Genes from a wild plant, Solanum bulbocastanum,
used to resist potato blight fungus

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Genes from a wild plant, Solanum bulbocastanum,
used to resist potato blight fungus

Prof. Joe Cummins

The German company BASF Plant Science GmbH is planning to test genetically modified (GM) potatoes  in Ireland by deliberately releasing the modified potatoes into the environment. The field trial is planned to be undertaken during a five year period. The GM potatoes are modified using a gene from a wild Mexican plant, Solanum bulbocastanum, related to potato along with marker genes  including  a gene for resistance to a herbicide. The potatoes are modified to be resistant to the fungus causing late blight disease No environmental and health studies appear to be planned (1). Animal feeding studies on the GM potatoes do not appear to have been done. Release to the environment of untested  GM crops  to the environment  are unwise  because even related plant to related  plant single gene transfers have resulted in unexpected toxicity which appeared in the  transgenic gene product resulting from altered structure and immunogenicity of  the modifying  gene product (2).When single genes from plants such as bean are used to modify another plant, the pea, it was assumed that such transfers could not produce toxic products in the plant being modified. However, the unexpected prevailed. The people and animals of the townland of Arodstown should not be exposed to inadequately tested genetic constructions.

Late blight is one of the most devastating plant diseases. It is caused by the fungus, Phytophora infestans, a pathogen of potato and to a lesser degree tomato. In potato, Solanum tuberosum, there are four main dominant genes for resistance to blight infection, R1 through  R4,  an additional  7 genes were identified  5 of which are alleles of the complex  R3 locus (for a total of 11 dominant R genes). Hybridization with wild Mexican species began in 1909  and continues to the present. However, in spite of constant effort the fungus rapidly developed strains that overcame the genetic resistance. Chemical fungicides have been developed to control blight but these to succumbed  to the versatility of the fungus. The fungus has two mating types (A1 and A2) both of which appeared first in Mexico, however, only the A1 mating type was present in European potatoes until 1978 when the A2 mating type appeared in Britain. The presence of the two mating types greatly enhances gene exchange  leading to accelerated loss of genetic resistance and fungicide control (3,4).

Early resistant potatoes were obtained using true sexual  hybridization with  wild Mexican species but the resistant  strains  soon succumbed to  mutants of the  blight fungus. A wild Mexican plant, Solanum bulbocastanum,  was  stably resistant to blight but could not be sexually crossed with  potatoes.  A process called somatic hybridization was used  to create sexual hybrids. Somatic hybridization includes fusing cells from cell cultures of  Solanum bulbocastanum and potato, fused cells contain nuclei of both potato and  Solanum bulbocastanum. When the fused cells undergo mitosis  the chromosomes of the two species are mixed and a single hybrid nucleus is formed in the cell. The cells can be cultured on solid media to form solid callous  (tumor) which when treated with plant growth hormones  produces plantlets that  produce  flowering plants . The somatic hybrids have irregular meiosis and irregular chromosome pairing but relatively stable blight resistant lines can be obtained (5,6,7) Along with the  11 potato blight resistance genes  that produce broad spectrum resistance are very effective against blight, these  .include the gene RB (8) along with the genes Rpi-blb1 (9) and Rpi-blb 2 (10) which are active in both potato and tomato.  The somatic hybrids are useful in identifying resistance genes and transmitted into potato breeding lines by crossing. Nevertheless,  genetic modification of potato breeding lines is presently preferred  because resistance can be introduced into commercial lines with greater speed.

The BASF proposal for field testing GM potatoes (11) involves  the use of  two broad spectrum resistance genes, Rpi-blb1 and Rpi-blb2,  These two genes have a structure associated with regulatory genes called  nucleotide binding site-leucine rich repeat (NBS-LRR) class of  regulatory proteins. Many disease resistance genes code for proteins of that class.  Numerous  plant  NBS-LRR  genes are present  are present  in the typical plant genome,  each protein is specific for a particular pathogen signaling a defense response frequently a localized plant cell death called a hypersensitive response. The  C terminus of the protein containing LRR recognizes  a ligand feature of a pathogen activating the NBS signaling module to initiate the defense response (12). The blight fungus  suppresses  the potato defense genes in sensitive plants but thwarted by successful defense genes. The NBS-LRR  resistance genes in plants  are localized in the cell cytoplasm and do not span the cell membrane but are activated by pathogen signals  that penetrate the cell (13). The plant  NBS-LRR proteins  generally  produce antibodies when injected  into mammals but the modifications of  the disease resistance proteins by glycosylation or mryistylation  which contribute to the immune response  are not  yet studied.

The BASF proposal (11)  indicates that  the potatoes being studied were transformed using two plasmids each containing   copies of the S. bulbocastanum resistance genes Rpi-blb1 and Rpi-blb2 both of which contained an intron. The two genes were each driven by  Rpi-blb 1 or 2 promoter with  including an intron as an enhancer and accompanied  by a transcription terminator  from Rpi-blb1 or 2.  The plasmids also contained a mutant  acetohydroxy acid  synthetase (ahas) gene from the tiny mustard plant Arabidopsis  that conferred resistance to  the herbicides of the imidazolines group

( which are not approved for use on potatoes in Ireland).  The ahas gene was driven by the nopaline synthase gene promoter  of Agrobacterium  and its transcription was terminated using  the nopaline terminator.  The transformed potatoes aare herbicide tolerant but the herbicide is only used during  selection of transformed potato cells and not during cultivation of the potato.  All GM lines intended for the release contain one or two copies  of the plasmid inserts.  Neither the resistance genes nor the ahas gene is expected  to effect pollen or seed dispersal of the potato. The possibility that the GM potatoes  will outcross to field potatoes was not expected to be effected by the genetic modifications. Interestingly , the expression  of the modifying genes was not studied under extreme conditions of stress such as drought, water logging, heat, cold, nitrogen excess or starvation in glass house experiments. In the past, gm crops have been tested under optimum conditions for growth prior to commercial or test release into real environments. Certainly, stress conditions may lead to unexpected toxicity in gm crops.

The BASF proposal (11) indicate that  the resistance genes are not expected to exert any toxic , allergenic or harmful effects on human health arising for genetic modification. The genetic modifications are assume to be safe because plants contain numerous NBS-LRR proteins and cultivated potatoes  contain R genes from the wild species  S. demissum. The assumptions of safety are specious.  The S. demissum genes in commercial potatoes are   NBS-LRR genes but are not from the broad spectrum  NBS-LRR genes used  in the BASF potatoes.  Mainly, however,  observed finding that  transfer of genes between related species may actually  lead to proteins with powerful  (sometimes fatal) immune responses.(2).  The procedure used to scan DNA sequences  for  epitope specifying codes  for allergic responses  (IgE) would overlook  the powerful immune responses  leading to  fatal or near fatal inflammation . It is only sensible to test glass house grown GM potatoes for  not only allergenicity but for inflammation before releasing the GM potatoes to the environment. The immune response that  triggered the immune response  described in reference 2  was triggered to altered protein modification following transfer between species . However, little information is available on the modification of plant NBS-LRR genes.  It sems a simple matter to conduct animal experiments on glasshouse grown  GM potatoes prior to release of the potatoes to the environment yet that does not seem to have been done. Impact of the site on non-target organisms seems to be based on an assumption safety and does not provide for an adequate monitoring scheme. If the GM potato proves immunologically active the impact on both human and animals may be severe.

In the proposal the handling , release controls and  disposal of Pytopthora infestans  innocula  and infected plants was alluded to but not described in detail. That should be done. The isolation distance  20 meters  to cultivated potatoes  does not seem adequate.  Control of GM seeds and tuber escape from the site did not seem to be adequately described in the proposal. Post release treatment of the test site did not seem adequately monitored nor will it achieve a clean post harvest site. There does not seem to be any reason that a round the clock guard cannot be kept over the test sites.  In conclusion, the considerations of  human and environmental safety seem primarily based on wishful thinking not on serious efforts to gather or obtain factual information on the safety of the gm constructs. Monitoring also seems based on wishful thinking rather than serious efforts to detect  negative impacts.

BASF  petitioned for  field test release of  resistant potatoes modified with Rbi-blb1 and 2  beginning 2005 in the Netherlands. The notice of petition indicated that  the GM potato would be released in Germany, United Kingdom and Sweden. (14). In the United States  five field tests have been undertaken using GM potatoes modified with  RB1 and RB2 broad spectrum NBS-LRR  blight resistance genes obtained from Solanum bulbcastanum. The releases were undertaken in  Minnesota and Wisconsin, by  USDA or the university of Minnesota (15)  The isolation and deployment of the RB genes in potato has been  described (16,17)

Field testing of  broad spectrum .NBS-LRR genes has begun with the potato blight resistant strains. Broad spectrum  pest resistant  strains  of rice, maize, soybean, and numerous food crops will soon follow.  It is imperative that the  safety of these genetic modifications to humans and the environment be fully evaluated before the GM crops are commercialized. The proposition that the NBS-LRR  family of  plant pest resistance genes and their products  provide safe transgenes  for  human consumption and for   environmental release because they are found in food crops  and for that reason require no further testing is simply fool hardy. The suggestion that NBS-LRR genes must be assumed safe until proven hazardous certainly appeals to greedy promoters  of GM crops but does not serve the public good.

References

1.   O’Callaghan,M  BASF plans 5 year  GMO potato  experiment near Hill of Tara  2006  http://www.indymedia.ie/article/73989

2.   Prescott VE, Campbell PM, Moore A, Mattes J, Rothenberg ME, Foster PS, Higgins TJ and  Hogan SP. Transgenic expression of bean alpha-amylase inhibitor in peas results in altered structure and immunogenicity. J Agric Food Chem. 2005 Nov 16;53(23):9023-30 

3.   Deacon,J.  The Microbial World: Potato blight-Phytohthora infestans  2006 http://helios.bto.ed.ac.uk/bto/microbes/blight.htm

4.   Bradshaw JE, Bryan GJ, Lees AK, McLean K and  Solomon-Blackburn RM

Mapping the R10 and R11 genes for resistance to late blight (Phytophthora infestans) present in the potato (Solanum tuberosum) R-gene differentials of Black. Theor Appl Genet. 2006 Jan 5;:1-8 [Epub ahead of print]  

5.  Helgeson,J.,Pohlman,J,Austin,S,Haberlach.G,Wielgus,S,Ronis,D,Zambolim,L,Tooley,P,McGrath,J,James,R and  Stevenson,W. Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight  TAG Theoretical and Applied Genetics 1998 , 96, 738 – 42

6.   Masuelli,R,Tanimoto,E,Brown Cand Comai,L. Irregular meiosis in a somatic hybrid between S. bulbocastanum and S. tuberosum detected by species-specific PCR markers and cytological analysis   TAG Theoretical and Applied Genetics  1995, 91, 401 – 8

7.   Naess SK, Bradeen JM, Wielgus SM, Haberlach GT, McGrath JM and  Helgeson JP. Analysis of the introgression of Solanum bulbocastanum DNA into potato breeding lines. Mol Genet Genomics. 2001 Jun;265(4):694-704

8.   Staples,R. Race nonspecific resistance for potato late blight Trends in Plant Sciences 2004,9, 5-6

9.   van der Vossen E, Sikkema A, Hekkert BL, Gros J, Stevens P, Muskens M, Wouters D, Pereira A, Stiekema W and Allefs S. An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato. Plant J. 2003 Dec;36(6):867-82

10. van der Vossen EA, Gros J, Sikkema A, Muskens M, Wouters D, Wolters P, Pereira A and  Allefs S. The Rpi-blb2 gene from Solanum bulbocastanum is an Mi-1 gene homolog conferring broad-spectrum late blight resistance in potato. Plant J. 2005 Oct;44(2):208-22

11.  BASF Plant Sciences Notification for the release into the environment of genetically modified potatoes with imporoved resistance to Phytopthera infestans  (2006-2010)

12. Belkhadir Y, Subramaniam R and  Dangl JL. Plant disease resistance protein signaling: NBS-LRR proteins and their partners. Curr Opin Plant Biol. 2004 Aug;7(4):391-9

13. Huitema E, Bos JI, Tian M, Win J, Waugh ME and  Kamoun S. Linking sequence to phenotype in Phytophthora-plant interactions. Trends Microbiol. 2004 Apr;12(4):193-200

14. SeedQuest - Central information website for the global seed industry Deliberate release into the E.U. environment of GMOs for any other purposes than placing on the market:Potato with improved resistance to Phytophthora infestans - BASF Plant Science GmbH  2005 http://www.seedquest.com/News/releases/2005/october/13818.htm

15. Information systems for biotechnology  Search Results of the Field Test Release Permits Database for the U.S  late blight disease potato http://www.nbiap.vt.edu/cfdocs/fieldtests3.cfm

16. Song J, Bradeen JM, Naess SK, Raasch JA, Wielgus SM, Haberlach GT, Liu J, Kuang H, Austin-Phillips S, Buell CR, Helgeson JP and  Jiang J. Gene RB cloned from Solanum bulbocastanum confers broad spectrum resistance to potato late blight. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9128-33 

17. Helgeson,J,Austion-Phillips,S,Naess,S,Jiang,J,Bradeen,J and Buell,C. Potato genes for late blight  US patent Applicarion Publication  2004 , US2005/0204419A1

January 28, 2006