Molecular Biology of the Citrus Tristeza Virus

CTV is a member of the genus Closterovirus of the Closteroviridae, and has a positive single-stranded RNA genome of approxi- mately 20 kb. Different strains of the virus can cause diverse disease syndromes that vary from mild, with no visible symptoms; to quick decline and death of scions grafted on sour orange; to stem pitting and fruit size reduction of most species and varieties of citrus (Bar-Joseph et al., 1989). Virions are long fl exuous particles encompassed by two CPs, a major CP of 25 kDa (referred to in this work as CP) and a minor CP of 27 kDa (p27) that encapsidates about 5% of the terminal region of the particle (Febres et al., 1996). Including these two genes, the single- stranded, positive-sense genomic RNA encodes 12 open reading frames (ORFs) (Pappu et al., 1994; Karasev et al., 1995). The 5¢ -proximal ORF 1a encodes a 349 kDa pro- tein containing two papain-like protease domains, plus methyltransferase-like and helicase-like domains; the virus RdRp is encoded by ORF 1b and is expressed by a +1 frameshift (Karasev et al., 1995; Cevik, 2001). The 3¢ half of the genome contains ten ORFs that are expressed via 3¢ -co-terminal subge- nomic RNAs. One of the most highly tran- scribed genes is that for a 20 kDa protein (p20) that accumulates as a cytoplasmatic amorphous inclusion body in infected cells (Gowda et al., 2000) and appears to be a repressor of local and systemic RNA silenc- ing (Lu et al., 2004). The most 3¢ gene of CTV, p23, that codes for a 23 kDa protein, is also highly transcribed and has been found to control asymmetric accumulation of CTV RNAs, in addition to being a suppressor of local silencing (Lu et al., 2004). It downregu- lates negative-stranded RNA accumulation and indirectly increases the expression of 3¢ genes (Satyanarayana et al., 2002). The 107 bp 5¢ - and 271 bp 3¢ -untranslated regions (UTRs) of the genomic RNA are necessary for virus replication (Mawassi et al., 2000). Several strains of CTV have been fully sequenced, and they reveal a higher degree of nucleotide sequence homology in the 3¢

end (> 97% identity) than in the 5¢ end (< 40% identity) of the genomic RNA (Mawassi et al., 1996; Vives et al., 1999; Yang et al., 1999; Albiach-Marti et al., 2000). In the past, sour orange (Citrus auran- tium L.) and Mexican lime (C. aurantifolia (Christm.) Swing.) have been transformed with the CP gene of CTV (Gutierrez et al., 1997; Dominguez et al., 2000; Ghorbel et al., 2000). Only the Mexican lime plants were evaluated for resistance against CTV. Some of the clonal lines showed some resistance (in a proportion of 10–33%), while others showed delay in virus accumulation (Dominguez et al., 2002). However, by the third fl ush, the resistance was largely overcome. In another published experiment where citrus plants have been transformed with a CTV sequence, Mexican lime transgenic plants expressing the CTV p23 protein showed virus-like symptoms such as vein clearing and stem pit-

ting (Ghorbel et al., 2001).