A Dual-Reporter System for the Analysis of Phloem Structural and Signaling Responses In Vivo.

Plants have evolved an effective defense mechanism in the phloem to prevent the spread of pathogens and minimize the loss of phloem sap following injury. Specific structural phloem proteins known as P-proteins rapidly seal affected sieve elements by plugging the sieve plates, a phenomenon defined as sieve element occlusion. This chapter describes a live cell imaging method for the analysis of P-protein responses and signal propagation in vivo without tissue sectioning or mechanical manipulation. It is based on an Arabidopsis thaliana dual-reporter line where P-proteins are labeled with fluorescent tags in a complementation background, allowing real-time visualization of the parietal protein network during sieve element occlusion. The calcium sensor Yellow Cameleon 3.6 is specifically expressed and anchored in the sieve elements, enabling the detection of calcium waves and their effects on P-protein structure over longer distances in vivo. Using this protocol, a wide range of external triggers-including chemical treatments, buffers, and wounding-can be applied with precision, allowing the analysis of P-protein functions and long-distance signaling in the phloem. The method is readily adaptable to other genetically encoded sensors and can be used to investigate P-protein-independent processes as well as the diverse signaling and structural responses of additional sieve element components.