Svetlana Oard and Zhi-Yuan Chen

Every crop can be damaged by many different diseases and pests throughout its life cycle. In common practice, growers apply pesticides to control different pathogens and insect pests and protect a crop from all possible diseases and pests. This entails enormous economic and environmental costs. Developing broad disease resistance in plants instead of developing resistance for individual pathogens can save time and resources. This is why for the past several years LSU AgCenter researchers have been using biotechnology to find ways to control various crop diseases, such as Asian soybean rust and cercospora leaf blight. Both diseases, especially the former, have the potential to cause severe soybean yield losses in the southern United States.

Thionins are basic plant peptides with broad-spectrum, anti-microbial activity. Enhancing thionin production and accumulation in plants has potential to promote broad disease resistance against bacterial and fungal pathogens. Thionins are excellent candidates for developing a broad-range plant defense system. They exhibit broad activity against bacteria and fungi at low concentrations and have the ability to rapidly damage microbial cells. Seed thionins are abundant in wheat and barley flour and are safe for human consumption. Introducing antimicrobial peptides through plant transformation could offer a solution for creating crops with targeted resistance to a wide range of bacterial and fungal pathogens.

Developing antimicrobial and especially antifungal resistance is a challeng   ing task that requires effective levels of thionin production and a delivery system to ensure thionin accumulation where pathogens enter a plant. LSU AgCenter researchers have addressed several challenges in developing a strategy for transgenic expression of thionins. These include producing edible, seed-derived thionins in leaf tissues; expressing a highly effective peptide without damaging plant cells; and reaching levels of activity in plant leaf tissues sufficient for inhibiting bacterial as well as fungal pathogens.

The result of this work was a hybrid signal peptide and an improved thionin gene that provides antimicrobial resistance in leaf tissue. Successful production of biologically active, seed-derived thionin in leaf tissues requires a specific signal peptide that has three main functions: to protect plant cells from the harmful activity of thionin, to excrete thionin outside the plant cell, and to deliver it to the plant cell wall where thionin awaits for pathogenic microbes. The hybrid signal peptide does it all, providing antimicrobial resistance in plant leaf tissue. Up to 60 percent increase in antifungal resistance was demonstrated in a true-breeding line of a model plant – Arabidopsis – that was charged with the fungus Fusarium oxysporum.

The Asian soybean rust pathogen is capable of devastating soybean fields. Currently, no commercial soybean varieties in the United States have genetic resistance to this pathogen. Introducing the new thionin gene could protect soybeans from Asian soybean rust as well as from other fungal and bacterial pathogens. A successful transformation vector to introduce the seed-derived thionin into soybeans demonstrated high levels of transgene production. The next step is to use the vector for a formal soybean transformation experiment to select a line with high levels of production and test it for antimicrobial activity. The created gene has been given to the U.S. Department of Agriculture for additional testing in tobacco and cotton to demonstrate broad-range resistance.
 
In addition to transgenic approaches, researchers have been using proteomics, a state-of-the-art technique to study the full set of proteins in a plant to find soybean proteins either highly present in disease-resistant soybean varieties or are induced upon pathogen invasion. Some of these differentially produced proteins are believed to play important roles in enhancing host resistance to various pathogens. This approach is more cost- effective and less time-consuming than the traditional plant-breeding approach in identifying candidate genes to enhance host resistance.

Several such proteins have been identified in soybeans infected by rust. The importance of these proteins in host resistance to Asian soybean rust, however, needs to be first verified.

The normal approach is to either over-produce the target protein or silence it through genetic engineering and soybean transformation, which can take two to three years. Instead, researchers are taking a novel approach called virus-induced gene silencing to reduce the production of these proteins one at a time so their importance in host disease resistance can be determined in a matter of several months. Through collaboration with scientists at Iowa State University, several candidate genes have been inserted into a mild version of bean pod mottle virus. These modified, nonpathogenic viruses will be introduced into soybean plants in a controlled growth chamber in the laboratory to see whether reducing the production of candidate proteins can cause a significant increase in susceptibility to fungal pathogens. If the answer is yes, it indicates this particular protein plays a critical role in soybean resistance to the fungal infection. Then, the production of this candidate protein can be increased through breeding or genetic engineering to enhance soybean resistance to these pathogens.

Researchers also are applying biotechnology to combat Cercospora leaf blight disease. They have developed specific DNA probes that can reliably detect the presence of the cercospora pathogen in field-grown soybean plants one to two months before the disease symptoms appear. This procedure is called real-time polymerase chain reaction, and it can detect the presence of target pathogen DNA with extreme sensitivity. This early detection method has allowed LSU AgCenter plant pathologists to develop a more effective fungicide regimen to control leaf blight disease by following the increase of fungal DNA after the treatments.

In addition, researchers are exploring ways to make fungal pathogens less aggressive on soybeans, such as by reducing cercosporin toxin production through targeted gene disruption or reducing pathogen growth through "transgene silencing," a phenomenon that has only been discovered in a few plant systems.

Although all these studies are still in their early trial stages, the knowledge generated from these studies can provide soybean growers with more effective and environmentally friendly strategies to control diseases in soybeans and other crops.

Svetlana Oard, Associate Professor, School of Plant, Environmental & Soil Sciences, LSU AgCenter, Baton Rouge, La., and Zhi-Yuan Chen, Associate Professor, Department of Plant Pathology & Crop Physiology, LSU AgCenter, Baton Rouge, La.

(This article was published in the spring 2011 issue of Louisiana Agriculture.)