HS1 Is Involved in Hygromycin Resistance Through Facilitating Hygromycin Phosphotransferase Transportation From Cytosol to Chloroplast.

Yanzhong Luo,Miaoyun Xu,Weiwei Li,Lei Wang,Chunyi Zhang,Lan Zhang

doi:10.3389/fpls.2020.00613

Abstract

The transportation of proteins encoded by nuclear genes from plant cytosol to chloroplast is essential for chloroplast functions. Proteins that have a chloroplast transit peptide (cTP) are imported into chloroplasts via translocases on the outer and inner chloroplast envelope. How proteins lacking transit sequence are imported into chloroplast remains largely unknown. During screening of an Arabidopsis population transformed with a hairpin RNA gene-silencing library, we identified some transgenic plants that had active expression of the selectable marker gene, hygromycin phosphotransferase (HPT), but were sensitive to the selection agent, hygromycin B (HyB). Mutant and complementation analysis showed that this HyB sensitivity of transgenic plants was due to silencing of the HS1 (Hygromycin-Sensitive 1) gene. HS1 is localized in the chloroplast and interacts physically with HPT in yeast cells and in planta. Fluorescence and immunoblotting analysis showed that HPT could not be transported effectively into chloroplasts in Aths1, which resulted in Aths1 is sensitivity to hygromycin on higher HyB-containing medium. These data revealed that HS1 is involved in HyB resistance in transgenic Arabidopsis through facilitating cytosol-chloroplast transportation of HPT. Our findings provide novel insights on transportation of chloroplast cTP-less proteins.

Highlights

Chloroplasts are essential organelles of endosymbiotic origin in plants and function in photosynthesis and other important physiological processes (Whatley, 1978; Nelson and BenShem, 2004; Reinbothe et al, 2010)
The proteins initially targeted to the secretion (Sec) translocon at the endoplasmic reticulum (ER)/Golgi apparatus and co-translational proteins are subsequently delivered to the chloroplast via vesicle trafficking or other unknown pathways (Chen et al, 2004; Villarejo et al, 2005; Nanjo et al, 2006; Radhamony and Theg, 2006), which may be a vestige of an early endosymbiotic event; a set of proteins that lack cleavable targeting signals are imported into chloroplast by a largely uncharacterized system (Kleffmann et al, 2004; Nada and Soll, 2004; Miras et al, 2007)
Once in hygromycin B (HyB)-free Murashige and Skoog (MS) medium, these plants grew normally. These plants differed from untransformed plants, which were dwarfed on the MS-HyB medium after 10 days, and normal transgenic lines, which grew vigorously under high concentrations of HyB (Figures 1A,B)

Summary

Introduction

Chloroplasts are essential organelles of endosymbiotic origin in plants and function in photosynthesis and other important physiological processes (Whatley, 1978; Nelson and BenShem, 2004; Reinbothe et al, 2010). The transport of nucleus-encoded plastid proteins require the core translocon at the outer envelope membrane of chloroplast (TOC) complex formed by TOC159, TOC75 and TOC34, and chloroplast translocon at the inner envelop membrane of chloroplast (TIC) complex such as TIC110, TIC20, TIC21, and TIC236 These complexes are suggested to function as a translocation channel for preproteins (Inaba et al, 2003; Soll and Schleiff, 2004; Inaba et al, 2005; Kessler and Schnell, 2009; Li and Chiu, 2010; Chen et al, 2018). The proteins initially targeted to the secretion (Sec) translocon at the ER/Golgi apparatus and co-translational proteins are subsequently delivered to the chloroplast via vesicle trafficking or other unknown pathways (Chen et al, 2004; Villarejo et al, 2005; Nanjo et al, 2006; Radhamony and Theg, 2006), which may be a vestige of an early endosymbiotic event; a set of proteins that lack cleavable targeting signals are imported into chloroplast by a largely uncharacterized system (Kleffmann et al, 2004; Nada and Soll, 2004; Miras et al, 2007)

Methods

Results

Conclusion