The nanovirus-encoded Clink protein affects plant cell cycle regulation through interaction with the retinoblastoma-related protein

Abstract

Nanoviruses, multicomponent single-stranded DNA plant viruses, encode a unique cell cycle link protein, Clink, that interacts with retinoblastoma-related proteins (RBR). We have established transgenic Arabidopsis thaliana lines that conditionally express Clink or a Clink variant deficient in RBR binding. By controlled induction of Clink expression, we demonstrated the capacity of the Clink protein to alter RBR function in vivo. We showed that transcription of both S-phase-specific and G2/M-phase-specific genes was up-regulated depending on the RBR-binding proficiency of Clink. Concomitantly, ploidy levels increased in a substantial fraction of leaf cell nuclei. Also, leaf epidermis cells of transgenic plants producing Clink were smaller and more numerous, indicating additional cell divisions in this tissue. Furthermore, cytogenetic analyses following induction of Clink expression in mature leaves revealed the presence of metaphasic and anaphasic nuclei, clear evidence that Clink-mediated RBR inactivation is sufficient to induce quiescent cells to reenter cell cycle progression and, for at least a fraction of them, to pass through mitosis. Expression of Clink had no effect on genes transcribed by RNA polymerases I and III, suggesting that, in contrast to its mammalian homologue, A. thaliana RBR is not involved in the repression of polymerase I and polymerase III transcription. The results of these in vivo analyses firmly establish Clink as a member of the diverse class of multifunctional cell cycle modulator proteins encoded by small DNA viruses.

FIG. 1.

Expression of viral sequences in transgenic plants, showing the impact of Clink on cell cycle-regulated gene expression. Following Dex (D) or ethanol (E) treatment of detached, fully expanded leaves, total RNAs (panel A) or proteins (panel B) were extracted and analyzed. (A) Six micrograms of total RNAs extracted from the expanded rosette leaves was used for Northern blot analysis. The time (T) of the treatment and the identity of the transgenic plant line (C, Clink; C*, Clink^mut) are given above the panels. The two membranes were hybridized successively with probes specific for the classes of genes given on the left (see Materials and Methods). The transcript level of actin was used as a control for quantification. Six micrograms of total RNA extracted from cells from an actively growing A. thaliana T87 suspension culture (6) was also loaded and served as a positive control for hybridization with the cell cycle-specific probes. (B) Typical immunoblot result obtained with the anti-Clink antibodies. Molecular masses of protein markers (in kilodaltons) are given on the right. As previously reported (5), the Clink^mut protein migrates slightly faster than the Clink protein. Equivalent amounts of proteins were loaded in each lane, as confirmed by the similar intensities of the ≅45-kDa bands resulting from cross-reactions to endogenous proteins.

FIG. 2.

Representative DAPI-stained nuclei isolated after 4 days of Dex treatment on detached, expanded 6- to 7-week-old rosette leaves from Clink-transgenic plants. The relative proportions of these different types of nuclei in Clink-expressing tissues were as follows: 33% for oval nuclei with irregular contours and ∼10 CCs (A), 10% for larger nuclei with more CCs (B), 19% for smaller, rounded nuclei with >10 CCs (C and D), 7% for nuclei with highly condensed euchromatin (E), and 1% for dividing (e.g. anaphasic) nuclei (F). For Dex-treated Clink^mut plant leaves and ethanol-treated Clink or Clink^mut plant leaves, only nuclei of the types shown in panels A and B were observed, in proportions of about 95% and 5%, respectively. In each case, the relative proportions are based on the observation of 800 to 1,100 nuclei. Bars, 15 μm.

FIG. 3.

Effect of Clink expression on cell size. (A) SEM of the leaf adaxial epidermis of Clink-expressing, Clink^mut-expressing, and nontransgenic Col4 plants. Bars, 200 μm. (B) Cell area distribution of Clink-expressing (open bars), Clink^mut-expressing (shaded bars), and nontransgenic (solid bars) Col4 plants. (C) Number of cells per square millimeter of leaf epidermis of Clink-expressing (open bar), Clink^mut -expressing (shaded bar), and nontransgenic (solid bar) Col4 plants. (D) Immunoblot analysis of total proteins from Col4 plants and from the two transgenic plant lines using Clink-specific antibodies. The identity of the plant line is given above the panel. As explained for Fig. 1B, the Clink^mut protein migrates faster than the Clink protein. Plants for which results are shown in panels A and D were grown and induced under the same conditions.

FIG. 4.

Effect of Clink expression on endoreduplication in rosette and cauline leaves. (A) Flow cytometry profiles of the DNA contents of nuclei from third and fourth expanded rosette leaves of 4-week-old Clink-expressing, Clink^mut-expressing, and nontransgenic Col4 plants grown on plates in the presence of Dex. (B) Comparison of the DNA contents in nuclei of rosette leaves derived from Clink-expressing (open bars), Clink^mut-expressing (shaded bars), and nontransgenic (solid bars) Col4 plants. (C) Comparison of the DNA contents in nuclei of detached cauline leaves following 4 days of Dex treatment. For panels B and C, three independent experiments with six replicates were performed; representative results for one replicate are shown in panel A.

FIG. 5.

FBNYV infection of A. thaliana plants. (A) Symptoms of an infected plant (left) compared to a healthy plant (right). (B) Ploidy levels in cauline leaves of FBNYV-infected plants (shaded bars) or healthy control plants (solid bars) 29 days postinfection.