The seeds of the wild-type, and transgenic Arabidopsis were sterilized and grown in a greenhouse at 22˚C, 16 h light/8 h dark, and 70% relative humidity. Nicotiana benthamiana (N. benthamiana) plants were grown in a greenhouse at 24˚C, 12 h light/12 h dark, and 70% relative humidity. Transient expression analysis was performed using 5-week-old N. benthamiana plants.

Total RNA was obtained from 4-week-old wild-type Arabidopsis leaves using Trizol reagent and chloroform. Precipitate RNA with isopropanol and dissolve it in RNase-free water. Remove DNA contamination by treating RNA solution with RNase-free DNase at 37˚C for 30 minutes. First strand cDNA was synthesized from total RNA using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific) at 42˚C for 60 minutes. Amplify target CDS from cDNA for cloning and transient expression.

In brief, using wild-type Arabidopsis leaf cDNA as the initial template. Transfer the recovered PCR products into the entry vector PENTR/D through a one-step cloning method. Subsequently, the target fragment was transferred to the expression vector using Gateway cloning technology, such as yeast vector pGBDKT7 and pGADT7 or plant vector pEarleygate101 for YFP-HA or Myc tagged or pGWB3 for GUS labeled.

In a word, after two minutes of vacuum treatment at 80,000 Pa, 4-week-old transgenic Arabidopsis plants were stained with 2 mM 5-bromo-4-chloro-3-indoyl-β-d-glucuronide (X-Gluc) in 100 mM phosphate buffer at 37˚C for 24 hours and washed with 70% ethanol to remove chlorophyll.

Linking BOI with the bait vector pGBKT7 for screening 4-week-old wild-type Arabidopsis leaves cDNA library constructed in the prey plasmid pGADT7. The transformation, mating, screening, and interaction determination of yeast were strictly executed in accordance with the corresponding chapters in the Clontech Yeast Protocols Handbook.

Agrobacteria carrying the constructs were cultivated overnight in LB medium containing Rifampicin and kanamycin. Agrobacterium culture was centrifuged and resuspended in MES buffer, incubated at room temperature for 1 hour and infiltrated into 4-week-old N. benthamiana leaves at a specific OD₆₀₀ values.

Total proteins were harvested using extraction buffer (20 mM Tris-HCl pH 8.0, 5 mM EDTA, 1% SDS, 10 mM DTT). The protein samples mixed with the loading buffer were denatured at 100˚C for 10 min. Equal amounts of total proteins were separated through SDS-PAGE and transferred to NC membranes. Immunoblotting was performed using the following antibodies: anti-HA (Roche, #11867423001), anti-Myc (Genscript, #A00704), anti-β-actin (Abbkine, #A01050-2) at a 1:10,000 dilution.

The expression localization and stage of BOI gene are likely to play a role in specific functions during plant growth. To investigate the BOI gene expression pattern, transgenic plants carrying the GUS reporter gene under the control of the BOI gene promoter, including the region 1.8 kb upstream of the transcription start sites, were generated through Agrobacterium-mediated genetic transformation. Several independent transgenic lines were then screened in the T₂ generation for GUS activity at multiple life stages and representative examples are shown in Figure 1 . The results of the GUS reporter gene assay with X-Gluc staining indicated that native expression of the BOI gene in Arabidopsis was demonstrated in all tissues investigated, containing roots, stems, mature leaves, flowers, and siliques. The results of BOI gene promoter triggers GUS gene expression were consistent with the observation of expression data collected from The Arabidopsis Information Resource (TAIR) website (TAIR - Home (arabidopsis.org)), indicating that BOI gene may be involved in multiple processes in plant growth and development in Arabidopsis.

Previous articles have shown that the bait vector pGBDKT7-BOI did not have self-activation effect [ 6 , 8 ]. Eight positive colonies that may interact with BOI were acquired through yeast two-hybrid library

screening and the number of repetitions and functional descriptions of each protein in the screening library are shown in Figure 2 and Table 1 . Functional analysis of these candidate interacting proteins shows that they participate in multiple pathways, including biological and abiotic stress, programmed cell death, protein degradation, material metabolism and transcriptional regulation. EXO70E2 participated in regulation of protein targeting and positive regulation of extracellular exosome assembly, which acts upstream of or within immune response and defense response by callose deposition. WRKY7 encodes a Ca-dependent calmodulin binding protein, which sequence similarity to the WRKY transcription factor gene family and involved in regulation of DNA-templated transcription. WRKY11 related to defense response to bacterium, induced systemic resistance, regulation of DNA-templated transcription, regulation of jasmonic acid (JA) mediated signaling pathway and response to bacterium. WRKY17 concerned with regulation of DNA-templated transcription, which acts upstream of or within defense response to bacterium. UBP20 encodes a ubiquitin-specific protease, which involved in regulation of protein stability. L5, a nucleotide-binding site and leucine-rich repeat receptors (NBS-LRR/NLR), localized to plasma membrane. Overexpression triggers cell death, anchored to the plasma membrane due to the presence of the myristoylation site Gly2, which involved in defense response to other organisms. SAUR9 participated in response to abscisic acid, response to auxin, response to brassinosteroid, response to light intensity and response to red or far-red light. TCP21 belongs to the circadian oscillator protein and interacts with bZIP63 to regulate the circadian oscillator's response to sugar. In addition, together with TCP7, 8, 14, 15, 21, 22, and 23, promote the expansion of cells dependent on internal replication in leaves.

We have previously demonstrated that BOI possesses E3 ubiquitin ligase activity, and introducing the H273Y mutation in its RING domain can abolish its E3 ubiquitin ligase activity [ 9 ]. Additionally, BOI degrades the L5 receptor through ubiquitination, thereby abolishing the cell death caused by it in N. benthamiana [ 9 - 11 ]. Next, we would like to know the impact of BOI on the stability of the other eight candidate interacting proteins. We fused YFP-HA tags at the C-terminus of these candidate proteins and co-expressed them with BOI-Myc in N. benthamiana leaves. The results of transient expression indicated that BOI can not only reduce the content of NLR receptor L5, but also negatively regulate the protein abundance of EXO70E2, and its degradation behavior also depends on its E3 ubiquitin ligase activity ( Figure 3(a) and Figure 3(b) ).