Production of polyclonal and monoclonal antibodies specific against membrane proteins of Candidatus Phytoplasma asteris , chrysanthemum yellows isolate (CY))

Three genes encoding membrane proteins of chrysanthemum yellows phytoplasma were cloned and sequenced: secY, amp and artI encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, re-spectively. Alignments of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16SrDNA-based classification, while Amp sequences resulted highly variable. Five CY partial sequences were cloned into pRSetC expression vector and two (Amp 64-224 and ArtI 131-512) were expressed as fusion antigens in E. coli for the production of CY-specific polyclonal antisera (A416 against Amp 64-224; A407 against ArtI 131-512) and monoclonal antibodies. A416 recognized, in Western blots, the full length Amp from CY-infected plants (periwinkle, daisy) and insect vectors (Euscelidius variegatus, Macrosteles quadripunctulatus). A416 also reacted to European aster yellows (EAY) and primula yellows (PY) phy-toplasmas and to Northern Italian strains of “Ca. P. asteris” from lettuce (LY163) and gladiolus (GLA), but not to American aster yellows (AAY) and clover phyllody (CPh) phytoplasmas.