3.4 Prediction and Identification of CsHSP24.6 Target Genes
Based on the analysis described in 3.3, the protein interactions ofTEA033542 and TEA017741 were validated by constructing anA. thaliana association model using STRING software (version 10.5, https://string-db.org/). The amino acid sequence of TEA033542 was used to retrieve the homologous gene AtHSP21 . The subnetwork of the hub geneAtHSP21 is displayed in Figure 5A , Figure S3.displays the subnetwork of the hub gene AtHSP22.0 , which is similarly homologous with TEA017741 . In the two interaction networks, the protein–protein relationship scores and the HSP family genes were evaluated, and the correlation between HSP21 andpTAC5 had the highest score. On the basis of the gene sequences of AtpTAC5 , CspTAC5 was amplified with BLAST to compare cDNA sequences from the NCBI. A full-length ORF of 1200 bp was cloned from young leaves and was observed to encode 399 amino acids.CspTAC5 contains a peptidoglycan binding-like domain (PG binding) and a DnaJ domain46 (Figure S4 ). On the basis of the TEA033542 nucleotide sequence, a full-length ORF consisting of 657 bp was amplified and was named CsHSP24.6 in accordance with its molecular weight.
To verify the relationship between CsHSP24.6 and CspTAC5 , the expression profiles of CsHSP24.6 and CspTAC5 in tea plants were characterized by using qRT-PCR analysis. The results revealed that the expression levels of CsHSP24.6 andCspTAC5 were higher in mature leaves than in top buds (Figure5B ). The expression levels of CsHSP24.6 andCspTAC5 in mature leaves were approximately 4.28 and 5.17 times higher, respectively, than those in buds (Figure 2B ). GFP fusion is an efficient approach for subcellular protein localization47. For subcellular localization ofCsHSP24.6 and CspTAC5 , a GFP ORF was fused to the C-terminus of an ORF fragment and injected into N. benthamiana . Negative GFP controls were also injected into N. benthamiana . Confocal microscopy observation was used to determine that the CsHSP24.6 -GFP fusion protein was localized in the chloroplast (Figure 5C ). These results suggest that there is an interaction between CsHSP24.6 andCspTAC5 .
Pull-down and BiFC experiments showed the interaction ofCsHSP24.6 with CspTAC5 in vitro and in vivo. GST-CsHSP24.6 could pull down MBP-CspTAC5 , but free GST protein could not capture CspTAC5 protein or free MBP protein. Therefore, CsHSP24.6 and CspTAC5 interact in vitro (Figure5E ). A strong fluorescence from YFP was observed whenCsHSP24.6 and CspTAC5 were co-expressed in A. thaliana protoplasts; however, when the combination ofCsHSP24.6 -YFPN and YFPC(CsHSP24.6 -YFPC and YFPN) or the combination of CspTAC5 -YFPN and YFPC(CspTAC5 -YFPC and YFPN) was co-transformed into protoplasts, no YFP fluorescence was observed. This result demonstrates thatCsHSP24.6 also interacts with CspTAC5 in vivo (Figure5D ).