Transcomplementation and synergism in plants: implications for viral transgenes?

In plants, viral synergisms occur when one virus enhances infection by a distinct or unrelated virus. Such synergisms may be unidirectional or mutualistic but, in either case, synergism implies that protein(s) from one virus can enhance infection by another. A mechanistically related phenomenon is transcomplementation, in which a viral protein, usually expressed from a transgene, enhances or supports the infection of a virus from a distinct species. To gain an insight into the characteristics and limitations of these helper functions of individual viral genes, and to assess their effects on the plant-pathogen relationship, reports of successful synergism and transcomplementation were compiled from the peer-reviewed literature and combined with data from successful viral gene exchange experiments. Results from these experiments were tabulated to highlight the phylogenetic relationship between the helper and dependent viruses and, where possible, to identify the protein responsible for the altered infection process. The analysis of more than 150 publications, each containing one or more reports of successful exchanges, transcomplementation or synergism, revealed the following: (i) diverse viral traits can be enhanced by synergism and transcomplementation; these include the expansion of host range, acquisition of mechanical transmission, enhanced specific infectivity, enhanced cell-to-cell and long-distance movement, elevated or novel vector transmission, elevated viral titre and enhanced seed transmission; (ii) transcomplementation and synergism are mediated by many viral proteins, including inhibitors of gene silencing, replicases, coat proteins and movement proteins; (iii) although more frequent between closely related viruses, transcomplementation and synergism can occur between viruses that are phylogenetically highly divergent. As indicators of the interoperability of viral genes, these results are of general interest, but they can also be applied to the risk assessment of transgenic crops expressing viral proteins. In particular, they can contribute to the identification of potential hazards, and can be used to identify data gaps and limitations in predicting the likelihood of transgene-mediated transcomplementation.

The mutational consequences of plant transformation.

Plant transformation is a genetic engineering tool for introducing transgenes into plant genomes. It is now being used for the breeding of commercial crops. A central feature of transformation is insertion of the transgene into plant chromosomal DNA. Transgene insertion is infrequently, if ever, a precise event. Mutations found at transgene insertion sites include deletions and rearrangements of host chromosomal DNA and introduction of superfluous DNA. Insertion sites introduced using Agrobacterium tumefaciens tend to have simpler structures but can be associated with extensive chromosomal rearrangements, while those of particle bombardment appear invariably to be associated with deletion and extensive scrambling of inserted and chromosomal DNA. Ancillary procedures associated with plant transformation, including tissue culture and infection with A tumefaciens, can also introduce mutations. These genome-wide mutations can number from hundreds to many thousands per diploid genome. Despite the fact that confidence in the safety and dependability of crop species rests significantly on their genetic integrity, the frequency of transformation-induced mutations and their importance as potential biosafety hazards are poorly understood.

Microarray analysis of changes in bone cell gene expression early after cadmium gavage in mice.

We developed an in vivo model for cadmium-induced bone loss in which mice excrete bone mineral in feces beginning 8 h after cadmium gavage. Female mice of three strains [CF1, MTN (metallothionein-wild-type), and MT1,2KO (MT1,2-deficient)] were placed on a low-calcium diet for 2 weeks. Each mouse was gavaged with 200 microg Cd or vehicle only. Fecal calcium was monitored daily for 9 days, beginning 4 days before cadmium gavage, to document the bone response. For CF1 mice, bones were taken from four groups: +/- Cd, 2 h after Cd and +/- Cd, 4 h after Cd. MTN and MT1,2KO strains had two groups each: +/-Cd, 4 h after Cd. PolyA+ RNA preparations from marrow-free shafts of femura and tibiae of each +/- Cd pair were submitted to Incyte Genomics for microarray analysis. Fecal Ca results showed that bone calcium excreted after cadmium differed for the three mouse strains: CF1, 0.24 +/- 0.08 mg; MTN, 0.92 +/- 0.22 mg; and MT1,2KO, 1.7 +/- 0.4 mg. Gene array results showed that nearly all arrayed genes were unaffected by cadmium. However, MT1 and MT2 had Cd+/Cd- expression ratios >1 in all four groups, while all ratios for MT3 were essentially 1, showing specificity. Both probes for MAPK 14 (p38 MAPK) had expression ratios >1, while no other MAPK responded to cadmium. Vacuolar proton pump ATPase and integrin alpha v (osteoclast genes), transferrin receptor, and src-like adaptor protein genes were stimulated by Cd; other src-related genes were unaffected. Genes for bone formation, stress response, growth factors, and signaling molecules showed little or no response to cadmium. Results support the hypothesis that Cd stimulates bone demineralization via a p38 MAPK pathway involving osteoclast activation.

Cadmium pathways during gestation and lactation in control versus metallothoinein 1,2-knockout mice.

Effects of metallothionein (MT) on cadmium absorption and transfer pathways during gestation and lactation in mice were investigated. Female 129/SvJ metallothionein-knockout (MT1,2KO) and metallothionein-normal (MTN) mice received drinking water containing trace amounts of (109)CdCl(2) (0.15 ng Cd/ml; 0.074 micro Ci (109)Cd/ml). (109)Cd and MT in maternal, fetal, and pup tissues were measured on gestation days 7, 14, and 17 and lactation day 11. In dams, MT influenced both the amount of (109)Cd transferred from intestine into body (two- to three-fold higher in MT1,2KO than MTN dams) and tissue-specific (109)Cd distribution (higher liver/kidney ratio in MT1,2KO dams). Placental (109)Cd concentrations in MT1,2KO dams were three- and seven-fold higher on gestation days 14 and 17, respectively, than in MTN dams. Fetal (109)Cd levels were low in both mouse types, but at least 10-fold lower in MTN fetuses. MT had no effect on the amount of (109)Cd transferred to pups via milk; furthermore, 85-90% of total pup (109)Cd was recovered in gastrointestinal tracts of both types, despite high duodenal MT only in MTN pups. A relatively large percentage of milk-derived intestinal (109)Cd was transferred to other pup tissues in both MT1,2KO and MTN pups (14 and 10%, respectively). These results demonstrate that specific sequestration of cadmium by both maternal and neonatal intestinal tract does not require MT. Although MT decreased oral cadmium transfer from intestine to body tissues at low cadmium exposure levels, MT did not play a major role in restricting transfer of cadmium from dam to fetus via placenta and to neonate via milk.

A Nest Box to Facilitate Excreta Collection From Mouse Dams Through Pregnancy, Parturition, and Lactation.

Standard metabolism cages are inadequate for collecting excreta from dams during parturition because newborn pups can fall through the grating into the excreta collection area and out of reach of the dam. A nest box was designed that facilitates excreta collection from mouse dams continuously housed in metabolism cages from conception, through parturition, and into lactation and provides a safe, warm environment for pups during their first week of life. The nest box was tested by using pregnant and lactating mice of two varieties of strain 129/SvJ, metallothionein-normal and metallothionein-knockout; non-pregnant mice were used as controls. Pregnant mice (with nest box) and non-pregnant mice (without nest box) each twice received a solution of 109CdCl2 by gavage. Dams with nest boxes fastidiously urinated and defecated outside the nest box. The percentage of gavage 109Cd dose recovered in dam feces was the same after the first gavage (mean6SE, with nest box through parturition, 95%66%; n=5) as after the second gavage (mean6SE, without nest box, 95%66%; n=5). Weights and percentage weight gain of mouse dams were independent of housing conditions (metabolic cage with next box vs. conventional polycarbonate caging). Furthermore, pup growth and survival were unaffected by the inclusion of the nest box or by its removal at 1 week after birth. Therefore, the described nest box provides for the first time a way to quantitatively collect excreta from mouse dams through pregnancy, parturition, and the early postnatal period. Additional experiments are needed to test its application to other animal species and strains of mice, including those with poor mothering behavior.