By Katharina Schoebi, Checkbiotech

Crown rust (Puccinia coronata) is the most serious foliar fungal disease in ryegrass species. A new genetically engineered Italian ryegrass (Lolium multiflorum Lam.) produces an enzyme that destroys the fungus and thereby increases resistance against crown rust disease.

Italian ryegrass is one of the most important forage grasses in temperate regions. The infection of the grass with the fungal pathogen crown rust results in decreased yields and lower grades of animals feed. That is why ryegrass breeding programs try to develop plant varieties that show high resistance to crown rust.

Conventional breeding of Italian ryegrass by selection proceeds very slowly, because the plant is self-infertile and thus, each cultivar has a diverse genetic background. Genetic engineering, in contrast, can be a promising tool for efficient breeding of ryegrass plantlets that are resistant to the fungus crown rust.

When plants are infected by a fungal pathogen, some of them produce an enzyme called chitinase. Chitinase hydrolyzes a structural component of the fungal cell wall, called chitin. When chitin is broken apart, the cell wall of the fungus – and thus the fungus, too - are destroyed. Furthermore, the small degradation products of chitin elicit the defense system of the plant. However, most of plant species do not contain a chitinase gene and are thus at the mercy of fungal pathogens.

By introducing the chitinase gene into tobacco, canola, strawberry, cucumber, rice, wheat, grape and cotton plants, their resistance to a wide range of fungal pathogens can be enhanced. “Thus, we hoped that the chitinase gene would also confer resistance to crown rust on Italian ryegrass as well,” explained Dr. Takahashi of the Forage Crop Research Institute in Higashiakada, Japan.

For this purpose, the researchers introduced the rice chitinase gene in Italian ryegrass varieties, which gave them the ability to produce the chitin-destroying enzyme. The evaluation of the experiment indicated that the transgenic plants exhibited higher chitinase activity than untransformed ryegrass plants.

However, detached leaves of both untransformed and transgenic leaves showed disease symptoms which gradually progressed. And even though detached leaves of the genetically modified plants showed a somewhat increased resistance to crown rust disease, after more than ten days following infection, the transgenic and nontransgenic ryegrass plants were indistinguishable. Thus, resistance to crown rust disease with Dr. Takahashi’s Italian ryegrass is only partial for now. Though chitinase prevented fungal growth, it does not completely hinder the invasion of an attacking pathogen into the host plant cell.

In the meantime, however, Dr. Takahashi and his colleagues have found some true resistance genes for crown rust disease. The researchers are now attempting to isolate these genes. “We expect that, in the future, we will be able to completely protect the ryegrass by introducing the true resistance genes in it,” Dr. Takahashi told Checkbiotech.

According to the researchers, the resistance against crown rust shown by transgenic ryegrass would not be strong enough to carry out greenhouse and field trials. “We will do such experiments not before having produced genetically engineered plants that show stronger resistance to the fungal pathogen,” emphasized Dr. Takahashi.

However, before these experiments can be realized, some practical ideas for the prevention of gene flow have to be established, because “Italian ryegrass is an outcrossing plant that is pollinated by the wind,” Dr. Takahashi explained. And there are many naturally growing ryegrass plants that could cross with the transgenic ryegrass plants.

“We are now evaluating the disease resistance of the male sterile offspring of the plants we have genetically engineered,” continued Dr. Takahashi. Male sterility is advantageous, as it prevents gene flow from transgenic plants to the environment.

When asked if the overproduction of chitinase by genetically engineered plants would have an effect on herbivores, such as grass-eating domestic animals, Dr. Takahashi told Checkbiotech, “I think this possibility is very low because chitinase commonly exists in plants.” However, a possible allergic reaction in a small number of individuals could not be ruled out. “Indeed, in humans, products of some plant defence-related genes are known to be suspicious allergens.”

A further aspect that has to be considered is how to prevent other (beneficial) fungi, for example fungi that the plants need for nutrient uptake, from the effects of chitinase in transgenic plants. Dr. Takahashi supposes that this could be achieved by controlling the chitinase production. By inducing special signal sequences in the plant genome, the enzyme could be produced only at rust infection sites, he explained.

Dr. Takahashi’s work will bring the research community one step closer to understanding how to produce crops that are resistant to fungal pathogens. He is now engaged both in the development of molecular markers that are linked to resistance genes for the ryegrass blast and in the production of blast-resistant ryegrass varieties.

Katharina Schoebi is a biologist and Chief Science Writer for Checkbiotech.

Tadashi Takamizo et al. Increased resistance to crown rust disease in transgenic Italian ryegrass (Lolium multiflorum Lam.) expressing the rice chitinase gene. Plant Cell Rep (2005) 23:811–818 (DOI 10.1007/s00299-004-0900-1)

Link to the abstract:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15599752&dopt=Citation

Contact:
Wataru Takahashi
Forage Crop Research Institute
Japan Grassland Farming and Forage Seed Association
388-5 Higashiakada
Nishinasuno
Tochigi
329-2742
Japan
e-mail: takahashi@jfsass.or.jp
Tel.: +81-287-376755
Fax: +81-287-376757