*2.2. Generation of Transgenic Plants*

The *sl1* mutant was generated by CRISPR/Cas9 technique. For CRISPR vector construction, the target sequence used as sgRNA on the only exon of *Sl1* was searched by the CRISPR-P program (http://cbi.hzau.edu.cn/cgi-bin/CRISPR (accessed on 10 May 2019)). For deletion of a large fragment, two targets were selected (sgRNA-*Sl1*-254: TCATTAAAGGGTCTTCAACA and sgRNA-*Sl1*-564: GTTGAACTTGGAGCAATGAT), primers were then generated by adding adapter sequence to two ends of target sequences (Figure S1A). The sense and antisense sgRNA primers were annealed and inserted into the *Bbs*I site of the AtU6-sgRNA-AtUBQ-Cas9 vector, then positive clones were confirmed by sequencing. The two clones were named sgRNA-*Sl1*-254 and sgRNA-*Sl1*-564. The fragment was amplified using the clone of sgRNA-*Sl1*-564 as a template, the fragment was then inserted into the backbone vector with Cas9 and sgRNA-254. pCAMBIA1301 was used as a binary expression vector, and the sgRNA-254-sgRNA-564-Cas9 was inserted into the *EcoR*I and *Hind*III site of pCAMBIA1301. Positive clones were transformed into *Agrobacterium tumefaciens* strain GV3101 for transgenic plant generation.

For the construction of *Sl1* overexpressing transgenic lines, the full-length coding sequences (CDS) of *Sl1* were amplified with primers (Forward primer 5 -TTACAATTACCAT-GGGGCGCGCCATGGATCTTGTTAGACTAAAGTATTTTGAA-3 , Reverse verse primer 5 -AACATCGTATGGGTAGGTACCTGACTCTAACTGAATAGGTAAAACTACATTTC-3 ), then inserted the PCR products into the *Asc*I and *Kpn*I site of pFGC1008-HA vector. The positive vector was confirmed by sequencing and then transformed into *A. tumefaciens* strain GV3101.

The detailed method of generation of transgenic lines was described previously [35]. Two independent homozygous lines of the F2 generation of *Sl1* overexpressing plants were used in further experiments. Two homozygous lines of *sl1* mutants without CRISPR/Cas9 DNA were selected for further research. The special primers for mutant detection were designed as follows: Forward primer 5 -GCAGAGAGACAACATTCACCA- 3 , Reverse primer 5 -AAAGTTGTCGATCCGTCGCT-3 .

#### *2.3. E3 Ubiquitin Ligase Activity Assay*

The full length CDS of *Sl1* were amplified with the primers (Forward primer 5'- GAGGGAAGGATTTCAGAATTCATGGATCTTGTTAGACTAAAGTATTTTGAA-3', Reverse primers 5'-CAGGTCGACTCTAGAGGATCCTGACTCTAACTGAATAGGTAAAACT-ACATTTC-3'). The PCR products were digested with restriction endonuclease *EcoR*I and *BamH*I and were then inserted into the pMAL-2c vector (New England Biolabs, Ipswich, MA, USA). The maltose-binding protein-empty vector (MBP-EV) and MBP-fused Sl1 protein were expressed in *Escherichia coli* strain BL21 (DE3) and purified with instructions of the manufacturer (New England Biolabs, Ipswich, MA, USA). The in vitro ubiquitination assay of Sl1 was performed by instructions described previously [36]. The reaction system was prepared as described previously [19].

After the reaction, a Western blot was used to detect whether the Sl1 protein has E3 ubiquitin ligase activity. Anti-His (A5C12; HUABIO, Hangzhou, China) and anti-MBP (MBP61R; Thermo Fisher Scientific, Waltham, MA, USA) antibodies were used in Western blot assay.
