Transcriptomic analysis of Dubas bug (Ommatissus lybicus Bergevin) infestation to Date Palm

Date palm (Phoenix dactylifera L.) and its fruit possess sociocultural, health and economic importance in Middle East. The date palm plantations are prone to Dubas bug (DB; Ommatissus lybicus DeBergevin; Homoptera: Tropiduchidae) attacks that severely damages the tree’s growth and reduces fruit production. However, the transcriptome related datasets are not known to understand how DB activates physiological and gene regulatory mechanisms during infestation. Hence, we performed RNA-Seq of leaf infected with or without DB to understand the molecular responses of date palm seedlings. Before doing that, we noticed that DB infestation significantly increase superoxide anion and malondialdehyde production to two-folds as compared to healthy control. Stress-responsive genes such as proline transporter 2, NADP-dependent glyceraldehyde and superoxide dismutase were found significantly upregulated in infected seedlings. The infection repercussions were also revealed by significantly higher contents of endogenous phytohormonal signaling of jasmonic acid (JA) and salicylic acid (SA) compared with control. These findings persuaded to dig out intrinsic mechanisms and gene regulatory networks behind DB infestation to date palm by RNA-Seq analysis. Transcriptome analysis revealed upregulation of 6,919 genes and down-regulation of 2,695 genes in leaf during the infection process. The differentially expressed genes were mostly belongs to cellular functions (calcium and MAPK), phytohormones (auxin, gibberellins, abscisic acid, JA and SA), and secondary metabolites (especially coumarinates and gossypol). The data showed that defense responses were aggravated by gene networks involved in hypersensitive responses (PAR1, RIN4, PBS1 etc.). In conclusion, the results revealed that date palm’s leaf up-regulates both cellular and phytohormonal determinants, followed by intrinsic hypersensitive responses to counter infestation process by Dubas bug.

Scientific RepoRtS | (2020) 10:11505 | https://doi.org/10.1038/s41598-020-67438-z www.nature.com/scientificreports/ other constituents. This become the onset of mainstay problem by development of pathogenic infection (black sooty mold on the foliage), further damaging the leaf parts via chlorosis 12 . This consequently cause reduction in the photosynthetic rates 9,13 . Prolonged and high intensity of infestation results in the flagging and destruction of palm plantations 14 . Thus, overall there are various factors that influence the infestation patterns, however, this needs detailed in-depth molecular approaches ensure proficient datasets for further studies. Dealing with DBs infestations, various approaches have focused on the use of insecticides. In Iraq and Israel directly inject dichlorvos (DDVP), and systemic carbamates (e.g., aldicarb and butocarboxim) respectively, into infected palms that are found successful in controlling infestations 3,15 . However, these strategies are consider harmful for environment, human and to other species e.g., Oligosita sp. (Hymenoptera: Trichogammidae, Aprostocetus sp. (Hymenoptera: Eulophidae), and Runcinia sp. (Aranae: Thomsidae) 3 . In addition to that, studies have shown that after application of insecticides, some pesticide residues remain on the date palm fruits for up to sixty 16 . Although some recent work has been carried to understand the DB infestation and life cycle, however, how the date palm responds to the infection process has not been well understood. Elucidating such infestation based genetic responses by the host itself will help to explain the innate immunity mechanism against prolong infections and to give alternatives and specific targets for date palm breeder in developing resistant cultivars.
Although fungal infestation and host physio-molecular responses have been well studied in various crop plants, however, studies related to arid land date palm has been frequently overlooked. Broadly, insect attack on leaf is preset of wounding or injury to the tissue, also in case of DB, which follows with the fungal infection-a duo synergistic action triggering a race for feed and reproduction 17 . This initiate activation of defense related mechanisms such as production of antioxidants and signaling cascades of endogenous phytohormones such as jasmonic acid and salicylic acid, whereas some trees tends to produce volatile and resinous components to counteracts such attacks [17][18][19][20][21][22] . However, such responses could vary among different species whereas it depends on insect infestation mode and intensity. Particularly, the way DB attack might be similar to other insect; however, the post-infection process is restricted only to the species.
In case of date palm tree, there are a few previous studies [23][24][25][26] explaining the physiology and genomics, however, very few studies have also shown the differential gene expression of date palm during abiotic stress conditions 27 . There are recent studies performed to understand the infestation process and related gene's regulation in aphid feedings to susceptible plants 28 , Ostinia furnacalis leaf feeding to maize 29,30 , soldier fly on sugarcanes in Australia, aphid attacks on wheat crops, and Lepidoptera species infection to cotton 31,32 . These studies have used detailed RNA-Seq based method to point out various underlying mechanisms during herbivory infection. Such studies utilizing the 'omics-based approaches could help in finding out resistance and attack mechanisms that could broadly improve the control of infection strategies. Contrarily, there are no studies till date on the transcriptomic analysis of DB infection to date palm. Hence, in current study, we aimed to understand the underlying mechanisms of DB infection on the leaf of date palm. For this purpose, healthy control and infected date palm samples were assessed initially for their responses against oxidative stress and regulation of endogenous phytohormonal during DB attack (Fig. 1A). These intriguing results persuaded further to perform in-depth nextgeneration sequencing (RNA-Seq) approaches to identify and elucidate the gene expression network(s) during infection process. This study was performed for the first time to usher the gene expression patterns, activation of defense related pathways, triggering of endogenous phytohormones and generating transcript datasets for future studies during DB attack to date palm. This will address how the date palm respond to DB infection and what molecular pathways are activated during defense mechanisms.

Results
Oxidative stress and gene expression during Dubas bug infection process. To understand the level of effects in stress inception on date palm leaf under DB infection, initial assessments were made by analyzing several key biochemical and molecular determinants. The results showed that DB infestation significantly increased (p < 0.001; two folds) superoxide anion (O 2 − ) as compared to healthy control (Fig. 1B). Lipid peroxidation, a stress indicative process during biotic and abiotic stresses, showed that malondialdehyde (MDA; a biproduct of lipid peroxidation) was significantly (p < 0.001; two and half fold) higher in infected leaf as compared to healthy (Fig. 1C). We noted a significantly higher (p < 0.001; 83%) amount of superoxide dismutase in infected plants as compared to control. Hence, the results reveal that DB infection predominantly cause severe damages to the leaf tissues whereas date palm in turn intercept stress factors by activating antioxidant apparatus. Similarly, the photosynthetic pigments including Chl a, b, Chl a + b and carotenoid (Car) decreased significantly (17.02, 23.89, 18.78 and 13.85% respectively) in DB infected (Figure S1A-D). Whilst, phenolic acid increased significantly in DB infected leaf tissues ( Figure S1E). However, polyphenol level did not change significantly in infected leaves as compared to healthy ( Figure S1F).
In molecular determinants, protective osmolytes such as proline are activated to protect cellular osmotic balance, cell-wall modification and synthesis 33 and the results of proline transporter 2 gene expression was significantly (p < 0.05) up-regulated in DB infected leaves as compared to control (Fig. 1D). Whereas, NADPdependent glyceraldehyde-3-phosphate dehydrogenase was significantly (p < 0.001) up-regulated during DB infection (Fig. 1E) and was previously shown to mobilize ascorbate-glutathione pathway and NADPH-dependent thioredoxin reductase during apoplastic oxidative burst during biotic stresses 34 . A similar perspective was also noted for SOD synthase related gene with exponentially significant activation in DB infection in date palm leaves as compared to control (Fig. 1F). The abscisic acid receptor PYL4-Like was significantly (p < 0.02) upregulated than control ( Figure S2), indicating higher stress incursion in infected leaf. endogenous phytohormonal regulation. To counteract the negative impact of herbivory, endogenous phytohormones such as jasmonic acid (JA) and salicylic acid (SA) have been known signaling players in defense Scientific RepoRtS | (2020) 10:11505 | https://doi.org/10.1038/s41598-020-67438-z www.nature.com/scientificreports/ responses 17 . We found that JA was significantly (p < 0.001) higher (double-fold; 90%) in DB infected date palm leaves as compared to control ( Fig. 2A). SA was also significantly higher (p < 0.001; one and half fold) during the infection process (Fig. 2B). However, the amount of SA synthesized was extremely negligible as compared to JA, suggesting a more potent role in DB infestation process 35 .
Transcriptome sequencing, assembly and annotation. Persuaded by significant biochemical and gene expression results, further in-depth next-generation sequencing (RNA-Seq) approaches were used to elucidate the intricate gene expression networks during infection process. Using standard protocols of RNA-Seq, we generated total of 92 million reads from healthy and DB infected date palm leaves. For downstream gene expression analysis, after filtration only high-quality reads (ranging from 1,600 to 1,900 MB) were mapped to the date palm (DPV01 pdS000001) genome. The sequences of healthy and infected sample were analyzed for differential  Figure S3). A total of 140 genes were deferentially expressed in infected and control of these 31 were up-regulated and 109 were down-regulated ( Figure S3). In the healthy leaves, although many genes have a log fold ratio higher than 1, but these were non-significant FDR. In the control/infected comparison a considerable portion of the analyzed genes were significantly (p < 0.05) regulated ( Figure S3).  (Fig. 3D).

Calcium and MAPK signaling cascades during Dubas bug interactions. According to KEGG path-
ways analysis, about 73 DEGs were found related to plant-pathogen interaction pathways, among that 17 were commonly deferentially expressed across the time points ( Fig. 4A; Supplementary dataset 2). About 64 DEGs were found up-regulated while 13 DEGs were found down-regulated (Fig. 4A). Cytoplasmic Ca 2+ precipitously aggregates during the perception of pathogen associated molecular patterns (PAMPs). We revealed that cyclicnucleotide-gated channels (CNGCs), Rboh and CaM/CML have been up regulated while CDPK is found up as well as down-regulated after DB infection. Similarly, the LOC103717884, LOC103723419, LOC103703874, LOC103701286 and LOC10371780 were found related to calcium dependent protein kinase (CDPK) family. CDPKs, on the other hand, are phosphorylated to reciprocate the hyper-sensitive response (HR). Similarly, calcium related locus (LOC103707714, LOC103713163, LOC103697431, LOC103705017, LOC103722965 and LOC103722132) were related to calmodulin and calmodulin-like (CaM/CML) genes (Fig. 4A). The up-regulation of CNGCs, Rboh and CDPK indicated that Ca 2+ plays very important role in signal transduction in date palm We have detected eleven genes related to effector triggered immunity (ETI) pathways during DB infection. Among these genes RPM1-interacting protein 4 (LOC103710585), PRM1-interacting protein 4 (RIN4), two disease resistant associated genes (SGT1, LOC103708998; HSP90, LOC103722381), three R genes (RPM1, PBS1, and RPS5) and pto-interacting protein 1-like (LOC103707679; encoding serine-threonine kinase 36 ) were found up-regulated. However, one SGT1 gene was found down-regulated during infection. This suggests the participation of expressed genes in either HR or defense-response strategies, followed by cell-death (Fig. 4A). The DEGs expressed in healthy and infected samples associated with plant-pathogen interaction are shown in the heat map (Fig. 4B).

Secondary metabolites activation during Dubas bag infection for improved defense responses.
In the biosynthesis of phenyl-propanoid pathway, up-regulation of genes that leads to synthesis of coumarinate from coumarin are also considered important in herbivory. Results showed that 38 DEGs out of 48 were up-regulated that were mainly associated with phenyl-propanoid pathway during DB infection (Fig. 6A). One of the key enzyme phenylalanine ammonia-lyase-like (PAL; LOC103703950) was up-regulated in infected samples. As DB attacks the leaf part by herbivore lignin, we found related genes viz. cinnamyl alcohol dehydrogenase 1 (CAD; LOC103702778), caffeoyl-CoA O-methyltransferase (COMT; LOC103713767), 4-coumarate-CoA ligase 2-like (4CL; LOC103701563), and trans-cinnamate 4-monooxygenase-like (C4H; LOC103702142) activated in infected samples compared with control (Fig. 6A).
Results showed that genes involved in MVA pathways were relatively more expressed than MEP pathway. The results also showed a sole DEG related to IDI (LOC103705690) (Fig. 7A). Furthermore, the transcriptome data analysis revealed two DEGs related to GPPS and FPPS. The highly expressed DEG for FPPS (LOC103708154; LOC103709296) compared with GPPS also suggest a broader involvement of sesquiterpenes production in date palm. During DB infection, 48 genes were deferentially expressed in relation to photosynthesis process (Fig. 7A). In conjunction, about 110 DEGs were either up or down-regulated in response to oxidative stress metabolism for example NADH dehydrogenase (7 DEGs) and NADH-ubiquinone oxidoreductase (5 DEGs) during DB infection (Fig. 7B).

Discussion
Studies have shown that herbivory induces oxidative stress by generating reactive oxygen species in infected leaf part 39,40 . Current results showed DB infection significantly increased O 2 − as compared to control which is perceived as signaling molecules during plant innate immunity responses 41 . However, this oxidative burst could cause multitudinal damages, e.g. oxidation of lipid bi-layer 19 especially by anionic oxides. Current results showed increased contents of malondialdehyde in infected leaf tissues suggesting a clear indication of increased peroxidation in the tissues 42 43 . To avoid such damages, plant recruit oxidative stress related enzymes such as superoxide dismutase (SOD), whereas it has been directly correlated with the production of O 2 − and H 2 O 2 19 . An increased level of SOD in DB infection predominantly suggest that date palm activates its antioxidant apparatus like other plants such as rice during herbivory 44 . We noted that mRNA gene expression of PT2 and NADPH-TR were significantly activated during DB attack, suggesting their involvement in cell-wall modification and osmoprotectant activity 33,34 .
To counteract the negative impact of herbivory, phytohormones e.g. JA are identified as key signaling molecule to trigger defense responses in plants 17,45 . This has been well-attributed to pre and post herbivory attacks in plants [17][18][19] . Besides, SA has also been coined for its resilient role in enhancing resistance against insect induced pathogenic attacks 35 . The current results showed that JA and SA contents were not antagonistic to each other and both were higher as compared to control. This could be inter-correlated to the dual action that DB infection causes (a) herbivory and (b) pathogenesis. Since the process of dual antagonisms of such attacks could be highly complex, therefore, we performed transcriptome of infected and control seedlings to usher more molecular insights. Current study showed 10,042 DEGs, where among 69% (6,919) were up-regulated genes. GO annotation showed that 4,341 DEGs belonging to cellular, bio-chemical and molecular components. Recent study of Norway spruce tree's transcriptome showed involvement of molecular function, biological process but no cellular component term during needle bladder rust, suggesting a variable responses of DB infection 46 . During DB infection, it was noted that 73 DEGs were related to plant-pathogen interaction, suggesting a post-herbivory genetic cascade.
In these interaction, calcium are considered vital for regulating plant responses to various pathogens and herbivory 47 46 . A similar conclusion was also revealed by Trujillo-Moya et al. 46 Norway spruce. www.nature.com/scientificreports/ It was reported that during pathogen-associated molecular pattern (PAMPs) 55 , a considerable activation of MAPK and WRKY related genes are used to induce defense responses 36,56 . These orthologues have been noted in current studies and are known to regulate pathogen-associated molecular pattern triggered-immunity (PTI) pathways. However, plants, in some cases can use effector trigger immunity (ETI) defense response strategy to cope with pathogenic attacks 36,56 . Both the pathways induce signaling cascades of ET, JA, and SA 57 whereas, some of the studies also suggest the involvement of BRs and GA as well 58,59 during infection process as was noted in case of Flg22 and BR biosynthesis 60,61 . Furthermore, auxin is associated with plant defense response 62 . During pathogenic fungi attack, plant activate defense signaling, in which auxin signaling role is well known, however it does not show this kind of relation under pathogens attack 63,64 . In the current study, we found that DEGs related to auxin biosynthesis were expressed significantly. We conclude that date palm can response to DB attack by modulating auxin, BR, GA, JA and ET signaling. Based on speculation that how several pathogen invades other plants 63 , we suggest that DB might secrete effectors into date palm tissues to improvise the infestation process.
Other than auxin, GA are regulated through DELLA protein family 69,70 that also help to maintain SA and JA homeostasis 65-67 during infection process. Our findings are in synergy to Zhang et al. 68 that these are upregulated during fungal infection in grapes plants. Since, SA is involved in systemic acquired resistance (SAR) and regulation of plant defense responses 69 , we noted activation of PR1 genes. SA is activate tolerance in plants against pathogenic attacks while ET and JA are centric against bio-necrotrophic pathogenesis 65,70,71 . In addition to sole ABA, SA, and JA role, their antagonism such as SA vs JA has been reported in various plants 72 . We noticed that upon DB attack on date palm, most of upregulated DEGs were related to JA and SA signaling. The results suggest that the interaction of DB and date palm based on alternative processes as compared to that described in Arabidopsis and rice. Current study showed that DB has considerably novel infection process than other plants. Similarly, ABA also paly important role in plant experienced different biotic and abiotic stress conditions 69 . ABA trigger various expression of various genes responsible for physiological and developmental process in plants 73 . Likewise, several studies have reported the up-regulation of ABA under pathogen attack, suggesting its essentiality to reprogram JA dependent-defense responses 74 . Here, the increased ABA and SA level possibly indicates stress tolerance and SAR activities. In addition, BR confers pathogenesis related stress tolerance 60,75 , for example, exogenous BR substantially diminished the fungal-induced pathogenic attacks in potato 61,76 , whilst www.nature.com/scientificreports/ correlative impacts were also revealed in sugar beet, cucumber, rice and tomato etc 77 . Herein, we have found four up-regulated DEGs in the ET transduction pathways, suggesting preemptive role of ET in initial DB signaling during infection process 78 . The phenyl propanoid biosynthesis pathway plays a vital role in plant physiology including plant-defense system by producing various chemical barriers against infection process 79 . Our results showed 38 upregulated DEGs that were associated with phenyl propanoid biosynthesis pathway during DB infection. Similar results were reported by Zhang et al. 68 in grapevine under fungal (Lasiodiplodia theobromae) attack. Accumulation of these metabolites in infected samples suggest active contribution during plant-defense responses in date palm during DB infection. The metabolites of phenyl propanoid pathways leading into coumarinate was found in the current results. Coumarinate have been noted for their contribution in defense responses against phytopathogen, oxidative stresses and endogenous phytohormonal responses 80 . Chloropgenat and caffeate are phenolics, synthesized in the pathway with potential benefits in defense mechanism 79 and scavenging ROS 81 . The results of the current study were consistent with previously reported study 82 . Similarly, increased expression of 4CL and PAL genes in date palm are in synergy to grapevine after infection 68 . Moreover, similar results were recorded in Arabidopsis 83 . Hence, current results suggest that date palm can adopt PB pathways to regulate lignin synthesis and by activating secondary metabolites to hinder the DB infestation process.
In case of secondary metabolites, terpenoids are considered to play very important role against herbivores either directly or by attracting natural enemies of the attacking insects. Current results revealed 22 DEGs associated to terpenoid biosynthesis pathway were expressed significantly. We noted the expression of gossypol and its derivate that are known phytoalexin, lead to constitutive and inducible tolerance toward variety of pests 84 . Not only secondary metabolisms but primary metabolism was also active during DB infection. Numerous studies have demonstrated that herbivory can results in down-regulation of primary metabolic processes while simultaneously activating defense-related processes including secondary defense metabolism. As the result showed that DB infection showed 48 highly expressed genes, responsible for photosynthesis as compared to control. This indicate that date palm reactivate both primary and secondary metabolism under DB infection 85,86 . conclusion Dubas bug infection has been noted to enormously influence the endogenous plant innate immune responses, where a continued exposure leads to increased cell death. The results showed that the Dubas bug infection leads to the activation of endogenous JA and or SA initiating signaling cascades via the expression of 140 genes mostly belonging to cellular functions (calcium and MAPK), endogenous phytohormones (auxin, GA, ABA, ET, BR, JA and SA), and secondary metabolites (phenylpropanoids-coumarinates and mevalonate-gossypol). The hypersensitive responses of the leaves were significantly expressed during the infection process. This study performed for the first time detailed transcriptomic analysis of the infection and defense related responses of the date palm. These findings and the transcript dataset generated through this study would act as a future resource for qualitative trait loci of disease and insect resistance mechanisms in date palm.

Material and methods
Plant material, Dubas bug infection and RNA extraction. Healthy date palm (Phoenix dactylifera L., Khalas cultivar) seedlings were grown for one year in greenhouse conditions. The seedlings, in a randomized block design experiment, were used for two treatments (1) healthy control and (2) DB infected with each comprising of 30 seedlings. The disease infestation process was followed as mentioned previously 87 . To avoid dispersion of insect attack to neighboring plants or environment, the pots were shifted into environmental growth chamber (12 h of light at 30 °C (08:00-19:00; 12 h of dark at 30 °C 20:00-07:00; relative humidity 60%; Excelsior Scientific, UK) for rest of the experiment. The control seedlings were also grown at the same temperature conditions. After twenty days of infestation, leaf samples (three biological replicates) from each healthy control and infected seedlings were collected in liquid nitrogen for high molecular weight RNA extractions. RNA extraction buffer was prepared (Tris-HCl; 0.25 M, NaCl; 0.05 M, 20 mM; EDTA; (pH = 7.5); 1% (w/v) sodium dodecyl sulphate (SDS), 4% PVP w/v) as mentioned in the Liu et al. 88 . The quality of RNA was checked on formaldehyde agarose gel electrophoresis and quantified using Qubit 3.0 with dsRNA broad range kit (Thermo Fisher, USA). To remove the DNA contamination, the resulting RNA were treated with DNase I. The RNA was further checked on Bioanalyzer (Agilent, Germany), where a standard 7.0 to 7.5 RIN value was used to affirm suitability of RNA for onward NGS workflow and molecular analysis.
Gene expression of infected sample by quantitative real time PCR. For cDNA synthesis, 1 µg RNA was used. High-Capacity cDNA Reverse Transcription Kit from ThermoFisher was used for cDNA synthesis. Specific conditions (25 °C for 10 min, 37 °C for 2 h and 85 °C for 5 min) of Polymerase Chain Reaction (PCR) was performed in thermo-cycler. The cDNA was stored in − 80 °C refrigerator. In healthy and disease samples, the expression of 4 genes (Table S1) were analyzed in replicate by qRT-PCR (QuantStudio 5.0 by Applied Biosystems Life Technologies), by using "SYBR" green Master Mix. The PCR reaction was carried out in triplicate. For all the primers, Actin gene was used is reference 42,89  www.nature.com/scientificreports/ (pH 3.5), and 1.5 mL of 0.81% thiobarbituric aqueous acid (TBA) solution in a reaction tube. Thereafter, the mixture was heated in boiling water for 60 min. After cooling to room temperature, 5 mL butanol: pyridine (15:1 v/v) solution was added. The upper organic layer was separated and the optical density of the resulting pink solution was recorded at 532 nm using a spectrophotometer. Tetramethoxypropane was used as an external standard. The level of O2 ·− was estimated using the method described by Gajewska and Skłodowska 91 . The homogenate for the reaction was prepared by immersing 1 g of fresh plant sample in 10 mM phosphate buffer (pH 7.0) , containing nitrobluetetrazolium (NBT) (0.05%; w/v), and sodium azide (NaN 3 ) (10 mM), followed by incubation at room temperature for 1 h. Then, 5 mL of the mixture was taken in a new tube and heated for 15 min at 85 °C. Thereafter, the mixture was cooled and vacuum filtered. The absorbance was read at 580 nm with a spectrophotometer. photosynthetic pigments determination. Chlorophyll and carotenoid contents were determined spectrophotometry according to Lichtenthaler 92 . Exactly 400 mg of fresh tissue of plant was mixed with 10 mL 80% acetone, the absorption was read with spectrophotometer at 663.2, 646.8 and 470 nm against acetone 80% blank. The concentration of chlorophyll (Chl) and carotenoid (Car) was determined by the following formulas:

Phenolic acid and polyphenol quantification. The established protocol of the Zavala-López and
García-Lara 93 , were used for the quantification of phenolic acid. Briefly, phenolic acid was extracted with 80% methanol. Exactly 0.7 mL were 80% methanol were used for 50 mg sample, after mixing the sample were vortex vigorously. Followed by incubation at 25 °C for 15 min, followed by centrifugation for 10 min at 5,000 rpm. The supernatant was decanted and stored at − 20 °C until analysis. The total phenolics were extracted and quantified using the improved extraction methods of Urias-Peraldí et al. 94 . To quantify total polyphenol, both healthy and disease sample (200 mg) were extracted with 80% ethanol. For assay, 100 µl extract was combined with 1 ml Folin-Ciocalteau reagent and 1 ml of 10% Na 2 CO 3 . After incubation for 1 h, tubes were centrifuge at 15,000 rpm for 10 min at 4 °C. The supernatant was taken and read at 760 nm in spectrophotometer. The Polyphenol content were expressed as mg/g. transcriptomic sequencing of Dubas bug infected date palm. Looking at the initial results on the activation of endogenous hormones and antioxidants, transcriptome analysis was performed of infection and control samples to understand various underlying mechanistic pathways in date palm. Total RNA was extracted from the infected and control leaves collected (three biological replicates), using the Pure Link Plant RNA reagent kit (Life Technologies, USA) with some modifications to the extraction method. Quality, integrity and quantity of RNA were checked using gel electrophoresis and Qubit 3.0 fluorophotometer and Bioanalyzer (Agilent Technologies, Santa Clara, CA). The defined criterion for qualification of RNA for RNAseq is that the RIN value of a sample is higher than 7.6. The RiboMinus Plant Kit for RNA seq Kit (Invitrogen) was used to remove large ribosomal RNA (rRNA) from the total RNA, which was followed by the concentrating the rRNA depleted RNA using the RiboMinus concentration module, post-ribominus the RNA was evaluated on the Bioanalyzer (Agilent Technologies, Santa Clara, CA). The cDNA Libraries were constructed using the Ion total RNA-seq kit V2, ERCC RNA spike-in control mixes were added according to the manufacturer's protocol using 100 ng of RNA. The total RNA was fragmented using RNase III followed by purification, and quantification on the Bioanalyzer. The resulting cDNA was amplified and quantified on the Bioanalyzer. The Ion One Touch 2 system (Life technologies USA) were used for the emulsion PCR of cDNA, which was followed by the Ion One Touch ES for the enrichment of template. The enriched template was loaded into Ion 540 Chips for the transcriptome sequencing on Ion Torrent S5.
Sequencing and transcriptomic data analysis. The FastQC program (V 0.11.5), and Trimmomatic (V 0.36) 98 were used for the assessment of Quality control, and adapter and poly-N contamination (low-quality sequences; phred ≤ 20) were removed. The remaining high-quality read were utilized for downstream analysis. From NCBI genome database, a reference genome and gene model annotation were downloaded (ftp:// ftp.ncbi.nih.gov/genom es/Phoen ix_dacty lifer a/GFF/). The Bowtie (v2.2.3) was used to developed index of the reference genome, and TopHat v2.0.12 used for paired-end reads were aligned to the reference genome Broad Institute), and number of reads mapped to each gene were determined by using the software HTSeq v0.6.1. The TopHat alignment results ware further investigate for construction and identification of both known and novel transcripts by using Cufflinks (v2.1.1) Reference Annotation Based Transcript (RABT) assembly approach 99,100 . Go ontology of DeGs. The Cuffdiff v.2.2.156 and DESeq R package v1.18.0 were used to identify DEGs in control (healthy) and infected treatments ( Figure S3; Supplementary Dataset 1). The resulting p values were adjusted using Benjamini and Hochberg's approach to control the false discovery rate (FDR)_ENREF_107 102 . Genes with an adjusted p value < 0.05 found by DESeq were assigned as differentially expressed. Most of the time, Gene Ontology (GO) analyses were conducted in order to investigate large-scale transcription data 103 . For the genetic studies, in order to determine the information related to the network among genes are usually determined by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database 104 . In the current study, we used DAVID (version 6.8) 105 to enrich the GO functions and pathways of specific DEGs in the KEGG (https :// www.genom e.ad.jp/kegg/) and GO (https ://www.geneo ntolo gy.org) databases and the R package 3.5.2 Goplot 106 with an adjusted p value (q-value) of < 0.05.