Arabidopsis thaliana Remorins Interact with SnRK1 and Play a Role in Susceptibility to Beet Curly Top Virus and Beet Severe Curly Top Virus

Abstract

Remorins, a family of plant-specific proteins containing a variable N-terminal region and conserved C-terminal domain, play a role in various biotic and abiotic stresses, including host-microbe interactions. However, their functions remain to be completely elucidated, especially for the Arabidopsis thaliana remorin group 4 (AtREM4). To elucidate the role of remorins in Arabidopsis, we first showed that AtREM4s have typical molecular characteristics of the remorins, such as induction by various types of biotic and abiotic stresses, localization in plasma membrane and homo- and hetero-oligomeric interaction. Next, we showed that their loss-of-function mutants displayed reduced susceptibility to geminiviruses, Beet Curly Top Virus and Beet Severe Curly Top Virus, while overexpressors enhanced susceptibility. Moreover, we found that they interacted with SnRK1, which phosphorylated AtREM4.1, and were degraded by the 26S proteasome pathway. These results suggest that AtREM4s may be involved in the SnRK1-mediated signaling pathway and play a role as positive regulators of the cell cycle during geminivirus infection.

Keywords: Arabidopsis; SnRK1; geminivirus; remorin.

Fig. 1

The amino acid alignment and expression level of AtREM4s. (A) The amino acids sequence alignment of AtREM4s were constructed using the ClustalW software. Black boxes represent conserved amino acids and remorin C-terminal domains that were predicted by the Marcoil software. (B) RT-PCR was performed for stress responses using 2-week-old plants treated with 300 mM mannitol, 150 mM NaCl, drought and 100 μM ABA at indicted time. Actin-2 is used for a loading control and RD29A as a stress marker. (C) Promoter activity for stress response analysis. T3 transgenic plants were treated with MES, 100 μM ABA and 150 mM NaCl at 3 h, and then GUS-staining was performed. The 1,622-bp upstream region of AtREM4.1 is indicated by p4.1, and p4.2 represents the 1,544-bp upstream region of AtREM4.2. (D) RT-PCR analysis for various tissues. Actin-2 was used for loading control. B, bud; S, stem; R, root; F, flower; Si, silique; RL, rosette leaf; CL, cauline leaf; SL, senescence leaf.

Fig. 2

Subcellular localization and oligomeric interaction of AtREM4s. (A) Confocal images of 35S::AtREM4.1-CFP and 35S::AtREM4.2-CFP in 5-day-old seedlings. FM4-64 was used for PM staining marker. Bars = 50 μm. (B) Y2H analysis for interaction among AtREM1.4, AtREM4.1, and AtREM4.2. (C) BiFC analysis of homo- and hetero-oligomeric interactions between AtREM4s in N. benthamiana, as described in the Materials and Methods. The fluorescence indicates the interaction between the indicated partner proteins. BiFC cloning vectors, NE and CE, and AtREM1.4 were used as control. Scale bar = 50 μm.

Fig. 3

Geminivirus susceptibility of AtREM4s. (A) The gene structures and T-DNA insertion sites. Black boxes are exons, and gray boxes are untranslated regions. Intergenic regions or introns are marked with lines. Expression levels of AtREM4s in the double mutants as assayed by RT-PCR. (B) For the geminivirus infection experiment, Agrobacterium stains containing BCTV, BSCTV and control vector, pMON, were inoculated in the crown of the rosette of four-week-old WT and double mutant plants using a needle. (C) BSCTV infection experiments were performed using WT, AtREM4.1-1, AtREM4.2-1, AtREM4.1-1/4.2-1, 35S::AtREM4.1-CFP and 35S::AtREM4.2-CPF. (D) For severely infected plants, plant symptom severity rates were classified as previously described (Park et al., 2011). (E) For RT-PCR, total RNA was isolated from the plants that were infected with BSCTV, and RT-PCR was performed. The Actin-2 gene was used as a loading control for PCR.

Fig. 4

Phosphorylation of AtREM4s by SnRK1.2 in vitro. (A) Schematic representation of AtREM4 domains. Putative PEST domain and phosphorylation site numbers are shown. Numbers indicate amino acid positions. (B) Y2H analysis to determine interacting domains of AtREM4s with SnRK1. Constructs of full length and truncated N and C-terminal ends of AtREM4s were used. (C) In vitro kinase assay for recombinant proteins of AtREM4s, SnRK1.2 and GRIK1. The reactions were resolved by SDS-PAGE gel and visualized by autoradiography. (D) Quantification analysis for the band signal intensity was measured by ImageJ, and the signal intensity rates were calculated such as (GST-AtREM4 intensity/GST intensity) × 100.

Fig. 5

Cell free degradation assay of recombinant AtREM4 proteins. (A) Immunoblot analysis for GST-AtREM4s degradation in the presence or absence of the proteasome inhibitor MG132. Recombinant GST-fusion proteins were expressed and purified from E. coli and then added to the total proteins that were extracted from Col-0 for the indicated times. Immunoblots were probed with anti-GST antibody. Ponceau stain is shown as a loading control. (B) Quantification of the immunoblot analysis was performed using ImageJ.