(reduced sporulation, etc.) that are potentially dis-
advantageous in a biocontrol strain, reducing its en-
vironmental fitness. It may be possible, therefore, to
manipulate the cDNA in vitro so that it has only the
most desirable traits for biocontrol.
It is notable that the cDNA, when transformed into
Cryphonectria(causing the fungus to be hypovirulent),
became stably integrated in the chromosomal genome
so that it was replicated along with the other chromo-
somal genes. This cDNA was maintained throughout
the life cycle, even entering the sexual spores, whereas
dsRNA seldom enters the sexual spores of Cryphonectria
or other Ascomycota. This would mean that biocontrol
strains could be produced with permanent, stable hypo-
virulence, subject to proofreading like the rest of the
chromosomal genes.
Recent developments
The question arising from the stable integration of
“hypovirulence” cDNA is: can there still be cytoplas-
mically transmitted hypovirulence? The answer
seems to be “yes” because the chromosomally integrated
cDNA is transcribed into dsRNA rather than into
single-stranded mRNA (as in the rest of the genome)
and this dsRNA accumulates in the cytoplasm, where
it can be transmitted between strains during hyphal
anastomosis. This still leaves unanswered the question
of how dsRNA suppresses virulence. Preliminary evid-
ence suggests that it does so by downregulating some
of the normal chromosomal genes, including the gene
for production of laccase, an enzyme involved in
lignin breakdown (Chapter 11) and which is likely to
be significant for a fungus that colonizes wood.
When the hypovirulence cDNA (derived from dsRNA
of C. parasitica) was transformed into other canker-
forming Cryphonectriaspecies, and into a less closely
related Endothiaspecies, it failed to convert these to
hypovirulence. However, when the same cDNA was used
as a template to produce RNA and this was transfected
into the other fungi, it gave rise to full-length dsRNA
in the cytoplasm and caused both a marked reduction
of virulence and an alteration of the growth rate and
pigmentation (Chen et al. 1994). The success achieved
with RNA but not cDNA seems to be explained by the
fact that C. parasiticaproduces RNA from the cDNA but
then splices this RNA to delete a 73-base sequence before
the RNA can act as a template for dsRNA production.
In summary, this work on the hypovirus system of
Cryphonectriahas raised many issues of fundamental
interest in fungal genetics as well as holding the
prospect of developing new approaches to plant dis-
ease control. However, recent studies in Switzerland
(Hoegger et al. 2003) suggest the need for a cautious
approach, because the only way of transmitting the
hypovirus is by anastomosis, and both the nuclear DNA
and the mitochondrial DNA of the donor (biocontrol)
strain are introduced into the environment during
this process. The mitochondrial DNA of the donor
strain was found to be transmitted in nearly one-half
of the treated cankers. The nuclear DNA also persisted
in the treated cankers but it did not spread beyond
them. The major issue that remains to be resolved is
the long-term safety of introducing genetically modified
strains of pathogenic fungi into the environment,
especially if the nuclear DNA is transferred and increases
the genetic diversity of the pathogen.And back to the genomeThe term genomicswas coined in 1986 to describe
the mapping, sequencing and analysis of genomes, the
ultimate goal being to understand the structure, func-
tion, organization, relationships, and evolution of
genes. While many sequenced genomes of bacteria
and archaea have been published, there are relatively
few published genome sequences of fungi, although
several draft sequences are available. All the genome
sequences generated by governmental or public fund-
ing bodies are made available on the internet (see
Online resources for a list of sequenced genomes) so
that researchers can compare different genomes and
search for similarities and differences.
The basic techniques of genome sequencing are
relatively simple, and many of the procedures are
automated. Essentially, the DNA to be sequenced
(termed the template) is denatured by heat or alkali
to produce single-stranded DNA, and DNA polymerase
is used to synthesize DNA based on the nucleotide
sequence in the template. DNA polymerases require a
region of double-stranded DNA to initiate synthesis. This
is provided by adding a short single-stranded DNA
molecule – a primer, with a DNA sequence comple-
mentary to the template DNA. The primer binds to the
template to form a short region of double-stranded
DNA, from which the rest of the template DNA is
synthesized.
For sequencing projects, relatively long template
DNA sequences are prepared by cutting the DNA
randomly with restriction enzymes and inserting
them into plasmids or other vectors such as cosmids
(cloning vectors that resemble plasmids but are pack-
aged into λphage capsids which can carry inserts up
to 40 kb). The many lengths of sequenced DNA from
different regions of the genome are then searched
for overlapping regions. In this way the DNA can be
assembled into contigs, representing continuous cov-
erage of the nucleotide sequence of whole chromosomes
or regions of chromosomes. The genome sequence
is then searched for characteristic features such as