Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin

Abstract

Little is known about the division of eukaryotic cell organelles and up to now neither in animals nor in plants has a gene product been shown to mediate this process. A cDNA encoding a homolog of the bacterial cell division protein FtsZ, an ancestral tubulin, was isolated from the eukaryote Physcomitrella patens and used to disrupt efficiently the genomic locus in this terrestrial seedless plant. Seven out of 51 transgenics obtained were knockout plants generated by homologous recombination; they were specifically impeded in plastid division with no detectable effect on mitochondrial division or plant morphology. Implications on the theory of endosymbiosis and on the use of reverse genetics in plants are discussed.

Figure 1

Alignment of amino acid sequences of two eukaryotic and one prokaryotic FtsZ proteins in the one-letter-code. Aligned are PpFtsZ from the moss Physcomitrella patens (GenBank AJ001586), cpFtsZ from the dicotyledon Arabidopsis thaliana (SwissProt Q42545) and FtsZ from the enterobacterium Escherichia coli (SwissProt P06138). Where applicable a consensus is given below the alignment. Two PROSITE motifs, FTSZ 1 and FTSZ 2, are marked by underlining the Physcomitrella sequence once. A third PROSITE motif, the tubulin signature motif, is marked by underlining the consensus sequence twice. An amino acid position is boxed within this motif, where serines in the eukaryotic proteins match perfectly to the tubulin signature motif while a threonine in the prokaryotic protein deviates from this consensus motif. Note the different lengths of the N-terminal extensions of the two eukaryotic FtsZ proteins as compared with the bacterial sequence.

Figure 2

Schematic representation of the cloning strategy to generate, and of PCR primer specifications to analyze ΔPpftsZ knockout plants. (A) The 1775 bp PpftsZ cDNA (open bar) comprises an ORF for 378 amino acids (ORF). A 1,173 bp subclone (hatched bar) was used for subsequent experiments. (B) A HindIII (Hd) fragment (cross-hatched bar) containing the 35S promoter-driven nptII gene as a selectable marker (23) was cloned into the HincII (Hc) digested subclone (C) The hybrid EcoRI/SacI fragment (E/S), containing the nptII cassette flanked by 247 and 658 bp PpftsZ cDNA sequence, was subsequently used for transformation of Physcomitrella protoplasts. (D) Pairs of PCR primers were specific for nptII sequences (PT 1/PT2), 5′-integration site (ppf4/RT1) and 3′-integration site (RT4/ppf5), respectively. Dimensions are drawn to scale.

Figure 3

Phenotypes of transgenic Physcomitrella patens generated by the construct outlined in Fig. 2C. Light microscopy from different tissues from transgenic plants with wild type-like plastids (A–C), and macrochloroplasts (D–F), respectively. The tissues depicted are chloronema (A and D), caulonema (B and E), and leaves (C and F). See ref. for a review on moss development and tissue definition. Bar = 100 μm. As judged by transmission electron microscopy, division of chloroplasts was impeded in ΔPpftsZ knockouts, whereas form and number of mitochondria remained unchanged (G). (Bar = 1 μm.)

Figure 4

Molecular analyses of transgenic Physcomitrella generated by the construct outlined in Fig. 2C. Analyzed were water (lane 1), untransformed wild type (lane 2), three different transgenics with macrochloroplasts (lanes 3–5; compare Fig. 3 D–G), and a transgenic with wild type-like plastids (lane 6; compare Fig. 3 A–C), respectively. (A–C) DNA gel analysis of representative PCR reactions. Each pair of primers was specific for sequences from nptII (A), 5′-integration site (B) and 3′-integration site (C), respectively. See Fig. 2D for primer specifications. Sizes of PCR fragments were 700 bp (A), 373 bp (B), and 826 bp (C). (D) Representative RNA blot analysis. About 10 μg of total RNA (19) was isolated from plants and probed with the 1,173 bp PpftsZ subclone indicated in Fig. 2A. (E) RNA blot analysis. The same filter was reprobed with the nptII cassette indicated in Fig. 2B. Transcript sizes in nucleotides.