Feature: Strengthening plant defensins

At Melbourne’s La Trobe University, Marilyn Anderson’s research group in the Department of Biochemistry has made promising progress towards boosting plant resistance to pathogenic Ascomycota, filamentous fungi responsible for many of serious diseases of agriculture and forestry.

Anderson is executive director and chief science officer of the plant biotechnology company Hexima Limited, which she co-founded with Professor Adrienne Clarke and Dr Robyn Heath, of the School of Botany at the University of Melbourne.

Ascomycete diseases include verticillium wilt of potatoes, tomatoes, many herbaceous ornamentals, stone fruit trees, lychees and avocados, and fusarium wilt of grain crops including maize, wheat and barley.

A member of Anderson’s group, postdoctoral researcher Dr Nicole van der Weerden, was awarded a Victoria Fellowship in 2006 for her research into defensins, which are naturally produced by plants for protection against fungal disease.

Anderson says the project began with an intriguing question: why do fungus diseases have great difficulty invading flowers compared to other parts of plants such as leaves, stems and roots?

Van der Weerden showed that flowers accumulate high levels of antifungal proteins, particularly defensins, which protect the vital reproductive organs against damage by disease. The floral defensin, NaD1, recognises an invading fungus and locks onto the cell wall of the invader’s thread-like hyphae.

NaD1 then punches holes in the hyphal cell membrane and enters the cytoplasm, where it engages components of the cell’s autolysis system, triggering collapse and cell death.

Anderson said plant defensins are structurally similar to defensins secreted by cells in the innate immune system of animals and insects, and they function in a similar way. “What is really exciting about defensins is that they open gates in the fungal cell membrane and allow other molecules to enter that accelerate cell death,” he said.

In the mid-2000s, Anderson’s team isolated the major defensin gene from the ornamental tobacco, Nicotiana alata, and working with Robyn Heath’s team, introduced it into cotton to see if it enhanced resistance to fusarium wilt in plants in the field. “We did three years of field trials, and the results were stunning,” says Anderson. “The plants with the defensin gene were much less susceptible to disease.

“We are now working with Pioneer/DuPont as a major partner to transfer our antifungal technology into maize and have built a $2.5 million maize transformation and testing facility here in Australia. Our collaboration with Pioneer/DuPont has created a path to market for our technology which is essential for small biotechnology companies like Hexima.”

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Starving the enemy

Anderson’s La Trobe University team is also developing a novel, and potentially generic technology to protect crop plants against insect attack using proteinase inhibitors, anti-feedant molecules that bind and inactivate enzymes in the insect gut that break down plant proteins, causing insect larvae to starve.

Monsanto’s proprietary Bacillus thuringiensis (Bt) technology currently dominates the market for transgenic pest resistance. The insecticidal Bt proteins, isolated from insect-killing strains of B. thuringiensis, bind to receptors on cells lining the gut of insect pests, triggering apoptosis.

But the many different strains of B. thuringiensis tend to be specialised for killing particular classes of insect: the Bt proteins Cry1Ac and Cry2Ab, for example, target different receptors in the gut of Helicoverpa caterpillars, which include the world’s most damaging crop pests.

Plants produce proteinase inhibitors naturally as one of their lines of defence against insect pests. According to Anderson, the PinII family of proteinase inhibitors is particularly fascinating. Washington State University researcher Clarence “Bud” Ryan and his colleagues showed the genes that code for these inhibitors are finely tuned and respond to insect attack.

“Bud Ryan found that when a pest chews on a tobacco or tomato plant, the plant sends a mobile signal from the wounded leaf to other leaves that have not yet come under attack. This mobile signal is a small peptide called systemin, and it turns on the inhibitor genes in unwounded leaves, pre-arming them before the insects start chewing,” Anderson said.

“Even more amazing, the plants then produce a volatile molecule, methyl jasmonate, that spreads the alarm to neighbouring plants, heightening the collective defences of the plant population.”

The complex pattern of regulation of the PinII gene family suggests a central role in insect protection. However, the Hexima teams led by Anderson and Heath discovered that the PinII genes alone are not sufficient to confer sustainable insect protection in transgenic cotton.

“Biologically this make sense because plants and insects have coevolved over millennia and thus are in an intricate sparring match where one contestant develops a defensive shield and the opponent develops a strategy to bypass that defence,” Anderson said.

Dr Kerryn Dunse in Anderson’s group discovered that insects respond to ingestion of the PInII inhibitors by producing a different group of digestive proteinases that are not affected by the PinII inhibitors, thus allowing the insect to survive and reproduce.

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“We purified the new proteinases and used them to identify a second class of inhibitors that work well,” Anderson said. “These inhibitors are produced by a different family of genes, but they are regulated by wounding and methyl jasmonate in the same way as the PinII inhibitors. Thus the plant ‘punches back’ and defeats the new resistant insect proteinases with another family of inhibitors.”

Even though the two classes of proteinases work by the same mechanism, Kerryn Dunse was able to identify modified domains in the resistant proteinases that prevented the PinII inhibitors from working effectively. Using this information, the Hexima teams have produced transgenic tobacco plants with both classes of inhibitors.

They exhibit much better resistance to pest attack than transgenic plants expressing only one class of inhibitor. “Nevertheless, we don’t see the level of protection that would be required of a commercial technology. We’re now looking for genes for proteins that interact with proteinase inhibitors. Stacking the proteinase inhibitor genes with other transgenes for pesticides, like Bt genes, that have different targets in the insects should provide more sustainable resistance.”

Anderson said there are already reports of emerging resistance to Bt in Helicoverpa caterpillars from several Asian cotton-growing nations. “We’re more concerned about resistance emerging in places like South America, where there is more pressure from insects,” she said.

“Helicoverpa larvae attack at least 100 different crop species, and they’re a major pest of maize. We’ve identified some candidate genes and we’re looking for proof of concept in greenhouse trials before we test them under field conditions. We’re being cautious with PI technology because farmers are looking for something as effective as Bt technology. They don’t want to see slow-growing insects, they want to see dead ones.”