Fig 1.
Phylogenetic and conserved protein motifs of TCTPs.
The phylogenetic tree of TCTP protein sequences included Homo sapiens TCTP (P13693), Mus musculus TCTP (P63028), Drosophila melanogaster TCTP (Q9VGS2), Poeciliopsis prolifica TCTP (JA088771), Cucumis sativus TCTP1 (XP-004134215), Cucumis sativus TCTP2 (XP-004135602), Cucurbita maxima TCTP (ABC02401), Cucumis melo TCTP1 (AAF40198), Cucumis melo TCTP2 (XP_008450611), Arabidopsis thaliana TCTP 1 (NP-188286), Arabidopsis thaliana TCTP 2 (NP-187205), Zea mays TCTP (Q8H6A5), Triticum aestivum TCTP (Q8LRM8), Oryza sativa TCTP (XP_015617060), Hevea brasiliensis TCTP (Q9ZSW9), Brassica oleracea TCTP (Q944W6), Larix kaempferi TCTP (AGW01241), Pseudotsuga meziesii TCTP (Q9ZRX0), Schizosaccharomyces pombe TCTP (Q10344), and Physcomitrella patens TCTP (Q10344). The construction of the tree was conducted with Mega 6.0. Numbers above the branches indicate bootstrap values.
Fig 2.
Putative structural domains of CsTCTP1 and CsTCTP2.
Fig 3.
Relative expression levels of CsTCTP1 in various tissues of the susceptible variety (a) and resistant variety (b). Relative expression levels of CsTCTP2 in various tissues of the susceptible variety (c) and resistant variety (d).
The expression level in the root was normalized as 1. Data represent means ± SE of three biological replicates. Letters indicate significant differences at P < 0.05 compared with the root by Student’s t-test.
Fig 4.
Relative expression levels of CsTCTP1 and CsTCTP2 in resistant (B21-a-2-1-2) and susceptible (B21-a-2-2-2) varieties inoculated with S. fuliginea.
The expression level in B21-a-2-2-2 was normalized as 1. Data represent means ± SEs of three biological replicates. Asterisk or asterisks indicate significant differences at P < 0.05 or P < 0.01, respectively, compared with B21-a-2-2-2 by Student’s t-test.
Fig 5.
Relative expression levels of CsTCTP1 and CsTCTP2 in a resistant (B21-a-2-1-2) variety under various abiotic stresses.
The expression level at 0 h was normalized as 1. Data represent means ± SEs of three biological replicates. Letters indicate significant differences at P < 0.05 compared with 0 h treatment by Student’s t-test.
Fig 6.
Relative expression levels of CsTCTP1 and CsTCTP2 in a resistant (B21-a-2-1-2) variety under various treatments.
The expression level of the water treatment (12 h) was normalized as 1. Data represent means ± SEs of three biological replicates. Asterisk or asterisks indicate significant differences at P < 0.05 compared with water treatment (12 h) by Student’s t-test.
Fig 7.
Overexpression of CsTCTPs in E. coli.
(A) Diagram of prokaryotic expression vector construction, (B) the enzyme digestion to identify the constructed prokaryotic expression vector, M. 2000 DNA marker, 1, 2. pET30a-CsTCTP1/Kpn I+Sac I, 3, 4. pET30a-CsTCTP2/Kpn I+Sac I, (C) CsTCTP1 protein expression in E. coli BL21, M. protein marker, 1. pET30a without induction, 2. pET30a induction for 6 h, 3. pET30a-CsTCTP1 without induction, 4, 5, 6. pET30a-CsTCTP1 induction for 3 h, 6 h, 12 h, respectively, 7, 8, 9. Repeat 4, 5, 6, (D) CsTCTP1 protein expression in E. coli BL21, M. protein marker, 1. pET30a without induction, 2. pET30a induction for 6 h, 3. pET30a-CsTCTP2 without induction, 4, 5, 6. pET30a-CsTCTP2 induction for 3 h, 6 h, 12 h, respectively, 7, 8, 9. Repeat 4, 5, 6.
Fig 8.
Spot assay of BL21+pET30a, BL21+pET30a-CsTCTP1 and BL21+pET30a-CsTCTP2 on LB plates containing 50 μg/mL kanamycin at 37°C, 45°C and 55°C (for heat stress) or on LB plates with NaCl (250 mM, 500 mM and 750 mM, for salt stress), mannitol (0.4 M, 0.8M and 1.2M, for drought stress), or HgCl2 (15 μM, 25 μM and 35 μM, for mercury stress) at 37°C overnight. Liquid culture assay on LB liquid medium containing 50 μg/mL kanamycin at 45°C or on LB liquid medium with 500 mM NaCl, 0.8M mannitol, or 25 μM HgCl2 at 37°C. All data points are mean ± SE (n≥3).