Volume 2, Issue 6, Pages 1223-1232 (November 2009) The Chlamydomonas Chloroplast HLP Protein Is Required for Nucleoid Organization and Genome Maintenance  Karcher Daniel , Köster Dietrich , Schadach Anne , Klevesath Anja , Bock Ralph   Molecular Plant  Volume 2, Issue 6, Pages 1223-1232 (November 2009) DOI: 10.1093/mp/ssp083 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Identification of a Chloroplast-Targeted HLP Protein in the Green Alga Chlamydomonas reinhardtii. (A) Alignment of the Chlamydomonas HLP protein with the homologs from Escherichia coli (E.c.), the cyanobacterium Synechococcus elongatus (S.e.), and the cryptomonad alga Guillardia theta (G.t.). Amino acid residues identical or similar in all species listed here are shown in red. Note presence of an N-terminal extension in the Chlamydomonas protein (presumably harboring the transit peptide for protein import into the chloroplast) and a C-terminal extension of unknown functional significance. (B) Analysis of sub-cellular localization of the HLP protein from Chlamydomonas using a transgenic strain expressing a FLAG-tagged HLP. Total cellular protein (total), purified chloroplast protein (cp), and purified mitochondrial protein (mt) were analyzed by Western blotting using a monoclonal anti-FLAG antibody (α-FLAG; left panel) or an antibody against the mitochondrial carbonic anhydrase (α-CA; kindly provided by Dr Mats Eriksson, University of Umea, Sweden; right panel). The HLP–FLAG expressing transgenic strain is compared with the untransformed wild-type control (cw15). (C) Analysis of sub-cellular localization and nucleoid association of the HLP protein using YFP and the Chlamydomonas expression strain UVM4 (Neupert et al., 2009). A YFP fusion protein containing the first 34 amino acids of HLP at its N-terminus (cpYFP) is targeted to the chloroplast (upper panel). Diffuse YFP fluorescence within the chloroplast suggests stromal localization. A full-length HLP–YFP fusion protein is also targeted to the chloroplast but displays punctate localization of YFP fluorescence (middle panel) tentatively suggesting association of the fusion protein with chloroplast nucleoids. As an additional control, an algal strain expressing an unfused cytosolic YFP (Neupert et al., 2009) is also shown (cyYFP; lower panel). YFP fluorescence (YFP), chlorophyll fluorescence (Chl) and the overlay of the two fluorescences (YFP + Chl) are shown for each transformed algal strain. Molecular Plant 2009 2, 1223-1232DOI: (10.1093/mp/ssp083) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Isolation of RNAi Strains with Down-Regulated Expression of the HLP Gene (HLP–RNAi). A northern blot analysis of 10 independently transformed HLP–RNAi strains, the untransformed wild-type strain (cw15), and a control strain (Co-A) transformed with the empty RNAi vector (Rohr et al., 2004) are shown. As a loading control, the 28S rRNA band in the ethidium bromide-stained agarose gel prior to blotting is also shown. Molecular Plant 2009 2, 1223-1232DOI: (10.1093/mp/ssp083) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 Altered Nucleoid Morphology and Abundance in Chlamydomonas HLP–RNAi Strains. Nucleoids were visualized by DAPI staining in cw15 wild-type cells (A) and the RNAi strains HLP–RNAi-39 (B) and HLP–RNAi-43 (C). Note weaker and more diffuse nucleoid staining in the RNAi strains compared to the cw15 control. Stronger chlorophyll fluorescence in the mutants is due to the need for longer UV exposure times to visualize the nucleoids. Molecular Plant 2009 2, 1223-1232DOI: (10.1093/mp/ssp083) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Reduced Chloroplast DNA Content in Chlamydomonas HLP–RNAi Strains. (A) Southern blot analysis of four independently transformed HLP–RNAi strains, the untransformed wild-type strain (cw15), and a control strain transformed with the empty RNAi vector (Co-A). Total cellular DNA was digested with the restriction enzyme PstI and co-hybridized to a chloroplast genome-specific probe (rbcL) and a nuclear genome-specific probe (β-tubulin). As there are two highly homologous β-tubulin genes present in the Chlamydomonas nuclear genome (Merchant et al., 2007; JGI Chlamydomonas Genome 4.0 accession numbers: 129876 and 129868), the probe recognizes a double band (TUB1 and TUB2). (B) Quantitation of relative chloroplast DNA amounts in the algal strains by measuring the hybridization signal intensities and determining the ratio of the rbcL signal to the β-tubulin (TUB1 + 2) signal. As the nuclear genome copy number can be assumed to stay constant (in vegetatively grown haploid algae), the decreased rbcL/TUB1 + 2 ratio in the strong RNAi strains HLP–RNAi-24, HLP–RNAi-39, and HLP–RNAi-43 indicates reduced copy numbers of the chloroplast genome. Values represent the averages of three biological replicas; the bars indicate the standard deviation. Molecular Plant 2009 2, 1223-1232DOI: (10.1093/mp/ssp083) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Unaltered Expression of Chloroplast Genes in Chlamydomonas HLP–RNAi Strains. RNA accumulation of selected chloroplast genes was determined by slot blot hybridization and normalized to the hybridization signals of the constitutively expressed nuclear β-tubulin genes TUB1 and TUB2. Four independently transformed HLP–RNAi strains, the untransformed wild-type strain (cw15), and a control strain transformed with the empty RNAi vector (Co-A) were analyzed. (A) Slot blot hybridization with a β-tubulin-specific probe. (B–D) Slot blot probes for three chloroplast genes and determination of their relative expression levels after normalization to the β-tubulin signal. (B) Analysis of rbcL encoding the large subunit of Rubisco. (C) Analysis of psbD encoding the reaction center protein D2 of photosystem II. (D) Analysis of rpl2 encoding the L2 protein of the large subunit of the chloroplast ribosome. Molecular Plant 2009 2, 1223-1232DOI: (10.1093/mp/ssp083) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions