Abstract
East Asian Passiflora virus (EAPV) causes passionfruit woodiness disease, a major threat limiting passionfruit production in eastern Asia, including Taiwan and Vietnam. In this study, an infectious cDNA clone of a Taiwanese severe isolate EAPV-TW was tagged with a green fluorescent protein (GFP) reporter to monitor the virus in plants. Nicotiana benthamiana and yellow passionfruit plants inoculated with the construct showed typical symptoms of EAPV-TW. Based on our previous studies on pathogenicity determinants of potyviral HC-Pros, a deletion of six amino acids (d6) alone and its association with a point mutation (F(8)I, simplified as I(8)) were conducted in the N-terminal region of the HC-Pro gene of EAPV-TW to generate mutants of EAPV-d6 and EAPV-d6I(8), respectively. The mutant EAPV-d6I(8) caused infection without conspicuous symptoms in N. benthamiana and yellow passionfruit plants, while EAPV-d6 still induced slight leaf mottling. EAPV-d6I(8) was stable after six passages under greenhouse conditions and displayed a zigzag pattern of virus accumulation, typical of a beneficial protective virus. The cross-protection effectiveness of EAPV-d6I(8) was evaluated in both N. benthamiana and yellow passionfruit plants under greenhouse conditions. EAPV-d6I(8) conferred complete cross-protection (100%) against the wild-type EAPV-TW-GFP in both N. benthamiana and yellow passionfruit plants, as verified by no severe symptoms, no fluorescent signals, and PCR-negative status for GFP. Furthermore, EAPV-d6I(8) also provided complete protection against Vietnam's severe strain EAPV-GL1 in yellow passionfruit plants. Our results indicate that the attenuated mutant EAPV-d6I(8) has great potential to control EAPV in Taiwan and Vietnam via cross-protection.