Author Contributions
Conceptualization, N.A.A. and T.S.; Methodology, N.A.A., T.S., M.H.; Software, N.A.A. and M.H.; Validation, N.A.A., M.H. and T.S.; Formal Analysis, N.A.A. and T.S.; Investigation, N.A.A. and T.S.; Resources, N.A.A. and T.S.; Data Curation, N.A.A. and T.S.; Writing-Original Draft Preparation, N.A.A. and T.S.; Writing-Review and Editing, N.A.A. and T.S.; Visualization, N.A.A. and T.S.; Supervision, T.S., M.H., S.T. and I.O.; Project Administration, T.S.; Funding Acquisition, T.S.
Figure 1.
Effect of Pst inoculation-timing on disease severity. (a) NT: No HST followed by Pst challenge by inoculation at 3, 6, 12, 24, 48, or 72 h after HST (45 °C, 2 min). (b) Partial HST tests, only the first leaf of tomato plants was treated by HS, followed by inoculation on both first and second leaves at 24 h after HST. (c) bacterial speck symptom caused by Pst. Vertical bars indicate the standard error of the mean (n = 3). Different letters indicate significant differences between treatments, based on Tukey’s test, p < 0.05.
Figure 1.
Effect of Pst inoculation-timing on disease severity. (a) NT: No HST followed by Pst challenge by inoculation at 3, 6, 12, 24, 48, or 72 h after HST (45 °C, 2 min). (b) Partial HST tests, only the first leaf of tomato plants was treated by HS, followed by inoculation on both first and second leaves at 24 h after HST. (c) bacterial speck symptom caused by Pst. Vertical bars indicate the standard error of the mean (n = 3). Different letters indicate significant differences between treatments, based on Tukey’s test, p < 0.05.
Figure 2.
Changes in (a) PR1a2, and (b) PR1b1 expression levels in the tomato cultivar Natsunokoma. Gene expression levels in the first leaf at 3, 6, 12, 24, 48, or 72 h after HST (45 °C, 2 min) were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 4).
Figure 2.
Changes in (a) PR1a2, and (b) PR1b1 expression levels in the tomato cultivar Natsunokoma. Gene expression levels in the first leaf at 3, 6, 12, 24, 48, or 72 h after HST (45 °C, 2 min) were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 4).
Figure 3.
Relative expression levels of PR1a2 and PR1b1 in the leaves of the tomato cultivar Natsunokoma after partial treatment by HS, Pst inoculation or combined HS + Pst inoculation. Only the first leaf was subjected to HST at 45 °C for 2 min (a,b) or dipped into Pst solution (c,d). Thereafter, total RNA from the first and second leaf was extracted at 24 h after HST or Pst inoculation. In combined treatment (e,f), Pst was inoculated at 24 h after HST. Total RNA from the first leaf was extracted at 48 h after Pst inoculation, which was 72 h after HST. Gene-expression levels were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 6).
Figure 3.
Relative expression levels of PR1a2 and PR1b1 in the leaves of the tomato cultivar Natsunokoma after partial treatment by HS, Pst inoculation or combined HS + Pst inoculation. Only the first leaf was subjected to HST at 45 °C for 2 min (a,b) or dipped into Pst solution (c,d). Thereafter, total RNA from the first and second leaf was extracted at 24 h after HST or Pst inoculation. In combined treatment (e,f), Pst was inoculated at 24 h after HST. Total RNA from the first leaf was extracted at 48 h after Pst inoculation, which was 72 h after HST. Gene-expression levels were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 6).
Figure 4.
Changes in the relative expression level of (a) HsfA2 and (b) HsfB1. Gene expression levels in the first leaf at 3, 6, 12, 24, 48, or 72 h after HST (45 °C, 2 min) were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 4).
Figure 4.
Changes in the relative expression level of (a) HsfA2 and (b) HsfB1. Gene expression levels in the first leaf at 3, 6, 12, 24, 48, or 72 h after HST (45 °C, 2 min) were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 4).
Figure 5.
Accumulation of (a) salicylic acid (SA) and (b) salicylic acid b-d-glucoside (SAG) at 6, 12, 24 and 48 h after HST. Vertical bars indicate the standard error of the mean (n = 3). Significant differences between groups (indicated by asterisks) were obtained from performing two-tailed t-tests, p < 0.05. FW, fresh weight.
Figure 5.
Accumulation of (a) salicylic acid (SA) and (b) salicylic acid b-d-glucoside (SAG) at 6, 12, 24 and 48 h after HST. Vertical bars indicate the standard error of the mean (n = 3). Significant differences between groups (indicated by asterisks) were obtained from performing two-tailed t-tests, p < 0.05. FW, fresh weight.
Figure 6.
(a) The location of possible heat shock elements (HSEs) in tomato PR genes. The numbers indicate the distance (in bp) from the proximal transcription start site (ATG). TATA represents the TATA box consensus sequence. (b) Comparison of the HSE from tomato PR genes (Sl-) and Arabidopsis apx1 (At-). Sequences matching the nGAAn, the basic 5 bp HSE motif are indicated in uppercase letters. Orientation of the nGAAn-like motifs are indicated by arrows.
Figure 6.
(a) The location of possible heat shock elements (HSEs) in tomato PR genes. The numbers indicate the distance (in bp) from the proximal transcription start site (ATG). TATA represents the TATA box consensus sequence. (b) Comparison of the HSE from tomato PR genes (Sl-) and Arabidopsis apx1 (At-). Sequences matching the nGAAn, the basic 5 bp HSE motif are indicated in uppercase letters. Orientation of the nGAAn-like motifs are indicated by arrows.
Figure 7.
The relative expression level of PR1b1 in the leaves of the tomato cultivar Natsunokoma at 24 h after geldanamycin (GDA) treatment. Different GDA concentrations (0.1, 1, 10, and 100 µM) and 0.1% (v/v) dimethyl sulfoxide (DMSO) were applied by foliar spray. Gene expression levels were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 4).
Figure 7.
The relative expression level of PR1b1 in the leaves of the tomato cultivar Natsunokoma at 24 h after geldanamycin (GDA) treatment. Different GDA concentrations (0.1, 1, 10, and 100 µM) and 0.1% (v/v) dimethyl sulfoxide (DMSO) were applied by foliar spray. Gene expression levels were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 4).
Figure 8.
Relative expression level of PR (a–f) and Hsfs (g,h) genes in the leaves of the tomato cultivar Natsunokoma at 24 h after treatment with DMSO, HST, and GDA. 0.1% (v/v) DMSO and 10 µM GDA were manually applied by foliar spray. Gene expression levels were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 4).
Figure 8.
Relative expression level of PR (a–f) and Hsfs (g,h) genes in the leaves of the tomato cultivar Natsunokoma at 24 h after treatment with DMSO, HST, and GDA. 0.1% (v/v) DMSO and 10 µM GDA were manually applied by foliar spray. Gene expression levels were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 4).
Figure 9.
The relative expression level of PR1b1 in the leaves of the tomato cultivar Natsunokoma at 24 h after HST combined with KB. Different KB concentrations (1, 10, and 100 µM) were manually applied by foliar spray at 6 h after HST. Gene expression levels were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 4).
Figure 9.
The relative expression level of PR1b1 in the leaves of the tomato cultivar Natsunokoma at 24 h after HST combined with KB. Different KB concentrations (1, 10, and 100 µM) were manually applied by foliar spray at 6 h after HST. Gene expression levels were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 4).
Figure 10.
The relative expression level of PR (a–f) and Hsfs (g,h) genes in the leaves of the tomato cultivar Natsunokoma at 24 h after HST combined with KB. 100 µM of KB was manually applied by foliar spray at 6, 12, and 18 h after HST. Gene expression levels were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 6).
Figure 10.
The relative expression level of PR (a–f) and Hsfs (g,h) genes in the leaves of the tomato cultivar Natsunokoma at 24 h after HST combined with KB. 100 µM of KB was manually applied by foliar spray at 6, 12, and 18 h after HST. Gene expression levels were quantified by qPCR and normalized to GAPDH expression. Vertical bars indicate the standard error of the mean (n = 6).
Figure 11.
Disease severity upon inoculation of Pst. 1% DMSO, 100 µM KB, HST, and HST combined with 100 µM KB manually applied by foliar spray at 6, 12, and 18 h after HST. Vertical bars indicate the standard error of the mean (n = 3). Different letters indicate significant differences among treatments based on Tukey’s test, p < 0.05.
Figure 11.
Disease severity upon inoculation of Pst. 1% DMSO, 100 µM KB, HST, and HST combined with 100 µM KB manually applied by foliar spray at 6, 12, and 18 h after HST. Vertical bars indicate the standard error of the mean (n = 3). Different letters indicate significant differences among treatments based on Tukey’s test, p < 0.05.
Figure 12.
Comparison between HSIR (a) and systemic acquired resistance (SAR) (b). Black lines indicate proved pathways and gray lines indicate proposed pathways as elucidated by the work reported herein. During HSIR, Heat shock induced Hsfs gene expression, followed by SA accumulation, which in turn enhanced Hsfs binding to HSE, thereby activating non-systemic stimulation of basic and acidic PR gene expression. Disease severity in the first leaf was significantly reduced concomitantly with upregulation of PR gene expression when Pst was inoculated.
Figure 12.
Comparison between HSIR (a) and systemic acquired resistance (SAR) (b). Black lines indicate proved pathways and gray lines indicate proposed pathways as elucidated by the work reported herein. During HSIR, Heat shock induced Hsfs gene expression, followed by SA accumulation, which in turn enhanced Hsfs binding to HSE, thereby activating non-systemic stimulation of basic and acidic PR gene expression. Disease severity in the first leaf was significantly reduced concomitantly with upregulation of PR gene expression when Pst was inoculated.
Table 1.
Primers used to amplify pathogenesis-related (PR), heat shock transcription factors (Hsfs), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from tomato leaves.
Table 1.
Primers used to amplify pathogenesis-related (PR), heat shock transcription factors (Hsfs), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from tomato leaves.
Target Gene | Location | Accession Number | Properties | Primer Name | Sequence (5′ to 3′) |
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PR1a2 | chr 9 | NM_001321040.1 | Pathogenesis-related leaf protein 4 Acidic | SLPR1a2 F | TGTTACTTATGACTTGTCTCATGGT |
SLPR1a2 R | CGACCCAATTGCCTACAGGA |
PR1b1 | unplaced scaffold | NM_001247385.2 | Pathogenesis-related leaf protein 6 Basic | SLPR1b1 F | ACATCTCATTGTTACTCACTTGTCT |
SLPR1b1 R | GACGTTGTCCGATCCAGTT |
GluA | chr 1 | NM_001247869.2 | Glucan endo-1,3-beta-d-glucosidase Acidic | SLGluA F | GGTCTCAACCGCGACATATT |
SLGluA R | CACAAGGGCATCGAAAAGAT |
GluB | chr 1 | NM_001247876.2 | β-1,3-glucanases Basic | SLGluB F | TCTTGCCCCATTTCAAGTTC |
SLGluB R | TGCACGTGTATCCCTCAAAA |
Chi3 | chr 2 | NM_001247475.2 | Class III endochitinase Acidic | SLChi3 F | TGCAGGAACATTCACTGGAG |
SLChi3 R | TAACGTTGTGGCATGATGGT |
Chi9 | chr 10 | NM_001247474.2 | Class I chitinase Basic | SLChi9 F | CTCCAATGGCTCTTCCACAT |
SLChi9 R | GAAATTGCTGCTTTCCTTGC |
HsfA2 | chr 8 | XM_010326728.2 | Heat shock transcription factor A2 | SLHsfA2 F | GCGAATGGAGGTTTTCTGGG |
SLHsfA2 R | GTCACAACAGAATCCGGCCT |
HsfB1 | chr 2 | NM_001321450.1 | Heat shock transcription factor B1 | SLHsfB1 F | CTATACGCCGTCGGAAGACC |
SLHsfB1 R | TACCACGTCCACCACAAGTC |
GAPDH | chr 5 | NM_001279325.2 | Glyceraldehyde-3-phosphate dehydrogenase | SLGAPDH F | GGTTTGGCATTGTTGAGGGTC |
SLGAPDH R | TCGACAACGGAGACATCAGC |