The antigenic membrane protein Amp of chrysanthemum yellows phytoplasma is involved in transmission by leafhopper vectors

Background and objectives The major antigenic membrane protein (Amp) of different isolates of 'Candidatus Phytoplasma asteris' interacts in vitro specifically with cytosolic (actin, myosin) and membrane (ATP synthase) proteins of leafhopper vector species (Suzuki et al., 2006; Galetto et al., 2011). Such interaction has not been observed with proteins of other phylogenetically-related, non-vector species. Once acquired by the vector upon feeding on an infected plant, phytoplasmas cross the gut epithelium and basal lamina, multiply in the hemolymph and colonize the salivary glands, before being transmitted to a new plant during a successive nutrition (Bosco and D'Amelio, 2010). As these bacterial pathogens lack a cell wall, one or more phytoplasma membrane proteins may interact with cells of the gut and salivary gland epithelia and may be involved in defining specificity of transmission. Aim of this work was to develop a system to evaluate in vivo the effect of the most abundant antigenic phytoplasma membrane protein on the acquisition and transmission capabilities by vector leafhoppers. Materials and Methods To study the in vivo role of Amp of the 'Candidatus Phytoplasma asteris' (chrysanthemum yellows strain, CYP) at the gut level, nymphs of two species (Macrosteles quadripunctulatus Kirschbaum ed Euscelidius variegatus Kirschbaum), before CYP acquisition on infected daisies, were fed on an artificial medium containing a partial fusion construct of CYP Amp (CYPfAmp, Galetto et al., 2008), specific antibody raised against CYPfAmp (AbfAmp), and a mix of CYPfAmp and AbfAmp. At the end of the latency period (LP), adults were singly caged on healthy daisies for the inoculation access period (IAP). After IAP, insects were collected and assayed for phytoplasma presence (Lee et al., 1993, 1994). To study the in vivo role of CYP Amp at the salivary gland level, adult E. variegatus were microinjected with a phytoplasma suspension (Bressan et al., 2006; Galetto et al., 2009) added with CYPfAmp (Galetto et al., 2008) and AbfAmp. Following microinjection, serial inoculation to healthy daisies were performed to establish the length of the LP. After the last IAP, vectors were collected and assayed by PCR to confirm phytoplasma presence (Lee et al., 1993, 1994). Following IAP, plants were treated with insecticide and kept under controlled conditions for symptom observation. Preliminary experiments were run to define the optimal protein concentration for the artificial feeding as well as the microinjection experiments, the minimal acquisition length on the infected plant as well as the minimal LP length to obtain efficient phytoplasma acquisition and transmission. ELISA was used to monitor CYPfAmp and AbfAmp persistence in the artificial medium and in the body of microinjected E. variegatus adults. Results and Discussion Sucrose 5% in TE was the artificial substrate which allowed the best survival of both vector species (100% survival for M. quadripunctulatus and 80% for E. variegatus after 24h), and the fusion protein was assayed at 1 mg/ml in the feeding medium. Under these conditions, survival of both species was over 50% following 24 h artificial feeding and about 80% following abdominal microinjection, in agreement with what previously reported for microinjection of a Spiroplasma citri protein in the Circulifer haematoceps vector (Labroussaa et al., 2011). CYPfAmp was detected by ELISA up to 24 h post abdominal microinjection and up to 30 h after the acquisition on the artificial medium. Preliminary experiments under artificial feeding conditions showed that 4 and 6 h AAP were enough to obtain high CYP transmission efficiencies with M. quadripunctulatus and E. variegatus, respectively. Moreover, LP in the vector in average lasted for 20 (