The yellow crookneck squash (Curcurbita pepo L.), line CZW-3, was developed through a specific genetic modification to be resistant to infection by three plant viruses WMV2, ZYMV and CMV. WMV2 and ZYMV are members of the potyvirus group and CMV is the type member of the cucumovirus group. The novel variety was developed by insertion of the coat protein (CP) encoding sequences from these three single-stranded RNA viruses. The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology. The genetically modified squash exhibits the trait of resistance to infection and subsequent disease caused by these viruses through a process that is related to viral cross-protection.<br>The CZW-3 squash line was created by Agrobacterium-mediated transformation in which the transfer-DNA (T-DNA) contained the coat protein genes from each of the three viruses. The constitutive expression of these genes was regulated by the 35S promoter from cauliflower mosaic virus. In addition, the T-DNA contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII) from the Tn5 transposon of Echerichia coli, strain K12. The expression of NPTII activity was used as a selectable trait to screen transformed plants for the presence of the viral CP genes. There was no incorporation of plasmid DNA sequences outside of the T-DNA region as verified by Southern blot analysis.<br>Constitutive expression of the CMV coat protein in transgenic squash was estimated to be 5.9-10.8 mg/kg fruit tissue. This level of expression was significantly lower than measured levels of ZYMV, WMV2 and CMV coat protein in zucchini squash, yellow crookneck squash, cantaloupe and honeydew melons from supermarket shelves. Human consumption of viral coat proteins occurs whenever virus-infected plant products are consumed, or where crops have been protected from virus infection by classical cross-protection using a mild or symptomless strain of the virus.