A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)1

Ap Applicati tions ons in in Pl Plant t Scien Sciences ces Protein structure/function relationships can be investigated using a structural model to generate hypotheses and an in vitro reconstitution assay for functional analysis of protein variants. However, this approach is only available for some proteins. For example, elucidation of the structure/function relationships for bacterial cellulose synthases has progressed due to two recent technical advances—solving of the crystal structure of Rhodo-bacter sphaeroides (van Niel 1944) Imhoff et al. 1984 cellulose synthase (Morgan et al., 2013) and in vitro reconstitution of functional cellulose synthase from purifi ed R. sphaeroides proteins (Omadjela et al., 2013). A computational model for the cytoplasmic region of a plant cellulose synthase (designated CESA) from Gossypium hirsutum L. (Sethaphong et al., 2013) has been used to postulate functions of the structural features that distinguish CESAs from bacterial cellulose synthases (Slabaugh et al., 2014a). However, methods for routine in vitro assay of CESA activity are not available for functional testing (Guerriero et al., 2010). In vivo complementation assays provide alternatives to in vitro assays for protein functional analysis. This approach was used effectively to test an engineered point mutation in an Ara-bidopsis thaliana (L.) Heynh. CESA that was predicted to impair protein function based on computational modeling (Slabaugh et al., 2014b). However, due to the time and space required for genetic transformation and backcrossing, A. thaliana is not ideal for screening large numbers of mutations. As in all mosses, the protonema and gametophores of Physcomitrella patens (Hedw.) Bruch & Schimp. are haploid. Thus, mutant phenotypes can be detected immediately without the need for backcrossing to produce homozygous diploid lines. This makes P. patens a faster alternative to A. thaliana for assays that involve genetic transformation (Cove, 2005 ; Cove et al., 2006). Physcomitrella patens cell walls contain the same classes of polysaccharides as vascular plant cell walls, with some differences in side-chain structure (Roberts et al., 2012). Also, P. pat-ens has rosette cellulose synthesis complexes (Roberts et al., 2012) and seven CESA genes (Roberts and Bushoven, 2007). However, consistent with the absence of lignifi ed vascular tissue in mosses, the PpCESA genes are not orthologous to the CESA genes that synthesize primary and secondary cell walls in seed plants (Roberts and Bushoven, 2007). We have previously generated ppcesa5 knockout lines that are defi cient in gameto-phore production. In these lines, the gametophore buds typically produce irregular clumps of …