Cloning and Characterization of Protein Prenyltransferase Alpha Subunit in Rice

doi:10.1016/j.rsci.2021.05.014

Abstract

Abstract:

Protein prenylation plays a crucial role in plant development and stress response. We report the function of prenyltransferase α-subunit in rice. Protein-protein interactions showed that the farnesyl- transferase (OsPFT)/geranylgeranyltransferase-I (OsPGGT I-α) protein interacted together with OsPFT-β and OsPGGT I-β. The α- and β-subunits of OsPFT formed a heterodimer for the transfer of a farnesyl group from farnesyl pyrophosphate to the CaaX-box-containing peptide N-dansyl-GCVLS. Furthermore, the tissue expression patterns of the OsPFT and OsPGGT I subunits were similar, and these subunits were localized in the cytoplasm and nucleus. Moreover, OsPFT/OsPGGT I-α-deletion homozygous rice mutants had a lethal phenotype, and the heterozygous mutants exhibited reduced pollen viability. These results indicated that prenylation plays an important role in rice development.

Key words: protein prenylation, plant development, rice, pollen viability

Tao Wang, Lijuan Lou, Zeyu Li, Lianguang Shang, Quan Wang. Cloning and Characterization of Protein Prenyltransferase Alpha Subunit in Rice[J]. Rice Science, 2021, 28(6): 557-566.

Figures/Tables 8

Fig. S1. The interaction between OsPFT/OsPGGT I-α and the three isoforms of OsPFT/PGGT I- α in Y2H.

Fig. 1. Multiple sequence alignment of deduced amino acid sequences of prenyltransferase subunit and molecular.A, Amino acid sequence alignment of OsPFT/OsPGGT I-α subunit with HsaPFT/HsaPGGT I-α and AtPFT/AtPGGT I-α subunits. Letters in the black boxes indicate highly conserved amino acid residues. The red solid frames represent the protein prenyltransferase alpha subunit repeat domain.B, Amino acid sequence alignment of OsPFT-β subunit with HsaPFT-β and AtPFT-β subunits. The blue solid frames represent the prenyltransferase and squalene oxidase repeat domain.C, Amino acid sequence alignment of OsPGGT I-β subunit with HsaPGGT I-β and AtPGGT I-β subunits. The blue dotted frames represent the prenyltransferase and squalene oxidase repeat domain.

Fig. S2. Molecular phylogenetic tree analysis of selected prenyltransferase subunit amino acids among different species.The construction of phylogenetic tree is based on the maximum likelihood method in MEGA7. Bootstrap value = 1000. The pentagram indicates the corresponding prenyltransferase subunit in rice.

Fig. 2. OsPFT/OsPGGT I-α interacts with OsPFT-β and OsPGGT I-β.A and B, Yeast-two hybrid analysis of OsPFT/OsPGGT I-α and OsPFT-β interaction (A) and OsPFT/PGGT I-α and OsPGGT I-β interaction (B). Bait of OsPFT-β or OsPGGT I-β was fused to the GAL4 DNA-binding domain, and prey of OsPFT/OsPGGT I-α was fused to the GAL4 activation domain. Colonies growth on the SD-L-W-H-A medium represent positive interaction.C and D, Firefly luciferase complementation imaging (LCI) assay in Nicotiana benthamiana. The N-terminal half of luciferase (Nluc) was fused into OsPFT/OsPGGT I-α or OsPGGT I-β and the C-terminal half of luciferase (Cluc) was fuse into OsPFT/OsPGGT I-α or OsPFT-β.E and F, Co-immunoprecipitation (Co-IP) assay for OsPFT/OsPGGT I-α and OsPFT-β (E) or OsPFT/OsPGGT I-α and OsPGGT I-β (F). OsPFT/OsPGGT I-α was fused to green fluorescent protein (GFP), OsPFT-β and OsPGGT I-β were fused to Nluc, respectively. These constructions and GFP control vector were transformed into Agrobacterium GV3101 strain and infiltrated into N. benthamiana. OsPFT/OsPGGT I-α-GFP or GFP were immobilized on GFP-trap beads and incubated with OsPFT-β-Nluc or OsPGGT I-β-Nluc. Input represents equal amounts of purified OsPFT/OsPGGT I-α-GFP or GFP fusions and OsPFT-β-Nluc or OsPGGT I-β-Nluc before Co-IP.

Fig. 3. In vitro continuous ?uorescence assay for OsPFT/OsPGGT I-α and OsPFT-β activity measurement.A, In vitro OsPFT/OsPGGT I-α and OsPFT-β activity assay using farnesyl pyrophosphate (FPP) as substrate. OsPFT/OsPGGT I-α + FPP and OsPFT-β + FPP were used as the negative controls.B, In vitro OsPFT/OsPGGTI-α and OsPFT-β activity assay using geranyl geranyl pyrophosphate (GGPP) as substrate. OsPFT/OsPGGT I-α + GGPP and OsPFT-β + GGPP were used as the negative controls.The fluorescence assays were performed in the Tecan Infinite Pro Microplate Reader and monitored using an excitation wavelength of 340 nm and emission wavelength of 505 nm.

Fig. 4. Gene expression pattern in different orgens and tissues.Gene expression levels of OsPFT/OsPGGT I-α, OsPFT-β and OsPGGT I-β in different rice organs or tissues. Rice Actin was used as the reference. Data are Mean ± SD (n = 3).

Fig. 5. Subcellular localization assay of GFP-tagging OsPFT/OsPGGT I-α, OsPFT-β and OsPGGT I-β in Nicotiana benthamiana.The assays were performed using mCherry-NLS as a nucleus-location signal by the confocal laser microscope scanning. The scale bar is 10 μm in the first column, and 25 μm in the next three columns.

Fig. 6. Mutation analysis in OsPFT/OsPGGT I-α by CRISPR/Cas9-mediated genome editing system.A, Schematic diagram of the designed targeted sites in OsPFT/OsPGGT I-α. 3′/5′ UTRs, exons and introns are indicated by green rectangles, blue rectangles and black lines, respectively. The target sequences are underlined, and the protospacer adjacent motif (PAM) is emphasized in red letters. Sequence analyses of the targeted regions in OsPFT/OsPGGT I-α are in T3 transgenic plants. The deletion sequence is shown by short solid line. CR-1 and CR-2 refer to the two OsPFT/OsPGGT I-α mutants; WT refers to the wild type.B, PCR assay of T3 transgenic lines in CR-2. Each lane represents an independent CRISPR/Cas-mediated editing line.C, Comparison of WT and Hetero CR-1/CR-2 in floret at the mature pollen stage. Hetero CR-1 and Hetero CR-2 represent the heterozygous OsPFT/OsPGGT I-αCR-1/2. Scale bars are 1 mm.D, Pollen was stained with I2-KI. The red arrows marked the sterile pollen. Scale bars are 30 μm.E, Statistic of sterile pollen. The error bars at each data point indicate the standard error obtained from three biological replicates. **, Significant difference at the 0.01 level.

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