A project undertaken at Australian National University, and supervised by Anthony Millar
The use of chemical pesticides to combat plant pathogens increases crop productivity and underpins food security, but comes at a cost to human health, ecosystems and is a costly farming input. Additionally, export markets are jeopardised when residue levels on produce exceed minimum standards. An alternative approach is to use genetics to breed disease-resistant crops that negate the use of chemical pesticides. However, the biology underpinning this is extremely complex, and most resistance genes only provide protection to a narrow range of pathogens.
This project builds on a newly discovered pathogen defense system. Central to this defense system is a gene that encodes a small regulatory RNA known as a microRNA (miRNA), which binds to the mRNAs of target genes and destroys them to prevent the gene’s activity. Present in all terrestrial plants is a miRNA known as miR159, where it specifically targets a family of GAMYB genes. These genes encode regulatory proteins known as transcription factors, that switch on genetic pathways. GAMYB mRNA is continually produced in a plant cell, however, no GAMYB protein is produced, as miR159 is always present to destroy the GAMYB RNA. Given this continual production GAMYB mRNA in a plant cell, only for it to be to be destroyed by miR159, raises the question of what is the function of this miR159-GAMYB genetic switch mechanism?
To explore the biological role of miR159, we inhibited miR159 function in Nicotiana tabacum (tobacco), Nicotiana benthamiana (Australian wild tobacco) and Arabidopsis thaliana, all model plant species. miR159 inhibition resulted in GAMYB activation in all three species, and in each case resulted in analogous deleterious growth defects (Figure 1). These conserved features are consistent with miR159 strong conservation throughout terrestrial plant species. Moreover, in Nicotiana tabacum plants in which miR159 was inhibited and GAMYB activated, it was found that genetic pathways controlling pathogen defense had been strongly activated, and that the diversity of activated genes would be predicted to confer defense to a broad-spectrum of pathogens. Such a large-scale activation of defense genes would explain the deleterious phenotype of plants in which GAMYB has been activated, as energy has been channelled from growth into defense. This suggests that this conserved miR159-GAMYB genetic switch mechanism is critical for plant defense, explaining its ubiquitous nature in plants.
Surprisingly, in both Nicotiana benthamiana and Arabidopsis, defense genetic pathways were not activated in miR159 inhibited/GAMYB activated plants, but rather repressed. This is especially confounding given how closely related Nicotiana tabacum and Nicotiana benthamiana are. This finding triggered a new hypothesis (Figure 2): given all three species had analogous deleterious phenotypes upon GAMYB activation, we hypothesised that defense genes are induced upon GAMYB activation in all species, but in Nicotiana benthamiana and Arabidopsis, defense gene activation is only transiently induced given the severe impacts it has on growth.
Figure 2
To test this hypothesis, we generated Nicotiana tabacum and Arabidopsis plants in which the GAMYB is only transiently induced (rather than continually expressed as occurred in the miR159 inhibited plants). In both species, we found that defense genes are induced upon GAMYB induction. This implies that the main function of this genetic mechanism is a conserved role in plant defense.
Research is ongoing to obtain more evidence for our hypothesis that the miR159-GAMYB genetic mechanism a molecular switch controlling a fundamental broad-spectrum defense mechanism that is widely conserved in terrestrial plants. We intend to address whether this miR159-controlled defense system can confer broad-spectrum resistance to pathogens. We believe the ever-present GAMYB mRNA implies a pre-existing mRNA (or primed system) awaits activation (GAMYB protein production), enabling a rapid and cell-specific response. Further understanding and exploiting this system for biotechnological purposes raises the possibility of generating crops with broad-spectrum disease-resistance or developing versatile spray-on RNA strategies to respond to pathogen outbreaks. Such outcomes would reduce the reliance on chemical pesticides, benefiting agriculture, food quality and security, and the environment.