The infectious full-length cDNA clone of zucchini yellow mosaic virus (ZYMV) isolate PA (pZYMV-PA), which was isolated from pumpkin, was constructed by utilizing viral transcription and processing signals to produce infectious in vivo transcripts. Simple rub-inoculation of plasmid DNAs of pZYMV-PA was successful to cause infection of zucchini plants (Cucurbita pepo L.). We further engineered this infectious cDNA clone of ZYMV as a viral vector for systemic expression of heterologous proteins in cucurbits. We successfully expressed two reporter genes including gfp and bar in zucchini plants by simple rub-inoculation of plasmid DNAs of the ZYMV-based expression constructs. Our method of the ZYMV-based viral vector in association with the simple rub-inoculation provides an easy and rapid approach for introduction and evaluation of heterologous genes in cucurbits.