Ryegrass Endophyte & CRISPR

AgResearch, Grasslanz Technology and PGG Wrightson Seeds are developing new pest resistant and animal safe endophytes for ryegrass. Using cutting edge gene editing technology, they can only work within New Zealand in highly regulated containment facilities with field trials held outside of New Zealand. So, why are they working to design ryegrass endophytes that are not yet able to be commercialized in this country? 

Perennial ryegrass is the most commonly sown grass on New Zealand farms. This makes it an important species for animal nutrition and farm profitability. Agricultural scientists are very interested in ryegrass endophytes. An endophyte is a microorganism, typically a fungus or bacterium, that lives inside the tissues of a plant in a symbiotic relationship.  

Ryegrass endophytes are fungi and can be found in various parts of plants. They play important roles in plant health and ecology. Endophytes can help protect plants from stress, such as that caused by pathogens (disease-causing organisms) and herbivores. They do this by producing compounds called alkaloids. These can be toxic or just taste bad to pests that attack the plant that the endophyte supports. (Some endophytes can also assist plants to absorb nutrients, such as nitrogen and phosphorus, from the soil, which can also contribute to the overall growth and development of the plant.)  

However, some compounds in endophytes can also harm farm animals. Toxic compounds can be overexpressed in warmer and drier weather, and this can cause problems such as grass staggers in sheep and cows, or heat stress in cows. 

Scientists and farmers understand the need for ‘futureproofing' pasture, especially in the face of our changing climate. AgResearch has a long history of working with ryegrass endophytes. Earlier research identified several natural endophyte strains to deter key insect pests of ryegrass. 

AgResearch scientist Dr Linda Jonson says of the current research that, “we’ve exhausted natural variations and we’re now working with cutting edge gene editing tools to design endophytes for the future.” They’re looking for a combination of attributes that will confer pest resistance against common pests, like the Argentine stem weevil, while suppressing other components in endophytes that can cause illness in animals. 

Linda says, “Instead of looking for natural endophyte strains, we’re looking at genes and metabolic pathways that determine the toxic and beneficial compounds. This knowledge is then used to design new endophytes with ​​the gene editing technique CRISPR-Cas9.”  

The work began by identifying and understanding genes that code for known metabolic pathways. They needed to understand the pathways that led to the expression of compounds that were toxic to sheep and cows. Understanding this was essential as they could not simply ‘knock out’ the entire pathway – they needed to retain some of it to ensure the plant could still resist pests. 

Once they had an idea of the pathways and genes, the scientists used CRISPR-Cas9 to silence those genes responsible for controlling the more toxic compounds that occur later in the pathways. This new ‘designer’ endophyte is ​​cultured on a plate. Then the cultured endophyte is inoculated into the base of a ryegrass seedling stem. All the work is carried out in high level containment facilities and a containment glasshouse, to meet New Zealand’s strict GM regulatory regime.  

Genome mapping of the endophyte and further tests are carried out on the seedlings to ensure: 

• take up of the new designer endophyte by the plant 
• no unintended consequences have occurred, and 
• the modification is a “site-directed nuclease one” (SDN-1). An SDN-1 modification is a regulatory term that describes the modification as being one where a gene has been silenced but no new material has been added to the organism genome.    

The grass with the new designer endophyte is also extensively tested to assess the plant resistance to key pasture insect pests. Researchers work with entomologists to harvest insects and test plant resistance to them. This is also carried out in containment glasshouses. 

Mice models are used to test the ‘tremorgenic compounds’ – the compounds that cause illness in animals. The most promising strains are then selected and induced to flower so the seeds can be harvested. This cycle can be repeated a few times to get a good number of seeds. 

In New Zealand, gene editing and genetic modification are considered ‘genetically modified organisms’, regardless of whether they have new genes added, genes simply ‘silenced’ or genes from different species added to another.  Therefore, seed having had gene edits (with no foreign DNA inserted) are sent to Australia to be planted for seed production and agronomic field trials outside containment. Large outdoor field trials enable the researchers to scale up testing to assess the efficacy of the grass in resisting pasture pests and in eliminating unwanted toxic effects on sheep and cows. 

Australian regulations do not define organisms as GMOs when they are the result of SDN-1 modifications. This makes field trialing easier in Australia. Organisms developed with SDN-1 modifications can be field trialed in New Zealand, but only with approval from the Environmental Protection Authority. This requires a lengthy application process and scientist engagement with many stakeholders, including mana whenua. 

The research aims to achieve an endophyte strain suitable for commercialization, with improved resistance to insect pests but little or no animal health and welfare concerns.  Concurrently researchers are developing ways to insert genes to create new traits and functionality – for example an endophyte that might also help reduce animal methane emissions. 

However, until changes occur in New Zealand, regulations on new gene editing techniques occur, these endophytes are unlikely to be able to be used by farmers in New Zealand.