Ni nta agarose beads

The CDSs of Sspep1 and pod-1a were amplified by PCR using the corresponding primer pairs pGEX-pep1-BamHI-F/pGEX-pep1-BamHI-R and pET30a-pod-BamHI-F/pET30a-pod-BamHI-R (Table S1) with cDNA generated from the whit part of a whip of infected sugarcane plants serving as a template. The fragments were cloned into the prokaryotic expression vectors pGEX4-1 and pET30a to yield the constructs pGEX4-Sspep1 and pET-Pod-1a. The plasmids were transformed into BL21 Competent Cells (Vazyme, Nanjing, China), and the expressed recombinant proteins were purified using Ni–NTA Agarose beads or GST-tag Purification Resin (both from Beyotime, Shanghai, China). In vitro horseradish peroxidase (HRP) activity was assayed by the Peroxidase (POD) Assay Kit (Solarbio, Beijing, China).

GST-Akt was cloned into the vectors pGEX-4T-2, and His-SOCS1 was cloned into pET-28a(+). E. coli BL21 were transformed with pGEX-4T-2-Akt and pET-28a(+)-SOCS1 vectors. GST-Akt and His-SOCS1 protein expression were induced with IPTG and purified using Glutathione Agarose 4B (Cytiva, #17075601) and Ni-NTA Agarose beads (Beyotime, #P2218) according to standard protocols. Purified GST-Akt and His-SOCS1 were incubated in reaction buffer at 4 °C for 1 h, and then with GST-beads overnight at 4 °C. Afterwards, the beads were washed four times with low-salt buffer, and bound proteins were eluted and subjected to immunoblot analysis.