TMAW - RESPONSE TO DR. LATHAM (26 May 2003)
"Can Genetic Material in GM Plants Transfer to Viruses?"

As a former research student at the John Innes Institute, Dr JR Latham is fully familiar with the detailed molecular biology of plant viruses and the many hundreds of examples, since 1986, of transgenic (GM) plant lines that express a variety of viral sequences, functional or dysfunctional, which can render a plant resistant or even immune to subsequent challenge virus inoculation. He will also be aware of the technical facility, durability, heritability and proven efficacy of this approach in many (hundreds of ) cases.

In addition to many thousands of contained laboratory, glasshouse and small-scale field trials, several GM crops (e.g. yellow crookneck squash, sweet potato and papaya) that express viral sequences which confer functional field resistance to a devastating wild-type virus have been grown commercially on large-scales, for 7 years or more, without any evidence of horizontal gene transfer from the plant to the target virus (or to any other virus, or vice versa).

Many artificial, laboratory-based recombinant-selection systems can and have been established between two or more debilitated (mutated) viruses in non-GM host plants, or between a more-or-less defective virus in a GM plant containing a transgene sequence capable of restoring wild-type virus. In most cases, usually depending on the strength of the evolutionary selection pressure applied, wild-type virus can be recovered in some plants (from <1% to approx. 30%) through homologous recombination (template strand switching) during RNA-RNA replication (as in most plant viruses), by RNA-DNA reverse transcription (in plant Pararetroviruses) or through DNA-DNA replication (in Geminiviruses). In nature, of course, by definition, all infecting viruses will be fully viable wild-type strains - and natural mixed infections commonly occur providing further opportunities for virus-to-virus gene reassortment, recombination, etc.

Recombination is well-documented and plays a key role in natural virus evolution (see Tepfer, M. 2002 Annual Review Phytopathology 40: 467-491). But, as yet, there is no evidence of such recombination (i.e. horizontal viral transgene transfer; HVTT) occurring in the field where single transgene resistance has been engineered against a wild-type virus.

In the past, the weakness or complete absence of any "natural" virus resistance gene in the breeding stock of many susceptible non-GM crops has required liberal use of pesticides to control the insect, fungus or nematode which naturally transmits a particular devastating virus. A specific virus-targeted resistance transgene in a GM crop variety thus provides a more selective, measurable and environmentally sustainable means to protect crop yield and quality.

The possibility that HVTT might occur and its possible virological, biological and/or ecological consequences have been discussed and speculated-on in every review article on this subject since the earliest days of the technology. The nature of any hazard, the probability of its occurrence and any possible consequences or lack thereof, remain real but manageable issues for those who design, produce and test virus-resistant GM crops.

In the seminal paper on this topic (AE Greene and RF Allison in Science 1994 263: 1423-1425), a deletion mutant of cowpea chlorotic mottle virus was restored to wild-type CCMV in 3% of inoculated plants following homologous recombination with a transgene transcript which had a perfect sequence overlap of 338 nucleotides and a fully functional 3'-replication origin. When the replication origin was removed (1996 paper) the frequency of recombination fell dramatically. Neither I, nor anyone else denies that such events can happen, if the experimental system is set-up appropriately. The real questions are: do similar events happen between wild-type viruses and transgenes in the field; and, if so (though so far not found), what is their possible significance, if any, on a case-by case basis?

In the short time available to introduce my (first) draft paper to the GM Science Panel Drafting Group I was, as Dr Latham confirmed, referring only to the impact of including a replication origin on transgene recombination events with RNA plant viruses. These represent the vast majority of plant viruses and hence have been targets for almost all virus-derived pathogen resistance transgene strategies in GM plants over 17 years. Indeed, reports of successful transgene-mediated resistance against DNA plant viruses are relatively rare. Thus while recombination-selection and rescue events through RNA-DNA reverse transcription (Pararetroviruses), or DNA-DNA replication (Geminiviruses) do not appear to be highly dependent on the presence of a viral replication origin [e.g. four papers cited by Latham - note Kiraly et al. 1998 not included in Latham & Steinbrecher, Econexus submission to the GM Debate, Royal Society of Edinburgh (27 January)], any GM-based field resistance strategy may offer limited success with these virus types anyway. Perhaps this is because RNA viruses are more susceptible to trangene-derived RNAi-mediated cellular silencing at an early stage of infection?

The recommendations which Dr Latham advocates so strongly in paragraph 4 of his note are precisely those contained in the Summary (para. 15) and Section 7 of the 1999 DETR Research Report (no.11) which he cites, which I and several senior molecular virologist colleagues at the Scottish Crop Research Institute authored! In fact, this mid-1990s review covered all 392 relevant papers published at the time, as well as surveying 23 UK and 49 overseas public and private sector organisations actively engaged in GM crop science and virology. With today's improved knowledge and understanding of RNAi and silencing defense pathways, many of the risk issues that were proposed last century can now be avoided when designing new viral resistance transgene strategies.

It is interesting, however, that the original Monsanto "New Leaf" potato was the subject of an extensive 6-year study (Thomas, PE et al., 1998 Molecular Breeding 4: 407-417). Over 25,000 plants in 442 lines transformed with 16 different coat protein gene constructs (with a Luteovirus replication origin), and 40,000 plants in 512 lines transformed with 7 different replicase gene constructs of potato leafroll virus were exposed to field infection over a 6-year period. Individual plants were inspected annually, and extensive molecular and biological studies done on any PLRV or heterologous viruses found to be infecting the crop. No changes in virus properties or evidence of recombination (HGT) were found.