Post-transcriptional gene silencing (PTGS) is an ancient eukaryotic regulatory mechanism (Bingham, 1997) in which a particular RNA sequence is targeted and destroyed. The process may be triggered when multiple copies of a transgene (or a transgene and a homologous endogenous gene) are present in the same genome (for recent reviews of PTGS see Depicker and Van Montagu, 1997; Grant, 1999). Double stranded RNA induces PTGS in many systems (Metzlaff et al., 1997; Montgomery and Fire, 1998; Waterhouse et al., 1999) and, in plants, it can also be triggered by cytoplasmically replicating viruses many of which produce double stranded RNA replication intermediates (Baulcombe, 1999; Kumagai et al., 1995; Ruiz et al., 1998). Once the mechanism has been triggered, any homologous RNA is degraded whether it is transcribed from the transgene, the endogenous gene or from the viral RNA. The fact that plant viruses can act both as inducers and as targets of post-transcriptional gene silencing (AJ-Kaff et al., 1998; Matzke and Matzke, 1995; Ratcliffet al., 1997) has led to the idea that PTGS may have evolved as an antiviral defense mechanism in plants. The related processes in other eukaryotic organisms may serve a similar function.
If PTGS is an antiviral defense mechanism in plants, then it is not surprising that some plant viruses have evolved a counter-defense. Our recent work and that of others has identified a plant viral protein, HC-Pro, that interferes with the induction of post-transcriptional gene silencing (Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau and Carrington, 1998). This result further supports the idea that PTGS may be linked to natural antiviral resistance systems in plants, and opens the door to a new approach to understanding gene silencing in plants.
The identification of a viral suppressor of PTGS stems from studies of synergistic viral disease, in which coinfection with two heterologous viruses leads to much more severe symptoms than does infection with either virus alone. Many such synergistic diseases involve a member of the potyvirus group of plant viruses (Vance, 1991; Vance et al., 1995). Previously, it was found that transgenic plants expressing the 5′ proximal region of the tobacco etch potyviral (TEV) genome (termed the P1/HC-Pro sequence) develop synergistic disease when infected with any of a broad range of plant viruses (Pruss et al, 1997). This result suggested that expression of the P1/HC-Pro sequence might interfere with a general antiviral system in plants, thereby permitting viruses to accumulate beyond the normal host mediated limits. The general antiviral system was hypothesized to be post-transcriptional gene silencing (Pruss et al., 1997). To test this hypothesis, the effect of P1/HC-Pro expression on post-transcriptional gene silencing was tested in two different silencing systems. The results indicate that P1/HC-Pro acts as a suppressor of both transgene-induced (Anandalakshmi et al., 1998; Kasschau and Carrington, 1998) and virus-induced gene silencing (Anandalakshmi et al., 1998; Brigneti et al, 1998). Further experiments indicated that the suppression of PTGS is mediated by the HC-Pro protein, that the RNA is not sufficient for suppression and that the P1 protein may act as a nonessential accessory protein in the suppression (Anandalakshmi et al., 1998; Brigneti et al., 1998).
Expression of P1/HC-Pro in transgenic plants confers a number of phenotypes, some potentially useful, others detrimental. As discussed above, P1/HC-Pro interferes with post-transcriptional gene silencing. This phenotype is potentially useful. PTGS limits the level of expression from transgenes and is a serious problem in many biotechnology applications, and HC-Pro can be used to directly counter PTGS. However, suppression of PTGS is not the only process affected by HC-Pro. The P1/HC-Pro transgenic plants have an altered response to viral plant pathogens (and possibly to other pathogens as well). Our early experiments showed that P1/HC-Pro-expressing transgenics respond to infection with potato virus X or tobacco mosaic virus with a systemic necrosis (Pruss et al., 1997). However, in other cases the response of these plants to a viral pathogen resembles systemic acquired resistance (SAR). Under conditions where the N resistance gene of tobacco is active, the plants respond to TMV inoculation with fewer and smaller lesions than wild control plants (the classic SAR response). The same lines are completely resistant to infection with tomato ringspot nepovirus, although the control plants are susceptible. The mechanism of this induced resistance is unknown. Expression of P1/HC-Pro in transgenic plants also confers several unique developmental characteristics. The first leaves of the germinating plants are pointed rather than being rounded (the pointed character is a mature leaf character while rounded is the appropriate immature character). The plants grow slowly with reduced root biomass, and a differentiated tumor develops at the junction of the stem and the root. Finally, the flowers show delayed and reduced pigmentation. This result suggests that PTGS is used by plants as one aspect of developmental gene regulation.
To fully utilize transgenic plants, the mechanisms behind gene expression and suppression need to be controlled. In particular, methods for modulating gene expression in plants are needed.
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