Test of previously reported SA-responsive genes in leaves of ‘Pei-Chiao’ plantlets
In order to identify the salicylic acid (SA) marker genes in banana that would enable the analysis of SA-induced signaling in ‘Pei-Chiao’, we first analyzed genes that had been previously reported to be SA-responsive (Table 1). Banana plantlets grown in potted soil were treated with buffer (Mock) or SA by foliar spray. Leaves of each treated plant were harvested at two time points, 0 (no-treatment) and 6 hour-post treatment (hpt) for SA concentration and gene expression analyses. We measured SA concentration by high-performance liquid chromatography-mass spectrometry, which revealed a significant increase in SA concentration in SA-treated plants compared to the mock or no-treatment control at 6 hpt (Fig. 1a).
Table 1
Salicylic acid responsive candidate marker genes selected from reference studies.
Name | Description | Reference | Primer Sequence |
MNPR1A | NPR1 | Endha et al., 2008 | F: 5′-GTCGGCATTGTACCAACACA-3′ R: 5′-CAGTGCAGGAGTCAGCAAAA-3′ |
MNPR1B | NPR1 | Endha et al., 2008 | F: 5′-AGGTTTGCCCGAACAAGAAG-3′ R: 5′-TGAGAGGCAACAACTCAGAGAG-3′ |
MaNPR1B | NPR1 | Wang et al., 2015 | F: 5′-CCATTCCCAGATTCAGATACA-3′ R: 5′-TCGACATTCTTCGCAACC-3′ |
MaNPR1D | NPR1 | Wang et al., 2015 | F: 5′-CTGAACGCCTTCCTAACA-3′ R: 5′-TGACCTGAACGAAACACC-3′ |
BanPR1 | PR1 | Van den Berg et al., 2007 | F: 5′-TCCGGCCTTATTTCACATTC-3′ R: 5′-GCCATCTTCATCATCTGCAA-3′ |
MaTGA1 | TGA | Wang et al., 2015 | F: 5′-ATGATGACGAGGAAGATAA-3′ R: 5′-AATGGACCTAAATGAAGC-3′ |
MaTGA2 | TGA | Wang et al., 2015 | F: 5′-ACATTCTGACATCCCTCCAC-3′ R: 5′-GCTGATCTGATCCCAACG-3′ |
We next examined the transcriptional responses of the known SA-responsive genes listed in the Table 1 with real-time reverse transcriptase quantitative PCR (RT-qPCR). Among them, only MaNPR1D showed significant and reproducible induction at 6 hpt of SA (Fig. 1b). Compared to mock treatment, MaNPR1D increased by 2-fold at 6 hpt (Fig. 1b). This gene was selected for further analysis.
Analysis of SA concentration and MaNPR1D expression in leaves and roots of ‘Pei-Chiao’ plantlet after SA treatment
Roots serve as the initial infection site for several hemi-biotrophic pathogen such as Fusarium oxysporium f. sp. cubense, Xanthomonas spp., and Ralstonia spp. [4, 37]. To analyze whether MaNPR1D could serve as a reliable SA marker in roots of ‘Pei-Chiao’, we first optimized our SA treatment. To this end, we used ‘Pei-Chiao’ tissue culture plantlets grown on sterilize agar medium to prevent the potential infection of soil microorganism and minimize the damage during root sampling. ‘Pei-Chiao’ tissue culture plantlets grown in agar were treated with buffer (mock) or SA by foliar spray. Since we do not know whether SA level is increased under the treatment method of foliar spray, we first quantified the SA concentration in mock- and SA-treated leaves and roots by the use of ultra-performance liquid chromatography chromatography (HPLC)-mass spectrometer (MS)/MS. (Fig. 2). Quantification of SA concentration revealed the increased levels of SA in the SA-treated ‘Pei-Chiao’ leaves and roots harvested at 6 hpt (Fig. 2a and b). Statistical significance was observed for the SA concentration in leaves of SA-treated plantlets at 6 hpt compared to that of no-treatment control or mock-treated plantlets (Fig. 2a); no statistical significance was found between no-treatment (0 hpt) and mock-treated plantlets (6 hpt) (Fig. 2a). In roots, the average SA concentration was also significantly higher in 6 h post SA-treated plantlets compared to no-treatment control plantlets or mock-treated plantlets (Fig. 2b). No statistical significance was found between no-treatment (0 hpt) and mock-treated plantlets (6 hpt) (Fig. 2b).
As expected, real-time RT-qPCR confirmed that MaNPR1D expression positively correlated with higher levels of SA in the leaves of ‘Pei-Chiao’ in repeated experiments (Fig. 2c). A 3.4-fold increase of statistical significance in MaNPR1D mRNA level was detected in the leaves of SA-treated plants compared to mock-treated plants at 6 hpt (Fig. 2c). However, only a slight induction of MaNPR1 gene expression level (2.3-fold) was observed in the roots (Fig. 2d).
Transcriptome Analysis Of Sa Responsive Genes In ‘pei-chiao’ Plantlet
Although MaNPR1 can be induced by SA treatment, only 2.3-fold induction was observed in our repeated experiments. To identify more sensitive SA-responsive genes that could be as reliable SA marker genes in roots of ‘Pei-Chiao’, we conducted 2 sets of RNA-seq for transcriptional response analysis of SA treatment vs. no-treatment at 0 hpt and mock treatment at 6 hpt of roots of banana grown in sterile conditions.
To identify differentially expressed genes under SA treatment, we first mapped the reads to the banana genome database (Musa acuminata DH Pahang v2; banana-genome-hub.southgreen.fr) after adaptor trimming and quality filtering. The mapping rates of the processed reads among the samples for RNA-seq analysis were similar and ranged from 83.29%-89.53% (Table 2). We then analyzed differentially expressed genes (DEGs) under SA treatment in ‘Pei-Chiao’ using statistic test with adjusted P value < 0.1 and obtained a total of 22 DEGs (Table 3). Among 19 SA up-regulated gene, M. acuminata Pathogenesis-Related protein 1-like (MaPR1-like; Ma04_g29640), WRKY transcription factor 40 (MaWRKY40; Ma07_g16310), WRKY transcription factor 70 (MaWRKY70; Ma07_g23510), and Downy Mildew Resistant 6 (DMR6)-Like Oxygenase 1 (MaDLO1; Ma00_g04490) are known to be important in the SA-mediated immunity pathway [38–42] and showed an induction > 2.5 fold at 6 h post treatment of SA in both replicates (Table 3 and Fig. 3). These 4 genes were selected for further analysis by real-time RT-qPCR (Table 3 and Fig. 3). Expression pattern of MaPR1-1, MaWRKY40, MaWRKY70, and MaDLO1 are shown in Fig. 3.
Table 2
Summary of RNA-seq data for analysis of SA responsive genes in ‘Pei-Chiao’ plantlet.
Sample | Repeat | Treatment | Raw reads | Clean reads | Mapped reads | Mapping rate (%) |
PC-M-0 h | 1 | Mock | 31,419,774 | 28,078,146 | 26,592,044 | 84.63 |
PC-M-0 h | 2 | Mock | 26,820,728 | 25,081,892 | 24,006,856 | 89.51 |
PC-M-6 h | 1 | Mock | 26,419,442 | 23,288,808 | 22,004,390 | 83.29 |
PC-M-6 h | 2 | Mock | 29,018,865 | 27,243,084 | 25,981,414 | 89.53 |
PC-S-6 h | 1 | SA | 28,817,447 | 25,838,512 | 24,202,029 | 83.98 |
PC-S-6 h | 2 | SA | 27,345,079 | 25,654,923 | 24,428,110 | 89.33 |
Table 3
List of differentially expressed genes in ‘Pei-Chiao’ plantlet at 6 hour post-salicylic acid-treatment.
Gene ID | Fold Change (log2) | Adjusted p-value | Description |
SA up-regulated genes (6 hpt) | | | |
Ma00_g02460 | 2.21 | 1.94E-03 | calmodulin-like |
Ma00_g03560 | 5.78 | 1.03E-11 | probable mannitol dehydrogenase |
Ma00_g04490 | 3.12 | 2.01E-09 | DMR6-like oxygenase 1 |
Ma01_g07400 | 1.99 | 3.43E-03 | monothiol glutaredoxin-S9-like |
Ma01_g07410 | 1.62 | 8.02E-02 | pentatricopeptide repeat-containing protein At4g02750 |
Ma01_g20340 | 2.50 | 1.38E-02 | Hypothetical protein |
Ma02_g01990 | 1.81 | 4.68E-02 | adenylate isopentenyltransferase 5, chloroplastic-like |
Ma03_g11500 | 4.27 | 1.16E-04 | 3-oxoacyl-[acyl-carrier-protein] reductase, chloroplastic-like |
Ma03_g12470 | 2.05 | 1.33E-02 | uncharacterized LOC103978016 |
Ma04_g29640 | 1.46 | 3.42E-02 | pathogenesis-related protein 1-like |
Ma05_g12600 | 1.55 | 5.16E-02 | flavanone 3-dioxygenase-like |
Ma07_g06750 | 5.24 | 6.70E-15 | L-lactate dehydrogenase A-like |
Ma07_g16310 | 2.25 | 1.73E-03 | probable WRKY transcription factor 40 |
Ma07_g23510 | 1.55 | 7.50E-02 | probable WRKY transcription factor 70 |
Ma09_g26520 | 1.90 | 5.85E-03 | Probable glutathione S-transferase parA |
Ma10_g12070 | 2.15 | 3.35E-03 | uncharacterized LOC104000673 |
Ma10_g28170 | 2.66 | 1.73E-03 | uncharacterized LOC103969442 |
Ma11_g04000 | 1.84 | 1.38E-02 | ankyrin repeat-containing protein At2g01680-like |
Ma11_g18240 | 1.76 | 2.98E-02 | non-specific lipid-transfer protein-like |
SA down-regulated gene (6 hpt) | | | |
Ma02_g24600 | -2.00 | 5.16E-02 | ribulose bisphosphate carboxylase small chain, chloroplastic |
Ma09_g31150 | -2.12 | 2.27E-02 | Putative germin-like protein 2 − 1 |
Ma11_g03350 | -1.55 | 9.97E-02 | methionine gamma-lyase-like |
Verification of SA Induction By Real-time Rt-qpcr
Expression levels of ‘Pei-Chiao’ MaPR1-like, MaWRKY40, MaWRYK70, and MaDLO1 in the roots and leaves of mock- and SA-treated samples by foliar spray were analyzed by real-time RT-qPCR (Fig. 4) with the use of gene specific primers (Table 4). In the roots, all 4 genes exhibited a higher expression level in the SA treatment compared to the mock treatment at 6 hpt, MaPR1-like increased by 2-fold, MaWRKY40 increased by 56-fold, MaWRKY70 increased by 31-fold, and MaDLO1 increased by 487-fold; however, the induced expression of MaPR1-like expression did not reach a statistical significance (Fig. 4a).
Table 4
Primers for real-time RT-qPCR of salicylic acid responsive marker genes in banana
Name | Description | Primer Sequence | Gene ID | NCBI Accession |
MaPR1-Like | Pathogenesis-related protein 1-like | F: 5’-GAAGCAGGACTACGACTACAAC-3’ R: 5’-GGACGAACGCCACACAA-3’ | Ma04_g29640 | XM_009400035.2 |
MaWRKY40 | WRKY transcription factor 40 | F: 5′-CGGGATGATGTGTACGGTTT-3′ R: 5′-TAAGGTTGCAGGTGTGTTC − 3′ | Ma07_g16310 | XM_009411509.2 |
MaWRKY70 | WRKY transcription factor 70 | F: 5′-CTGCAGCTTGGACATGGA − 3′ R: 5′-CCCTCTTAACTGATGTACTCATCG-3′ | Ma07_g23510 | XM_009412236.1 |
MaDLO1 | DMR6-like oxygenase 1 | F: 5′-GAGAGCTTGGGACTTGAGAAG − 3′ R: 5′-CTGTGGGCATGGTGGATAG-3′ | Ma00_g04490 | XM_009389881.2 |
Consistent with the RT-qPCR results of the roots, MaWRKY40, MaWRYK70, and MaDLO1 mRNA levels in the leaves were significantly higher by 92-fold, 34-fold, and 1332-fold, respectively, at 6 hour post-SA-treatment compared to mock-treatment (Fig. 4b). No obvious difference was observed for MaPR1-like expression after the SA treatment in roots (Fig. 4b).
MaWRKY40, MaWRYK70, and MaDLO1 are induced by SA treatment in all tested cultivars
To determine whether MaWRKY40, MaWRKY70 and MaDLO1 could represent a core set of SA-responsive genes across bananas of various genomic groups, we further compared the gene expression profiles of these genes in between mock- and SA-treated samples of ‘Pisang Awak’ (ABB genome) and ‘Lady Finger’ (AA genome) by real-time RT-qPCR at 6 hpt (Fig. 5–6).
In concordance with the real-time RT-qPCR result of ‘Pei-Chiao’, MaWRKY40, MaWRKY70, and MaDLO1 expression was significantly induced (> 20 folds) by SA in ‘Pisang Awak’ plantlet at 6 hpt (Fig. 5a). In ‘Pisang Awak’, roots of treatment group compared to mock treatment, MaWRKY40, MaWRKY70, and MaDLO1 significantly increase by 179-fold, 21-fold, and 2672-fold, respectively (Fig. 5a). In the leaves of ‘Pisang Awak’ plantlet, SA treatment also significantly increased MaWRKY40 (188-fold), MaWRKY70 (25-fold), and MaDLO1 (1650-fold) mRNA levels (Fig. 5b).
Similarly, in the diploid ‘Lady Finger’ banana, MaWRKY40, MaWRKY70, and MaDLO1 mRNA levels are significantly higher in SA-treated leaves and roots of plantlet compared to that of mock treatment (Fig. 6a and b). In the roots, MaWRKY40, MaWRKY70, and MaDLO1 significantly increase by 11-fold, 7-fold, and 122-fold, respectively, compared to mock treatment (Fig. 6a). MaWRKY40, MaWRKY70, and MaDLO1 significantly increase by 146-fold, 24-fold, and 855-fold, respectively, in SA-treated leaves compared to mock treatment (Fig. 6b).