ENGINEERING BROAD-SPECTRUM DISEASE RESISTANCE
Santosh Misra
October, 2005

The global production of agricultural and food products is the world’s largest industry, with total revenues of ~$5 trillion (US) per year. Diseases caused by phytopathogens are economically important, resulting in multibillion-dollar losses annually, and it has been estimated that more than 25% of all crop plants worldwide are lost to fungal, bacterial, and viral diseases. In the developing world, up to 40% of all crop destruction can be directly attributed to plant diseases, with occasional catastrophic, life-threatening losses. The increased reliance on chemical solutions to diseases has compromised environmental quality, engendered a negative impact with consumers, and resulted in a rise of fungicide resistant microorganisms.

Conventional breeding, though relatively successful in the development of disease resistant plant cultivars, has become increasingly difficult because of the limitations of resistance genes within usable gene pools. However, advances in plant genetic engineering have facilitated the integration of beneficial "designer" genes into plants. This technology has been successfully applied to generate plant resistance to herbicides, some insects, and occasionally some phytopathogens. Strategies to improve the health of crops through genetic engineering have included transgenic expression of plant, fungal, or bacterial hydrolytic enzymes, pathogenesis-related proteins, components of plant defense-response pathways, antimicrobial proteins, and peptides. Although food safety is of utmost importance and disease resistant plants are required, the commercial application of this technology is lagging. The protection offered in some cases is limited to specific diseases, which means other pathogens can thrive and plants must still be sprayed with fungicides.

To address this problem, we have developed a platform methodology of protecting plants from a broad-spectrum of bacterial and fungal diseases. Work is in progress to extend this spectrum to viruses and insects. The technology is based on engineering antimicrobial peptides in plants. Because of their wide spectrum of antimicrobial activity and, at low concentrations, lack of toxicity to eukaryotic cells, the antimicrobial peptides represent promising candidates for transgenic application in plants.

Candidate antimicrobial peptides
Recently-discovered antimicrobial peptides are a class of potent, natural antibiotics found widely in nature from insects to plants and animals¹. Several related antimicrobial peptides have also been found in humans, where they are part of our early defense "innate" immune system. Depending on the source, these natural products vary greatly in their killing activity and spectrum, which extends from various types of bacteria to fungi, viruses, parasites, and even to such destructive pests as nematodes. The mechanism for killing microbial cells is multi-targeted and highly effective, and involves rapidly killing the microbe by drilling a molecular "hole" in its membrane, then inactivating and destroying its genome. Because the primary target of membrane-active, positively charged antimicrobial peptides is the cell membrane and not specific receptors or substrates, these peptides usually confer their activity against a broad spectrum of pathogenic microorganisms, and there is less probability of resistance arising by variation of its metabolic pathways.

Antimicrobial peptides of plant, insect, and amphibian origin have been expressed in transgenic plants, but only a few provide the target plants with any degree of broad-spectrum antimicrobial resistance. We have pioneered the development of unique, candidate target peptides (probiotics) with powerful broad-spectrum antimicrobial activities but with reduced cytotoxicity to plant and animal cells. This was achieved by using Insight II molecular modeling, and 2-D NMR structural determinations, combined with extensive screening against a variety of plant pathogens (collaboration: REW Hancock, UBC). These approaches have yielded three new target families of probiotics with fungal, bacterial, and viral disease resistance.

Shown below is a predicted model of temporin A and its modified analogue MsrA3². Using the Insight II (version 97.2) molecular modeling program, Homology (Molecular Simulations Inc., San Diego, USA), the temporin structure was drawn as an α-helix, based on the known α-helicity of temporin. The structure was then energy minimized using the Discover Program of Insight II.

Engineering select peptide / probiotics in plants
We used one of these peptides, MsrA1, to engineer broad-spectrum disease resistance in potato, including resistance to ‘late blight’, a disease of pandemic proportions². Potato is an ideal crop for the introduction of disease-resistance technology due to its severe and growing problems with bacterial, fungal, and viral diseases and accompanying storage losses.

The probiotic-enhanced potatoes demonstrated a phenomenal degree of broad-spectrum disease resistance to pathogens, including late blight and pink rot, and a variety of post-harvest pests. Potato tubers stored for over 27 months remain in nearly pristine condition when compared to unprotected tubers. These results have profound implications not only for diseases in the field but also with respect to the mitigation of storage crop losses (>$600B worldwide).

We have continued to test new peptides in potato and tobacco. Our recent data shows that MsrA3, a temporin analogue³, as well as MsrA2, derived from Dermaseptin⁴, and combinations thereof, provide a greater degree of disease resistance against a broad range of pathogens. When MsrA2 was used to inhibit the growth of agronomically important fungal pathogens, including Fusarium, Alternaria, Rhizoctonia, Phytophthora, and Pythium sp., its activity was far superior to the activities of other peptides tested. The estimated concentration of MsrA2 in leaf tissue was ~ 1 – 5 µg/g of fresh tissue, which, although not high, seems to be sufficient to protect the plants from the attack of pathogen(s)⁴. Constitutive expression of MsrA2 at this level is apparently non-toxic to transgenic plants, as no deleterious effects on the morphology or yield of plants and tubers could be seen.

A. Probiotic-expressing potato plant growing through mycelia of Fusarium sp.

B. Leaf derived from transgenic potato plant (right) expressing probiotic is resistant to P. infestans challenge. Leaf from control plant (left) is dead.

Regulated transgene expression, whereby a promoter is specifically activated in response to pathogen invasion or pest attack, has distinct advantages for genetic engineering disease/pest resistant traits in plants. We used a truncated 823 bp downstream fragment of the win3.12 promoter from poplar and showed that the win3.12 promoter-regulated expression of the antimicrobial peptide was sufficient to confer resistance against F. solani in transgenic tobacco.

This technology is considered a flexible platform, which can be extended to a host of economically important food and non-food crops. Equally important, plants can be used for cost-effective production of the peptides/probiotics destined for human pharmaceuticals and veterinary applications. We have extended this work to include canola, soybean, wheat, and even poplar, a model tree species.

Mycotoxins and food/feed safety
In addition to managing diseases, probiotic-enhanced plants can address the problem of food and feed safety. Phytopathogenic fungi not only cause a decrease in quantity and quality of crops, they often cause acute toxicity in animals and humans. Widely different genera of fungi (Fusarium, Penicillium, Aspergillus, Claviceps, Stachybotrys sp. etc.) produce mycotoxins in a wide variety of grains and foods. Mycotoxins are highly stable and cannot be destroyed by boiling, pressing, or processing, and infested produce has to be destroyed. Toxicological manifestations are both acute and chronic, such as cancer, immunosuppression, mutagenicity, and estrogenicity, and gastrointestinal, urogenital, vascular, renal, and nervous disorders. These mycotoxins can also be metabolized by animals fed contaminated grains and passed into milk, eggs, and other organs, thus reentering the food chain. Mycotoxin contamination is a worldwide problem affecting staple crops such as corn and small grains, as well as tree nuts, peanuts, sorghum, and many others. Maize, wheat, barley, and rice are high-risk commodities, and it is estimated that approximately 16,000 tons of maize, 123,000 tons of wheat and barley, and 12,000 tons of rice are affected by mycotoxins in southeast Asia alone. According to FAO estimates, world losses of foodstuffs due to mycotoxins are in the range of 1 B tons per year. One strategy to control mycotoxin levels is by controlling the growth of fungi.

We are testing the ability of our peptide probiotics to control Fusarium Head Blight (FHB) of wheat and barley caused by Fusarium graminearum. FHB has emerged as one of the most serious and damaging diseases of small grains. Trichothecenes are the virulence factors produced by the fungus. We are introducing probiotic genes into wheat in order to increase the FHB defense mechanism in wheat spikes and hence reduce or prevent the initial infection.

Summary
Our current growing reliance on antimicrobial chemicals remains unabated and is compromised due to the rise of fungicide and bactericide resistant microorganisms. We believe that the widespread incorporation of probiotic-enhanced plants and crops will make a large contribution to the general reduction in chemical solutions to disease control, increase yields, and reduce losses during storage. Furthermore, the antimicrobial peptides do not readily lead to microbial resistance and would help stem the rise of antibiotic and fungicide resistant microorganisms directly threatening human and animal health. The importance of feed or food microbial contamination in the industry is expected to increase with time. The emergence of pathogen strains resistant to conventional antibiotics and pesticides currently in use warrants application of new approaches to the containment of pathogenic microbes and to enhance food safety.

1. Hancock REW & Lehrer R (1998) Trends Biotechnol 16: 82–88

2. Osusky M et al. (2000) Nature Biotechnol 18: 1162–1166

3. Osusky M et al. (2004) Transgenic Research 13: 181-190

4. Osusky M et al. (2005) Theoretical & Applied Genetics DOI:10.1007/s00122005-2056-y

Santosh Misra
Department of Biochemistry & Microbiology
University of Victoria
smisra@uvic.ca