Imaging systemic calcium response and its molecular dissection using virus-induced gene silencing

Many biotic and abiotic stimuli arrive locally on the plant. For example, attack by an insect or invasion by a fungal pathogen generally starts with a single leaf. However, the responses that are then elicited are often systemic, triggering effects throughout the entire plant body. One of the rapid signaling systems that helps coordinate these plant-wide response networks is changes in cytoplasmic Ca²⁺ that rapidly propagate throughout the plant. These Ca²⁺ signals are readily visualized using plants expressing green fluorescent protein-based Ca²⁺-sensitive bioreporters, such as those of the GCaMP and GECO families. Dissecting the underlying molecular machinery behind this systemic spread of information is often approached by imaging the Ca²⁺ response in mutants in candidate genes. Introducing the GFP sensor into the relevant genetic backgrounds and then selecting lines usable for imaging can be very time consuming. An alternative, more rapid approach to screening these candidates is through virus-induced gene silencing (VIGS), where target genes are suppressed in the wild-type bioreporter expressing plants. This chapter describes how to generate VIGS constructs targeted to candidate genes and then how to visualize wound-induced, systemic Ca²⁺ signaling in the VIGS suppressed plants.