Synopsis of the studies described in the text that involved experiments with transgenic plants resistant to viruses and the influence to the resistance of the various factors examined; n/t, not tested.
Keywords
1. Introduction
Plant viruses constitute one of the main problems of the agricultural production worldwide (Kang et al., 2005). To date, there are not therapeutical measures available for the control of plant-virus diseases in the field and the main control strategy used in practice is based on prevention measures. Genetic resistance is by far the most effective way to control plant viruses. However, ‘traditional’ genetic sources of resistance to viruses are rare (Lecoq et al., 2004) and due to the high rate of mutation of the viral genomes this resistance even when applicable, is frequently broken under field conditions. The era of
2. Strategies for engineering resistance to plant viruses
After the revolutionary work that was carried on
Besides the PDR strategy, alternative biotechnological approaches for the manufacturing of plants resistant to viruses include the expression of plant virus-resistance genes in other plants than those from which they were isolated (Farnham, 2006; Seo et al., 2006; Spassova et al., 2001) and the expression of peptides (Lopez-Ochoa et al., 2006; Rudolph et al., 2003; Uhrig, 2003) or antibodies. After the first successful application of the later strategy in 1993 by Tavladoraki and co-workers, with antibodies that reduced the susceptibility to
3. RNA-silencing based transgenic resistance against plant viruses
RNA silencing constitutes a vital element of the innate antiviral ‘immune’ response in plants. It uses cytoplasm-associated small interfering RNAs (siRNAs) to specifically target and inactivate invading nucleic acids. Besides siRNAs, a vast population of small RNAs (sRNAs) accumulates in plant tissues, which includes microRNAs (miRNAs),
RNA silencing, is activated as a response to double-stranded RNA (dsRNA). Viruses, as well as transgenes, arranged as inverted repeats (IR), can directly produce dsRNA (which at a subsequent stage will give rise to primary siRNAs), whereas highly transcribed, sense orientated, single copy transgenes produce aberrant transcripts that serve as a substrate for producing dsRNA (subsequently processed to secondary siRNAs). In the latter case dsRNA is synthesized by one member of a family of cellular RNA-depended RNA polymerases (RdRPs) which counts six members in
RNA silencing impedes viral multiplication in plants by two major ways. First it degrades the dsRNA intermediates of virus replication themselves as well as the cognate mRNAs (referred to as cell-autonomous silencing), a procedure that leads to the increase of accumulation of the respective siRNAs. Second, it generates a mobile signal that triggers the degradation of homologous mRNAs in distant cells (systemic silencing). This systemic branch of antiviral RNA silencing is related to siRNA population or their dsRNA precursors that move between neighboring cells through plasmodesmata and over long distances through the phloem (Kalantidis et al., 2008).
RNA-silencing based resistance against viruses was first reported by Lindbo et al. (1993) and was shown to be related to the previously observed co-suppression mechanism (Napoli et al., 1990; Van der Krol et al., 1990). The following years, engineering of transgenic plants to harbor single-stranded sense and to a less extend antisense viral sequences became a common strategy to pre-activate the silencing machinery and obtain resistance against the homologous virus from which the introduced sequence has derived (Ritzenthaler, 2005). Further exploiting this knowledge led to constructing IR transgenes from which long double-stranded (ds) RNA precursors of siRNAs were directly generated. The utilization of such IR transgene constructs has become the method of choice for providing genetically engineered resistance to viruses because a single copy is sufficient to provide immunity, there is no expression of viral proteins, short genome incomplete sequences can be used and efficiencies of up to 90% of all transgenic plants produced to be resistant to the homologous virus were achieved (Lin et al., 2007; Tenllado et al., 2004; Ritzenthaler, 2005). In contrast to the situation with RNA viruses, the use of RNA silencing against DNA viruses most often resulted in delays in symptom development and did not always prevent virus replication (Lin et al., 2007). However, immune lines against
In order to overcome the weakness of RNA-silencing based resistance [ineffective against viruses whose sequence differs from that of the transgene by more than 10% (Bau et al., 2003; Jones et al., 1998)], Bucher et al. (2006) fused 150-nt fragments of viral sequences of four tospoviruses in a single small chimeric IR construct. This strategy resulted in a high frequency of produced resistant plants. A most recent approach used modified plant miRNA cistrons to produce a range of antiviral artificial miRNAs (amiRNAs) (Niu et al., 2006; Qu et al., 2007; Schwab et al., 2006; Simon-Mateo & Antonio Garcıa, 2007; Zhang et al, 2011).
4. Factors that influence the RNA-silencing based transgenic resistance
4.1. Heterologous viruses
Since 1998 where the first viral suppressor of silencing was discovered it has been established that most known virus species carry at least one RNA silencing suppressor (Dıaz-Pendon & Ding, 2008; Ding & Voinnet, 2007). The awareness of this viral counter-defensive strategy against the innate antiviral defense system of plants guided several groups to investigate the effect that could invoke on transgenic resistance of plants that were immune to a virus, the infection with a different virus carrying a strong silencing suppressor.
The first studies were presented in 2001 by Savenkov and Valkonen, and Mitter and co-workers. Savenkov and Valkonen produced transgenic tobacco plants resistant to
Simon-Meteo et al. (2003) performed similar experiments on
Taking into consideration these first studies several of the numerous succeeding reports (Fuentes et al, 2006; Germundsson & Valkonen, 2006; Praveen et al, 2010; Kawazu et al, 2009; Yang et al, 2004) of engineered transgenic resistance to plant viruses have examined the possible effect of heterologous virus infection in the resistance. However, not always an influence on resistance was observed. Missiou et al. (2004) in transgenic potato plants resistant to PVY examined the effect on the resistance of
In contrast, in
The results from the studies that examined the effect of heterologous virus infection on the silencing-based transgenic resistance indicated that this kind of resistance, despite the immunity that can confer to the plants against a specific virus, could be compromised to some degree if applied in the field where mixed virus infections occur frequently. However, it became evident that the outcome of the interference between the heterologous viruses and the silencing machinery of the plant is not so easily predictable (Table 1).
The reasons for the discrepancies are unclear, but could be related to the mode of action of the viral suppression proteins of the different virus tested. Viral silencing suppressors are highly diverse in sequence, structure and activity, and could target multiple points in RNA silencing pathways whereas viruses with large genomes may encode several functionally distinct proteins to achieve silencing suppression (Dıaz-Pendon & Ding, 2008; Ding & Voinnet, 2007). It is considered that suppressor proteins interfere either with siRNAs biogenesis or siRNA function without a multifunctional nature to be excluded. For instance, most studies agree that the potyviral HC-Pro probably specifically blocks accumulation of secondary siRNAs and leaves primary siRNA accumulation unimpaired, whereas P25 blocks accumulation of primary siRNAs (Dıaz-Pendon & Ding, 2008). In contrast, the 2b protein of cucumoviruses directly sequestrate siRNAs duplexes using a pair of hook-like structures that interact more promiscuously with long and short dsRNA (Dıaz-Pendon & Ding, 2008; Ding & Voinnet, 2007; Ruiz-Ferrer & Voinnet, 2009). Additionally, it binds AGO1 and blocks slicing without interfering with sRNA loading
Additional antiviral plant defense pathways could also be involved in the interference between the heterologous virus infection and the transgenic resistance or as yet unknown factors involved in specific virus species interactions. Thus, the CMV 2b protein has been shown also to block silencing indirectly by interfering with the salicylic acid mediated defense pathway (Li & Ding, 2001). Moreover,
4.2. Temperature
It has been well known to plant virologists that temperature strongly influences plant-virus interactions. In high temperature, symptoms are frequently attenuated and virus titers in infected plants are decreased. In contrast, outbreaks of virus diseases are frequently associated with low temperatures (Hull, 2002).
Kalantidis and co-workers (2002) examined the influence of elevated temperature on siRNAs in CMV-resistant transgenic tobacco plants. Two transgenic lines, one expressing very high and the other very low levels of siRNAs, were tested for siRNAs concentration at 25 ºC and 32 ºC and at two time points, 20 and 30 days post-germination. At the early time point, transgene derived siRNAs could be detected only in the first line at 25 ºC and in both lines at 32 ºC. However, in the first line transgene specific siRNAs were at 32 ºC in a significantly higher concentration compared to that of 25 ºC. The analysis of samples taken at the second time point revealed the presence of transgene derived siRNAs in both lines at 25 ºC. However, at 32 ºC, siRNAs were detected in both plant lines at a higher concentration. Apparently, in these experiments, except for temperature the developmental stage of the plants also influenced the siRNA concentration (discussed further below).
Factor | Transgenic Plant | Engineered resistance against | Effect on the resistance | Reference | |
Heterologous viruses | PVY | N. tabacum | PVA | Suppressed | Savenkov & Valkonen, 2001 |
CMV | N. tabacum | PVY | Reduced | Mitter et al., 2001; 2003 | |
CMV TVMV |
N. benthamiana | PPV | Suppressed Unaltered |
Simón-Mateo et al., 2003 | |
PVX | Potato | PVY | Unaltered | Missiou et al., 2004 | |
ZYMV ZFMV CVYV CMV |
Cucumber | CFMMV | Unaltered | Gal-On et al., 2005 | |
BMYV BYV BSBV BVQ |
Sugar beet | BNYVV | Unaltered | Lennefors et al., 2007 | |
SPFMV-C | Sweetpotato | SPCSV SPFMV-Uganda |
Suppressed | Kreuze et al., 2008 | |
GVB, PVY | N. benthamiana | GVA | Suppressed | Brumin et al., 2009 | |
TYRV-s | N. benthamiana | TSWV GRSV TCSV WSMoV TYRV-t |
Suppressed | Hassani-Mehraban et al., 2009 | |
CMV | N. tabacum | TMV | Unaltered | Hu et al, 2011 | |
TMV TYLCV |
Tomato | CMV | Unaltered | Zhang et al, 2011 | |
CMV, PVY TSWV |
N. tabacum | TRV | Unaltered | Vassilakos (unpublished) | |
Temperature | 320C | N. tabacum | CMV | n/t | Kalantidis et al., 2002 |
15 ºC | N. benthamiana | CymRSV | Suppressed | Szittya et al., 2003 | |
N. tabacum | TMV CMV |
Unaltered | Hu et al, 2011 | ||
N. tabacum | TRV | Suppressed locally | Vassilakos (unpublished) | ||
Light | High/Low Intensity | N. benthamiana | PPV | n/t | Kotakis et al., 2010 |
Early developmental stage | N. benthamiana | PMMoV | Reduced | Tenllado & Dıaz-Ruız, 1999 | |
Squash | SqMV | Suppressed | Jan at al., 2000 | ||
Papaya | PRSV | Suppressed | Tennant at al., 2001 | ||
N. tabacum | CMV | n/t | Kalantidis et al., 2002 | ||
N. tabacum | TRV | Reduced | Vassilakos et al., 2008 |
Szittya and associates (2003) provided further insight into the mechanism that is involved in these observations. Through a set of delicate experiments they demonstrated that RNA silencing induced by viruses or transgenes is inhibited at low temperatures and enhanced with rising temperatures. They used wild type
Chellappan and co-workers (2005) expanding the above findings quantified gemini virus-derived siRNAs at different temperatures and evaluated their distribution along the virus genome for isolates of five species of cassava geminiviruses, consisting of recovery and non-recovery types. In cassava plants, geminivirus-induced RNA silencing increased by raising the temperature from 25 ºC to 30 ºC and the appearance of symptoms in newly developed leaves was reduced, irrespectively of the nature of the virus. Consequently, high temperature rendered non-recovery type geminiviruses to recovery-type viruses. The distribution of virus derived siRNAs on the respective virus genome at three temperatures (25 ºC, 25 ºC-30 ºC and 30 ºC) remained unaltered only for recovery-type viruses. siRNAs derived from recovery-type viruses accumulated at moderately higher levels during virus-induced silencing at higher temperatures. However, siRNAs from non-recovery-type viruses accumulated six times higher than those observed for infections with recovery-type viruses at high temperature. Thus, the decreased symptom severity and virus concentration that were recorded at higher temperature indicate a similar effect of temperature on ssDNA and RNA viruses although there was a differential effect of temperature on the level of virus-derived siRNAs between recovery and non-recovery types of ssDNA viruses.
As with the effect of heterologous viruses, inhibition of RNA silencing or decreasing of siRNAs concentration in low temperature has not always been observed. Thus, transgene anti-sense induced RNA silencing was not inhibited in potato plants at low temperature (Sos-Hegedus et al., 2005). Moreover, tomato plants carrying an IR construct derived from
In summary (Table 1), the well-known temperature effect on the development of viral diseases is closely associated to the RNA silencing antiviral pathway and consequently influences the efficiency of silencing-based transgenic resistance. However, it appears that the low temperature effect on the transgenic resistance depends on additional factors that remain to be identified, fact supported by inconsistencies in the results of the diverse studies described here. Importantly, although at low temperature the siRNA-based silencing machinery is partially inactivated as an adaptive response of plants to adverse conditions, the miRNA-mediated, which is essential for regulatory functions, continues to operate ensuring plant growth (Szittya et al., 2003).
4.3. Light
Studies on the effect of light on transgenic resistance to viruses are not available, however light has been implicated as one of the factors that affect RNA silencing initiation and maintenance in several studies. Although in most of them light effect on silencing was not clearly isolated from that of temperature (Nethra et al., 2006; Vaucheret et al., 1997) recently, Kotakis et al. (2010) investigated solely the role of light intensity in physiological ranges on RNA silencing. They used as a system
4.4. Plant developmental stage
Quite a few studies with plants carrying sense transgenes and displaying RNA-silencing mediated resistance have suggested an influence of plant developmental stage on the degree of the expressed resistance. Tenllado and Diaz-Ruiz (1999) reported that a higher percentage of transgenic
As mentioned already, Kalantidis and associates (2002) showed that siRNA accumulation in transgenic tobacco, incorporating an IR construct carrying CMV sequences, was higher at later developmental stages. No significant differences in the siRNA concentration were observed between leaves of different age from a single plant or from the seven-leaf stage on, while the siRNA concentration reached a plateau that remained stable in the course of further development.
In a more recent work,
In conclusion (Table 1), most studies agree that younger transgenic plants accumulate reduced amounts of transgene specific siRNAs compared to older ones, or correspondingly accumulate higher amount of transgene specific transcripts suggesting a reduced efficiency of transgenic resistance against plant viruses. However, the resistance phenotype was not always affected in younger plants, possibly due to reasons associated with the type of the transgene construct used, its integration into the plant genome or the viral sequences that are targeted.
5. Conclusion
A great deal of progress has been made towards comprehension of plant virus biology and the ways in which plants defend themselves against these pathogens. RNA silencing has provided a promising potential for generating virus-resistant transgenic plants and this potential is certainly not cancelled by the awareness of factors that may affect under specific conditions the acquired resistance. However, as with any other pathogen control strategy, RNA silencing does not constitute a panacea and a number of issues should be taken into consideration before being applied in the field. Noticeably, silencing based transgenic resistance is not influenced solely by the factors that were presented in this review. However, planting into areas where endemic virus diseases occur and mixed virus infections are expected especially during early stages of the vegetation period, time intervals of low air temperature and greenhouse or open field cultivation practices could affect the stability of transgenic resistance against plant viruses.
Further exploitation of our knowledge on RNA-silencing pathways is essential to improve the efficiency of the existing strategies or for the development of potential new strategies which will hopefully lead to a better reception by the public. Recent advances like the construction of chimeric IR constructs incorporating sequences derived from different virus species if combined with epidemiological data and pest risk analyses could reduce the effect of mixed virus infections on the resistance (Bucher et al., 2006; Dafny-Yelin & Tzfira, 2007; Kung et al., 2009). Recently, virus resistance was achieved through expression of amiRNAS against viral coding sequences (Ding & Voinnet, 2007; Duan et al., 2008; Niu et al., 2006; Qu et al., 2007; Simon-Mateo & Antonio Garcia, 2006; Zhang et al, 2011). Although there was evidence that amiRNA-mediated virus resistance may not be inhibited by low temperature (Niu et al., 2006) this possibly depends on the plant species examined (Qu et al., 2007). Moreover, the durability of this approach, which resulted in relatively few antiviral small RNAs compared with those of the long dsRNA approach, needs to be further demonstrated (Duan et al., 2008; Simon-Mateo & Antonio Garcia, 2006).
Acknowledgments
The author wishes to thank Christina Varveri for helpful discussions and for critically reading the manuscript, and to express his apologies to all those, whose papers were not cited.
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