Outlook

Real-world application of obtained mutations Multiple precise gene editing methods can be used to introduce effector-resistant mutations to PRRs in crops.

There are three main technologies available to perform precise gene editing in plants, :

• Base editing (Hua et al., 2022)
• Prime editing (Li et al., 2022)
• Homology-directed repair (Chen et al., 2022)

When choosing the methods, there are several considerations: whether this method is established or efficient in the target crop; whether this method can violate the regulations of gene-edited plants in certain countries.

Advantage and disadvantage of different gene-editing system (Chen et al., 2022)

Loosening regulations and increasing public acceptance on gene-edited plants make our strategy feasible in the real-world agriculture Despite the identification of multiple resistance genes and the fact that many innovative genetic approaches have been developed to confer disease resistance to crops, few of them have been subjected to real-world application. One important reason is that the use of these resistant genes/genetic approaches involves transgenic processes, resulting in GMO (genetically modified organism) crops that are strictly regulated in many countries.

However, our strategy of introducing effector-resistant mutations relies on genome editing, rather than transgenes. Many countries have already introduced regulations or guidelines for gene-edited (GE) crops that enable the use of such edited lines in agriculture in a similar way as conventionally bred crops

Therefore, crops engineered using our strategy can enter following countries without being counted as a GMO (up to January 2023) (Buchholzer & Frommer, 2022):

Africa: Kenya, Nigeria Asia & Pacific: Australia, China, India, Israel, Japan, Philippines Europe: Switzerland, the UK, Russia, Latin America: Argentina, Brazil, Colombia, Chile, Ecuador, Guatemala, Honduras, Paraguay North America: the USA, Canada

In certain countries, such as the USA and Brazil, Gene-edited crops are treated under the same regulatory framework as conventional plants, while in others, regulatory approval is still needed (Tachikawa & Matsuo, 2023).

Many other countries that currently regulate gene-edited plants as GMOs are also considering loosening the regulations on GE plants (Nature Plants, 2023).

Tips: Using the Global Gene Editing Regulation Tracker, you can check the regulation status of genome edited plants in the countries you are interested in.

Stack several mutations to avoid pathogen’s evolution

Once a new disease resistance strategy is employed, it imposes evolutionary pressure on pathogens to overcome the resistance. The concern arises that pathogens might evolve their effectors to regain interaction with PRRs.

As multiple mutations can be stacked to generate durable resistance (Schultink et al., 2021), we propose that combining several effector-resistant mutations could serve as an effective method to counteract the rapid evolution of pathogens.

Susceptibility genes

Our strategy can be used to engineer not only the resistance genes (R genes, like PRR genes), but also the susceptibility genes (S genes), which are exploited by pathogens (often via physical interactions between effectors and proteins encoded by S genes) to promote their growth. Since S genes are often involved in physiological processes of plants, complete knock-out or disruption of a S gene sometimes leads to fitness cost (e.g., growth defects) (Koseoglou et al., 2022). Therefore, using rY2H technology to design gene variants resistant to effectors meanwhile retaining its physiological functions can be another potential application scenario of our strategy.