Tamarillo mosaic potyvirus: characterization and resistance

Abstract

The export of tamarillo is an important component of the New Zealand, exotic fruit industry. However, the quality of tamarillo fruit is severely decreased by tamarillo mosaic potyvirus (TaMV) which detrimentally affects fruit colour resulting in the fruit being unacceptable on the international marketplace. Virus incidence was surveyed in four tamarillo growing regions. Viruses were detected by indicator plant symptomology, and the incidence confirmed by dot blot analysis or ELISA. TaMV was present in 100% of tamarillo trees analyzed in ten of the twelve orchards surveyed. Incidence of potato aucuba mosaic potexvirus, cucumber mosaic cucumovirus, alfalfa mosaic virus and arabis mosaic nepovirus, which had all been previously reported in tamarillo was also determined. Tomato spotted wilt tospovirus was identified for the first time in tamarillo plants in New Zealand. TaMV RNA was purified from infected tamarillo leaves and a 1600 base pair cDNA clone generated to the 3'-terminus. The clone was sequenced and the location of the gene for the coat protein was identified by direct amino-terminal sequencing of purified TaMV coat protein. Comparison with other potyvirus coat protein sequences established that TaMV represents a new member of the potyvirus group. Seven chimeric transgenes containing TaMV sequences were constructed in three different binary vectors suitable for Agrobacterium-mediated transformation of plants. Of the twenty-six independent Nicotiana benthamiana plant lines generated expressing either TaMV coat protein or TaMV RNA sequences modified to block translation, eight plant lines demonstrated resistance to virus infection (more than 10% of resistant plants/line). All plants of one line, PT#25, were resistant to TaMV infection using dilute inocula; 40% were also resistant after inoculation with more concentrated inocula. The level of accumulation of CP in transgenic plant lines did not correspond with the degree of resistance to TaMV infection. In eight of the twenty-six transgenic lines a proportion of plants demonstrated recovery from systemic infection. Recovery was manifested as an absence or significant reduction of virus symptoms in newly developed leaves of plants that had previously shown symptoms of systemic TaMV infection. The mechanism of recovery from systemic infection is discussed. The protocol for the regeneration of transgenic tamarillo by Agrobacterium-mediated transformation was improved. The efficiency of transformation was optimized and the rate of transgenic shoot elongation was increased. Two tamarillo plants transformed with a transgene designed to express the TaMV coat protein were produced and were demonstrated to express the neomycin phosphotransferase gene and TaMV coat protein gene. However, following challenge with a low concentration of inoculum, micropropagants of these tamarillo plants failed to demonstrate resistance to TaMV infection. Three in vitro enzymatic activities of the virus encoded cytoplasmic inclusion protein were studied. The cytoplasmic inclusion protein was purified to near homogeneity using differential centrifugation and sucrose gradient purification. RNA-stimulated ATPase activity was demonstrated and the Km and Vmax determined. RNA binding and energy-dependent dissociation were characterized. RNA helicase activity of the cytoplasmic inclusion protein was demonstrated in the presence of NTP using RNA duplexes with single-stranded overhangs. These results have confirmed and extended the previous findings of the likely RNA helicase function of the potyvirus cytoplasmic inclusion protein, and suggest possible future mechanisms for obtaining resistance to potyviruses.