Stability of small ubiquitin-like modifier (SUMO) proteases OVERLY TOLERANT TO SALT1 and -2 modulates salicylic acid signalling and SUMO1/2 conjugation in Arabidopsis thaliana

Abstract

Small ubiquitin-like modifier proteases 1 and 2 (SUMO1/2) have been linked to the regulation of salicylic acid (SA)-mediated defence signalling in Arabidopsis thaliana. In order to define the role of the SUMO proteases OVERLY TOLERANT TO SALT1 and -2 (OTS1/2) in defence and to provide insight into SUMO1/2-mediated regulation of SA signalling, we examined the status of SA-mediated defences in ots1/2 mutants. The ots1 ots2 double mutant displayed enhanced resistance to virulent Pseudomonas syringae and higher levels of SA compared with wild-type (WT) plants. Furthermore, ots1 ots2 mutants exhibited upregulated expression of the SA biosynthesis gene ICS1 in addition to enhanced SA-responsive ICS1 expression beyond that of WT. SA stimulated OTS1/2 degradation and promoted accumulation of SUMO1/2 conjugates. These results indicate that OTS1 and -2 act in a feedback loop in SA signalling and that de novo OTS1/2 synthesis works antagonistically to SA-promoted degradation, adjusting the abundance of OTS1/2 to moderate SA signalling. Accumulation of SUMO1/2 conjugates coincides with SA-promoted OTS degradation and may play a positive role in SA-mediated signalling in addition to its repressive roles reported elsewhere.

Keywords: Arabidopsis thaliana; SUMO protease; SUMOylation.; defence; pathogen; salicylic acid (SA); small ubiquitin-like modifier (SUMO).

Fig. 1.

The ots1 ots2 double mutant displays enhanced resistance to virulent Pst. (A) Colony-forming unit counts of Pst DC3000 in the leaves of 4-week-old WT, ots1 ots2 double mutant, and OTS1-overexpressing lines (OTS1-HOx1 and OTS-HOx2) on the day of infiltration (day 0) and on day 3. (B) Quantitative PCR analysis of gene expression from 4-week-old WT, single ots1 and ots2 mutants, and the double ots1 ots2 mutant of PATHOGENESIS-RELATED1 (PR1), PR2, and PR5 genes (normalized to ACTIN7). Error bars represent SEM. P values for differences between WT and mutants: *P<0.05, **P<0.01, and ****P<0.0001 (one-way ANOVA with Tukey post hoc test ).(one-way ANOVA with Tukey post hoc test).

Fig. 2.

The ots1 ots2 double mutant displays spontaneous lesions. Trypan blue staining for cell death within comparable leaves from 2-week-old WT and ots1 ots2 double mutant plants. (A) Representative images of stained leaves. (B) Analysis of the percentage of cell death per 4mm² across biological replicates using ImageJ. Error bars represent SEM. P values for differences between WT and mutants: **P=0.001–0.01 (unpaired Student’s t-test).

Fig. 3.

SA biosynthesis is upregulated in the ots1 ots2 double mutant. (A) qPCR analysis of gene expression from 4-week-old WT, single ots1 and ots2 mutants, and the double ots1 ots2 mutant, of SA biosynthesis genes ICS1 and PAL1 to -4 (normalized to ACTIN7). (B) Liquid chromatography/mass spectrometry quantification of SA and glycosylated SA (SAG) in WT, single ots1 and ots2 mutants, and the double ots1 ots2 mutant. Internal standards were unavailable for SAG; hence, values are given as relative abundances (one-way ANOVA with Tukey’s post hoc test). Error bars represent SEM. P values for differences between WT and mutants: **P<0.01, ***P<0.001 and ****P<0.0001, respectively (one-way ANOVA with Tukey’s post hoc test).

Fig. 4.

SA-related defence gene expression is upregulated in the ots1 ots2 double mutant. qPCR analysis of gene expression from 4-week-old WT, single ots1 and ots2 mutants, and the double ots1 ots2 mutant, of SA signalling pathway components, the TGA transcription factors TGA1, -2, -3, -4, -5, and -6 and NPR1 and its paralogues NPR1-LIKE PROTEIN3 (NPR3) and NPR4 (normalized to ACTIN7). Error bars represent SEM. P values for differences between WT and mutants: *P=0.01–0.05, **P=0.001–0.01, and ****P< 0.0001, respectively (one-way ANOVA with Tukey’s post hoc test).

Fig. 5.

OTS1 and -2 restrict ICS1 gene expression. qPCR gene expression analysis of ICS1 in (A) 10-d-old WT, ots1 ots2 double mutant and transgenic OTS1-overexpressing (OTS1-HOx1 and -2) plants grown in the presence of INA (40mg ml^–1) or solvent (control) (A) and in 10- and 28-d-old WT and the ots1 ots2 double mutant (B). Error bars represent SEM. P values for differences between WT and mutants: *P=0.01–0.05, ****P<0.0001, respectively (multi-way ANOVA with Tukey test post hoc).

Fig. 6.

OTS1 and -2 degradation are promoted by SA. Western blots probed with anti-HA monoclonal antibodies showing OTS1 and -2 stability using the Arabidopsis transgenic line OTS1-HOx2 (A, B) or Agrobacterium-mediated transient expression in N. benthamiana of OTS2 (C, D). (A) OTS1: 4-week-old plants sprayed with SA or solvent (control) over a 6h time course. (B) OTS1: 10-d-old plants, grown in liquid ½ MS treated with SA (+SA) or solvent (–SA) following pre-incubation with the 26S proteasome inhibitor MG132 (+) or solvent (−). (C) OTS2: 4-week-old plants infiltrated with SA or solvent (control) over a 3h time course. (D) OTS2: 4-week-old plants infiltrated with SA (+SA) or solvent (–SA) following pre-incubation with MG132 (+) or solvent (−). Asterisks (*) indicate the HA-OTS1/2 bands. Coomassie blue (Coom. Blue) staining of the blots is shown as a loading control.

Fig. 7.

SUMO1/2 conjugation is promoted by SA. Western blots probed with anti-SUMO1/2 polyclonal antibodies showing SUMO1/2 and their conjugates in 4-week-old plants sprayed with salicSAylic acid or solvent (control). (A) Effect on WT over 6h. (B) Effect on WT compared with the ots1 ots2 double mutants over 6h. Coomassie blue staining (Coom. Blue) of the blots is shown as a loading control.