs dissected and incubated in the same amount of CYP cells, for the same time) the very early stages of phytoplasma colonization of salivary glands. This assay enabled us to detect phytoplasma cells in the organs by confocal microscopy as well as qPCR, overcoming the undetectable phytoplasma titer in salivary glands dissected from insects soon after microinjection. Confocal microscopy observation showed that, after 4 h incubation with CYP suspension, CYP cells were visible in two forms: as isolated cells (in four salivary glands out of 15 observed ones; Fig. 2a, b) and packed within vesicles (in two salivary glands out of 15 observed ones; Fig. 2c-f). On the other hand, after 4 h incubation of salivary gland with CYP suspension added with A416 antibody, phytoplasmas were visible only as isolated cells in three salivary glands out of 15 observed organs (Fig. 2i-n). Interestingly, CYP cells packed intracellularly within vesicles were never observed following incubation in the presence of A416. . As negative control, glands were incubated for 4 h with an extract of healthy insects and no signal was detected (Fig. 3g, h). CYP titer in the salivary glands incubated with phytoplasma suspension was very low (less than 100 CYP cells/ng of insect DNA) and similar irrespective of the antibody A416 presence (Additional file 4).Fig. 2


Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species.

Rashidi M, Galetto L, Bosco D, Bulgarelli A, Vallino M, Veratti F, Marzachì C - BMC Microbiol. (2015)

Putative mechanism of phytoplasma adhesion to and internalization in the epithelium of insect vector midgut (a, b) and salivary glands (c, d). Following infectious nutrition (a, b) or abdominal microinjection (c, d), phytoplasma cells reach the microvilli of the brush border membrane of vector midgut epithelium (a) or the salivary gland epithelium (c) of the vector, where native antigenic membrane protein (Amp) molecules within the phytoplasma cell membrane may specifically interact with putative vector receptors, and start vesicle-mediated colonization of host salivary glands (c). Masking of native Amp by its antibody A416 (b, d) impedes the interaction with putative vector receptors, therefore blocking midgut crossing and decreasing acquisition efficiency (b) or affecting salivary gland colonization and decreasing inoculation efficiency (d)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4589916&req=5

Fig3: Putative mechanism of phytoplasma adhesion to and internalization in the epithelium of insect vector midgut (a, b) and salivary glands (c, d). Following infectious nutrition (a, b) or abdominal microinjection (c, d), phytoplasma cells reach the microvilli of the brush border membrane of vector midgut epithelium (a) or the salivary gland epithelium (c) of the vector, where native antigenic membrane protein (Amp) molecules within the phytoplasma cell membrane may specifically interact with putative vector receptors, and start vesicle-mediated colonization of host salivary glands (c). Masking of native Amp by its antibody A416 (b, d) impedes the interaction with putative vector receptors, therefore blocking midgut crossing and decreasing acquisition efficiency (b) or affecting salivary gland colonization and decreasing inoculation efficiency (d)
Mentions: The reduced transmission efficiency of E. variegatus, microinjected with CYP suspension plus A416, is the long-lasting effect of an interaction that presumably occurs at early stages after microinjection. To address this hypothesis, an internalization assay was developed (modified according to [33]), consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with A416 antibody. The organs were then either observed in confocal microscopy or subjected to DNA extraction and phytoplasma quantification by qPCR, to visualize and quantify possible differences among treatments in localization/presence/concentration of CYP cells. This approach was selected to investigate under standardized conditions (organs dissected and incubated in the same amount of CYP cells, for the same time) the very early stages of phytoplasma colonization of salivary glands. This assay enabled us to detect phytoplasma cells in the organs by confocal microscopy as well as qPCR, overcoming the undetectable phytoplasma titer in salivary glands dissected from insects soon after microinjection. Confocal microscopy observation showed that, after 4 h incubation with CYP suspension, CYP cells were visible in two forms: as isolated cells (in four salivary glands out of 15 observed ones; Fig. 2a, b) and packed within vesicles (in two salivary glands out of 15 observed ones; Fig. 2c-f). On the other hand, after 4 h incubation of salivary gland with CYP suspension added with A416 antibody, phytoplasmas were visible only as isolated cells in three salivary glands out of 15 observed organs (Fig. 2i-n). Interestingly, CYP cells packed intracellularly within vesicles were never observed following incubation in the presence of A416. . As negative control, glands were incubated for 4 h with an extract of healthy insects and no signal was detected (Fig. 3g, h). CYP titer in the salivary glands incubated with phytoplasma suspension was very low (less than 100 CYP cells/ng of insect DNA) and similar irrespective of the antibody A416 presence (Additional file 4).Fig. 2

Bottom Line: Pre-feeding of E. variegatus and M. quadripunctulatus on anti-Amp antibody resulted in a significant decrease of acquisition efficiencies in both species.Inoculation efficiency of microinjected E. variegatus with CYP suspension and anti-Amp antibody was significantly reduced compared to that of the control with phytoplasma suspension only.The possibility that this was due to reduced infection efficiency or antibody-mediated inhibition of phytoplasma multiplication was ruled out.

View Article: PubMed Central - PubMed

Affiliation: Istituto per la Protezione Sostenibile delle Piante, CNR, Torino, Italy. rashidii.mahnaz@gmail.com.

ABSTRACT

Background: Phytoplasmas are bacterial plant pathogens (class Mollicutes), transmitted by phloem feeding leafhoppers, planthoppers and psyllids in a persistent/propagative manner. Transmission of phytoplasmas is under the control of behavioral, environmental and geographical factors, but molecular interactions between membrane proteins of phytoplasma and vectors may also be involved. The aim of the work was to provide experimental evidence that in vivo interaction between phytoplasma antigenic membrane protein (Amp) and vector proteins has a role in the transmission process. In doing so, we also investigated the topology of the interaction at the gut epithelium and at the salivary glands, the two barriers encountered by the phytoplasma during vector colonization.

Methods: Experiments were performed on the 'Candidatus Phytoplasma asteris' chrysanthemum yellows strain (CYP), and the two leafhopper vectors Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum. To specifically address the interaction of CYP Amp at the gut epithelium barrier, insects were artificially fed with media containing either the recombinant phytoplasma protein Amp, or the antibody (A416) or both, and transmission, acquisition and inoculation efficiencies were measured. An abdominal microinjection protocol was employed to specifically address the interaction of CYP Amp at the salivary gland barrier. Phytoplasma suspension was added with Amp or A416 or both, injected into healthy E. variegatus adults and then infection and inoculation efficiencies were measured. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with A416 antibody. The organs were then either observed in confocal microscopy or subjected to DNA extraction and phytoplasma quantification by qPCR, to visualize and quantify possible differences among treatments in localization/presence/number of CYP cells.

Results: Artificial feeding and abdominal microinjection protocols were developed to address the two barriers separately. The in vivo interactions between Amp of 'Candidatus Phytoplasma asteris' Chrysanthemum yellows strain (CYP) and vector proteins were studied by evaluating their effects on phytoplasma transmission by Euscelidius variegatus and Macrosteles quadripunctulatus leafhoppers. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with anti-Amp antibody. To visualize possible differences among treatments in localization/presence of CYP cells, the organs were observed in confocal microscopy. Pre-feeding of E. variegatus and M. quadripunctulatus on anti-Amp antibody resulted in a significant decrease of acquisition efficiencies in both species. Inoculation efficiency of microinjected E. variegatus with CYP suspension and anti-Amp antibody was significantly reduced compared to that of the control with phytoplasma suspension only. The possibility that this was due to reduced infection efficiency or antibody-mediated inhibition of phytoplasma multiplication was ruled out. These results provided the first indirect proof of the role of Amp in the transmission process.

Conclusion: Protocols were developed to assess the in vivo role of the phytoplasma native major antigenic membrane protein in two phases of the vector transmission process: movement through the midgut epithelium and colonization of the salivary glands. These methods will be useful also to characterize other phytoplasma-vector combinations. Results indicated for the first time that native CYP Amp is involved in vivo in specific crossing of the gut epithelium and salivary gland colonization during early phases of vector infection.

Show MeSH
Related in: MedlinePlus