Semi-In-Vivo Pull-Down Assay for Blue Light-Dependent Protein Interactions.

Cryptochromes are photolyase-like blue-light receptors found in all major evolutionary lineages (Ahmad and Cashmore, Nature 366:162-166, 1993; Lin, Plant Physiol 110:1047, 1996; Cashmore, Cell 114:537-543, 2003; Partch and Sancar, Methods Enzymol 393:726-745, 2005). Arabidopsis cryptochrome 1 (CRY1) and cryptochrome 2 (CRY2) mediate primarily blue-light inhibition of hypocotyl elongation and photoperiodic control of floral initiation (Ahmad and Cashmore, Nature 366:162-166, 1993; Somers et al., Science, 282:1488-1490, 1998; Guo et al., Science 279 (5355):1360-1363, 1998; Yu et al., Arabidopsis Book 8:e0135, 2010). It has been proposed that phototransduction of cryptochromes involves the blue-light-dependent protein interactions, such as AtCRY2-CIB1 (CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1), AtCRY1-PIF4 (PHYTOCHROME INTERACTING FACTOR 4) modules, sequentially mediate gene expression and plant growth (Liu et al., Science 322 (5907):1535-1539, 2008; Ma et al., Proc Natl Acad Sci U S A 113 (1):224-229, 2016; Wang et al., Science 354:343-347, 2016). Cryptochromes also showed blue light response in vitro when expressed in Sf9 insect cells using the baculovirus expression system, thus the wavelength-specific CRY2-CIB1 interaction can also be observed in Semi-in-vivo pull-down assay (Li et al., Proc Natl Acad Sci U S A 108 (51):20844-20849, 2011; Liu et al., EMBO Reports, 2018). Here, we describe the detailed process of blue light-dependent CRY2-CIB1 interaction in Semi-in-vivo conditions.