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

Abstract

Premise of the study: A method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens.

Methods and results: A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins.

Conclusions: Compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.

Keywords: Physcomitrella patens; cellulose synthase; complementation assay; gametophore development; protein structure/function relationships.

Fig. 1.

Functional analysis of engineered CESA proteins by complementation in Physcomitrella patens. A. Colonies, gametophores, and gametophore leaves from lines resulting from stable transformation of ppcesa5KO-2 protoplasts transformed with a vector driving expression of wild-type PpCESA5 (positive control), an empty vector control or vectors driving expression of PpCESA5 carrying engineered point mutations (R453K and R453G). Scale bars shown in the first column apply to all images in the row. B. ppcesa5KO-2 complementation rates for vectors driving expression of PpCESA5 carrying engineered point mutations compared to positive and negative control vectors from the same experiment. Means for two (R453K and R453D) or three (R453G) experiments are shown with P values determined using a two-tailed Fisher’s exact test of independence.

Fig. 2.

Western blot analysis of protein expression for P. patens lines derived from transformation of ppcesa5KO-2 protoplasts with vectors driving expression of wild-type PpCESA5 (positive control) or PpCESA5 carrying engineered point mutation (R453K, R453G, and R453D). Western blots probed with anti-HA are shown above the same blot stained with Ponceau S as a loading control. Protein loading per lane was 7.1 μg (PpCESA5), 9.9 μg (R453G), 33 μg (R453K), or 7.4 μg (R453D). Lines that produced gametophores are indicated by “G,” and those producing no gametophores are indicated by “g.” Positive (+) and negative (-) control lines are included with lines from each test transformation.

Fig. A1.

A schematic of site-directed mutagenesis through PCR fusion. Fragments F1 and F2 of the gene of interest are amplified using the primers attB5 and SR1 and attB2 and SF1, respectively. Fragments F1 and F2 are combined in a single-cycle PCR fusion reaction to create the mutated gene of interest, which is cloned into pDONR P5-P2 for MultiSite Gateway cloning. Primers are depicted as arrows, attB sites appear in blue, coding sequence is in red, and the point mutation appears as a green star.

Fig. A2.

PCR products separated on a 1% agarose gel (100 V, 25 min). Concentrations of F1 and F2 were both estimated at ∼240 ng/μL. However, F1 is ∼2500 bp and F2 is ∼1000 bp. Thus, 2 μL of PCR reaction F1 was combined with 1.33 μL of PCR reaction F2 for the PCR fusion reaction.

Fig. A3.

Selection of stable transformants. A. Colonies derived from ppcesa5KO-2 protoplasts that survived the first round of antibiotic selection following transformation with a positive control vector that drives constitutive expression of wild-type PpCESA5; stable and unstable transformants are present. B. The plate shown in A after the second round of antibiotic selection showing vigorously growing stable transformants among dead colonies. C. Stable transformants from the plate shown in B after being arrayed with colonies from two other plates from the same transformation. D. Colonies ready to be harvested for protein extraction.