Group 3. Molecular Plant-Pest Interactions

C. EXPERIMENTAL PLAN

1. Molecular basis of plant resistance to pests and pathogens. The molecular basis of plant defense against insects and pathogens in Brassica and Arabidopsis is being studied in the Mitchell-Olds lab. Highly replicated quantitative genetic experiments were conducted on more than 9,500 plants. Selection for resistance to fungal pathogens (Leptosphaeria, Oomycetes; Peronospora, Oomycetes) showed genetic variation for generalized plant defense. Selecting for resistance to one pathogen brought correlated increases in resistance to the other pathogen, and vice versa, indicating positive genetic correlations among levels of disease resistance (Mitchell-Olds et al. 1993a; 1993b; 1993c). Genetic variation for generalized plant defense also extends to insect resistance: selection for disease resistance caused significant increases in insect resistance (p < 0.001 by ANOVA, Siemens and Mitchell-Olds, unpublished). Disease resistance genes are now being mapped by bulk segregation analysis (Michelmore et al. 1992) in a cross between resistant and susceptible genotypes of Brassica rapa. This approach is fast and efficient: in a comparable study of flowering time in Arabidopsis, it took 1 person less than two weeks to screen 120 RAPD primers (Williams et al. 1990) and identify six markers linked to flowering loci (Mitchell-Olds, unpublished). The Mitchell-Olds laboratory will map and study genes causing resistance to diamondback moth in B. rapa using bulk segregation analysis and RAPD markers. After linked markers are identified, they will be converted to single locus codominant PCR-RFLPs by cloning and end-sequencing polymorphic bands, designing pairs of locus-specific primers, and screening polymorphic restriction sites within PCR products (Mitchell-Olds, unpublished). This permits intensive physiological and resistance studies of plant defense genes, and marker assisted selection to manipulate generalized plant defense. The effects of insect resistance genes on disease resistance and physiological defense pathways will be characterized. Effects of resistant and susceptible genotypes and induced defenses will be studied on activities of proteinase inhibitors, myrosinase, lignin biosynthetic enzymes, and general phenylpropanoid and shikimic acid pathway enzymes. Comparable studies of fungal resistance are ongoing in the Mitchell-Olds lab, with recent support from NSF and USDA-NRI.

2. Pest resistance using pest-derived genes. Three members of the group are investigating the use of compounds produced by a pathogen or pest in order to control that organism. Anderson's lab is investigating replicase-mediated resistance to barley yellow dwarf virus in barley. Sherwood's lab is analyzing the potential for controlling fungal pathogens by interrupting their sexual cycle with fungal pheromones. McCoy's lab is transforming alfalfa with insect-derived protease inhibitors in order to discourage feeding on the plant by insect pests.

  • a. Viral replicase-mediated resistance. Barley yellow dwarf virus (BYDV) strains infect a very wide range of monocotyledonous hosts and are collectively regarded as the most economically important viral pathogens of small grain cereals world-wide. Traditional plant-breeding strategies have been quite successful in obtaining genetic resistance to many viral plant pathogens. However, this approach has not been successful in obtaining resistance to the various strains of BYDV because of the lack of host resistance genes which are effective against all BYDV strains and the year to year variation in the proportions of BYDV strains. Molecular biology offers an alternative approach to these traditional control methodologies. The most completely explored molecular biology strategy has been to obtain transgenic plants containing viral coat protein genes (Beachy et al. 1990). This approach has at least two potential shortcomings: a different coat protein gene may be required for each BYDV type and, historically coat protein-mediated resistance does not give rise to completely resistant plants. Recently, virus resistant plants have been obtained by expressing a portion of the viral encoded replicase (Anderson et al. 1992). The resulting plants were completely resistant to virus infection. This replicase-mediated resistance has been shown to be effective against the four viruses to which it has been applied, i.e. cucumber mosaic virus (Anderson et al. 1992), potato virus X (Braun and Hemenway 1992), tobacco mosaic virus (Golemboski et al. 1990), and pea early browning virus (MacFarlane and Davies 1992). These results suggest that this replicase-mediated virus resistance can be effective against a wide range of plant viruses including BYDV. Consequently, this is the strategy that will be used to engineer resistance to BYDV in barley.

    • The objectives of this research are to:

    1) construct vectors in which cDNA clones of the polymerase
genes of the two major groups of BYDV are fused to the 35S
cauliflower mosaic promoter in single or separate constructs,
and
 2) transform barley with these replicase genes. The
transgenic plants will be analyzed for resistance or
susceptibility to virus inoculation.  BYDV polymerase gene cDNA
clones representing the two groups of BYDV strains will either be
obtained from other researchers or will be cloned.  These cDNA
clones will be subcloned into a plant expression vector
containing the 35S CaMV promoter and the NOS terminator.  The
second component to this work involves transforming barley with
these resistance-inducing constructs.  Because transformation of
barley is not yet commonplace, we will first determine if
resistance is effective at the protoplast level. Resistance to
TMV is expressed in tobacco protoplasts in which the resistance
encoding gene and viral RNA were electroporated into the
protoplasts (Carr and Zaitlin 1991). Once the vectors have been
shown to be effective in protoplasts, methods being developed for
barley transformation will be attempted. These will include
injecting DNA into tillers just above the developing
inflorescence (Rogers and Rogers 1992), particle bombardment,
protoplast electroporation and regeneration, and PEG-based
transformation protocols.

  • b. Disruption of the fungal disease cycle using pheromones. An alternate strategy for control of pests which has not yet been used with fungi is the disruption of a critical stage in the development of that pathogen, rendering it incapable of completing its life cycle. This strategy has been used with great success with insect pests using sex pheromones. As with insects, one potential "Achilles heel" in the disease cycle of many fungi is the sexual cycle. Fungal sexual spores often allow the long term survival of the pathogen, and mating increases genetic variability. The sexual cycle is particularly important in the disease cycle of the smut-causing fungi, such as Ustilago hordei, (Thomas 1991). Mating in U. hordei, which is controlled by a single bipolar mating locus, results in the conversion of haploid, nonpathogenic, yeast-like sporidia into pathogenic, dikaryotic mycelium (Thomas 1991). The Sherwood lab has found that U. hordei synthesizes small molecular weight pheromones which act as signals to prepare sporidia of the opposite mating type for mating. It is possible that the U. hordei pheromones, if presented to the fungus at an inappropriate time or concentration, will disrupt mating, and thus pathogenicity, of this pathogen. The U. hordei pheromones are being purified and characterized using methodologies similar to those described for the Saccharomyces cerevisiae mating factors (Anderegg et al. 1988). Concurrently, the genes for the pheromone and their receptors are being cloned using the a locus genes from U. maydis, which appear to encode pheromones in that related fungus (Bolker et al. 1992). These results will help define the nature of the mating factors and the regulation of the mating process. The effects on mating and pathogenicity of adding excess exogenous pheromone, which inhibits mating in S. cerevisiae (Marcus et al. 1991), to sporidia will be determined.

  • c. Pest resistance using protease inhibitor genes. Transfer of plant protease inhibitor genes to transgenic plants has been shown to be an effective means of controlling some insects (Hilder et al. 1987; Johnson et al. 1989). As an alternative to proteinase inhibitors of plant origin the McCoy lab has been working with a family of proteinase inhibitors isolated from the tobacco hornworm (Manduca sexta) by Kanost et al. (1989). Using Agrobacterium-mediated gene transfer, hundreds of transgenic alfalfa plants that express the protease inhibitor genes from the tobacco hornworm have been produced, and preliminary results demonstrated an effect on at least one insect pest of alfalfa (Thomas et al. 1992). Current research objectives include an in-depth analysis of expression of these genes throughout the growth cycle of alfalfa. We are testing the effects of promoter sequences on expression of these protease inhibitors in transgenic alfalfa, and we will be examining alternative constructions for increasing protease inhibitor concentration in the plant. In addition, extensive evaluations of insect resistance will be conducted, in order to determine the feasibility of this approach in forage production system.

    • 3. Pest control using non-pest derived genes. Another approach for the control of plant pests is the use of non-pest derived genes to provide the plant with a trait which will allow it to preferentially survive its competitors. This strategy is being used by the Dyer lab, who have developed a regeneration and transformation procedures for domestic safflower cultivars (Ying et al. 1992). Transformation experiments have been carried out using several combinations of Agrobacterium tumefaciens strains and Ti plasmid constructions. Transgenic shoots have been shown to contain one or both transgenes, NPT II and - glucuronidase, by growth on selective media, PCR assay, and Southern hybridization. A major constraint to safflower production is the lack of effective weed control. The introduction into safflower of the gene conferring resistance to glufosinate, a nonselective environmentally safe herbicide with excellent weed control properties would allow control of all competing weeds. Because glufosinate is quickly metabolized by soil microorganisms, substitution of this compound for herbicides now used will reduce environmental pesticide load. Resistance to glufosinate is mediated by the bar gene product, an acetyltransferase that acetylates and thereby inactivates the herbicide. The bar gene from Streptomyces hygroscopicus has been fused to a high level expression promotor and introduced into sugar beet, tobacco, tomato, potato, oilseed rape, alfalfa, and poplar (De Block et al. 1987). The resulting transgenic plants were physiologically normal in all respects and highly resistant to glufosinate (Botterman et al. 1991). Ti plasmids containing the bar gene under the control of several plant promoters are currently being constructed. Transformation of these constructions into safflower explants will allow the selection and regeneration of glufosinate-resistant plants.