Abstract
Banana Xanthomonas wilt disease, caused by Xanthomonas campestris pv. musacearum (Xcm), is a major threat to banana production in east Africa. All cultivated varieties of banana are susceptible to Xcm and only the progenitor species Musa balbisiana was found to be resistant. The molecular basis of susceptibility and resistance of banana genotypes to Xcm is currently unknown. Transcriptome analysis of disease resistant genotype Musa balbisiana and highly susceptible banana cultivar Pisang Awak challenged with Xcm was performed to understand the disease response. The number of differentially expressed genes (DEGs) was higher in Musa balbisiana in comparison to Pisang Awak. Genes associated with response to biotic stress were up-regulated in Musa balbisiana. The DEGs were further mapped to the biotic stress pathways. Our results suggested activation of both PAMP-triggered basal defense and disease resistance (R) protein-mediated defense in Musa balbisiana as early response to Xcm infection. This study reports the first comparative transcriptome profile of the susceptible and resistant genotype of banana during early infection with Xcm and provide insights on the defense mechanism in Musa balbisiana, which can be used for genetic improvement of commonly cultivated banana varieties.
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Introduction
Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is one of the most devastating disease endangering the livelihood of millions of farmers in east Africa, which is the largest banana-producing and -consuming region in Africa1. The impacts of BXW disease are both rapid and extreme, unlike those of other diseases, which cause gradually increasing losses over years. The disease has caused estimated economic losses of about $2-8 billion over the decade and significant reductions in production have resulted in major price increases1. The disease affects all banana cultivars grown in east Africa2. Only diploid Musa balbisiana, which is a wild type banana native to Asia and one of the progenitors of modern cultivated bananas, was found to be resistant2.
Resistant cultivars could play an important role in controlling the BXW disease in east Africa, where the consumption of bananas is highest in the world at 220 to 460 kg per person annually3. There is an ongoing project for developing transgenic bananas resistant to BXW using sweet pepper genes4. However, knowledge of resistance mechanism in Musa balbisiana against Xcm can be utilized for developing resistant varieties through cis-genesis using Musa genes associated with defense, or editing of genes related to susceptibility and/or negative regulation of plant immunity. Currently, there is no understanding about the molecular mechanism for disease resistance or susceptibility in response to Xcm infection. Therefore, to obtain insight into the molecular basis of disease resistance, the transcriptome-wide differential gene expression was investigated between the BXW-resistant genotype Musa balbisiana and BXW-susceptible genotype Pisang Awak in response to Xcm. Further the differentially expressed transcripts were mapped to biotic stress pathways using Mapman to identify genes associated with defense mechanism.
Results
RNA-Seq and mapping of sequences to reference genome
In this study, RNA-Seq data from Xcm challenged and mock-inoculated BXW-susceptible banana genotype Pisang Awak and BXW-resistant genotype Musa balbisiana were analyzed for transcriptome comparison.
In total, about 559 million raw reads were generated from sequencing of 30 libraries using Illumina HiSeq™ 2500. The majority of samples had average of 5–10 million reads that uniquely mapped to the reference Musa acuminata genome version 2. The raw reads were deposited in the National Center for Biotechnology Information Sequence Read Archive under project SRPRJNA401071 (https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP116676).
Differentially expressed genes (DEGs)
Readcounts for 35,238 annotated genes were loaded in R program and genes with low expression were filtered out. This filtering step reduced the dataset to 26,676 genes. The DEGs were identified in DESeq2 using filtered readcount data. The data from two time points, 12 h post inoculation (hpi) and 48 hpi, were analyzed and a model was created to identify genes that were responding to the treatment with pathogen and also had an interaction with the genotypes. The design parameter (~genotype + treatment + genotype:treatment) was used for DESeq2. The genes with fold change of greater than 1.5 or <−1.5 were identified.
The numbers of DEGs were higher in BXW-resistant genotype Musa balbisiana in comparison to BXW-susceptible genotype Pisang Awak at both 12 hpi (1749 vs 4) and 48 hpi (245 vs 88) (Fig. 1A, Supplementary Tables S1–S4). Out of 1749 DEGs in Musa balbisiana, only 540 DEGs showed up-regulation and remaining 1209 were down-regulated, whereas all four DEGs in Pisang Awak were found to be up-regulated (Fig. 1B).
Graphs showing differentially expressed genes (DEGs) in BXW-resistant wild type banana genotype Musa balbisiana and BXW-susceptible banana genotype Pisang Awak at 12 and 48 hours post-inoculation with Xanthomonas campestris pv. musacearum. (A) Venn diagram showing comparison of DEGs in two genotypes at 12 hpi and 48 hpi, (B) Graphs showing number of up or down-regulated DEGs.
The number of DEGs for Musa balbisiana was considerably higher at 12 hpi in comparison to 48 hpi (1749 vs 245), in contrast a greater number of genes were differentially expressed at 48 hpi than at 12 hpi in Pisang Awak (88 vs 4) (Fig. 1B). In Musa balbisiana, 107/245 DEGs at 48 hpi were up-regulated, whereas only 33/88 DEGs were up-regulated in Pisang Awak at 48 hpi (Fig. 1B).
Several DEGs were observed due to genotype:treatment interactions (Supplementary Tables S5–S6). The difference in genotypes due to differences in treatment i.e. genotype:treatment interaction were observed to be higher at 12 hpi than 48 hpi.
GO-term analysis
To know the functional categories of DEGs, GO enrichment analysis was performed using GO-terms provided by the Banana Genome Hub and the GOStats. A Gene Set Enrichment dataset was created using Gene-to-GO association in the Banana Genome Hub and used for identifying GO-terms that were over-represented in a given gene list. Out of 26,676 filtered genes set, 13,699 corresponding proteins were associated with at least one GO term. The GO-term analysis was performed for all the DEGs in both banana genotypes at 12 hpi and 48 hpi. At 12 hpi, the GO-terms were mainly due to interaction of bacterial pathogen with host plants (Supplementary Table S7).
No significant GO-term enrichment was seen for Pisang Awak at 12 hpi, however, many GO-terms were enriched for Musa balbisiana at 12 hpi (Supplementary Table S7). The majority of GO-terms in Musa balbisiana were associated with ‘Defense response’ suggesting that BXW-resistant genotype respond to pathogen attack at very early stage. At 48 hpi, several GO-terms were found in Pisang Awak, whereas only one significant GO-term was obtained for Musa balbisiana (Supplementary Table S7).
Gene annotation and metabolic pathway analysis
The transcripts were annotated based on the genome of Musa acuminata version 2 and mapped to the biotic stress pathway in Mapman using the Mercator tool. Table 1 lists number of annotated genes aligned to different functional categories (BINs) in metabolic pathways in Mapman.
More number of genes were differentially expressed in BXW-resistant genotype in comparison to BXW-susceptible genotype due to interaction with Xcm at different time post inoculation (Table 1). The overview of genes mapped onto the biotic stress pathway at 12 hpi and 48 hpi for BXW-resistant and BXW-susceptible genotypes is illustrated in Fig. 2.
Diagram showing differentially expressed genes in BXW-resistant and BXW-susceptible genotypes of banana after inoculation with Xanthomonas campestris pv. musacearum mapped onto known biotic stress pathway bins using Mapman. (A) BXW–resistant genotype Musa balbisiana at 12 hpi, (B) BXW-susceptible genotype Pisang Awak at 12 hpi, (C) BXW–resistant genotype Musa balbisiana at 48 hpi, (D) BXW-susceptible genotype Pisang Awak at 48 hpi. Figures were generated from Mapman. The scale is from down-regulated (blue) to up-regulated (red).
Stress associated genes were differentially expressed in both banana genotypes in response to Xcm. About 25 genes associated with stress were up-regulated in Musa balbisiana at 12 hpi, whereas only one gene, Germin-like protein (GER1), was activated in Pisang Awak at 12 hpi (Tables 1 and 2). Germin-like protein was also up-regulated in Musa balbisiana at 48 hpi. All stress-associated genes were suppressed in Pisang Awak at 48 hpi (Table 1).
Several pathogenesis-related (PR) proteins were up-regulated and also many PR genes were suppressed in response to pathogen interaction with Musa balbisiana at 12 hpi (Fig. 2, Table 2).
Disease resistance (R) gene in leucine-rich repeat (LRR) family protein and putative disease resistance protein RPM1 were activated in Musa balbisiana as early response to pathogen at 12 hpi (Fig. 2, Table 2). Lipase-like PAD4 gene was also activated in Musa balbisiana at 48 hpi (Table 2). None of the R or PR genes were differentially expressed in BXW-susceptible genotype Pisang Awak as early response to pathogen infection (Fig. 2).
Transcript aligned with MLO-like protein 13 (MILDEW RESISTANCE LOCUS O 13) was suppressed in BXW-resistant Musa balbisiana at 12 hpi (Table 2).
Heat shock related transcripts were differentially expressed in Musa balbisiana at 12 hpi (Fig. 2). HSP20-like chaperones superfamily associated genes were up-regulated in Musa balbisiana at 12 hpi and as well as at 48 hpi (Table 2). However, genes associated with heat shock proteins were either not differentially expressed or suppressed in Pisang Awak at 12 hpi and 48 hpi respectively (Fig. 2).
Transcript associated with apoptosis regulator, BAG family molecular chaperone regulator 6-like was activated in Musa balbisiana upon pathogen attack at 12 hpi (Table 2).
The genes related to transcription factors were differentially expressed in both genotypes at 12 hpi and 48 hpi (Fig. 2, Table 2). The genes associated with transcription factors like MADS-box transcription factor family, MYB, WRKY, C2H2 zinc finger family, AP2/EREBP, heat shock transcription factor C1 (HSFC1), GRAS transcription factor family, auxin response factor (ARF) family, C2C2 family, homeobox transcription factor family and basic helix-loop-helix family (bHLH) were activated in Musa balbisiana at 12 hpi. Some of the genes associated with transcription factors like MYB, trihelix, AP2/EREBY), bHLH and C2H2 were also up-regulated in Musa balbisiana at 48 hpi. The MYB transcription factor was also activated in Pisang Awak at both 12 hpi and 48 hpi (Fig. 2, Table 2).
As early response to pathogen interaction in Musa balbisiana, several receptor-like kinases such as wall associated receptor kinases, LRR receptor kinases, LRK10 like receptor kinase, receptor kinase DUF26, were up-regulated at 12 hpi and similarly receptor kinases were also activated at 48 hpi (Fig. 3, Table 2). Other genes involved in signaling pathways like mitogen-associated protein kinase (MAPK), phosphatidylinositol-4-phosphate 5-kinase family protein, G protein were also found to be up-regulated in Musa balbisiana (Table 2). However, none of signaling pathway-associated gene was differentially expressed in Pisang Awak at 12 hpi, whereas LRR receptor kinases and G protein were up-regulated at 48 hpi (Table 2).
Diagram showing mapping of the transcripts associated with receptor-like-kinases in the BXW-resistant genotype Musa balbisiana at 12 hours post-inoculation with Xanthomonas campestris pv. musacearum. Figure was generated from Mapman. The scale is from down-regulated (blue) to up-regulated (red).
Transcripts involved in redox regulation like glutaredoxins, peroxiredoxin, thioredoxins, ascorbate, glutathione, catalases and heme family were up-regulated in Musa balbisiana at 12 hpi (Fig. 2, Table 2). Also superoxide dismutase gene was induced in Musa balbisiana as response to pathogen interaction at 48 hpi. However, upon pathogen infection either the redox state was not affected or suppressed in Pisang Awak (Fig. 2). Peroxidases and glutathione S transferases (GSTs) were also differentially expressed in Musa balbisiana at 12 hpi. Genes associated with oxidative burst such as polyamine oxidase-like gene was induced in Musa balbisiana as early response (12 hpi) to Xcm.
As a result of plant-pathogen interaction, hormone metabolic pathways were activated in Musa balbisiana. Abscisic acid (ABA) metabolism was activated in Musa balbisiana at both 12 and 48 hpi (Fig. 2, Table 2). However, genes associated with ABA metabolism were not differentially expressed in Pisang Awak at 12 hpi and suppressed at 48 hpi (Fig. 2, Table 2). Genes associated with ninja family protein AFP3 was activated in Musa balbisiana at 12 hpi. Genes associated with ethylene synthesis and signal transductions were differentially expressed in Musa balbisiana at 12 hpi (Table 2). Ethylene pathway was suppressed in both genotypes at 48 hpi. Several genes associated with jasmonate (JA) metabolism were up-regulated in Musa balbisiana at both 12 hpi and 48 hpi (Table 2). The salicylic acid (SA) pathway was repressed in Musa balbisiana at 12 hpi (Fig. 2). However, no change was observed in JA and SA metabolism in Pisang Awak. Several genes associated with auxin metabolism were up-regulated in Musa balbisiana at both 12 hpi and 48 hpi (Table 2).
Genes associated with beta-glucanases were found to be differentially expressed in Musa balbisiana. Only one transcript associated with β-1,3-glucan hydrolase was activated in Musa balbisiana at 12 hpi and 48 hpi (Table 2). All other β-1,3 glucan hydrolase genes were suppressed in Musa balbisiana at 12 hpi. Upon pathogen attack, transcripts associated with proteolysis were also differentially expressed in Musa balbisiana at both 12 hpi and 48 hpi and Pisang Awak at 48 hpi.
Also, production of several secondary metabolites was activated in Musa balbisiana in response to pathogen infection at both 12 hpi and 48 hpi. Activation of isoprenoids, lignin biosynthesis, phenylpropanoids, flavonoids, terpenoids was observed in Musa balbisiana at 12 hpi (Fig. 4, Table 2). The wax synthesis was activated at 48 hpi and suppressed at 12 hpi in Musa balbisiana. Lignin biosynthesis, phenylpropanoids, flavonoids pathways were suppressed in Pisang Awak (Fig. 4).
Diagram showing differentially expressed genes mapped to secondary metabolic pathways in BXW-resistant and BXW-susceptible genotypes of banana in response to interaction with Xanthomonas campestris pv. musacearum. (A) BXW–resistant genotype Musa balbisiana at 12 hpi, (B) BXW–resistant genotype Musa balbisiana at 48 hpi, (C) BXW-susceptible genotype Pisang Awak at 48 hpi. Figures were generated from Mapman. The scale is from down-regulated (blue) to up-regulated (red).
In response to bacterial infection, vicilin-like antimicrobial peptide was activated in Musa balbisiana at 12 hpi (Table 2).
Genes associated with transporters of peptides and oligopeptides, proteins, aminoacids, sugars, metals, potassium, metabolites and ABC transporters and multidrug resistance systems were differentially expressed in Musa balbisiana at 12 hpi and 48 hpi. Gene associated with bidirectional sugar transporter SWEET14-like was significantly suppressed in Musa balbisiana at 12 hpi (Table 2). Transporters for v-ATPases and ABC transporters and multidrug resistance systems were also differentially expressed in Pisang Awak at 48 hpi.
Early nodulin like proteins were suppressed in Musa balbisiana at 12 hpi (Table 2).
Genes associated with E3 ubiquitin-protein ligases were differentially expressed in Musa balbisiana at 12 hpi and 48 hpi (Table 2). Several of the E3 ubiquitin ligases such as PUB22, PUB23, RMA1H1, Ring 1, ATL-4, RHA1B, RHA2B, were down-regulated in Musa balbisiana. E3 ubiquitin ligase BIG BROTHER-like was down-regulated in Pisang Awak at 48 hpi.
Genes involved in metabolic pathways like cytochrome P450 were found to be up-regulated as well as down-regulated in Musa balbisiana at both 12 hpi and 48 hpi. However cytochrome P450 pathway was suppressed in Pisang Awak at 48 hpi.
Validation of RNA-Seq data for selected genes by qRT-PCR analysis
The results of RNA-Seq analysis was validated by quantitative RT- PCR (qRT-PCR) using a set of 30 annotated genes differentially expressed in response to Xcm infection in Musa balbisiana and Pisang Awak (Table 3). The differential expression was confirmed by qRT-PCR for some of the genes involved in plant defense such as antimicrobial peptide vicilin, R genes (RPM1, LRR disease resistance family), PR genes, transcription factors (MYB, MADS-box, WRKY), receptor like kinases (wall associated receptor kinases and LRR receptor kinase), redox associated genes (polyamine oxidases), genes involved in signaling pathways (phosphatidylinositol 4-phosphate 5-kinase 6 like), E3 ubiquitin ligase and germin-like protein. The expression of genes involved in hormone metabolism such as ABA pathway (carotenoid cleavage dioxygenase 4), ethylene pathway (1-aminocyclopropane-1-carboxylate oxidase), auxin metabolism (auxin-induced protein 15A-like) and jasmonic acid pathway (linoleate 13S-lipoxygenase 2-1), alpha-galactosidase, sugar transporter gene (SWEET14), early nodulin-like protein, and genes involved in secondary metabolites like lignin biosynthesis (cinnamyl alcohol dehydrogenase 1) and starch biosynthesis (glucose-1-phosphate adenylyltransferase large subunit 1-like) was also corroborated.
The comparison of RNA-Seq and qRT-PCR showed similar trends of expression for candidate transcripts but difference in magnitude (Fig. 5). Pearson correlation indicated that the RNA-Seq and qRT-PCR were strongly correlated (r = 0.64, P = 0.00012). The differences in the fold change observed between the RNA-seq and qRT-PCR results might be due to analysis of independent samples and using different algorithms.
Comparison of RNA-Seq and qRT-PCR analysis for differential expression (log2 fold change) of selected genes. Details of selected genes are provided in Table 3.
Discussion
BXW is the most important production constraint of banana affecting livelihood of millions of people in east Africa. The wild type banana progenitor Musa balbisiana has resistance to BXW, whereas all cultivated banana varieties including commercial Cavendish varieties are susceptible. Currently, molecular basis of resistance and susceptibility of banana to Xcm is unknown. Therefore, transcriptome profiles of BXW-resistant and BXW-susceptible banana genotypes challenged with Xcm were compared to better understand molecular mechanism of resistance or susceptibility of banana upon bacterial infection. In presence of Xcm, resistant genotype showed higher number of DEGs at 12 hpi in comparison to 48 hpi, suggesting that defense pathways were activated by pathogen infection as early as 12 hpi.
Plants defend themselves against pathogens by activating defense mechanisms, including hypersensitive response (HR), induction of genes encoding PR, antimicrobial peptides, generation of reactive oxygen species (ROS), and enforcement of the cell wall.
Up-regulation of vicilin-like AMP in Musa balbisiana after artificial inoculation with Xcm suggested its involvement in plant defense and suppression of bacterial population like action of other AMPs. Vicilin-like AMPs are plant derived α-amylase inhibitors having antibacterial and antifungal activity5.
Once plants recognize pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRRs), series of reactions are activated leading to PAMP-triggered immunity (PTI) as first line of plant immunity or basal defense. E3 ubiquitin ligase plays important role in PTI6. Suppression of E3 ubiquitin ligases PUB22 and PUB23 in BXW-resistant genotype Musa balbisiana in response to Xcm was in agreement with previous report demonstrating role of E3 ubiquitin ligases (PUB22, PUB23, and PUB24) as negative regulators of PTI in Arabidopsis6.
Receptor-like kinases act as PRRs, which recognize pathogens as the first layer of inducible defense. Our results showed activation of several receptor like kinases and receptor kinases in BXW-resistant genotype, indicating their role in plant defense (Table 2). WAKs are known to be involved in plant development and defense. AtWAK1 and AtWAK2 are reported to be involved in cell wall expansion7. OsWAK14, which is similar to WAK2, was reported to be positive regulator of blast resistance in rice8. LRR-RKs recognizes the pathogen effectors and induces innate immunity. Receptor kinase DUF26 also might have been involved in induction of disease resistance as previously reported that cysteine-rich receptor-like kinases (CRKs) containing DUF26 motif in their extracellular domains are involved in stress resistance9. The previous report demonstrating that AtLRK10L1.2, Arabidopsis ortholog of wheat LRK10 is involved in ABA-mediated signaling10, suggested that up-regulation of LRK10 like receptor kinase in Musa balbisiana should have activated the ABA signaling pathway.
PTI involves activation of oxidative burst, MAPK activity and transcription factors, preventing colonization of pathogen in host cells and providing disease resistance. MAPK cascades are major plant plasma membrane-localized PRRs, signaling early basal defense responses against pathogen infection11. In this study, activation of MAPK in Musa balbisiana indicated that up-regulation of MAPK cascades in the BXW-resistant genotype might have signaled early defense responses, including generation of ROS and HR leading to program cell death.
Upon pathogen attack, plant cell changes its redox state as one of the earliest immune responses and induces HR and programmed cell death12. In this study, differential expression of several genes associated with redox state was observed in BXW-resistant genotype Musa balbisiana at 12 hpi as early response to Xcm infection, whereas no change in redox state was detected in BXW-susceptible genotype Pisang Awak at 12 hpi (Table 2). In response to pathogen, plants produce ROS including superoxide, hydroxyl radicals and hydrogen peroxide, which trigger HR and also induces lignification and signal transduction pathways and leading to programmed cell death13. Several heat shock proteins were also activated in Musa balbisiana in response to ROS particularly H2O2 accumulation.
Our results demonstrated activation of polyamine oxidases and peroxidases involved in ROS14,15 in BXW-resistant genotype Musa balbisiana as early response to Xcm attack indicating induction of programmed cell death. Similar increase in polyamine enzymatic activity is reported in barley (Hordeum vulgare) during HR in response to powdery mildew16.
During plant-pathogen interaction, the response starts from the cell wall followed by activation of signaling pathway leading to disease resistance. In this study, lignin biosynthesis was activated in Musa balbisiana at 12 hpi, indicating cell wall reinforcement (Fig. 4). The cytochrome P450 involved in the lignin biosynthetic pathway was also up-regulated in the BXW-resistant genotype leading activation of lignin accumulation. Lignin plays important role in plant defense as demonstrated that deposition of lignin provided resistance to Verticillium dahliae in Camelina sativa and to Sclerotinia sclerotiorum in cotton17,18. Lignin or lignin-like phenolic polymers are rapidly deposited upon pathogen attack creating physical barrier to pathogen invasion and makes the cell wall more resistant to cell wall degrading enzymes19. We also observed up-regulation of phenylpropanoid pathway responsible for lignin biosynthesis in Musa balbisiana (Fig. 4).
Our result was similar to the previous reports, in which genes encoding cytochrome P450 were highly induced in disease resistant cauliflower, pepper and grapevines in response to bacterial pathogen infection20,21,22. The cytochrome P450 superfamily is involved in several biochemical pathways leading to the production of primary and secondary metabolites. Our result confirmed induction of cytochrome P450, which might have led to significantly high production of secondary metabolites in Musa balbisiana in response to pathogen infection.
We also observed significant up-regulation of peroxidases as early defense response against Xcm in Musa balbisiana (Fig. 2). Plant peroxidases play important role in defense mechanism by reinforcing the cell wall in response to pathogen attack, lignin and suberin formation, catalyzing cross-links between phenolic compounds in the secondary walls and polysaccharides, synthesis of phytoalexins, participating in the metabolism of ROS, activating HR at the infection site and restricting the spread of pathogen23.
Our results demonstrated up-regulation of several transcription factors in Musa balbisiana (Fig. 2, Table 2). Transcription factors respond to biotic stress by altering the expression of a cascade of defense genes. C2H2 zinc-finger transcription factors are known to function as a pathogen-induced early-defense gene in Capsicum annuum24.
In response to pathogen infection, MYB106 and MYB88 were up-regulated in BXW-susceptible genotype Pisang Awak. MYB106 is reported to be a negative regulator of trichome branching and positive regulator photosynthesis and growth25,26. MYB88 is involved in stomatal development as well as regulation in abiotic stress responses and female reproductive development27. However, role of MYB88 and MYB106 in plant defense is not known. Up-regulation of MYB4, MYB61 and MYB96 was observed in Musa balbisiana after infection with Xcm (Fig. 2). MYB4 is associated with protection against UV and MYB61 regulates several aspects of plant growth and development28,29. MYB96 was reported to be involved in regulation of lateral root growth in response to drought stress by activating the ABA-auxin signaling network30.
In this study, ERF transcription factor was also activated in the BXW-resistant genotype in response to pathogen infection (Fig. 2). The APETALA2/ethylene responsive factor (AP2/ERF) family is one of the largest transcription factor families involved in defense responses against various pathogens31. AP2-mediated disease resistance was demonstrated in Arabidopsis and tomato against Botrytis cinerea and Ralstonia solanacearum, respectively32,33,34. AP2 mediated defense responses are linked to hormones such as jasmonic acid (JA) and ethylene (ET)31, which was also noticed in Musa balbisiana at 12 hpi in response to pathogen attack (Fig. 2).
WRKY transcription factor family also plays an important role in regulating genes associated with plant defense responses. WRKY transcription factors are involved in phosphorylation sites for MAP kinases, which is part of PTI35. Our results showed up-regulation of WRKY4 and WRKY75 in BXW-resistant genotype in response to Xcm attack. It has been reported that overexpression of WRKY4 have provided resistance to fungal pathogens like Botrytis cinerea, but enhanced susceptibility to bacterial pathogens like Pseudomonas syringae36. WRKY75 transcription factor act as positive regulators of defense during interactions with bacterial pathogens and demonstrated to activate basal and R-mediated resistance in Arabidopsis and strawberry37.
In this study, the majority of WRKY (6, 11, 17, 22, 40, 41, 49, 65 and 72) transcription factors were suppressed in the BXW-resistant genotype indicating their involvement as negative regulator (Table 2). Several of WRKY transcription factors (AtWRKY7, 11, 17, 18, 23, 25, 27, 38, 40, 41, 48, 53, 58, 60, and 62) were also reported as negative regulators of defense signaling35.
ABA plays important role in plants responding for abiotic as well as biotic stress. Although ABA is known as a negative regulator of defense against biotic stresses, a number of reports have also shown its role in induction of disease resistance38. Our results demonstrated activation of ABA metabolism in BXW-resistant genotype in Musa balbisiana in response to pathogen attack (Fig. 2). ABA has been reported to play important role in plant defense by callose deposition in response to pathogen attack39. ABA suppresses β-1,3-glucanase, an enzyme that degrades callose, ensuring callose accumulation. Our results also showed suppression of the majority of genes associated with β-1,3-glucanases in Musa balbisiana after inoculation with Xcm at 12 hpi, suggesting cell wall enforcement by callose deposition.
The phytohormones SA, JA and ET play key roles in defense responses against pathogens40. Generally, SA-dependent plant defense system is activated by biotrophic bacterial pathogens, whereas necrotrophic fungal pathogens and chewing insects trigger JA-dependent plant defenses. However, we observed suppression of SA and activation of JA pathway in Musa balbisiana in response to interaction with Xcm. Similar to our results, there are reports demonstrating antagonistic interactions between ABA and SA metabolism and ABA production contributing to JA accumulation and activation for resistance against pathogen infection38,39. In this study NPR1-like gene was also suppressed confirming no involvement of SA pathway for systemic acquired resistance (SAR) pathway in Musa balbisiana at 12 hpi.
A second line of defense is Effector-Triggered Immunity (ETI), which involves R-gene families. The R-genes interactwith virulence factors of pathogens and trigger defense response characterized by rapid calcium fluxes, oxidative burst, transcriptional reprogramming within and around the infection sites and localized programmed cell death, which leads to suppression of pathogen growth41. Resistance proteins protect the plant against pathogens after recognition of virulence factor, however, in the absence of specific recognition of virulence factors, basal defense response occurs through PAMPs. Similar to our studies, it has been reported overlapping of the PAMP-triggered defense with R-protein-mediated defense42.
In response to Xcm infection, R-gene in LRR family protein and putative disease resistance protein RPM1 in NBS-LRR domain were up-regulated in Musa balbisiana at 12 hpi inducing immune response providing resistance against pathogen (Table 2). LRR family proteins provide the important structural framework required for molecular interactions, and pathogen recognition42. Similar to our results, Arabidopsis thaliana RPM1 have shown to trigger the defense system against Pseudomonas syringae43.
Activation of R-gene lipase-like PAD4 gene was also observed in Musa balbisiana (Table 2). Lipase like gene is involved in SA signaling and function in R-gene-mediated and basal plant disease resistance44. However, activation of SA signaling was not observed in this study.
A typical class of susceptibility (S) genes, MLO-like protein 13 was found to be suppressed in Musa balbisiana at 12 hpi. MLO is postulated to act as a negative regulator of plant defenses and resistance to powdery mildew was demonstrated by knocking out susceptibility S-genes at MLO loci45.
Induction of PR genes in BXW-resistant genotype Musa balbisiana in response to Xcm inoculation indicates their role in innate immune responses like HR and systemic acquired resistance (SAR) in plants against pathogen infection. Osmotin-like protein, a PR5, is reported to be involved in plant defense responses to several pathogens and abiotic stresses46. Transgenic sesame overexpressing osmotin-like PR gene demonstrated resistance against Macrophomena phaseolina infection by activating JA/ET and SA pathways46.
Germin-like protein (GER1 or GLP1) was activated in both BXW-susceptible and BXW-resistant genotypes at 12 hpi and 48 hpi, respectively. Germin-like protein are reported to be increased in several plants after pathogen infection and involved in plant defense47. It has been reported that differences between susceptibility and resistance are associated with differences in the timing and magnitude of the induced response rather than just with the expression of various genes48. Further investigation is required to understand the function of GER1 in banana.
This study also showed activation of proteolysis in Musa balbisiana in response to pathogen at both 12 hpi and 48 hpi, whereas proteolysis was also induced at 48 hpi in Pisang Awak (Fig. 2). Generally, activation of proteolysis enables host plant cells to trigger defense response upon recognition of an invading pathogen49.
Our results also showed differential expression of transmembrane transporters in Musa balbisiana. Upon attack, pathogens use transporters to send signals to modify host cellular mechanisms promoting virulence and facilitating their own proliferation within the host tissues50. This study showed suppression of sugar transporter SWEET14-like protein in Musa balbisiana as early response to bacterial attack. However, no differential expression of this transcript was noticed in BXW-susceptible genotype Pisang Awak, indicating that SWEET14 facilitate bacterial colonization in susceptible interaction. Bacterial pathogens manipulate SWEET14 transporter for virulence by direct binding its effector to the SWEET promoter and inducing its expression leading to susceptibility to pathogen51,52.
Upon pathogen attack, early nodulin-like proteins were repressed in Musa balbisiana at 12 hpi. Normally, nodulin-like proteins are present in legumes and play an important role in symbiosis with Rhizobium bacteria53. Nodulin-like proteins are involved in transport of nutrients, amino acids, hormones and solutes required for plant development. As nodulin-like proteins assist pathogens to colonize on the host plants53, suppression of these proteins in BXW-resistant Musa balbisiana suggested restricted colonization of Xcm.
In conclusion, comparative transcriptome of the BXW-resistant and the BXW-susceptible genotypes of banana provided some understanding of molecular basis of response against Xcm. The DEGs mapped to biotic stress pathways allowed us to identify number of candidate genes involved in banana-Xcm interaction. Our results demonstrated activation of both PAMP-triggered defense and R protein-mediated defense in the BXW-resistant genotype Musa balbisiana as response to Xcm infection. Upon Xcm attack, pathogen-recognition receptors trigger cascade of responses leading to PTI as first line of defense. Further pathogen effectors were recognized by disease resistant genes inducing R-gene-mediated resistance. In this study transcripts associated with HR and programmed cell death were found to be activated in BXW-resistant genotype as early response to Xcm attack. RNAseq and qRT-PCR results also indicated activation of antimicrobial peptide in the BXW-resistant genotype. The antimicrobial activity of vicilin-like peptide from Musa balbisiana needs to be further tested against Xcm.
The differential expressions of several genes involved in plant defense were validated by qRT-PCR (Fig. 5 and Table 3). Further functional genomics need to be performed to understand in-depth molecular mechanism of defense against Xcm. The significant genes should be knocked out or over-expressed to better understand their role in plant defense against Xcm.
To the best of our knowledge, this study is the first transcriptome analysis of the banana genotypes for the response to Xcm pathogen. Our data provide insights on the defense mechanism in the BXW-resistant wild type banana Musa balbisiana to the most damaging pathogen Xcm. This information can be used in crop improvement program to transfer the disease resistance trait from wild type banana to farmer-preferred banana cultivars commonly grown in Africa.
Materials and Methods
Plant material, inoculation and sampling
The BXW disease resistant genotype Musa balbisiana (BB) and highly susceptible banana cultivar Pisang Awak (ABB, commonly known as Kayinja) were obtained from in vitro collection at IITA-Kenya. The in vitro plantlets were micropropagated in tissue culture.
Pure culture of Ugandan isolate of Xcm isolate collected from Pathology Laboratory, IITA was maintained on YTSA medium (1% yeast extract, 1% tryptone, 1% sucrose and 1.5% agar) at 4 °C. A single bacterial colony was inoculated into 25 ml of YTS medium (1% yeast extract, 1% tryptone and 1% sucrose) and cultured at 28 °C with shaking at 150 rpm for 48 h. The bacterial culture was centrifuged at 5000 rpm for 5 min and pellet was re-suspended in sterile double distilled water. The optical density (OD 600 nm) of the bacterial suspension was checked and bacterial concentration was adjusted to 108 cfu/ml with sterile water. Fresh inoculum was used for all the experiments in order to have high virulent potential of the pathogen.
One month-old in vitro rooted plantlets with 3–4 leaves were artificially inoculated with fresh culture of Xcm. About 100 μl of bacterial suspension (108 cfu ml−1) was artificially injected using insulin syringe into the midrib of the second fully open leaf. This method of artificial inoculation is similar to natural infection through injury by contaminated cutting tools, commonly practiced by farmers. Previous study in our laboratory showed development of BXW symptoms and complete wilting of Pisang Awak plantlets artificially inoculated with Xcm54. The inoculated leaf of M. balbisiana also showed necrotic and chlorotic patches due to hypersensitive response, but these symptoms did not progress and the plants were subsequently healthy54.
Additional plantlets were mock inoculated with sterile water as control in order to nullify the effect of wounding through injection.
The inoculated leaf samples were collected at 0, 12 hpi, 48 hpi. These time points were selected as the expression of defense genes during early infection with Xcm was observed to be high at 12 hpi and then decreasing at 48 hpi55. Three biological and three technical replicates were used for each time point.
The samples were:
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1.
Pisang Awak inoculated with bacterial culture at 0, 12 hpi, 48 hpi
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2.
Musa balbisiana inoculated with bacteria at 0, 12 hpi, 48 hpi
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3.
Pisang Awak mock inoculated with water at 12 hpi, 48 hpi
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4.
Musa balbisiana mock inoculated with water at 12 hpi, 48 hpi
RNA extraction and library preparation
Total RNA was extracted from 100 mg leaf samples using the RNeasy plant mini kit (Qiagen, GmbH, Hilden, Germany) and treated with DNase (RNeasy Plant Mini kit, Qiagen). The quality of RNA was assessed using denaturing agarose gel stained with gelred (Biotium, USA) and quantified using a Nanodrop 2000 (Thermo Fisher Scientific, MA, USA).
A total of 30 cDNA libraries were prepared in each of the two treatments [three biological replicates at each of the three time points (0, 12 hpi and 48 hpi) per genotype inoculated with bacterial culture and two time points (12 hpi, 48 hpi) inoculated with sterile water] using Illumina TruSeq RNA Sample Preparation Kit v2. Poly (A) mRNA was obtained from 1 µg of the total RNA using poly-T oligos attached to magnetic beads. The purified mRNA was fragmented and used to synthesize first strand cDNA using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) and random primers. Second strand cDNA was synthesized from the resultant first strand and this was end-repaired by adding ‘A’ (A-tailing) at the 3′ ends to allow the ligation of indexed adaptors. The adaptor indexes were used in such a way that it allowed for multiplexing in sequencing. The libraries were enriched through twelve cycles of PCR amplifications. Quantitative and qualitative assessments of the libraries were done using QuBit DNA assay (Thermo Fisher Scientific) as well as with Agilent DNA 1000 assay (Agilent technologies, CA, USA) on an Agilent 2100 bioanalyzer. All the assays on quality and quantity were performed according to the respective manufacturer’s protocols.
RNA sequencing (RNA-Seq) and differential gene expression analysis
The cDNA library was sequenced pair-ends wise using the Illumina HiSeq™ 2500, and 100-bp reads were generated. The sequencing was performed by Center for Genomics and Systems Biology, New York University.
The reads were trimmed for quality using Trimmomatic and aligned against the reference Musa acuminata genome using Hisat256. The reference genome for Musa acuminata DH Pahang annotation version 2 and annotations were downloaded from Banana Genome Hub57 (http://banana-genome-hub.southgreen.fr/download). This genome sequence is considered to be the reference genome for Musa species due to its completeness and quality of annotation58. The quality of annotation has recently been quantified through a BUSCO analysis, which scores the genome of Musa acuminata DH Pahang at 96.5% and the genome of Musa balbisiana Pisang Klutuk Wulung at 66.5%59. This genome sequence has been extensively used in other transcriptome studies on Musa including genotypes having B-genome60.
The protein sequences from the Musa acuminata DH Pahang and Musa balbisiana Pisang Klutuk Wulung genomes were downloaded and the reciprocal best BLAST hits were obtained using a perl script. The gene identifiers (IDs) from Musa balbisiana Pisang Klutuk Wulung are reported in all tables based on the results of the reciprocal best BLAST search. The best BLAST hit is reported for the genes where the reciprocal best BLAST search hit was not available and it is denoted with the symbol “*” after the gene identifier.
HTSeq-count was used to count the number of reads that map to the different annotated genes. Readcounts for annotated genes were loaded in R and genes with low expression were filtered out. The genes were removed from the dataset if none of its groups had a median of 10 reads mapping to the genome. The filtered readcount data was used as input for DESeq2 to determine differentially expressed genes. The genes with fold change of greater than 1.5 or <−1.5 and also an adjusted p-value less FDR corrected p-value of 0.1 were identified.
The change in gene expression due to bacterial inoculation was calculated for both genotypes at each time point by comparing them with mock-inoculated plants of the same genotype using the contrasts function in DESeq2. Also the interaction between genotypes and pathogen i.e. genotypes:treatment interaction was recorded.
Go-term enrichment analysis
A Gene Ontology (GO) Enrichment Analysis of differentially expressed genes was performed using GO-terms provided by the Banana Genome Hub and the GOStats package in R61. GOStats has a functionality that allows individuals to create their own GO annotation database for species that are currently not supported in the annotation databases. Using the Banana Genome Hub Gene-to-GO association a Gene Set Enrichment dataset was created and used for identifying GO-terms that were over-represented in a given gene list. The function has been enhanced to provide the FDR corrected p-values. All go-terms were adjusted for p-value of less than 0.05.
Biotic stress and metabolic pathway analysis
The DEGs were mapped to different pathways using the Mapman62 version 3.5.1R2. The mapping to the Mapman functional categories (BINs) was done for all the protein sequences from the Musa acuminata version 2 genome. The protein sequences were uploaded to the Mercator tool63. The pathway analysis used the overall metabolic pathway and the biotic stress pathways for this study.
Quantitative RT-PCR validation
To verify the results of RNA-Seq analysis, qRT-PCR analysis was performed with 30 selected DEGs using gene specific primers (Supplementary Table S8). The primers were designed using the PrimerQuest Tool (Integrated DNA Technologies, Iowa, USA) using the 2 primers, intercalating dyes qPCR design type.
The samples for qRT-PCR were independently prepared as described above for RNAseq. One month-old in vitro rooted plantlets were artificially inoculated with 100 μl of bacterial culture (108 cfu ml−1) of Xcm. The plantlets mock inoculated with sterile water were used as control. The inoculated leaf samples were collected at 0, 12 hpi, 48 hpi and RNA were isolated using the RNeasy plant mini kit (Qiagen, GmbH, Hilden, Germany) and treated with DNase (RNeasy Plant Mini kit, Qiagen). First strand cDNA was synthesized using reverse transcriptase of the Maxima H Minus First Strand cDNA synthesis kit with oligo DT primers (Thermo scientific). The primer specificity for each gene was confirmed by PCR and melting curve in qRT-PCR.
The qRT-PCR was performed using Maxima SYBR green/ROX PCR kit (Thermo Scientific) on 7900 Real Time PCR System (Applied Biosystems, USA). The experiment was set up using three biological and three technical replicates. Relative expression data were normalized using the Musa25S ribosomal gene as reference. Mock inoculated control plant was used as calibrator to calculate log2 fold change of target gene using the ΔΔCt method64.
The means and standard error were calculated for three replicates per experiment using Minitab 16 statistical software, 2012.
The log2 fold change by RNA-Seq and qRT-PCR analysis were compared using Pearson correlation analysis in Minitab 16 statistical software.
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Acknowledgements
This research was supported by the United States Agency for International Development (USAID) and CGIAR Research Program on Roots, Tubers and Bananas (CRP-RTB).
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L.T. conceived idea, designed study and drafted manuscript. J.N.T. prepared samples, extracted RNA and performed qRT-PCR analysis. T.S. performed bioinformatics analysis and pathway analysis and wrote that section. K.S.M. prepared libraries for sequencing. M.K. performed bioinformatics analysis. All authors contributed in writing and critically reviewed the manuscript.
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Tripathi, L., Tripathi, J.N., Shah, T. et al. Molecular Basis of Disease Resistance in Banana Progenitor Musa balbisiana against Xanthomonas campestris pv. musacearum. Sci Rep 9, 7007 (2019). https://doi.org/10.1038/s41598-019-43421-1
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DOI: https://doi.org/10.1038/s41598-019-43421-1
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