The WRKY family of transcription factors orchestrate the reprogrammed expression of the complex network of defense genes at various biotic and abiotic stresses. Within the last 96 million years, three rounds of Musa polyploidization events had occurred from selective pressure causing duplication of MusaWRKYs with new activities. Here, we identified a total of 153 WRKY transcription factors available from the DH Pahang genome. Based on their phylogenetic relationship, the MusaWRKYs available with complete gene sequence were classified into the seven common WRKY sub-groups. Synteny analyses data revealed paralogous relationships, with 17 MusaWRKY gene pairs originating from the duplication events that had occurred within the Musa lineage. We also found 15 other MusaWRKY gene pairs originating from much older duplication events that had occurred along Arecales and Poales lineage of commelinids. Based on the synonymous and nonsynonymous substitution rates, the fate of duplicated MusaWRKY genes was predicted to have undergone sub-functionalization in which the duplicated gene copies retain a subset of the ancestral gene function. Also, to understand the regulatory roles of MusaWRKY during a biotic stress, Illumina sequencing was performed on resistant and susceptible cultivars during the infection of root lesion nematode, Pratylenchus coffeae. The differential WRKY gene expression analysis in nematode resistant and susceptible cultivars during challenged and unchallenged conditions had distinguished: 1) MusaWRKYs participating in general banana defense mechanism against P.coffeae common to both susceptible and resistant cultivars, 2) MusaWRKYs that may aid in the pathogen survival as suppressors of plant triggered immunity, 3) MusaWRKYs that may aid in the host defense as activators of plant triggered immunity and 4) cultivar specific MusaWRKY regulation. Mainly, MusaWRKY52, -69 and -92 are found to be P.coffeae specific and can act as activators or repressors in a defense pathway. Overall, this preliminary study in Musa provides the basis for understanding the evolution and regulatory mechanism of MusaWRKY during nematode stress.
Citation: Kaliyappan R, Viswanathan S, Suthanthiram B, Subbaraya U, Marimuthu Somasundram S, Muthu M (2016) Evolutionary Expansion of WRKY Gene Family in Banana and Its Expression Profile during the Infection of Root Lesion Nematode, Pratylenchus coffeae. PLoS ONE 11(9): e0162013. https://doi.org/10.1371/journal.pone.0162013
Editor: Thumballi R. Ganapathi, Bhabha Atomic Research Centre, INDIA
Received: November 19, 2015; Accepted: August 16, 2016; Published: September 7, 2016
Copyright: © 2016 Kaliyappan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the Indian Council for Agricultural Research, grant no. 2000711005.
Competing interests: The authors have declared that no competing interests exist.
Dynamic cellular reprogramming is a plant defense response to pathogen infection . The WRKY family of proteins function as transcriptional regulator and orchestrate the reprogrammed expression of the labyrinthine network of defense genes as an induced defense response. Also, WRKY transcription factors function in regulating multiple processes ranging from seed germination to secondary metabolism [2, 3]. In general, WRKY transcription factors control the gene expression by binding to the TTGAC(C/T) W-box cis-element in the promoter region of target genes and function as activators or repressors. Several members of WRKY transcription factors had been shown to modulate expression of genes involving in pathogen-associated molecular pattern (PAMP) triggered immunity [4, 5] and effector-triggered immunity [6, 7]. High-affinity DNA binding takes place via their highly conserved amino acid sequence WRKYGQK at the N-terminus and the zinc finger-like motif Cys(2)-His(2) or Cys(2)-HisCys at the C-terminus, which are the characteristic features of WRKY family of proteins. WRKYs may also contain HARF, LZ, LXXL, LXLXLX motifs  and proline-directed serine (SP) clusters making them suitable for modifications like phosphorylation , acetylation  and dimer formation . Identifying and manipulating of this master transcriptional factor during a pathogen attack in banana, the only crop grown in most number of countries  with a huge economic importance especially for developing countries, would provide a foundation for deciphering the role of WRKYs in Musa defense mechanisms. Musa has the highest number of transcription factors among all sequenced plant genomes with a tremendous expansion of WRKY gene family during evolution. Musa contains the second largest WRKY family (153 members) next to Glycine max (176 members) . These numbers signify the need for WRKY transcription factors by Musa for a fittest survival amidst various biotic and abiotic factors.
Pratylenchus coffeae is a major destructive nematode of banana  that is widespread in tropical regions where 78% of bananas were grown . P. coffeae penetrate and migrate through root causing extensive necrosis of root cortical parenchyma and endodermal cells by direct feeding and this root damage also leads to an avalanche of infections via invasion of other phytopathogenic microorganisms. Intense P. coffeae root damages hinder the absorption of water and nutrients from the soil causing stunting, chlorosis in leaves, even toppling of the plant due to weak anchorage by extensively damaged root system and significant yield loss in banana production . P. coffeae is an obligate parasite and compulsorily needs to exploit a host for its reproduction to complete the lifecycle. These nematodes feed on the contents of host cells without the luxury of forming specialized feeding structures like synctia, cysts and giant cells as formed by other root knot nematodes and cyst nematodes [14, 15]. Such traits are the reflection of its remarkably smaller genome sized at only ~20Mb, the smallest known animal genome ever. However, with high gene density these obligatory nematodes originating >600 million years ago (MYA) have evolved well for their existence in banana plants that originated around 75 MYA. Interestingly, plant parasitism has spiked up at least four times independently within the nematode clade Tylenchida comprising P. coffeae . For example, P. coffeae uses stomatostylet, a unique stylet type of the suborder Tylenchina, to puncture holes for entering into cells and ingests cytoplasm directly. For modifying and loosening plant cell walls, an array of cell wall-degrading enzymes like cellulases, xylanases, polygalacturonases, β,1,3- endoglucanase, pectate lyases and expansins are putatively secreted by the subventral gland cells of nematodes to assist the stylet insertion [17–19]. As a counter attack, nematode secreted effector proteins/peptides are recognized by the root cells followed by initiation of defense signaling cascade and regulation of defense-related genes. Adaptive responses of Musa to P.coffeae include triggering lignin production, ROS burst and induced defense signaling events like ethylene response . WRKYs are well known for the activation and suppression of such defense signaling mechanisms. Many WRKYs had been reported to show resistance against various nematodes in rice, arabidopsis, tomato and soybean (S1 Table) and known for triggering a multitude of defense response genes. To gain an insight into nematode-Musa interactions, genome-wide expression pattern of MusaWRKY in both nematode resistant and susceptible cultivars were analyzed during the infection of a mixed life stage population of P. coffeae, by performing Illumina sequencing. The expression analysis of MusaWRKY genes showed that MusaWRKYs are involved in the nematode stress response. Along with the study on evolution and duplication of MusaWRKY genes, this paper provides a foundation for further functional studies on WRKY genes in banana.
Materials and Methods
MusaWRKYs and Phylogenetic analysis
The 153 Musa acuminata WRKY protein and nucleotide sequences were obtained from the Banana Genome Hub (http://banana-genome.cirad.fr/). Arabidopsis WRKY protein sequences were obtained as reference from The Arabidopsis Information Resource (TAIR). Complete CD sequences were separated from the partial fragment sequences available at the Banana Genome Hub. The 60 amino acid long complete WRKY and Zinc finger domains of 81 MusaWRKY proteins were used to create multiple protein sequence alignments using ClustalW. The neighbor-joining method was used to construct the phylogenetic tree based on amino acid sequence of WRKY domains using MEGA 5.2.1 software (Bootstrap value 1000). Analysis for conserved motifs in the WRKY proteins was carried out using MEME database (http://meme.sdsc.edu/meme/cgi-bin/meme.cgi). It was observed that most conserved domains are limited to a single subfamily of WRKY transcription factors and therefore MEME analyses were run for the members of each subfamily using the protein sequences (S11 Table). The online program 2ZIP (http://2zip.molgen.mpg.de/index.html) was used for predicting Leu zipper motifs, while HARF, LXXLL and LXLXLX motifs were identified by manual inspection.
Chromosomal location of MusaWRKYs and evolution
All MusaWRKY genes were mapped to Musa chromosomes based on information available at The Banana Genome Hub (http://banana-genome.cirad.fr/). Tandem clusters located within 50kb on individual chromosomes were identified from the gene positions on the scaffolds taken from the plant genome duplication database (http://chibba.agtec.uga.edu/duplication/index/files). To determine paralogous WRKY gene pairs between Musa chromosomes that resulted from the last three rounds of whole genome duplication (WGD), we used “Syntenic DotPlot” section of The Banana Genome Hub (http://banana-genome.cirad.fr/dotplot). To visualize the synteny, we did all chromosomes by all chromosomes analysis and obtained the dot plot. The list of co-localized paralogs with synonymous and nonsynonymous substitution rate values were obtained and the paralogous WRKY gene pairs were linked on the WRKY karyotype locus map obtained from the locus search on The Banana Genome Hub.
Nematode infected banana roots were collected from the research farm at the ICAR-National Research Centre for Banana, India. Roots were chopped and incubated in water. Then nematodes were extracted through sieves  and examined under light microscope (Olympus SD-STB3, Japan). Based on morphological appearance 100 number of female P. coffeae alone were picked out and inoculated into carrot discs on 1% agar medium. It was maintained at 26°C and after 45 days, nematodes were collected and used for inoculation in the banana roots.
Plant materials and sample collection
Uniformed sized suckers of banana resistant cultivar, Karthobiumthum (ABB) (NRCB-0050) and susceptible cultivar, Nendran (AAB) (NRCB-0615) were collected and treated with nematicide. These suckers were planted in the cement pots containing fumigated pot mixture in the ratio of 1:1:1 (sand:farmyard manure: red soil) and maintained in the glass house. One month after planting, individual plastic cup containing hole on the side was placed by removing the soil near the plants in pots and those cups were filled with potting mixture. Single root was selected in each plant and carefully inserted through the hole into the plastic cup. Each cup was inoculated with 3000 active root lesion nematodes. Inoculated roots were collected at various time points (2, 4, 6, 8 and 10 days post inoculation) in three replications for each time interval from independent plants of both resistant and susceptible cultivars. Similarly, the root samples were collected at the same time points from uninocluated plants. Collected roots were washed with DEPC water and frozen by using liquid nitrogen and kept in -80°C for later analysis.
Construction of cDNA library and Illumina deep sequencing
Total RNA was extracted from each sample (2 gram) using Agilent Plant RNA isolation mini kit (Agilent Technologies, Inc., USA) (product no. 5188–2780). The RNA was treated with DNase I and their integrity was tested. Equal amount of RNA corresponding to root samples collected at various time intervals (2, 4, 6, 8 and 10 days) were pooled together separately for resistant and susceptible cultivars from both inoculated and uninoculated plants. This pooled RNA samples of nematode inoculated and uninoculated resistant and susceptible cultivars were used for next generation sequencing and quantitative real time PCR validation (qRT-PCR). The library construction and sequencing were performed by the Genotypic Technology, Bangalore, India, according to the Illumina TruSeq RNA library protocol outlined in ‘‘TruSeq RNA Sample Preparation Guide” (Part # 15008136; Rev. A; Nov 2010). The prepared library was quantified using Nanodrop and validated for quality by running an aliquot on High Sensitivity Bioanalyzer Chip (Agilent). The library was loaded onto the channels of an Illumina HiSeqTM GAII Analyzer instrument for 4 gigabase in-depth sequencing, which was used to obtain more detailed information about gene expression. Each paired-end library had an insert size of 200–700 bp. The average read length of 90 bp was generated as raw data. Raw sequence data were generated by Illumina pipeline and were available in NCBI’s Short Read Archive (SRA) database (http://www.ncbi.nlm.nih.gov/sra/SRP071591) under accession number SRP071591. Information about the sequencing data like depth and number of contigs is listed in the S1 Table.
Genome assembly, sequence clustering and digital gene expression (DGE)
The clean reads were obtained from raw data by filtering out adaptor-only reads from reads containing more than 5% unknown nucleotides (nt), and low-quality reads. Then, clean reads were mapped to the reference AA genome (Musa acuminata) with TopHat (v2.0.4)  and their relative abundance were calculated using cufflinks (v2.0.1) and then annotated independently. Annotated results of WRKY genes were further shortlisted based on the reference genome ID. The differential expression of the genes among challenged and unchallenged libraries of resistant and susceptible cultivars was calculated based on fold value which was obtained from their respective FPKM values. The summation of FPKM values for every transcript associated with a particular gene gives the expression measurement in FPKM. The differential gene expression is calculated by the Cuffdiff program using the ratio of uninoculated vs. incoculated FPKM values for every gene. The following cutoff was assigned for upregulated and downregulated genes based on the P value < 0.05 (FC is log fold change to the base 2).
Quantitative Real Time PCR (qRT-PCR)
For qRT-PCR analysis, 1 μg of RNA was used for cDNA synthesis by first strand reverse transcription kit with oligo-(dT) primers according to manufacturer’s protocol (Promega). The first strand cDNA was diluted (1:10) with water and was used as template for qRT-PCR. Six MusaWRKYs (69, -52, -92, -41, -19 and -81) were selected for validation. Specific gene primers (S2 Table) were designed using PRIMER3 software. Three independent PCR reactions were carried out for each gene. The PCR reactions were performed in a 20 μL total reaction volume including 10 μL of Power SYBR Green PCR Master Mix (Applied Biosystem), 5 mM each of gene-specific primers, and 1 μL of cDNA templates. The detection was carried out by LightCycler 480 (Roche, Germany) system and the following programme was used: 10 min/94°C; then 40 cycles of 20 s/94°C, 30 s/56°C, and 20 s/72°C; and finally for melting curve analysis: 1 min/95°C, 30 s/56°C, and 30 s/95°C was used. The relative level of each target gene was normalized by comparing the copy numbers of target mRNA with RPS2. The relative expression level was calculated using the 2-ΔΔCt method.
Results and Discussion
MusaWRKY family and phylogeny
A total of 153 WRKY sequences were retrieved from the Banana Genome Hub (CIRAD, France), of which only 81 WRKYs had complete CDs. WRKY members were dispersed on 11 Musa chromosomes (Fig 1). Numbering of many published MusaWRKYs correspond to its orthologous Arabidopsis gene and may result in a WRKY to have two or more different names. So, we designated 153 WRKYs as MusaWRKY1 through MusaWRKY147 based on their orders on chromosomes and six MusaWRKY genes that may be located on unanchored scaffolds and could not be mapped to any chromosomes were designated as MusaWRKY148 through MusaWRKY153(S3 Table, Fig 1). To avoid false positives, only the 81 MusaWRKYs with complete CDs were classified into three major groups (Table 1, Fig 2) based on the number of WRKY domains (single or double), additional amino acid motifs and phylogeny . Accordingly, all group I members had two heptapeptide WRKY domains (WRKYGQK). The presence of SP clusters were also noticed among them which is a characteristic feature of group I WRKYs .Group I MusaWRKYs comprised 17% of the total MusaWRKY family and were comparable to group I WRKYs of Brachypodium distachyon (19%) and Oryza sativa (15%) belonging to commelinids clade of monocots [23, 24]. Based on the amino acid motifs outside the WRKY domain, Group II MusaWRKYs were further divided into 5 subgroups: Group IIa, IIb, IIc, IId and IIe. Unique motif and shared motif composition was observed among MusaWRKY members within the same subgroups (Fig 3, S4 Table). Group IIa is considered to be the recently evolved WRKY group  and in Musa, 7 members were present in this group. Noticeably, motifs 5 and 10 were limited to the members of group IIa and group IIb, whereas only group IIc members contained motifs 16 and 19. Group IIc had undergone a significant expansion containing more than 50% of the MusaWRKY family. The HARF motif was present only among the members of group IId. Motif 15 existed uniquely in group II-e. Group I and group II WRKYs shared the same C2-H2 type zinc finger motif (C–X4–7–C–X23–29–H–X1–H). In general, group III WRKY genes were believed to have specific roles in monocotyledonous plants  and in Musa, 6 WRKYs were present in this group with C2-HC-type zinc finger motif (C–X4–7–C–X23–29–H–X1–C). In group III, MusaWRKY43 showed divergence in the WRKY domain and had WRKYGYK. A variety of domains like LXXL (MusaWRKY18, -5, -2, -7, -80, -129, -75, -24 and -87), LZ (MusaWRKY10, -68, and -145), HARF (MusaWRKY135, -122, -90, -60, -46, -25, -17, -15 and -6) and LXLXLX (MusaWRKY6, -68, -81, -127, -62, -92, -140, -51 and -43) were present (S4 Table) and MusaWRKY family of proteins displayed fundamental physiochemical properties with wide ranges (S5 Table) together supporting that different MusaWRKYs can interact with different substrates to perform a wide variety of cellular functions. For example, the interaction of WRKY with MAPKs occurs at the SP (Serine-Proline) clusters located next to a D-domain, which is a MAPK docking motif. This interaction had been proved for effective phosphorylation of WRKY in plants and MusaWRKY18 belonging to group I have SP clusters and D-domain that can be phosphorylated by the MAPK for the activation of defense-related genes [26, 27].
Eleven synteny blocks of Musa chromosomes are represented as in the banana genome hub.The paralogous chromosome segments are represented in the same colors. The 17 paralogs are linked by the same colored lines. Number and size of the each chromosome are given at the top.
Amino acid sequences of WRKY genes were aligned with clustal W and 1000 bootstrap values in neighbor-joining method in MEGA 5.2.Branch name of sub trees are colored indicating different WRKY groups and subgroups. Triangle symbols indicated that significant expression in susceptible or resistant cultivars. Ο symbol shows that no genes were expressed, and arcs symbol showed that up-regulated in resistant and down-regulated in susceptible cultivar during nematode infection.
MEME was used to predict motifs and these motifs represented with boxes.
Expansion of MusaWRKY for resistance
Expansion of transcription factor families particularly in Musa is significantly greater than other plant genomes after genome duplication . Musa polyploidization events had occurred from selective pressure causing both variation in cis-regulatory elements (S6 Table and S2 Fig) and duplication of WRKYs with new activities . Based on the general WRKY functional role, we hypothesized that WRKY duplication events may contribute to the fittest survival amidst various biotic and abiotic stresses. First, we looked up for the duplicated paralogous WRKY gene clusters corresponding to the 12 synteny blocks developed by D’Hont and colleagues [13, 29] that represent the Musa ancestral genome within last three rounds of WGD: α, β and γ (Fig 1). Synteny blocks are homologous regions in chromosomes with a series of genes residing in same order corresponding to a common ancestral genomic region. Using synteny analyses data (S7 Table), paralogous relationships were derived based on the synonymous substitution rate (Ks), which is a function of genomic evolutionary events. Ks distribution among pairs of paralogous gene clusters indicates the α+ß WGD events (Ks ~ 0 to 0.45) and the γ WGD event (Ks ~ 0.45 to 0.85) that occurred 64.8MYA and 96MYA respectively in Musa lineage . Two paralog gene pairs, MusaWRKY40/MusaWRKY100 and MusaWRKY50/MusaWRKY143, were identified from the α+ß WGD events. Fifteen paralogous WRKY gene pairs were identified from the γ WGD event: MusaWRKY04/MusaWRKY08, MusaWRKY20/MusaWRKY111, MusaWRKY26/MusaWRKY121,MusaWRKY27/MusaWRKY39, MusaWRKY42/MusaWRKY97, MusaWRKY62/MusaWRKY92, MusaWRKY62/MusaWRKY140,MusaWRKY66/MusaWRKY137, MusaWRKY70/MusaWRKY130, MusaWRKY74/MusaWRKY116, MusaWRKY87/MusaWRKY133, MusaWRKY105/MusaWRKY146,MusaWRKY107/MusaWRKY134,MusaWRKY118/MusaWRKY108 and MusaWRKY118/MusaWRKY129. Among the 15 collinear relationships, only MusaWRKY62 and MusaWRKY118 have multiple paralogs (S7 Table and Fig 1). All duplicated genes were collinear with ancestral blocks and existed across chromosomes, suggesting that segmental duplication had significantly contributed to the expansion of WRKY gene family in Musa as similar to rice. Separately, we identified 15 MusaWRKY gene paralogs (Ks>0.85) attributing probably to older duplication events that may be inferred to have occurred along Arecales and Poales lineage of commelinids (S7 Table). Additionally, by Holub method  eight MusaWRKY genes could be placed in four tandem clusters (Fig 1 and S1 Fig), two on chromosome seven and one on chromosomes eight and ten. On chromosome seven, MusaWRKY79 and -80 are clustered together in opposite orientation to each other on a 30-kb fragment and similarly MusaWRKY96 and -97 are clustered in opposite orientation on a 37-kb fragment. MusaWRKY101 and -102 are clustered together in opposite orientation on a 36-kb fragment of chromosome eight whereas MusaWRKY127 and -128 are clustered together in the same orientation on a 7-kb fragment of chromosome ten (S1 Fig). Among the four tandem clusters, only three gene pairs were found to be tandemly duplicated: MusaWRKY79/MusaWRKY80, MusaWRKY96/MusaWRKY97 and MusaWRKY101/MusaWRKY102. Further by COGE analysis, MusaWRKY85/MusaWRKY86was also identified to be from tandem duplication. Thus, in Musa tandem duplication events are much lesser than the segmental duplication as similar to rice . In this transcriptome study, expression data were available only for four segmentally duplicated MusaWRKY pairs and the expression pattern of both copies of all paralogous MusaWRKY pairs was similar (either both upregulated or down regulated). Noticeably, there was expression divergence among one tandemly duplicated gene pair, MusaWRKY85/86, in which MusaWRKY85 was getting downregulated in contrast to its duplicated copy, MusaWRKY86, which was upregulated in susceptible cultivar after nematode challenge. We then predicted the divergence functions of duplicated genes from Ω ratio, the ratio of the number of nonsynonymous substitutions per nonsynonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks) . The Ω ratio (Ka/Ks) for all the paralog WRKY gene pairs was found to be less than one suggesting the fate of duplicated WRKY genes as sub-functionalization  (S7 Table) in which the duplicated gene copies continue maintaining a subset of theancestral gene function. Thus, WRKY gene copies were undergoing purifying selections in the Musa evolution thereby contributing to phenotypic variation in polyploids. Such evolutionary changes had a synergistic impact on WRKYs in acquiring new binding specificities thereby governing various signaling mechanism with respect to defense: JA, SA, ISR (Induced Systemic Resistance), SAR (Systemic Acquired Resistance) and PR genes (Pathogenesis-Related) like peroxidase and catalase [4, 5, 6, 7, 14].
Expression patterns of MusaWRKYs in compatible and incompatible interactions of Musa-P.coffeae
Several WRKYs have been directly associated with defense against nematodes such as rice OsWRKY59, -62, -70 and -11, tomato SlWRKY70 and Arabidopsis AtWRKY35, 45 and 48 (S8 Table) [15, 34–39]. To find MusaWRKYs that are involved in nematode stress response, the differential gene expressions of MusaWRKYs were shortlisted from the cuffdiff analysis of RNAseq data on nematode resistant and susceptible cultivars during nematode challenged and unchallenged conditions (Table 2). Altogether, during challenged and unchallenged conditions among 153 MusaWRKYs identified from Musa genome, 99MusaWRKYs were expressed in resistant cultivar (S9 Table) and only 91MusaWRKYs were expressed in susceptible cultivar (S10 Table). The non-detection of remaining MusaWRKYs in control and nematode challenged root tissues of resistant and susceptible cultivars may be due to insufficient depth and coverage or that these MusaWRKYs may not be associated with root development and/or nematode stress response. During unchallenged conditions, 93 and 79MusaWRKYs were found to be expressed in resistant and susceptible cultivars respectively (S9 and S10 Tables) and this constitutive expression indicates their functional role in root development.
Upon infection, 49 MusaWRKYs were found to be differentially regulated in resistant cultivar and 40MusaWRKYs were differentially regulated in susceptible cultivar significantly with majority of them from group IIc (42 MusaWRKYs). Among them, upregulation of 20 MusaWRKYs was common to both susceptible and resistant cultivars, indicating their participation in general banana response mechanism against P.coffeae.23 MusaWRKYs showed significantly increased transcript levels that were unique to the resistant line in P.coffeae infected root tissue as compared to control, indicating their role in resistance mechanism and 15 MusaWRKYs were uniquely upregulated only in susceptible cultivar pointing that some of these MusaWRKYs may aid in the pathogen survival as suppressors of plant triggered immunity. MusaWRKYs 31, -48 and -131 were uniquely downregulated only in resistant cultivar where as MusaWRKY85 was downregulated in susceptible cultivar. Interestingly, three MusaWRKYs,-52, -69 and -92 showed a contrasting regulation among the susceptible and resistant cultivar and hence can be considered as cultivar specific response. Mainly, MusaWRKY52 was twofold higher in resistant cultivar while getting significantly downregulated in susceptible cultivar, suggesting that MusaWRKY52 may be a P.coffeae specific response; MusaWRKY69 and MusaWRKY92 were highly upregulated in susceptible cultivar but downregulated in resistant cultivar speculating that it may be acting as a repressor in a defense pathway. To validate the differential expression patterns of DEGs, six functional genes were selected for RT-qPCR analysis: MusaWRKY52, MusaWRKY69, MusaWRKY92, MusaWRKY41, MusaWRKY19 and MusaWRKY81. Their expression trends were similar to the transcript abundance changes by RNA-Seq indicating the quality of illumina sequencing (Fig 4).
Relative quantification was carried out to measure changes of selective WRKY gene expression in P.coffeae resistant and susceptible root sample relative to an endogenous reference gene (RPS2). Red line shows the expression analysis in DGE. Data (technical triplicates of three biological experiments) are reported as means ± standard error.
WRKY transcription factors play multiple roles in plant growth and development, production of secondary metabolites and defense pathways. A large evolutionary expansion of this master transcriptional regulator in Musa may be a strategy for better performance of these plants to survive against biotic and abiotic stresses. After nematode attack, there was a significant change in expression patterns of many MusaWRKYs indicating their participation in the nematode stress response. In particular, MusaWRKY52, -69 and -92 showed a contrasting regulation between resistant and susceptible cultivar during nematode attack. Due to the limited availability of MusaWRKY studies, an in-depth study on the differentially regulated WRKYs is further needed to understand the actual WRKY gene regulatory networks during Musa- P.coffeae interaction. Our study based on the computational analysis and differential gene expression analysis of MusaWRKY gene family provide a foundation for further detailed studies on MusaWRKY.
S1 Fig. Tandem clusters of MusaWRKY79, -80, -96 and -97 (chr 7), -101, -102 (chr 8), -127, -128 (chr 10) from single ancestral genes on chromosomes.
This is shown in the screen shot from BGH database G-browser, the position and orientation indicated by arrows.
S2 Fig. Various cis-acting elements in MusaWRKY’s and Orthologs of rice WRKY’s.
S1 Table. Information about the illumina sequencing data.
S2 Table. Quantitative RT-PCR primer sequences of MusaWRKYs.
S3 Table. Chromosomal location of WRKY genes in Musa.
S4 Table. List of MusaWRKYs having LXXL, Leucine zipper, HARF and LXLXL motifs.
S5 Table. Physicochemical properties of Complete CD's of MusaWRKY's.
S6 Table. Comparison of commonly available cis-regulatory elements among MusaWRKYs with rice WRKYs orthologs.
S7 Table. Evolutionary expansion of MusaWRKY genes through duplicated events evidenced by synonymous and non synonymous substitution rate analysis.
S8 Table. Expression of WRKY gene family in various plant species against nematodes.
S9 Table. Differential Gene Expression (DGE) of MusaWRKYs in nematode resistant cultivar.
S10 Table. Differential Gene Expression (DGE) of MusaWRKYs in nematode susceptible cultivar.
- Conceived and designed the experiments: BS RK SV.
- Performed the experiments: RK SV.
- Analyzed the data: SV RK BS.
- Wrote the paper: SV RK BS US SMS MM.
- 1. Somssich IE, Hahlbrock K. Pathogen defence in plants a paradigm of biological complexity. Trends in Plant Science. 1998;3(3):86–90.
- 2. Grunewald W, De Smet I, De Rybel B, Robert HS, van de Cotte B, Willemsen V, et al. Tightly controlled WRKY23 expression mediates Arabidopsis embryo development. EMBO Reports. 2013;14(12):1136–42. pmid:24157946
- 3. Schluttenhofer C, Yuan L. Regulation of specialized metabolism by WRKY transcription factors. Plant Physiol. 2015;167(2):295–306. pmid:25501946
- 4. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu W-L, Gomez-Gomez L, et al. MAP kinase signalling cascade in Arabidopsis innate immunity. Nature. 2002;415(6875):977–83. pmid:11875555
- 5. Lippok B, Birkenbihl RP, Rivory G, Brümmer J, Schmelzer E, Logemann E, et al. Expression of AtWRKY33 Encoding a Pathogen- or PAMP-Responsive WRKY Transcription Factor Is Regulated by a Composite DNA Motif Containing W Box Elements. Molecular Plant-Microbe Interactions. 2007;20(4):420–9. pmid:17427812
- 6. Meng Y, Wise RP. HvWRKY10, HvWRKY19, and HvWRKY28 Regulate Mla-Triggered Immunity and Basal Defense to Barley Powdery Mildew. Molecular Plant-Microbe Interactions. 2012;25(11):1492–505. pmid:22809275
- 7. Deslandes L, Olivier J, Peeters N, Feng DX, Khounlotham M, Boucher C, et al. Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus. Proc Natl Acad Sci U S A. 2003;100(13):8024–9. pmid:12788974
- 8. Wang M, Vannozzi A, Wang G, Liang Y-H, Tornielli GB, Zenoni S, et al. Genome and transcriptome analysis of the grapevine (Vitis vinifera L.) WRKY gene family. Horticulture Research. 2014;1:14016. pmid:26504535
- 9. Ishihama N, Yamada R, Yoshioka M, Katou S, Yoshioka H. Phosphorylation of the Nicotiana benthamiana WRKY8 Transcription Factor by MAPK Functions in the Defense Response. The Plant Cell. 2011;23(3):1153–70. pmid:21386030
- 10. Le Roux C, Huet G, Jauneau A, Camborde L, Trémousaygue D, Kraut A, et al. A Receptor Pair with an Integrated Decoy Converts Pathogen Disabling of Transcription Factors to Immunity. Cell.161(5):1074–88. pmid:26000483
- 11. Xu X, Chen C, Fan B, Chen Z. Physical and Functional Interactions between Pathogen-Induced Arabidopsis WRKY18, WRKY40, and WRKY60 Transcription Factors. The Plant Cell. 2006;18(5):1310–26. pmid:16603654
- 12. Food and Agriculture Organization of the United Nations,FAOSTAT database 2013, 2014. Available: http://faostat3.fao.org/browse/Q/QC/E
- 13. D/'Hont A, Denoeud F, Aury J-M, Baurens F-C, Carreel F, Garsmeur O, et al. The banana (Musa acuminata) genome and the evolution of monocotyledonous plants. Nature. 2012;488(7410):213–7. pmid:22801500
- 14. Backiyarani S, Uma S, Arunkumar G, Saraswathi MS, Sundararaju P. Differentially expressed genes in incompatible interactions of Pratylenchus coffeae with Musa using suppression subtractive hybridization. Physiological and Molecular Plant Pathology. 2014;86:11–8.
- 15. Kyndt T, Denil S, Haegeman A, Trooskens G, Bauters L, Van Criekinge W, et al. Transcriptional reprogramming by root knot and migratory nematode infection in rice. New Phytol. 2012;196(3):887–900. pmid:22985291
- 16. Danchin EGJ, Rosso M-N, Vieira P, de Almeida-Engler J, Coutinho PM, Henrissat B, et al. Multiple lateral gene transfers and duplications have promoted plant parasitism ability in nematodes. Proceedings of the National Academy of Sciences. 2010;107(41):17651–6.
- 17. Haegeman A, Joseph S, Gheysen G. Analysis of the transcriptome of the root lesion nematode Pratylenchus coffeae generated by 454 sequencing technology. Molecular and Biochemical Parasitology. 2011;178(1–2):7–14. pmid:21513748
- 18. Bauters L, Haegeman A, Kyndt T, Gheysen G. Analysis of the transcriptome of Hirschmanniella oryzae to explore potential survival strategies and host–nematode interactions. Molecular Plant Pathology. 2014;15(4):352–63. pmid:24279397
- 19. Smant G, Jones J. Suppression of Plant Defences by Nematodes. In: Jones J, Gheysen G, Fenoll C, editors. Genomics and Molecular Genetics of Plant-Nematode Interactions: Springer Netherlands; 2011. p. 273–86.
- 20. Trapnell C., Pachter L. & Salzberg S. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 2009:25, 1105–1111 pmid:19289445
- 21. Eulgem T, Rushton PJ, Robatzek S, Somssich IE. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 2000;5(5):199–206. pmid:10785665
- 22. Huh SU, Lee G-J, Jung JH, Kim Y, Kim YJ, Paek K-H. Capsicum annuum transcription factor WRKYa positively regulates defense response upon TMV infection and is a substrate of CaMK1 and CaMK2. Scientific Reports. 2015;5:7981. pmid:25613640
- 23. Tripathi P, Rabara R, Langum T, Boken A, Rushton D, Boomsma D, et al. The WRKY transcription factor family in Brachypodium distachyon. BMC Genomics. 2012;13(1):270.
- 24. Wu K-L, Guo Z-J, Wang H-H, Li J. The WRKY Family of Transcription Factors in Rice and Arabidopsis and Their Origins. DNA Research. 2005;12(1):9–26. pmid:16106749
- 25. Rinerson C, Rabara R, Tripathi P, Shen Q, Rushton P. The evolution of WRKY transcription factors. BMC Plant Biology. 2015;15(1):66.
- 26. Adachi H, Nakano T, Miyagawa N, Ishihama N, Yoshioka M, Katou Y, et al. WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in Nicotiana benthamiana. The Plant Cell. 2015.
- 27. Ishihama N, Yamada R, Yoshioka M, Katou S, Yoshioka H. Phosphorylation of the Nicotiana benthamiana WRKY8 Transcription Factor by MAPK Functions in the Defense Response. The Plant Cell. 2011;23(3):1153–70. pmid:21386030
- 28. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002:30(1):325–7. pmid:11752327
- 29. Garsmeur Olivier, Denoeud France, Baurens Franc-Christophe, Aury Jean-Marc, Wincker Patrick, D'Hont Angélique Paleoploidization events in the Musa (banana) lineage. 2013. In: Polyploïde et cytogénétique, Rennes, France, 16–17.
- 30. Holub EB. The arms race is ancient history in Arabidopsis, the wildflower. Nat Rev Genet. 2001;2(7):516–27. pmid:11433358
- 31. Wang Y, Feng L, Zhu Y, Li Y, Yan H, Xiang Y. Comparative genomic analysis of the WRKY III gene family in populus, grape, arabidopsis and rice. Biology Direct. 2015;10(1):48.
- 32. Hurst LD. The Ka/Ks ratio: diagnosing the form of sequence evolution. Trends in Genetics.18(9):486–7. pmid:12175810
- 33. Flagel LE, Wendel JF. Gene duplication and evolutionary novelty in plants. New Phytologist. 2009;183(3):557–64. pmid:19555435
- 34. Ali MA, Wieczorek K, Kreil DP, Bohlmann H. The beet cyst nematode Heterodera schachtii modulates the expression of WRKY transcription factors in syncytia to favour its development in Arabidopsis roots. PLoS One. 2014;9(7):e102360. pmid:25033038
- 35. Bhattarai KK, Atamian HS, Kaloshian I, Eulgem T. WRKY72-type transcription factors contribute to basal immunity in tomato and Arabidopsis as well as gene-for-gene resistance mediated by the tomato R gene Mi-1. Plant J. 2010;63(2):229–40. pmid:20409007
- 36. Grunewald W, Karimi M, Wieczorek K, Van de Cappelle E, Wischnitzki E, Grundler F, et al. A role for AtWRKY23 in feeding site establishment of plant-parasitic nematodes. Plant Physiol. 2008;148(1):358–68. pmid:18599655
- 37. Ibrahim HM, Alkharouf NW, Meyer SL, Aly MA, Gamal El-Din AlK, Hussein EH, et al. Post-transcriptional gene silencing of root-knot nematode in transformed soybean roots. Exp Parasitol. 2011;127(1):90–9. pmid:20599433
- 38. Nguyn P, Bellafiore S, Petitot A-S, Haidar R, Bak A, Abed A, et al. Meloidogyne incognita—rice (Oryza sativa) interaction: a new model system to study plant-root-knot nematode interactions in monocotyledons. Rice. 2014;7(1):23. pmid:26224554
- 39. Petitot A-S, Lecouls A-C, Fernandez D. Sub-genomic origin and regulation patterns of a duplicated WRKY gene in the allotetraploid species Coffea arabica. Tree Genetics & Genomes. 2008;4(3):379–90.