A MANDA C OTTAGE 1 AND P ETER U RWIN 2 **
15.5. Targeting the Nematode Feeding Site
15.5.1. Multiple component strategies and cell death systems
Multiple component strategies can be achieved in a variety of ways, a few of which are outlined in Table 15.1. Figure 15.2 illustrates the general principles that underlie three of the mechanisms.
15.5.1.1. Barnase and barstar (enzyme and inhibitor)
Barnase is an extracellular ribonuclease produced by Bacillus amyloliquefaciens and barstar is its intracellular inhibitor. The tacit assumption has been that barnase serves the soil bacterium by degrading RNA for nutritional use. However, it could act as a toxin for predators or competitors within the soil environment. The bacterium pro- tects itself from the highly effective barnase before export by producing the inhibitor barstar. The barnase–barstar combination was utilized in the early 1990s to achieve plant sterility (Mariani et al., 1992). A male sterile line is maintained in a hemizygous state, which is with only one copy of the barnase gene under the control of an anther- specific promoter. Barnase expression in the anther results in cell death and no pollen is produced. To restore male fertility, the hemizygous line is crossed with a line homozygous for the gene encoding barstar, which is also under the control of an anther-specific promoter. Within the F1 population, plants hemizygous for both barnase and barstar will be able to produce pollen due to the neutralizing effect of barstar.
A similar system was utilized in A. thaliana to engineer resistance to H. schachtii; by using a promoter with an expression profile mostly limited to the syncytia to express barnase and constitutive expression of barstar a 70% reduction in susceptibility was achieved (Ohl et al., 1997).
Clearly expression of barnase and not barstar within the nematode feeding cell and nowhere else in the plant is necessary. However, expression of barstar alone may be elsewhere in the plant. A promoter that is turned off when a nematode feeding cell differentiates would be useful for driving expression of barstar.
452A. Cottage and P. Urwin
Table 15.1. Summary of targets of engineered nematode resistance, possible strategies and expected outcomes.
Resistance mechanism Engineered resistance Example Outcome
Natural resistance
Resistance (R) gene Transfer of R-gene Mi from resistant to susceptible cultivar HR response R-gene mediated signalling Modification of signalling activator Hsp90 activation in response
to nematode species
HR response Avirulence factor (Avr) recognition Modification of R protein or
Avr interacting protein
Gpa2 responds to all RBP-1 variants HR response
Targeting early nematode parasitism
Hatching and attraction Plants produce chemical deterrents Over-production of N-formylloline in roots Nematodes avoid plants Penetration and migration Disruption of chemical cues utilised
by nematodes during migration
Modification of auxin influx/ efflux carrier expression in response to nematode invasion
Migration disrupted, poor establishment
Targeting the nematode
Intoxication Expression of Bt crystal (Cry) protein Constitutive or local expression of Bt gene Nematode death Immobilization Production of antibodies against
nematode proteins
Plantibodies that bind to nematode cuticle proteins
Migration disrupted, poor establishment Sensory disruption Expression of a lectin Nematode sensory disruption
due to binding of ConA to amphids
Failure to establish Disruption of digestion Expression of a protease inhibitor CpTI inhibits nematode serine proteases Poor growth/reduced
fecundity Inhibition of protein
production (RNAi)
Expression of dsRNA homologous to target transcripts
Targeted destruction of nematode major sperm protein (MSP) transcripts
Impaired viable egg production Targeting the nematode feeding site
Cell death agent and inhibitor Expression of barnase within feeding cell
Expression of barnase at feeding sites and barstar elsewhere
Feeding cell death/death of nematode
Inactive cell death agent and activator
Expression of active RIP within feeding cell
Expression of inactive PAP-S and activator (TEV 1a protease) within the feeding cell
Feeding cell death/death of nematode
Fig. 15.2. Three examples of binary systems to induce nematode feeding cell death.
A: An active component is produced from two inactive components when both are co- localized within the feeding cell (e.g. maize RIP in Section 15.5.1.2). B: An inactive precursor is activated by removal of a blocking group by an activator when both are co-localized within the feeding cell (e.g. PAP-S with blocking group in Section 15.5.1.2). C: An active component, within the feeding cell and elsewhere, is inactivated by an inhibitor when both are co-localized outside of the feeding cell (e.g. barnase–barstar in Section 15.5.1.1).
Root tip
Root tip
Root tip
A
B
C
Nematode Feeding site
Nematode
Feeding site
Nematode
Feeding site inactive component 1
inactive component 2 active component
inactive precursor
activator blocking group active component
inhibitor
inhibited component active component
15.5.1.2. Ribosome-inactivating proteins (binary action and activation systems)
Ribosome-inactivating proteins (RIPs) have been linked to plant defence as long ago as 1925 when inhibition of viral infection by an extract of pokeweed, Phytolacca americana, was demonstrated. RIPs are N-glycosidases that depurinate the conserved a-sarcin loop of large rRNAs, which leads to the inactivation of ribosomes resulting in blocked protein synthesis. The RIPs have great potential as cell killing agents as their ability to inhibit translation is extremely effective. RIPs are classified into three types (Box 15.2).
Type 3 maize RIP is synthesized as an inactive precursor (RIPa–[25 AAs]–
RIPa), which is cleaved to its active form by the removal of 25 central amino acids (Walsh et al., 1991). The feasibility of using the two RIP polypeptide chains as a
‘binary’ cell death system was investigated by quantifying reporter gene (uidA) activity in tobacco protoplasts (patent number: WO 02/33106). The uncleaved pro- tein (RIPa–[25 AAs]–RIPa) did not affect reporter gene product activity, while a recombinant RIP (RIPa–RIPa) (with the central, cleavable residues removed) resulted in loss of activity indicating loss of tobacco ribosomal function. Both poly- peptide chains were evaluated separately; RIPa showed no effect whilst RIPa resulted in some loss of reporter gene activity. Loss of activity was also observed if both chains were synthesized separately and then combined. In transgenic tobacco plants both the recombinant RIP and the two component RIP systems (RIPa and RIPa), under the control of promoters predominantly active in the root and/or the nematode feeding site, appeared to have some inhibitory effect on the development of female M. javanica as measured by nematode size. RIP PAP-S, the seed form of the pokeweed antiviral protein of P. americana, was also shown to be capable of inhibiting reporter gene product activity, attaching a blocking group to the N-terminus of PAP-S inactivated RIP activity which could be partially restored by cleaving off the blocking group with a tobacco etch virus (TEV) NIa protease, thus offering potential as a binary cell death system. The maize RIP and the PAP-S two- component systems appear feasible as binary cell death mechanisms but require further development and are likely to be dependent on the identification of promot- ers to enable sufficient and more specific expression (A. Neelam, C. Thomas and M.J. McPherson, unpublished data).
Box 15.2. Types of ribosome-inactivating proteins (RIPs).
Type 1 RIPs, such as the pokeweed antiviral protein (PAP), are monomeric enzymes and most plant RIPs fall into this category. They are highly effective against ribosomes in vitro but are poorly transported in biological systems.
Type 2 RIPs, like ricin and abrin, are highly toxic heterodimeric proteins as their enzymatic chain is linked to a lectin, facilitating uptake into the cell.
Type 3 RIPs are synthesized as inactive precursors (proRIPs) and require proteolytic processing for activation.