SDS-PAGE and western blotting assay

Total proteins were extracted with RIPA lysis buffer (Beyotime Biotechnology, P0013B), and the protein concentrations were determined by the BCA protein assay kit (Sangon Biotechnology, C503021–0500). The total proteins were then mixed with a 5×protein loading buffer and boiled at 99°C for 10 min. After SDS-PAGE, the proteins were transferred to PVDF membranes by eBlot^TM L1 (GenScript) and incubated with specific primary antibodies, followed with HRP-conjugated goat anti-rabbit or anti-mouse IgG. The blotted membranes were developed using a chemiluminescence gel imaging system, and the gray value of each sample was calculated using ImageJ. The αTub served as the loading control.

To detect the fab1, ACTB, and αTub protein, the anti-PIKFYVE antibody (Solarbio Life Science, K004715P), anti-ACTB antibody (Beyotime Biotechnology, AF0003), and anti-αTub antibody (Beyotime Biotechnology, AF0001) were used respectively. Rabbit polyclonal anti-Atg8, mouse monoclonal anti-RSV, and mouse monoclonal anti-SRBSDV were used to detect Atg8, RSV NP, and SRBSDV P10 protein, respectively. Anti-RSV and anti-SRBSDV monoclonal antibodies were provided by Prof. Jianxiang Wu from Zhejiang University, and rabbit polyclonal anti-Atg8 was provided by Prof. Lili Zhang from Chinese Academy of Sciences. The RBSDV abundance at protein level was analyzed after the L. striatellus was treated with 3-MA (Aladdin, M129496), rapamycin (MedChemExpress, HY -10219), dseGFP, dsLsfab1, DMSO (Solarbio Life Science, D8371), YM201636 (TargetMol Chemicals Incorporated, TM-T6110), C8-PtdIns(3,5)P₂ (Echelon Biosciences, P-3508), or ML-SA1 (MedChemExpress, HY-108462) by western blotting using RBSDV P10 specific rabbit polyclonal antibody. RBSDV P10 specific rabbit polyclonal antibody was produced by Yonglong Biotechnology (Shanghai, China) using purified His-P10 protein. To prepare the RBSDV P10 specific rabbit polyclonal antibody, the pET15b-P10 plasmid was transformed into E. coli BL21 (DE3) Rosetta cells. Recombinant His-P10 protein was purified and used for immunization. Nilaparvata lugens Trpml gene sequence (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"KX249700","term_id":"1069777832","term_text":"KX249700"}}KX249700) and Recilia dorsalis ref(2)P/SQSTM1 gene sequence (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"MF038048","term_id":"1277384161","term_text":"MF038048"}}MF038048) [⁵⁵] were subjected to BLAST comparison with the L. striatellus genome (GenBank: GCA_017141395.1). The homolog cDNA sequences of Trpml and ref(2)P/SQSTM1 in L. striatellus were obtained and deposited in GenBank with the accession number {"type":"entrez-nucleotide","attrs":{"text":"MZ476564","term_id":"2172626267","term_text":"MZ476564"}}MZ476564 and {"type":"entrez-nucleotide","attrs":{"text":"MZ476565","term_id":"2172626269","term_text":"MZ476565"}}MZ476565, respectively. The corresponding protein sequences of Trpml (GenBank: {"type":"entrez-protein","attrs":{"text":"UIA40723","term_id":"2172626268","term_text":"UIA40723"}}UIA40723) and ref(2)P/SQSTM1 (GenBank: {"type":"entrez-protein","attrs":{"text":"UIA40724","term_id":"2172626270","term_text":"UIA40724"}}UIA40724) in L. striatellus were also deposited in NCBI. Rabbit polyclonal anti-Trpml antibody against LsTrpml peptide KGWDPTREVSSYPPC and polyclonal anti-ref(2)P/SQSTM1 antibody against ref(2)P/SQSTM1 peptide VIEIGKLGSKESPDC were produced by GenScript (Nanjing, China).