Virus-based transient-expression vectors are routine tools used in plant molecular biology laboratories throughout the world for rapidly expressing or silencing genes in plants. They also can be important tools in plant genomics to screen unknown sequences for function. Yet, available vectors have been developed from a limited number of rather similar viruses of herbaceous plants. Notable examples are the vectors based on Tobacco mosaic virus (TMV) (Dawson et al., 1989; Donson et al., 1991; Shivprasad et al., 1999; Rabindran and Dawson, 2001). Tree crops offer special challenges. Even if existing vectors could infect trees, the time required for systemic infection and analysis of the expressed genes in trees generally exceeds the stability of known virus-based vectors. Yet, the challenges of breeding restraints and the decades required for improving trees greatly increase the need for useful virus-based vectors.
Citrus tristeza virus (CTV) is a member of the complex Closteroviridae family that contains viruses with mono-, bi-, and tri-partite genomes transmitted by a range of insect vectors including aphids, whiteflies, and mealybugs (Bar-Joseph et al., 1979; Dolja et al., 1994; Agranovsky, 1996; Karasev, 2000). The long flexuous virions (2000 nm×10-12 nm) of CTV are encapsidated by two coat proteins: the major coat protein (CP) covering about 97% of the virion and the minor coat protein (CPm) completing encapsidation of the other terminus. The single-stranded RNA genome of CTV is approximately 19.3 kb, divided into twelve open reading frames (ORFs) (Pappu et al., 1994; Karasev et al., 1995) (FIG. 1). ORF 1a encodes a 349 kDa polyprotein containing two papain-like protease domains plus methyltransferase-like and helicase-like domains. Translation of the polyprotein is thought to occasionally continue through the polymerase-like domain (ORF 1b) by a +1 frameshift. ORFs 1a and 1b plus the nontranslated termini are all that is required for replication in protoplasts (Satyanarayana et al., 1999). Ten 3′ ORFs are expressed by 3′ co-terminal subgenomic (sg) mRNAs (Hilf et al., 1995; Karasev et al., 1997). In addition to the two coat proteins, p65 (HSP70 homolog) and p61 are required for efficient virion assembly, and are necessary for passage of the virus from protoplast to protoplast in order to amplify inoculum for infection of citrus trees (Satyanarayana et al., 2000). The p6 protein is needed for infection of plants as are the p20 and p23 proteins, which along with CP, are suppressors of RNA silencing (Lu et al., 2004).
CTV can infect and move throughout some citrus varieties with some of the viral genes deleted. CTV contains five genes, p6, p33, p18, p13, and p20, in the 3′ half of the genome that are not required for replication or formation of virions. p33, p18 and p13 are not conserved among other members of this virus group, and have been proposed to have evolved for specific interactions with the citrus host. We found that deletions within the p33, p18 or p13 ORF individually resulted in no significant loss of ability of the virus to infect, multiply, and spread throughout citrus trees (Tatineni et al., 2008). Furthermore, deletions in the p33, p18 and p13 genes in all possible combinations including deletions in all three genes allowed the virus to systemically invade citrus trees. Green fluorescent protein-tagged CTV variants with deletions in the p33 ORF or the p33, p18 and p13 ORFs demonstrated that the movement and distribution of these deletion mutants were similar to that of the wild-type virus.
Superinfection exclusion or homologous interference is a phenomenon in which a preexisting viral infection prevents a secondary infection with the same or closely-related virus, whereas infection by unrelated viruses can be unaffected. The phenomenon was first observed by McKinney (McKinney, 1926; 1929) between two genotypes of Tobacco mosaic virus (TMV) and later with bacteriophages (Dulbecco, 1952; Visconti, 1953). Since that time, the phenomenon has been observed often for viruses of animals (Adams and Brown, 1985; Bratt and Rubin, 1968; Delwart and Panganiban, 1989; Geib et al., 2003; Johnston et al., 1974; Karpf et al., 1997; Lee et al., 2005; Singh et al., 1997; Steck and Rubin, 1966; Strauss and Strauss, 1994; Whitaker-Dowling et al., 1983; Wildum et al., 2006) and plants (Bennett, 1951; Fulton, 1978; Gal-On and Shiboleth, 2005; Hull and Plaskitt, 1970; Hull, 2002; Lecoq et al., 1991; Salaman, 1933; Walkey et al., 1992). In plant virology, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were ‘strains’ of the same virus or represented different viruses (McKinney, 1929; Salaman, 1933). Subsequently, it was developed into a management tool to reduce crop losses by purposely infecting plants with mild isolates of a virus to reduce infection and losses due to more severe isolates, which is referred to as ‘cross-protection’ (reviewed in Gal-On and Shiboleth, 2005 and Hull, 2002).