Using CRISPR-Cas9 and calcium reporters to study arabidopsis interactions with the green peach aphid (A BSPP Summer Vacation Bursary Report)

Written by Emma Turley, John Innes Centre. This is the report from a BSPP Undergraduate Summer Vacation Bursary. Click here to read more/apply for one yourself.

As hemipteran pests and vectors of many significant plant viruses, aphids pose a substantial and increasing threat to plant health. Professor Saskia Hogenhout’s lab at the John Innes Centre, Norwich, is dedicated to exploring the interactions between such insect vectors of plant diseases and their interactions with host plants using techniques ranging from molecular biology to population genomics. With this group and under the supervision of Joshua Joyce, I spent 8 weeks exploring the molecular interplay between Arabidopsis thaliana (Arabidopsis) and the green peach aphid (Myzus persicae), a pest notorious for being polyphagous and able to transmit >100 plant viruses. In particular, we explored the signalling events that underpin aphid perception and defence responses in plants, as well as the suppression of such responses by aphid effectors.

Aphid feeding has been shown to induce rapid cytosolic Ca²⁺ signals in Arabidopsis plants expressing the fluorescent cytosolic [Ca²⁺] reporter, GCaMP3. These Ca²⁺ signals are thought to occur in epidermal and mesophyll cell layers around the feeding site but the mechanisms underpinning their induction and propagation remain unknown. Like bacterial elicitors such as flg22, aphid defence-inducing elicitors may contribute to these signals, with a purified aphid extract known to induce plant Ca²⁺ signals, defence gene expression and aphid resistance. By utilising pharmacological agents, Arabidopsis mutants in superoxide-generating NADPH oxidase genes (AtRboh), and GCaMP3 Arabidopsis, we asked whether reactive oxygen species (ROS) signalling contributed to aphid extract-induced defence signalling. Diphenyleneiodonium chloride (DPI), a chemical that suppresses ROS production, was found to inhibit both aphid- and flg22-induced Ca²⁺ signals, suggesting ROS signalling contributes to aphid extract-induced Ca²⁺ responses. However, mutants in AtRbohD and/or AtRbohF, previously shown to have impaired plant defences and ROS production in response to pathogens, displayed Ca²⁺ signals equivalent to those of wildtype plants. As such, ROS production appears to contribute to aphid extract-induced Ca²⁺ signals and perception, but the critical genetic components underpinning this response remain unclear.

In addition to investigating aphid-induced plant defence signalling, research in the host lab aims to characterise aphid effector proteins. Mp10, a conserved M. persicae effector, suppresses elicitor induced Ca²⁺ and ROS signals. However, it is unclear what underpins Mp10’s immunosuppressive activity. Yeast-two-hybrid and pull-down assays have revealed three key plant proteins as putative interactors of Mp10, referred to here as Protein Interactor of Mp10 1 (PIM1), PIM2 and PIM3. Expression of known variants of these proteins that do not interact with Mp10 in a pim1pim2pim3 null mutant background would help reveal the significance of these putative targets for Mp10 activity in planta and may limit Mp10’s effector activity. Thus, I generated constructs for simultaneous and independent CRISPR-Cas9-mediated knockout of these three genes in Arabidopsis using Golden Gate cloning. This required identification of target sites for Cas9 in the PIM genomic sequences, and incorporation of these into small guide RNA (sgRNA) scaffolds using PCR. To knockout three genes simultaneously, a plasmid carrying six sgRNAs, alongside Cas9 and a plant-selectable marker, was designed. With guidance from TSL SynBio and Dr Sam Mugford, we successfully used multiple rounds of Golden Gate cloning to assemble two multi-gene constructs for the CRISPR-Cas9-mediated knockout of our three putative Mp10 targets genes using alternative cut sites. Thus, this work generated tools to progress the research of how the aphid effector, Mp10, interferes with plant defence responses to aphid attack.

Having arrived in Norwich with very little lab experience, I have now partaken in a range of integral laboratory procedures in addition to those described above, including Agroinfiltration of Nicotiana, raising Arabidopsis from seed and conducting aphid fecundity assays. Importantly, I have also gained an appreciation for the rewards and difficulties associated with studying for a PhD in plant science and feel inspired to take on this challenge in future. I would like to say a huge thank you to Josh for being such an attentive supervisor, and to the BSPP for allowing me this valuable opportunity.