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[SANET-MG] gene therapy for chestnut blight



April 29, 2004
Prof. Joe Cummins
?Gene therapy for control of chestnut blight?
        Chestnut trees were ,at one time, the main canopy trees of the North Eastern
United States and Canada. The trees provide valuable hardwood and nuts for
animals and people. Early in the 1900s  a fungus, Cryphonectria parasitica,
was introduced from the orient producing cancerous canker growth on the
chestnut trees  which ultimately led to the death of the trees. The fungal
spores were spread by insects, gastropods, birds and animals that moved across
the bark cankers spreading sexual (asco)  spores. In addition wind born sexual
spores  were spread great distances on the wind  causing rapid spread of the
epidemic. Somewhat later an epidemic of the fungal disease spread through
Europe but that disease was less severe than in North America because resistant
trees appeared, The natural remedy that appeared in Europe  resulted from the
appearance of a fungal virus attenuated  disease (hypovirulent) and spread
among the fungal mycelia (mulitinucleate acelular threads) as they fused. In
the 1960s the chestnut blight epidemic was controlled by  treating disease
cankers with the hypovirus. However,  the treatment was not effective in North
America because the fungal mycelia (hyphae) did not fuse efficiently because
there was a genetic barrier to fusion among  North American strains of the
fungus that was not present in the European disease strains(1).
        Genetic modification  to provide hypoviulent virus that would spread widely
through out the infected trees was  first attempted in 1996. The hypovirulent
virus was  a double stranded RNA molecule that was converted to DNA using
reverse transcription then added to a transforming plasmid which was inserted
into a fungal chromosome. The chromosome inserted  virus was transcribed into a
single strand of RNA then converted to the double stranded virus in the
cytoplasm. The first attempt failed because the  hypovirulent virus
debilitated the  transformed virus so extensively that little or no
hypervirulent virus  was produced. Currently field tests  are being initiated
using a genetic modification  of the disease fungus  using a somewhat less
violent European strain of hypovirus that is expected to more effectively treat
the blight disease(1).
        Before discussing  the gene therapy further a  brief description of the
genetics of the ascomycete fungus  might be helpful. The ascomycete fungi
include may forms with long acellular filaments (mycelia or hyphae) containing
haploid nuclei. Fungi such as Cryphonectria parasitica have two mating types(1
and 2), when  filaments of the appropriate mating type meet they may fuse
followed by fusion of the nuclei to form a diploid zygote, the zygote undergoes
meiosis  to form 4 or 8 haploid sexual spores. Upon germination the spores form
hyphae with many nuclei.(2,3). Hyphae may  fuse to form heterokaryons with two
genetically different nuclei, Cryphonectria parasitica of North America have 45
vegetative compatibility types determining hyphal fusion (4). For that reason
meeting a fusion mate is a real challenge. In nature hypovirulent viruses are
spread by hyphal fusion , the difference between the effective European
treatments to disseminate hyopvirulent virus and ineffective North American
procedures  seems to be much lower number of vegetative compatibility types in
the European Cryphonectria parasitica community (1). Sexual (vertical)
transmission of the hypovirulence  virus integrated into the funagal chromosome
is  fully efficient while horizontal virus transfer by hyphal fusion alone is
limited by the genetic barrier to hyphal fusion (1,5). The hypovirulent virus
is not normally spread through sexual spores but is spread through fusion of
compatible  hyphae followed by dissemination of hyphal fragments and conidia
(asexual spores). Hypovirulent virus spreads extensively following hyphal
fusion as does the mitochondrial nucleic acid of the fungus but the nuclei
introduced into treated cankers persisted in the treated canker while the virus
and mitochondria spread further.into the resident Cryphonectria parasitica
population (6)  The  Cryphonectria parasitica genetically modified  to create
nuclear integration of the hypovirulent virus allows much wider dispersal
through sexual spores(1).
        There has  been extensive research into the  mechanism of attenuation of fungal
virulence by the virus.  The virus  was found to suppressed the accumulation of
 a G-protein ( a protein important in signal transduction) leading to reduced
fungal virulence (7). The virus also  contains and produces papain like protein
that enhances viral RNA accumulation and transmission  (8). Such research  may
lead to more effective control of the fungus, Cryphonectria parasitica.
        The basic question about the  field test of genetically modified  hypovirulence
virus is will the experiment lead to untoward consequences such as a virulent
fungus that spreads more widely than the original?  Even if the virus could,
somehow, enhance the virulence of  Cryphonectria parasitica  that virus does
not have a protein coat and depends for its dissemination of intact fungal
mycelia. However, the group of double stranded RNA viruses includes a number of
complete viruses that infect plants and animals including humans. Viral
recombination and transcapsidation (exchanging coats between related viruses)
was ignored in the proposal for field testing in a National Forest, surely a
serious oversight. The earlier, failed, experiment does not seem to have been
extensively monitored for long term genetic fall out. Can other elements in the
genetic construction, such as the selectable marker hygromycin B resistance
pose a significant  problem? The antibiotic hygromycin B is used mainly for
genetic engineering experiments and seems to have few clinical application,
however, the antibiotic is a member of a group of aminoglycoside antibiotics
used extensively that tend to have cross resistance. Finally, is undertaking
genetic engineering field tests in a National Forest a good idea? Frankly,
undertaking genetic engineering field tests on forest trees in a National
Forest seems to be a really bad idea, as indicated earlier, the virus is spread
by arboreal animals and the wind. Preliminary testing could have been done on
offshore islands to minimize the impact of untoward  genetic accidents. Viable
non-transgenic remedies for  control of chestnut blight such as the use of
bacterial endophytes (9) did not seem to have been considered before
undertaking  hazardous field trials  using gene therapy.
References
1.      United States EPA  ?OPP-2004-0128 Biotechnology Notification; Receipt of
application 04-28-2004 General Docket Notice 05-13-2004 Chestnut blight
control: Application For Permit or Courtesy Permit Under 7 CFR 340
Support-Background Notice? pp 1-23
http://cascade.epa.gov/RightSite/dk_public_home.htm

2.      Glass,N,Jacobson,D. and Shui,P.?The genetics of hyphal fusion and vegetative
incompatibility in filamentous ascomycete fungi? 2000 Ann Rev Genet 34,165-86
3.      Dawe,A, and Nuss,D. 2001 Ann. Rev. Genet. ?Hypoviruses and chestnut
blight:exploiting viruses to understand and modulate fungal pathognesis? 2001
Ann Rev Genet 35, 1-29
4.      Hogan,E. and Griffin,G. ?Spread of Creyphonectria hypovirus 1 into 45
vegitive compatibility types of Cryphonectria parasitica on grafted American
chestnut trees? 2002 For.path. 32, 73-85
5.      Cortesi,P, McCulloch,C,Song,H,Lin,H and Milgroom,M ?Genetic control of
horizontal virus transmission in the chestnut blight fungus Cryphonectra
parasitica? 2001 Genetics 159,107-18
6.      Hoegger,P,Heiniger,U,Holdenrieder,O. and Rigling,D. ?Differential transfer
and dissemination of hypovirus and nuclear and mitochondrial genomes of
hypovirus-infected Cryphonectria parasitica strain after introduction into a
natural population? 2003 Appl. Envir. Microbiology 69, 3767-71
7.      Choi,G,Chen,B. and Nuss,D. ?Virus-mediated or transgenic suppression of a
G-protein alpha subunit and attenuation of fungal virulence? 1995 Proc. Natl.
Acad. Sci.USA 92,305-9
8.      Suzuki,N,Maruyama,K,Mariyama,M. and Nuss,D. ?Hypovirus papain like protease
p29 functions in trans to enhance viral double stranded  RNA accumulation and
vertical transmission? 2003 J. Virol. 77,11697-707
9.      Wihelm,E,Arthofer,W,Schafleitner,R. and Krebs,B.  ?Bacillus subtilis and
endophyte of chestnut as antagonist against chestnut blight? 1998 Plant
Cell,Tissue and Organ Culture 52,105-8

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