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 ).