Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

The Citrus tristeza virus （CTV） uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strat- egies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA frag- ments from the three genes, and used them to construct vectors for expressing hairpin RNAs （hpRNAs）. To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Following transformation of sour orange （Citrus aurantium） with these constructs, 8 p20 hpRNA （hp20） and 1 p25 hpRNA （hp25） expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested following inoculation with CT14A and/ or TR-L5＂I4, both of which are severe strains. Results showed that 3 lines （hp20-5, hp20-6 and hp20-8） were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently challenged with CT14A. These results showed that expressing p20 hpRNAwas sufficient to confer sour orange with CTV resistance/tolerance.