Molecular Characterization of Bean – Infecting Geminiviruses and Antiviral Strategies:

GENOME FUNCTION AND ANTIVIRAL STRATEGIES

            Since the first report of genome organization of BGYMV by Howarth et al. (1985), many studies have contributed towards the understanding of gene function, ori region organization and function, and promoter region organization (see review Hanley-Bowdoin et al., 1999, Karkashian, 1998; Timmermans et al., 1994).  The Western Hemisphere, whitefly-transmitted geminiviruses are bipartite.  The functions associated with DNA replication and coat protein production are encoded by DNA-A, and virus movement, by DNA-B (Noueiry et al., 1994).  The availability of infectious clones of bean-infecting geminiviruses (Gilbertson et al., 1991a) has greatly facilitated the study of genome functions in these viruses.  The ac2 (trap) gene for BGYMV produces a trans-activator protein for the cp gene (Karkashian, 1998).  The coat protein for BGYMV was shown to be essential for whitefly transmission but not for infection (Azzam et al., 1994), and Briddon et al. (1990) showed that the coat protein determined the insect specificity of transmission for the whitefly- vs. leafhopper-transmitted geminiviruses.

            Geminiviruses replicate via a rolling-circle mechanism, which involves the specific binding of the Rep protein to the ori region (see Hanley-Bowdoin et al., 1999).  Extensive experiments with tomato golden mosaic virus and BGYMV determined that the Rep protein binding to the direct repeats 5' of the rep gene TATA-box is sequence specific for each geminivirus.  Thus, one Rep protein can not replicate heterologous geminiviruses.  After binding, DNA-nicking occurs between the underlined TA in the TAATATTAC sequence in the conserved loop of the stem-loop region (Laufs et al., 1995).  An antiviral mechanism based on this specific interaction between the Rep protein and the origin of replication was proposed.  It involves creating a non-functional Rep protein that will interfere with the binding of the wild-type protein (Hanson et al., 1991).  Mutational analysis of the Rep protein of BGYMV showed that single codon changes in the DNA-nicking (Hoogstraten et al., 1996) or the NTP-binding motifs (Hanson et al., 1995) are lethal.  These two motifs are conserved in the Rep proteins for all geminiviruses; and thus, are attractive targets for mutation when engineering trans-dominant rep gene constructs.  A transient DNA-A replication assay with NT-1 tobacco suspension cells was developed to measure viral replication (Hanson et al., 1995).  Trans-expressed mutant rep genes (rep gene to nucleotide number 1,472) with codon changes for Y103 to F or D262 to R prevented the replication of DNA-A of the homologous BGYMV, but not of the heterologous viruses, BGMV or BDMV (Hanson and Maxwell, unpublished data).  Interestingly, rep/trap/ac3 gene constructs with the same DNA-nicking or DNA-binding motif mutations inhibited replication of DNA-A of the homologous BGYMV isolates from the Dominican Republic and Guatemala, as well as that of the two heterologous viruses, BGMV from Brazil and BDMV (Hanson and Maxwell, 1999).  Similar mutations of the rep gene of tomato mottle virus were engineered into tomatoes and these plants were resistance when either agro-inoculated or exposed to high populations of viruliferous whiteflies (Stout et al., 1997).  Currently, transgenic beans with trans-dominant lethal rep gene constructs of BGMV are being engineered (F. Aragão and associates, personal communication).

            Other strategies for engineering resistance to geminiviruses have involved cp gene constructs and antisense strategies.  The first transgenic beans contained the cp gene for BGYMV, a herbicide resistance gene (bar gene) and the GUS gene (Russell et al., 1993).  Unfortunately, these transgenic beans did not express the coat protein for BGYMV and did not show any level of resistance (Azzam et al., 1996).  More promising results have been obtained with beans engineered with the antisense orientation of rep/trap/ac3 and bc1 genes, where beans were exposed to BGYMV viruliferous whiteflies showed delayed and attenuated symptoms (Aragão et al., 1998).

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