Proof of concept: control of grey leaf spot disease of maize using double-stranded RNA sprays.
Maize, a staple food for millions, faces a significant threat from a disease known as grey leaf spot (GLS). This disease is caused by the fungus Cercospora zeina in sub-Saharan Africa. It attacks maize leaves, creating necrotic lesions and drastically reducing crop yields. Traditional fungicides are becoming less effective due to resistance, leading to an urgent need for innovative solutions.
RNA interference (RNAi) is a natural process that organisms use to silence specific genes, essentially turning off endogenous genes when they are not needed or degrading genes of invading pathogens. Imagine RNAi as a molecular switch that can shut down unwanted or dangerous processes within cells. In this study, researchers explored using RNAi to target and disable essential C. zeina genes.
How does RNAi disease control work? Scientists create double-stranded RNA (dsRNA) molecules that match the nucleotide sequence of a target gene. These dsRNA molecules are taken up by the cells of a pathogen such as C. zeina when dsRNA is sprayed onto infected maize leaves in a method known as spray-induced gene silencing. Inside the fungus, the dsRNA binds to the messenger RNA (mRNA) of the target gene and triggers a pathway that cuts up the mRNA, preventing it from producing essential proteins. This process inhibits fungal growth, providing protection to the maize plants.
The research team conducted several experiments to test this innovative approach. First, they took a deep dive into the genome of the fungus to confirm it had the genetic machinery to carry out RNAi. Second, the fungal cells were studied under a microscope to confirm they could take up fluorescently labelled dsRNA. Third, they created a genetically modified fungal strain that expressed the jellyfish Green Fluorescent Protein (GFP) which glowed in the dark under UV light. They used this strain for a “test run” of RNAi in a petri dish by applying dsRNA targeting the GFP gene. The UV light torch was shone on the cultures treated with GFP-dsRNA, and “Voila!” there was a 50% reduction in fluorescence.
Encouraged by these results, the scientists set about testing this approach in maize plants. First, they had to decide on which C. zeina gene to target. The requirement was to select a DNA sequence unique to the fungus and not overlapping with any maize gene, to prevent damage to maize cells. Chitin is an essential building block of fungal cell walls so they selected the C. zeina chitin synthase gene as the RNAi target. Another “test run” in the lab proved this to be a good target, as treatment of C. zeina in culture with dsRNA targeting the chitin synthase gene reduced the fungal cell viability by 34%.
Finally, maize inoculation trials were carried out in a glasshouse. This is where practical knowledge of how grey leaf spot disease develops in the field became critically important. C. zeina is a fungus that is fastidious about the conditions required for infection. The pathogen “takes off” in mature maize plants before flowering when summer days start with misty mornings and the heat builds up to midday. In the experiments, these conditions were simulated in the glasshouse, and C. zeina spores were applied to the maize leaves.
The dsRNA targeting the C. zeina chitin synthase gene was applied in two different ways – either simultaneously with the fungal spores or the next morning. The maize plants were inspected for GLS disease symptoms over the next few weeks. Excitingly, the “morning after” treatment controlled GLS disease by 39-56% in two independent trials. This was a proof of concept for dsRNA as a fungicide against GLS disease in maize leaves.
Importantly, control treatments with dsRNA targeting the GFP gene or a water control did not reduce GLS disease. The scientists hypothesised that control was better after the fungal spores had germinated overnight on the maize leaves, as germ tubes may take up the dsRNA more readily than spores, which have a resilient coat. This was borne out by electron microscopy observations, which showed that a high proportion of the fungal spores germinated by the next day.
So, what is the future of this “green chemistry” RNAi fungicide? Scientists around the globe have demonstrated this type of proof of concept against a range of fungal pathogens, most notably those that infect leaves. The next phase will be product development to overcome several challenges, such as producing large amounts of the dsRNA cheaply, adding stabilising agents such as nanoparticles to prolong the “shelf-life” of the dsRNA on plant surfaces in the field, and optimising the dsRNA targets, to name a few. An important promise of this technology is the reduced risk of pathogens developing resistance to dsRNA fungicides since multiple genes can be targeted together. Furthermore, several pathogens can be targeted simultaneously with multiple dsRNAs.
IMAGE: Maize leaves inoculated with Cercospora zeina develop grey leaf spot disease when treated with a non-specific Gfp-dsRNA. In contrast, disease is reduced when treated with dsRNA targeting an essential fungal gene (chitin synthase).
Double-stranded RNA uptake for the control of the maize pathogen Cercospora zeina
TITLE IMAGE: (I) Cercospora zeina protoplasts take up fluorescently labelled dsRNA. (IV) Scanning Electron Microscopy of a conidium (C) with germ tubes (GT) on a maize leaf surface. The GT are oriented towards a maize stoma (ST) the usual entry site of the fungus C.zeina.
