The nuclear dynamic of CDC48 is affected during the immune response in plants

Abstract

Plants are continuously challenged by a myriad of pathogenic microorganisms, including bacteria, viruses, fungi and oomycetes, against which they must defend themselves. The protein Cell Division Cycle 48 (CDC48), a key player of ubiquitin-proteasome system which segregates and remodels ubiquitinated proteins for degradation, is known to be mobilized during plant immunity. Moreover, the characterization of the nuclear role of CDC48 is of interest, in particular its regulation in nuclear processes such as chromatin remodeling, DNA repair and gene expression. In this regard, its nuclear functions during plant immunity remain underexplored. This study investigates the dynamics of CDC48 during plant immune responses. The biophysical analysis using the Fluorescence Correlation Spectroscopy (FCS) on tobacco leaves stably overexpressing GFP-CDC48 revealed that the nuclear dynamics of CDC48 changed after treatment with cryptogein, an elicitor of immune responses. FCS analysis revealed an increase of the nuclear mobility of CDC48 and a faster interaction of CDC48 with a wide range of nuclear partners shortly after cryptogein treatment. Overall, our study shows a nuclear regulation of the interaction of CDC48 with its potential partners and sheds new light on potential nuclear involvements of CDC48 following the triggering of defense mechanisms.

Keywords: Cell division cycle 48; biophysics; cryptogein; fluorescence correlation spectroscopy; plant immunity.

Figure 1.

Nuclear functions of CDC48. (a) Chromatin Decondensation Regulation. In Arabidopsis, CDC48 interacts with sumoylated cenH3 to segregate it from chromatin, decondensing DNA regions required for ribosomal RNA synthesis, thereby promoting pollen tube growth. (b) Regulation of gene expression. CDC48 facilitates transcription by segregating the polyubiquitinated alpha2 transcriptional repressor and degrading it via the proteasome. (c) Replication and cell division regulation. CDC48 halts DNA replication by extracting ubiquitinated CMG helicase complexes through the recognition and segregation of the Mcm7 subunit. (d) DNA repair regulation. Following DNA damage repair, CDC48 segregates and targets to the proteasomal degradation the ubiquitin ligase RNF8. (e) INMAD. CDC48 plays a crucial role in the degradation of inner nuclear membrane proteins (INMAD), including SUN proteins, which are key regulators of nuclear morphology.

Figure 2.

Biophysical analysis of CDC48 nuclear dynamics, using the FCS technique, 30 minutes after the treatment with cryptogein. (a) Number of molecules of GFP-CDC48 within the analyzed volume. (b) Diffusion coefficient of CDC48 (D, µm².s⁻¹). (c) Association rate of CDC48 with its partners (Kon, µM⁻¹.s⁻¹). (d) Dissociation rate of CDC48 with its partners (Koff, en s⁻¹). (e) Dissociation constant of CDC48 with its partners (Kd, µM). All experiments were performed on leaf discs of tobacco plants stably overexpressing GFP-CDC48. Results are means of data from three independent biological replicates (± SD), n = 33 for the control condition and n = 30 for the condition treated with cryptogein for 30 min. (t-test, *p ≤ 0.005). Cry: cryptogein; n: number of measurements performed.

Figure 3.

Schematic representation of the nuclear dynamics of CDC48 before and after cryptogein treatment. (a) Before treatment with cryptogein, nuclear CDC48 exhibits slow diffusion within the nuclei and interacts with a limited set of nuclear partners in diverse nuclear processes. (b) After cryptogein treatment, the nuclear concentration of CDC48 decreased and its diffusion increased. Additionally, CDC48 interacts more rapidly or with more nuclear partners. This enhanced mobility and interaction of CDC48 potentially contributes to the expression of defense-related genes or the maintenance of nuclear protein homeostasis. CDC48 protein partners are represented in dark gray. The length of the red arrow represents CDC48 mobility.