Genes in the defence against virus

Viral diseases cause serious losses world wide in horticultural and agricultural crops in many parts of the world. They have been difficult to control and resistance has traditionally relied on either the use of pesticides to kill viral insect vectors or the introduction of natural resistance genes through conventional breeding programmes. More recently, viral resistance has been engineered by transforming susceptible plants with genes or sequences derived from viral sequences, the so-called pathogen-derived resistance (PDR) (Lomonossoff 1995). The mechanisms by which PDR is activated have not been well defined, but resistance may result from the expression of a viral protein (native or mutated) or an RNA-mediated mechanism that appears to be analogous to gene silencing (Baulcombe 1999). Interestingly, gene silencing appears to be the natural mechanism by which kohlrabi and Nicotiana clevelandii resist infection by cauliflower mosaic virus and tomato black ring nepovirus respectively (Ratcliff et al. 1997; Covey et al. 1997).

The first demonstration of PDR came from transgenic tobacco expressing the coat protein (CP) of tobacco mosaic virus (TMV) (Lomonossoff 1995). Since then, CP-mediated resistance (CPMR) has been engineered against a wide variety of viruses in many plant species. It is still unclear why CPMR sometimes confers resistance only to the viral strain from which the CP was derived, whereas in other cases it provides protection against related viruses. Nevertheless, as CPMR is effective in many plants against diverse viruses, it has been employed in different crops of agronomic importance. Transgenic lines of squash and papaya exhibiting CPMR have already been approved for commercial release and other crops are under development (Kaniewski et al. 1999). As more is known about the mechanisms associated with CPMR, it will be possible to manipulate the level and/or breadth of resistance, as in the case of TMV where mutant CPs that confer much greater resistance than the wild-type protein have been generated (Beachy, 1997).

Viral replicase genes in either wild-type or defective forms have also been employed to engineer PDR (Baulcombe 1996). Replicase-mediated resistance (RMR) can confer nearly full immunity to infection, but similarly to CPMR, it tends to be effective only against the virus strain from which the gene was derived and it is even more limited than CPMR (Baulcombe 1996). The mechanisms by which RMR is activated are not well understood but the high degree of resistance associated with RMR makes it attractive for engineering virus-resistant crops. Interestingly, two cases of broader-base RMR have been reported (Beachy 1997). Clearly, a better understanding of the mechanisms by which RMR is achieved is needed before immunity can be conferred against more than a single viral strain.

PDR has also been engineered by expressing mutant forms of viral movement proteins (MP). MP-mediated resistance (MPMR) represents a very interesting approach because it confers delayed symptoms and/or decreased systemic viral accumulation against a much broader spectrum of viruses than either CPMR or RMR (Baulcombe 1996). Furthermore, MPMR can also protect against viruses thought to move through plasmodesmata as well as tubules (Beachy 1997). It has been hypothesised that MPMR results from a dominant negative mutation in the MP, which obstructs the wild-type MP from either binding the viral genome or facilitating the cell-to-cell or systemic movement (Beachy 1997).

In addition to the above examples, PDR has been engineered using other viral genes as well as entire viral genomes (Baulcombe 1996; Beachy 1997; Song et al. 1999). The mechanisms through which viral genomes confer resistance are not well defined but it has been shown that a potato virus X (PVX) replicon provides high-level, strain specific resistance via an RNA-dependent silencing mechanism (Angell and Baulcombe 1997). Several strategies for engineering virus-resistant plants independent of PDR have also been developed. One employs ribosome-inactivating proteins (RIP), which cleave the N-glycosidic bond of adenine in a specific ribosomal RNA sequence thereby rendering them incapable of protein synthesis. One of the best studied RIPs is pokeweed antiviral protein (PAP), which exhibits potent nonspecific antiviral activity and has been used to engineer resistance in tobacco to a broad spectrum of plant viruses (Tumer et al. 1999).

Unfortunately, construction of these transgenic plants has been difficult probably due to the toxic effects of PAP. For that reason, the use of a carboxy-terminal deletion mutant of PAP (Zoubenko et al. 1997) and a recently isolated PAP isoform (PAP II) have resulted in reduced toxicity and a broad spectrum resistance to viral infection (Tumer et al. 1997; Wang et al. 1998). Interestingly, PAP overexpression also confers resistance to fungal infection (Wang et al. 1998).

Proteins that recognise and degrade double-stranded RNA, the replication intermediate for most plant viruses, have also been employed to engineer resistance. Tobacco plants expressing pac1, a yeast double-strand RNA-specific ribonuclease, exhibit modest levels of resistance after infection by several unrelated viruses (Watanabe et al. 1995), whereas plants expressing 2',5'- oligoadenylate system are either completely or partially resistant to various unrelated viruses (Ogawa et al. 1996).

Recently there have been a number of reports on the use of plant genes to confer resistance against viral diseases. They have used natural R genes (Van Der Vossen et al. 1997; Bendahmane et al. 1999; Cooley et al. 2000), genes coding for heat shock-like proteins (Whitham et al. 2000) or genes coding for unknown proteins (Kachroo et al. 2000; Mourrain et al. 2000).

A novel approach to engineer resistance against viral diseases has been the use of proteinase inhibitors. Proteinase activity is an important component of the processing mechanism of several groups of plant viruses (Spall et al. 1997). Therefore, introduction of the corresponding proteinase inhibitor may be one way to inhibit viral replication and confer resistance. Gutiérrez-Campos et al. (1999) have shown that transgenic tobacco plants containing a cysteine proteinase inhibitor (oryzacystatin I) exhibited immunity against more than one potyvirus (the most important virus group, from the standpoint of the economic losses that they cause) and presented several beneficial pleiotropic effects (Gutiérrez-Campos et al. 2000). Since the proteinases employed by the different groups of plant viruses may vary (serine, cysteine, etc.), an adequate choice of the inhibitor may provide resistance to a particular plant virus.

In addition to providing broad-based resistance, strategies involving nonviral, plant genes may be advantageous because they eliminate concerns that recombination between a virus and a related transgene or transcapsidation of a viral RNA might create a novel ‘superpathogen’ capable of massive crop infestation.