Localization by Immunogold Cytochemistry of Viral Antigen in Sections of Plant Cells Infected with Red Clover Mottle Virus

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Abstract
Red clover mottle virus (RCMV) was detected by light microscopy and EM in sections of systemically infected pea leaf cells at successive stages of early infection using the combination of a sensitive immunocytochemical method with colloidal Au as a marker of antigen and a low temperature embedding procedure. Au label was present in the cytoplasm at approximately the same time as the virus-induced membranous inclusions were established in the infected cells as judged by conventional EM of adjacent tissue. Labeling occurred simultaneously in the cytoplasm and among the membranes of the inclusion but not over the vesicles contained inside the inclusion. Viral antigen was localized by light microscopy with colloidal Au, nominally 8 nm, coupled to protein A and by EM with colloidal Au, nominally 5 nm, coupled to swine anti-rabbit Ig. Background staining was low and healthy tissue exposed to specific RCMV antibodies also had a very low level of Au particles.