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J. theor. Biol. (1985) 113, 395-405

The Concept of Parasite-Derived Resistance--Deriving

Resistance Genes from the Parasite's Own Genome

JC SANFORDt AND SA JOHNSTON~

t Department of Horticultural Science, Cornell University,

Hedrick Hall, Geneva, NY 14456 and $ Department of Botany,
Duke University, Durham, NC 27706, USA
(Received June 22 , 1984, and in revised form October 19, 1984)
A broadly-applicable strategy is proposed for genetically engineering resistance
to parasites. The strategy involves deriving resistance genes from the genome of
the parasite itself. Key gene products from the parasite, if present in a dysfunctional
form, in excess, or at the wrong developmental stage, should disrupt the function
of the parasite while having minimal affect on the host. Therefore, resistance might
be routinely achieved "by cloning the appropriate parasite gene, modifying its
expression if necessary, and transforming it into the host genome. The QB
bacteriophage is used to illustrate, specifically, how parasite-derived resistance
might be engineered. Examples are given of pathogen-derived resistance as it
already functions in nature, and potential applications of this strategy in agriculture
are discussed. The advantages and limitations of parasite-derived resistance are
outlined.

Introduction
A potentially important application of genetic engineering technology is in the area of
producing resistance to parasites. It should soon be possible to clone a gene conferring
resistance and transform it into the genome of a susceptible host. It is widely assumed that
resistance will be produced by identifying and isolating resistance-conferring genes within the
genomes of resistant hosts and transferring the gene to the plant or animal of interest.

This approach may prove effective but has several distinct disadvantages.
Resistant forms of the host may not exist, or may be very difficult to find for each new race of
parasite which arises. Such resistance may be polygenic, making the cloning and transfer of
the resistance genes difficult. If resistance is encoded by a single gene, the parasite is likely
to have evolved strategies for overcoming such host-derived resistances in a gene-for-gene
fashion.
Finally, the problem of identifying and isolating the resistance gene from within the large
genome of the host will generally remain very difficult. We propose an alternative strategy
which addresses these problems.
395
0022-5193/85/060395+11 $03.00/0 OR 1985 Academic Press Inc. (London) Ltd
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396 J c. SANFORD AND SA JOHNSTON

The Concept of Parasite-derived Resistance

The concept of parasite-derived resistance is that host resistance to a particular
parasite would best be engineered by introducing a gene of the pathogen into the host.
This approach is based upon the fact that in any parasite-host interaction, there are
certain parasite-encoded cellular functions which are essential to the parasite but not
to the host. These functions represent the "Achilles' heel" of the parasite. If one of these
functions is disrupted, the parasitic process should be stopped. Such essential functions,
which are under the control of the parasite's genes, might be disrupted by the presence
of a corresponding gene product in the host which is (1) dysfunctional; (2) in excess; or
(3) appears at the wrong developmental stage in the parasite's life cycle. If such "faulty
signals" were designed specifically for parasitic cell functions, they should have little
effect on the host. Therefore, resistance to a particular pathogen could be achieved by
cloning the appropriate parasite gene, if necessary modifying its expression, and
transforming it into the host genome.

This approach to engineering resistance has important advantages.

(1) The source of resistance genes would never be in question, since each
parasite would bring with it the genes necessary for deriving resistance.
(2) The stability of parasite-derived resistance should generally be greater than the
stability of simply-inherited forms of host resistance, for reasons that will be made clear.

(3) The difficulties involved in cloning genes from host organisms, which generally
have larger genomes relative to their pathogens, would be avoided.
(4) Parasite-derived resistance should have a minimal effect on the host, and should
not produce harmful substances to man.
As an example of how disease resistance might be engineered by this approach, we
discuss below how the genes of the bacteriophage, QB, could be used to make E. coli
resistant to QB infection.

The QB Bacteriophage as a Model

Viruses are a desirable model system for understanding host-parasite relations, owing
to their simplicity and accessibility to the molecular biologist. Because viruses are very
efficient in terms of utilizing the host's genes and cellular machinery, all viral genes tend
to be essential and unique to pathogenic functions. This means that nearly any viral
gene may be a suitable target for the genetic engineering of pathogen-derived resistance.

The QB bacteriophage is one of the best understood viruses and will serve to elucidate
how pathogen-derived resistance might be obtained. The biology of QB and other RNA
phages has been extensively documented
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CONCEPT OF PARASITE-DERIVED RESISTANCE 397

(Zinder, 1975), and the cDNA sequence of its genome has been determined.
The QB genome has only three basic cistrons. These code for a maturation protein
(involved in synthesis and phage binding to host pili), a coat protein, and a subunit of
the replicase enzyme. (A fourth gene product is a minor coat protein which is a read-
through product of the coat cistron.)
The life cycle of QB is basically as follows: the phage begins by binding to the sex
pili of F' E. coli, through which it enters the cell and begins to translate its replicase
subunit. Its replicase subunit polymerizes with three host subunits normally involved
in host protein translation. The resulting hybrid tetrameric enzyme has RNA replicase
activity specific for QB. This specificity is due to the affinity between the QB subunit of
the tetrameric replicase and a short segment of the QB genome within the replicase
cistron.
The replicase attaches to QB RNA at this binding site and replicates the viral RNA.
Late in the life cycle of QB, coat protein and maturation protein accumulate in the host.
The coat protein then binds to the replicase cistron, and thereby represses translation
of the replicase subunit. Termination of replication allows viral assembly and eventually
the maturation protein lyses the host, releasing a new population of infective QB.

From a conventional perspective, the life cycle of QB suggests two potential

mechanisms for developing resistance. Host-derived resistance might be developed
by: (1) blocking QB binding to sex pili; (2) producing variant host subunits lacking
affinity for the QB replicase subunit. Blocking QB binding is, in fact, a known mechanism
for producing QB resistance-- since non-F' mutants lacking pili are immune to infection
(Silverman et al., 1968). However, this strategy clearly disrupts a mechanism which is
relevant to the host's fitness as a species. The selection of variant forms of the host
subunits which help make up the replicase enzyme may also be a naturally occurring
mechanism conferring resistance. Since the host supplies three of the four subunits of
the viral replicase, one might expect mutations within these genes to confer resistance.
However, the extent to which these host subunits can be altered is clearly limited,
since these subunits are essential to host protein synthesis and the survival of the
host. Most of the variants of these host subunits would probably be lethal or sublethal
for the host.

Even non-lethal variants are likely to be suboptimal for protein translation efficiency.
Therefore, both of the host-derived resistance mechanisms suggested by the QB life
cycle would be obtained at the expense of disrupting crucial host functions.

The prospect of being able to transfer genes from parasite to host allows us to
approach resistance from an entirely new angle. Viewed from this perspective, the life
cycle of QB suggests at least as many mechanisms of pathogen-derived resistance as
host-derived resistance. Several strategies
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398 JC SANFORD AND SA JOHNSTON

seem promising: (1) deriving resistance from the QB coat protein; (2) deriving
resistance from a modified QB replicase; (3) deriving resistance by cloning the QB
replicase binding site; and (4) deriving resistance from expression of anti-sense strand
RNA sequences. Another strategy involving the maturation protein also appears
feasible.

RESISTANCE DERIVED FROM THE COAT PROTEIN

The QB coat protein is known to have a regulatory, as well as a structural role. Late
in the phage life cycle, coat protein binds to and represses the cistron coding for the
QB replicase subunit--stopping replication and allowing viral assembly (Bernardi &
Spahr, 1972). If cDNA to the coat protein translational sequence were linked to an E.
coli promoter and introduced into E. coli, the coat protein would be produced in the
host. In theory, expression of coat protein in the host should repress replication of any
infecting QB, thereby conferring resistance on the transformed host.

There is already precedence for a similar regulatory mechanism producing resistance,

since it was noted that overproduced A phage cro and repressor proteins make the E.
coli host resistant to infection by that phage (Flashman, 1978; Roberts, Kacich &
Ptashne, 1979).

RESISTANCE DERIVED FROM A MODIFIED REPLICASE

The QB replicase subunit has a dual affinity for a segment of the QB genome and
the three host replicase subunits (Kamen, 1970; Meyer, Webster & Weissmann, 1981).
If the QB replicase gene were cloned (as cDNA) and mutagenized, some variant forms
may be able to bind to the QB replicase site, and at the same time fail to polymerize
with the host subunits--a requirement to form a functional replicase. Alternatively, a
portion of the replicase gene could be cloned which produced a polypeptide containing
the functional domain for binding the replicase site but did not interact with the host
subunits. A transformed host producing such a modified replicase subunit may well be
QB-resistant if the modified QB replicase subunit or a portion of it could bind to the
replication sites of infecting QB and effectively compete with native QB replicase for
binding sites, thus disrupting QB replication .

RESISTANCE DERIVED FROM CONED REPLICASE BINDING-SITE

The above-mentioned replicase binds to a specific segment of the QB genome

which is roughly 100 base pairs in length. If this segment were cloned (cDNA) and
introduced into the host, it could be transcribed constitutively as mRNA if attached to
the appropriate promoter. The trans-
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CONCEPT OF PARASITE-DERIVED RESISTANCE 399

formed host might then be resistant to QB because the binding site, which has been
shown to compete for binding of the replicase enzyme in vitro (Meyer et al., 1976),
could limit the free replicase available for QB repli-cation.

ANTI-SENSE STRAND INTERFERENCE

The presence of an RNA complementary to QB RNA might allow forma-tion of an

RNA-RNA duplex which would block QB infection. This could be accomplished, for
example, by transcribing a cDNA clone of a portion of QB in the reverse orientation in
the E. coli host. The anti-sense strand RNA produced might then hybridize to the
infecting QB and interfere with its proper translation or packaging. Recently, Mizuno,
Chou & Inouye (1984) have shown that complementary RNA transcripts may regulate
ompF RNA translation in E. coli and Izant & Weintraub (1984) have shown that anti-
sense thymidine kinase RNA overproduced in mouse cells can inhibit thymidine
kinase expression . Both these reports suggest that using anti-sense RNA to block
viral infection is feasible. The advantages of this approach are that potentially any
fragment of the viral genome could be used without modification and it would be
extremely difficult for the virus to overcome this form of resistance.

RESISTANCE DERIVED FROM QB MATURATION PROTEIN

Although the maturation protein's mode of action is not yet well under-stood
(Karnik & Billeter, 1983; Winter & Gold, 1983), it might also be a potential source of
pathogen-derived resistance. It is conceivable that a modified maturation protein in
the host might block synthesis. Alternatively, a repressed operon containing a wild-
type maturation gene might be engineered in the host which would be activated by
QB infection. This would induce premature synthesis of a host cell upon initial infection
by QB, constituting on the population level a form of hypersensitivity.

Each of the specific mechanisms proposed above, may or may not prove effective
in practice. The point is that each of the three major cistrons of the QB genome, as
well as a fourth non-cistronic region, could conceivably be used for engineering
parasite-derived resistance. Clearly, this concept has rich possibilities.

Cross Protection--a Known Case of Pathogen-derived Resistance

It is rare for a useful strategy to be conceived of by man which is not already

operational somewhere in nature. It is well documented that closely
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400 JC SANFORD AND SA JOHNSTON

Related plant viruses, or different strains of the same virus, will cross-protect
(Hamilton, 1980). That is, an infected plant will not be subject to superinfection by
a second strain of that virus or by a related virus. In animals this resistance to
secondary infection is usually conditioned by a specific immune response to related
proteins encoded by the two viruses or by a generalized protection from interferon.
However, in plants, lacking as far as is known both antibodies and interferon, cross-
protection may result from a product of the primary-infecting virus directly interfering
with the propagation of the second virus. For example, the replicase of the primary
virus may bind to the replicase-binding site of the second virus, preventing its
replication (Gibbs, 1969). In contrast, in animals the protection arises from the
antigenic response to a product of the viral genome, although in at least one
instance a direct interference has been proposed (Marcus & Zuckerbraun, 1969,
1970).

Regardless of the specific mechanism underlying cross-protection it is likely in

plants and possible in animals that a direct form of parasite-derived resistance
occurs, as opposed to a resistance mediated through a reaction to a parasite via
antibodies or interferon. The mechanism postulated above suggests that the
resistance derives from the primary-infecting virus supplying the host with a
replicase which, through evolution, has been mutated and modified relative to the
second strain or related virus. While the replicase of the primary-infecting virus is
similar enough to the second virus' replicase to bind to its replicase attachment site,
it is sufficiently different to prevent functional replication. This would not only explain
why related plant viruses cross-protect, but also would explain why unrelated viruses
fail to cross-protect. As viruses diverge evolutionarily, they should develop new
replicase binding sites which are unrecognized by each other's "foreign" replicases.

If any cases of cross-protection result from such a mechanism, then a mutant form
of the virus' own replicase introduced into the host could produce the same result--
as we have proposed with QB.
Cross-protection is not the only example of naturally-occurring parasite-derived
resistance. Other examples include lysogenic bacteria bearing pro-phage (immune
to infection by vegetative phage), and transformed crown gall tissue in plants
(immune to superinfection by another strain of Agrobac-terium).

Potential Applications of Parasite-derived Resistance

VIRUS RESISTANCE

The most likely early application of the concept of parasite-derived resistance

would be in engineering plant virus resistance. This is because
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CONCEPT OF PARASITE-DERIVED RESISTANCE 401

the viral genome is small and, since viruses only propagate in the host, most
of the genome is involved in pathenogenicity. It should be possible in many
cases to clone portions of the viral genome and determine their potential for
conferring resistance. Alternatively, resistance-conferring genes might be
discovered empirically, by testing the biological effect of various DNA
restriction fragments of the viral genome. Most virus-derived resistances are
likely to involve a block in replication. While there has been some controversy
regarding whether plant viruses encode their own replicase, it seems likely
that most plant viruses do code for all or part of their replicases (Hall, Miller
& Bujarski, 1982; Dorssers et al., 1983 ) . The first plant virus to have its
replication mechanism characterized, turnip yellows mosaic virus, has proven
analogous to QB (Mouches, Candresse & Bove, 1984). This virus has been
shown to have a hybrid replicase, with its own subunit conferring specific
binding to its genome. This indicates that the approach described for QB
replicase should also apply to this virus. In our opinion, most or all RNA plant
viruses will code either for their own replicase, a subunit of the replicase, or a
protein modifying the specificity of the host's RNA polymerase. This means
that the replicase-derived resistance strategy outlined for QB may be directly
applicable to a wide range of plant viruses.
Regardless of the specific replicase mechanism of a plant virus, the general
concept of parasite-derived resistance should remain generally applicable.

NON-VIRAL RESISTANCE

The application of parasite-derived resistance to extracellular parasites will

be more complex than for viral parasites. Since "false signals" coded for by
the host must be recognized by the parasite, parasite-derived resistance will
only be useful where mechanisms exist which allow recognition or incorporation
of non-degraded macromolecules between the parasite and host. Van der
Plank (1978) has offered persuasive theoretical arguments for believing that
such an exchange of macromolecules between the host and the parasite
often occurs. There is at least one case where such incorporation has been
documented--in the malaria host/parasite system the parasite has been
shown to incorporate and utilize a host dismutase enzyme, indicating the
presence of a protein exchange mechanism (Fairfield, Meshnick & Eaton,
1983). The extent to which such mechanisms exist in other non-viral host/
parasite relationships needs to be determined.
Given a macromolecular exchange mechanism, a variety of approaches to
the engineering of parasite-derived resistance can be envisioned for either
viral or non-viral parasites. For example, in gene-for-gene host/parasite
systems (common in viral, fungal, and bacterial pathogens), it is generally
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402 Jc SANFORD AND SA JOHNSTON

found that the parasite's avirulence alleles are dominant to virulence alleles
(reviewed in Van der Plank, 1978). This suggests that the avirulence gene
products somehow override or block the activity of the virulence gene products--
thereby preventing infection. Thus, an avirulence allele from an avirulent strain
of the parasite, if expressed constitutively in a transformed host might enter the
parasite or act at the host-parasite interphase and override the infective capacity
of an otherwise virulent pathogen. In this way a parasite's avirulence gene
might be used to confer wide-spectrum resistance to the host. If this were
validated, it would introduce an entirely new dimension to the classical model
of gene-for-gene host/parasite interactions (Flor, 1971).

RESISTANCE FROM THE PARASITE'S REGULATORY GENES

A more general strategy for engineering parasite-derived resistance (appli-

cable with or without gene-for-gene interactions), would use specific regulatory
genes from the parasite. For example, fungal genes regulating haustorial
development or sporulation might be introduced into a host, thereby disrupting
the normal life cycle of the fungal pathogen. This type of regulatory approach
appears particularly exciting in the engineering of insect resistance. For
example, all insects depend on the regulated biosyn-thesis of juvenile and
molting hormones for precise timing of molting, metamorphosis and reproduction.
It may be possible to incorporate into the host, genes from the insect pest
encoding the activities necessary to produce such insects' hormones, or insect
pheromones or neurotransmitters. The host producing such insect growth
regulators pheromones or transmitters would be resistant by virtue of disrupting
the behavior or life cycle of the insect pathogen. There are examples in nature
where plants seemed to have exploited such a strategy for resistance by
evolving genes producing analogs to, or biosynthetic antagonists of, insect
hormones (Bowers, 1980).

Another potential application of parasite-derived resistance is where an insect

or other organism serves as an intermediate host, so that the disease cycle
could be disrupted by making the intermediate host resistant to the pathogen.
For example, efforts to control malaria have focused on eradication of the
intermediate host, the Anopheles mosquito. If, however, genes from the
Plasmodium pathogen could be introduced into the mosquito which conferred
resistance by disrupting the life cycle of the parasite, the disease cycle could
be broken. This approach would be most feasible if the resistance genes were
of selective advantage to the intermediate host, allowing resistance genes to
be maintained and propagated in natural populations after introduction of
modified individuals.
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CONCEPT OF PARASITE-DERIVED RESISTANCE 403

Advantages and Limits of Pathogen-derived Resistance

Parasite-derived resistance represents a systematic and generally-relevant approach

to the problem of how to genetically engineer insect and disease resistance. The rich
possibilities of this approach are illustrated by the fact that three different strategies for
deriving resistance from the QB bac-teriophage appear promising, in a parasite having
only three genes. There appear to be several distinct advantages of parasite-derived
resistance.
One of the most attractive features of parasite-derived resistance is that each new
parasite or race of parasite that becomes a problem simultaneously brings with it the
specific genes needed to engineer resistance to itself. These genes can be systematically
identified within the parasite's genome. Once such genes have been identified,
homologous genes in other parasite races or in related parasites will be readily
identifiable by DNA hybridization techniques. This eliminates the need for repeated and
exhaustive searches through the host's germplasm pools, seeking rare host resistance
genes.
Another major advantage of this strategy is that it should not generally be disruptive
of host functions. Van der Plank (1978), using evolutionary arguments and population
genetics data, has argued that host genes controlling "susceptibility" exist because
they involve essential host functions. Most hosts are genetically susceptible because
the "susceptible" allele is optimal relative to its natural function. Host-derived resistance
alleles, therefore, tend to disrupt the optimal functioning of the host. To the extent that
this is true, most host-derived resistances attack the pathogen indirectly by replacing an
optimal host gene product with a non-optimal host gene product which happens to be
incompatible with the parasite. This is seen in the QB system, where host-derived
resistance is likely to be achieved either by disrupting sex pili formation or by tampering
with the host's protein syn-thesis machinery. The beauty of the concept of pathogen-
derived resistance is that only pathogenic cell functions are attacked and are attacked
directly, which should have minimal subsequent effect on the host. The specificity of
parasite-derived resistance is not only desirable in terms of being non-disruptive to the
host, but also by being non-harmful to man. Resistance based upon production of
general toxicants, such as the "natural pesticides" of many resistant plant taxa, have
been shown to be potentially harmful to man when ingested (Ames, 1983). The
specificity of parasite-derived resistance should preclude, to a large extent, any such
harm to man.

There are reasons to believe that parasite-derived resistance should be relatively

durable compared to host-derived resistance. The ability of para-sites to circumvent
host-generated general toxicants is well known. Addi-tionally, specific host-derived
resistance genes are frequently overcome by
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404 JC SANFORD AND SA JOHNSTON

matching gene-for-gene mutations to virulence in the parasite (Flor, 1971).

In the case of host-derived QB resistance, alterations in the host replicase subunits
(making them incompatible with the viral subunit, thereby conferring resistance),
could easily be matched by mutations in the QB replicase subunit, which would
restore subunit compatibility, constituting a mutation to virulence. However, such
gene-for-gene mutations circumventing resistance should be relatively rare in the
case of parasite-derived resistance. In this case the parasite would usually be facing
a new form of resistance, which it had not previously faced in its evolution. These
types of resistances are likely to be very difficult for the parasite to overcome,
especially where regulatory genes are involved. For example, if resistance to QB
was derived from the QB coat protein gene, a new virulent QB strain could only
arise by first having a new binding site developed by mutation in the replicase
cistron (without disrupting replicase function) which would not bind the native coat
protein. Simultaneously a new coat protein would have to arise by mutation (without
disrupting coat protein function) which would bind to the new binding site. Such a
simultaneous and complementary set of mutations (which preserved both coat and
replicase functions) should be extremely rare.

Last, engineering parasite-derived resistance should be considerably more

approachable on the molecular level than engineering host-derived resistance-
ance. There are numerous reasons for this. (1) This strategy would generally focus
on the molecular biology of relatively simple organisms with short life cycles. (2) It
would generally require only the identification and isolation of individual genes from
small genomes. (3) Unregulated, constitutive expression of the parasite-derived
resistance genes would usually be effec-tive. (4) It would avoid the complex,
multigenic biosynthetic pathways which are the likely basis of many existing host-
derived resistances.
There do not seem to be any obvious disadvantages to the parasite-derived
approach to resistance, except that application of the strategy to non-virus parasites
is only possible where mechanisms exist for macromolecular exchange between
host and parasite. It has yet to be determined how pervasive such mechanisms
may be. In our opinion, most forms of parasit-ism, especially those forms displaying
gene-for-gene resistance, allow ample opportunity for gene product interactions and
should be suitable for engineering parasite-derived resistance.

We thank Dave Soderland, Bill Bowers, Mary Nijhout, Sharon Endow, Nick Gilham
and Sally Schuette for discussions and critical readings. We also thank Debbie
Gooch for typing the manuscript.
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CONCEPT OF PARASITE-DERIVED RESISTANCE 405

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