Resistance and susceptible reaction of genotypes
Two Isabgol genotypes DPO-14 and DPO-185 were evaluvated for DM resistance during 2014-2017 by artificial inoculation of PP spores under field condition. The genotype DPO-14 recorded mean score of 4.8 with the score ranging from 4.5 to 5.0 across seasons (Fig. 1). While the genotype DPO-185 recorded mean score of 1.1 with score ranging from 1.0 to 1.5 across seasons indicating the resistance or susceptibility of genotypes to the incidence of DM disease. DPO-14 has most prominent ash-coloured downy growth on the leaves, leaves turn yellowish at the time of flowering, ultimately leading to plant mortality as the disease progresses. A sporadic presence of ash-coloured downy growth on the leaves were found in DPO-185 at the time of flowering.
Illumina sequencing and annotation
Four cDNA libraries from susceptible infected (SI) and susceptible uninfected (SU) leaves of DPO-14 (DM susceptible) and resistant infected (RI) and resistant uninfected (RU) leaves of DPO-185 (DM resistant) genotypes were sequenced using Illumina Hi-Seq platform. Sequencing data included 94,02386 raw reads containing 2735985710 nucleotide bases for SI, 89,76119 raw reads containing 2628972037 nucleotide bases for SU, 108,14932 raw reads containing 3107632713 nucleotide bases for RI and 42,82684 raw reads containing 1267265127 nucleotide bases for RU (Table 1). The raw paired-end sequencing data in FASTQ format was deposited in the National Center for Biotechnology Information (NCBI) BioProject database (as Short Read Archive) under accession number PRJNA382334. De novo assembly yielded 38803, 40175, 45451 and 39533 non-redundant transcript contigs, respectively in SI, SU, RI and RU after filtering out those shorter than 200 bases. The Transcriptome Shotgun Assembly (TSA) project was deposited at DDBJ/EMBL/GenBank under the accession GFNS00000000. The version described in this paper is the first version, GFNS01000000. The N50 values of the assembly was 1205, 697, 377 and 1158bp respectively to SI, SU, RI and RU libraries, indicating fairly good transcriptome assembly. The total transcript length was 38098478 (38.09 Mb) bases, with average transcript length of 982 bases in SI, 23323465 bases (23.3 Mb), with average transcript length of 581 bases in SU,16682558 bases (16.6 Mb), with average transcript length of 367 bases in RI and 34131338 bases (34.13 Mb), with average transcript length of 863 bases in RU. A total of 26569, 18605, 28588 and 22599 CDS, respectively in SI, SU, RI and RU were predicted. The maximum CDS length was found to be 15279 bp, whereas minimum CDS length was 147 bp across libraries (Table 1).
Table 1. Raw reads, assembly and Coding DNA Sequence (CDS) statistics of leaf transcriptome of Isabgol (Plantago ovata Forsk.)
Read statistics
|
SI
|
SU
|
RI
|
RU
|
Total reads
|
9402386
|
8976119
|
10814932
|
4282684
|
Total number of nucleotides (bp)
|
2735985710
|
2628972037
|
3107632713
|
1267265127
|
Assembly statistics
|
Number of transcript contigs
|
38803
|
40175
|
45451
|
39533
|
Maximum length of transcript contig (bp)
|
24168
|
15034
|
15005
|
16394
|
Minimum length of transcript contig (bp)
|
400
|
200
|
180
|
300
|
N50 value
|
1205
|
697
|
377
|
1158
|
Total transcript contig length (in bases)
|
38098478
|
23323465
|
16682558
|
34131338
|
Average transcript contig length (in bases)
|
982
|
581
|
367
|
863
|
CDS statistics
|
Total number of CDS
|
26569
|
18605
|
28588
|
22599
|
Maximum length of CDS (in bases)
|
15279
|
14997
|
14970
|
15279
|
Minimum length of CDS (in bases)
|
297
|
297
|
147
|
297
|
Susceptible infected (SI), Susceptible uninfected (SU), Resistant infected (RI) and Resistant uninfected (RU).
Functional annotation
We first annotated the CDS through homologous search against green plant database (txid 33090) of NCBI using BLASTX search and threshold E-value as 1e-06. A total of 25154 (94.70%), 17768 (95.50%), 23605 (82.60%) and 21255 (94.10%), respectively in SI, SU, RI and RU had significant BLAST hits (Table 2). Out of the total CDS with blast hits, 5875 (23.3%) and 710 (3.0 %) CDS in SI and RI respectively were annotated as oomycetes sequences in the nr databases. Based on BLASTX annotation, top hit species with Isabgol transcriptome showed highest similarity to Sesamum indicum (41-59%) followed by Erythranthe guttata (10-13%) (Fig. 1) and Cofea conephorea (1-2%) genomes (Fig. 2). Blast hits were also obtained with Phytophthora parasitica, Phytophthora sojae, Phytophthora infestans and Phytophthora nicotianae, four closely related oomycete species of Peronospora plantaginis in the infected (SI and RI) samples.
Table 2. Distribution of BLAST results of CDS in the leaf transcriptome of Isabgol (Plantago ovata)
Description
|
Number of CDS
|
|
SI
|
SU
|
RI
|
RU
|
Number of CDS with BLAST hits and
Per cent of BLAST hits
|
25154 (94.70)
|
17768 (95.50)
|
23605 (82.60)
|
21255 (94.10)
|
Number of CDS without BLAST hits
|
1415
|
837
|
4983
|
1344
|
GO distribution of BLAST hits-Biological processes
|
10115
|
6945
|
10140
|
8014
|
GO distribution of BLAST hits-Molecular functions
|
7481
|
4977
|
7168
|
5915
|
GO distribution of BLAST hits-Cellular component
|
10745
|
7278
|
10674
|
8357
|
Database
|
|
|
|
|
KEGG
|
6247
|
5266
|
5016
|
6278
|
COG
|
17758
|
12124
|
13488
|
15069
|
Pfam
|
25274
|
15652
|
14134
|
21828
|
iTAK-Transcripton factors (TFs)
|
709
|
471
|
376
|
682
|
iTAK-Transcripton regulators (TRs)
|
331
|
201
|
151
|
283
|
iTAK-Protein kinases (PKs)
|
684
|
508
|
478
|
650
|
Susceptible infected (SI), Susceptible uninfected (SU), Resistant infected (RI) and Resistant uninfected (RU). Figures in the brackets indicate percentages.
Gene ontology (GO) was used for classification of predicted CDS into functional categories by Blast2GO searches. Function of predicted CDS were classified and each CDS were provided with ontology of defined terms. GO terms of 28341 in SI, 19200 in SU, 27982 in RI and 22286 in RU were enriched in the transcriptome of Isabgol. Of the enriched GO terms, 10115 (35.7%) terms were grouped to Biological process, 7481 (26.4%) for Molecular function and 10745 (37.9%) for Cellular component in SI (Table 2 and Fig. 3). While in SU, 6945 (36.2%) terms were grouped to Biological process, 4977 (25.9%) for Molecular function and 7278 (37.9%) for Cellular component. Of the enriched GO terms, 10140 (36.2%), 7168 (25.6%) and 10674 (38.1%) terms, respectively were grouped to biological process, molecular function and cellular component in RI. Similarly, in RU, 8014 (36.0%), 5915 (26.5%) and 8357 (37.5%) terms were grouped to biological process, molecular function and cellular component, respectively. In the biological process category, highest number of CDS were enriched in metabolic process (GO:0008152) group, followed by cellular process (GO:0009987) group (Fig. 3). In the molecular function category, “catalytic activity” (GO: 0003824) and “binding activity” (GO: 0005488) were most abundantly represented. Under the cellular component category, the highest number of CDS were associated with “membrane” (GO:0019898) and “cell” (GO: GO:0005623).
KEGG mapping
Ortholog assignment and mapping of the CDS to the biological pathways were performed using KEGG automatic annotation server (KAAS) with default score. A total of 6247, 5266, 5016 and 6278 CDS were enriched into 41 different functional KASS pathway categories respectively in SI, SU, RI and RU (Additional Table 1). The mapped CDS represented the genes involved in metabolism, genetic information processing, environmental information processing, cellular processes organizational systems and human disease. Metabolic pathways of major biomolecules such as carbon, carbohydrates, lipids, nucleotides, amino acids, glycans, cofactors, vitamins, terpenoids, polyketides and others were mapped in the transcriptome of Isabgol.
To further predict the function, CDS were subjected to classification into different protein families based on Clusters of Orthologus Groups (COG) of protein databases. Overall 17758, 12124, 13488 and 15069 CDS of SI, SU, RI and RU, respectively showed significant homology and assigned to the appropriate COG clusters. The COG annotated putative proteins were distributed functionally into 26 protein families (Fig. 4), of which the cluster for “Signal transduction mechanisms (T)” represented the largest group (2180, 1553, 1693 and 1944 respectively in SI, SU, RI and RU), followed by “General function prediction only (R)” (2007, 1454, 1447 and 1750), “Posttranslational modification, protein turnover, chaperones (o)” (1871, 1262, 1457 and 1505)” in transcriptome of Isabgol. The least represented groups include “multiple functions” (3, 1, 2 and 1 CDS respectively in SI, SU, RI and RU), “cell motility” (8, 5, 5 and 6 CDS) and “Extracellular structures” (87, 51, 47 and 77 CDS) in the transcriptome.
We used InterProscan to see protein similarity at domain level, in total, 25274, 15652, 14134 and 21828 transcripts in SI, SU, RI and RU respectively were annotated against the Pfam domains (Table 2). Pkinase (PF00069) domains represented the most which was followed by PPR (PF01535) and Pkinase_Tyr (PF07714) domains in the Isabgol leaf transcriptome indicating strong signal transduction mechanisms. (Additional Fig. S1, Additional file S1). Transcription factors (TFs) affect metabolic flux by regulating gene expression of key genes involved in the biosynthetic ways. We performed BLASTX search against the known Plant Transcription Factor database using the CDS from SI, SU, RI and RU transcriptomes. A total of 709, 471, 376 and 682 TFs belong to 63 families in SI, SU, RI and RU respectively were annotated (Table 2, Additional file S2). Similarly, we annotated 331, 201, 151 and 283 transcriptional regulators (TRs) belong to 25 families in SI, SU, RI and RU respectively indicates their role in gene expression in Isabgol.
Identification of differentially expressed genes
The relative expression level of commonly expressed CDS based on common ‘nr’ blast hit accession in terms of Fragments Per Kilobase of transcripts per Million (FPKM) was estimated by mapping all the clean reads from each library back to CDS. Subsequently, 6928 (15.33%) CDS were differentially expressed between RU and RI libraries of which 1123 (16.28%) were significantly up-regulated while 1019 (14.70%) CDS were significantly down-regulated with P values ≤ 0.05 solely in RI compared to RU (Table 3). There were 8779 (17.15%) CDS differentially expressed between libraries SU and SI of which 1408 (16.00%) CDS were significantly up-regulated while 1201 (13.60%) were significantly down-regulated with P values ≤ 0.05 in SI compared to SU (control) (Additional file S3). Heat map of top 50 up-regulated and top 50 down-regulated genes presented in Fig. 5 clearly shows both resistant and susceptible genotypes have some similar responses to PP infection for some DEGs. The data illustrated differences between resistant and susceptible genotypes up on infection of PP fungus and deep analysis of these genes may shed light on the resistance mechanism in Isabgol. However, some of the changes in gene expression may be due to the difference in genetic background of these genotypes. Furthermore, the functional classification of DEGs in leaf transcriptome was analysed using GO (Gene ontology). A total of 1519 and 3455 DEGs respectively in RU vs RI and SU vs SI were enriched to at least one GO term (Fig. 6; Additional file S4). Among the biological processes, where the term occurrences took place, metabolic process (11.06%) followed by oxidation-reduction process (6.30%) (Fig. 6) recorded maximum terms enriched. In addition, DEGs involved in biological processes like autophagy, response to stimulus and immune system were recognised through GO annotation. In cellular components, GO terms enriched were “integral to membrane” (37.29%) followed by “membrane” (7.97%). In molecular function, GO terms enriched were structural constituent of “ribosome” (2.61%), “protein kinase activity” (2.70%) and “protein serine/threonine kinase activity” (2.21%).
Table 3. Summary of deferentially expressed genes (DEGs) in the leaf transcriptome of Isabgol (Plantago ovata)
Control x treated
|
Total predicted CDS
|
Total DEGs (%)
|
Significant DEGs
|
Up-regulated genes
|
Down-regulated genes
|
RU vs RI
|
45174
|
6928 (15.33)
|
2142 (4.74)
|
1123 (52.42)
|
1019 (47.50)
|
SU vs SI
|
51187
|
8779 (17.15)
|
2609 (5.09)
|
1408 (53.96)
|
1201 (46.04)
|
Susceptible infected (SI), Susceptible uninfected (SU), Resistant infected (RI) and Resistant uninfected (RU). Figures in the parenthesis indicate percentages
Metabolic pathway analysis was performed for all DEGs using KEGG automatic server (KAAS) to explore the biochemical pathways in which DEGs involved. The results revealed that, 1209 and 2037 DEGs were assigned to 159 and 215 KEGG pathways in RU vs RI and SU vs SI, respectively. Most DEGs were assigned to ribosome [PATH: ko03010], carbon metabolism [PATH: ko01200], oxidative phosphorylation [PATH: ko00190], RNA transport [PATH: ko03013], spliceosome [PATH: ko03040], protein processing in endoplasmic reticulum [PATH: ko04141] and plant hormone signal transduction [PATH: ko04075] pathways. Most importantly, several pathways associated with defense were also enriched by KEGG analysis (Additional file S4).
Mining genes involved in Host-pathogen interaction pathway
Host plants perceive pathogens by different recognition systems which are accompanied by a set of induced defense to repel pathogen attack. Activation of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) at the surface of plant elicits a defense programme known as PAMP-triggered immunity (PTI). In plants, there are 25 genes identified to be involved in PTI [6-7]. In the present study, CDS were found for 18 (FLS2, EFR, BAK1, MEKK1, MKK1/2, MKK4/5, WRKY22/29, WRKY25/33, CNGC, CDPK, CaM, CML, RBOH, NOS, RLK1, NHo1, CERK and PTI6) genes involved in PTI indicating their role in plant-pathogen interaction (Additional Table 2 and 3). However, we did not find CDS for CEBiP, FRK1, PR1, Pto, PRF, PTI4 and PTI5 genes involved in conferring PTI in our dataset. Three (WRKY25/33, CDPK and RLK1) genes were significantly up-regulated while the genes (FLS2, EFR, CNGC and CaM) were significantly down-regulated in RU vs RI (Fig. 7) indicating their role in PTI in DPO-185 (Resistant) genotype. The genes EFR, BAK1, CaM, CML and RLK1 were significantly up-regulated in SU vs SI indicating their role in PTI in DPO-14 (susceptible) genotype.
Effector-triggered immunity (ETI) is thought to mount a second layer of defense or known as secondary immune response in plants. R-proteins can directly sense pathogen effectors, or they can detect pathogens through other cofactors, which are direct host targets for pathogens. There are genes reported conferring effector-triggered immunity in plants against various pathogens. In the present study, CDS were identified for 15 (RIN4, PBS1, RPM1, RPS1, RAR1, SGT1, HSP90, PIK1, EDS1, HCD1, WRKY1, Bs3, WRKY2, KCS1 and FDH) genes involved in ETI (Additional Table 4 and 5; Fig. 7) indicating their role in defense response ETI. However, we did not find CDS for RPS4, UPA20, RRS1-R, MLA10, L6, ATMYB30, UPA7 and RPS5 reported to be involved in conferring ETI in our dataset. Of the 15 genes detected in the leaf transcriptome, FDH gene was significantly up-regulated while the PIK1, WRKY1 and KCS1 genes were significantly down-regulated in RU vs RI in DPO-185 (resistant) indicating their role in ETI. Similarly, the genes RAR1, SGT1 and EDS1 were significantly up-regulated while KCS1 genes were significantly down-regulated in SU vs SI in the DPO-14 (Susceptible) indicating their role in conferring ETI in Isabgol.
Mining genes involved in Cell wall modifying enzymes
Cell wall forms a dynamic structure that determines the outcome of the interactions between host and pathogens. When pathogens start degrading the plant cell wall components, plants are capable of perceiving the loss of cell wall integrity and subsequently activate the defense signalling pathways. Pathogens try to escape the plant defense and sometimes take advantage of the host cell wall metabolism to facilitate their entry into the tissue. There were five [Lipid transfer protein 2 (LPT2), Glutathione S-transferase (GST), Callose synthase (CS), Cinnamyl alcohol dehydrogenase (CAD) and Lignin-forming anionic peroxidase (LAP)] genes encoding the enzymes involved in modifying the cell wall during host-pathogen interaction. In the present study, CDSs were found for LPT2, GST, CS and CAD and LAP genes in the leaf transcriptome indicating their role in host pathogen interaction (Additional Table 6 and 7). Further, the genes, LPT2, GST, CS, and CAD were significantly down-regulated in RU vs RI indicating their expression pattern up on DM infection in DPO-185 (resistant genotype). The gene LAP is significantly up-regulated while the genes LPT2 and CS were significantly down-regulated in SU vs SI in DPO-14 (susceptible genotype). However, the other genes GST and CAD did not shown difference in expression.
Genes involved in Phytohormone signalling
Phytohormones emerged as cellular signal molecules with key functions in the regulation of immune responses to microbial pathogens and insects. Phytohormones, such as abscisic acid (ABA), ethylene (ET), jasmonic acid (JA), salicylic acid (SA), brassinosteroids (BRs), gibberellic acids (GAs), auxins and cytokinins (CK) act as signalling molecules to triggering immune responses to pathogens in plants. There are 43 genes reported to be involved in phytohormone signalling in plants through various phytohormones (Additional Table 8 and 9; Fig. 8). In the present study, CDS were detected for 32 genes viz., three (PYR/PYL, PP2C and SNRK2) gene involved in ABA signalling, six (ETS/ETR, CTR1, SIMKK, EIN2, EBF1_2 and ERTF1/2) genes involved in ET signalling, four (JAR1, COI-1, JAZ, and MYC2) genes involved in JA signalling, two (NPR1 and TGA) genes involved in SA signalling, five genes in BR (BAK1, BRI1, BKI1, TCH4 and CD3) and four (GID, Della, GID2 and PIF3) gene in GA signalling, six (LAX3, TIR1, ARP, ARF, GH3 and SAUR) genes involved in auxin signalling and two (AHP5 and ARR-B) gene of cytokinins indicating their role in Isabgol and DM interaction. Further, the genes PYR/PYL, PP2C, SNRK2 of ABA signalling, TGA of SA signalling, GID and GID2 of GA signalling and GH3 of auxin signalling were significantly up-regulated, while NPR1 involved in SA signalling, TCH4 and CD3 of BR signalling, and TIR1, ARF of auxin signalling, and CRE1 and ARR-B of cytokinin were significantly down-regulated in RU vs RI indicating their role in DM infection in DPO-185 (Resistant genotype). Similarly, the genes ETS/ETR and CTR1 of ET signalling, COI-1 of JA signalling, NPR1 of SA signalling, BAK1 and CD3 of BR signalling, ARF of auxin signalling and ARR-B of cytokinin were significantly up-regulated while genes PYR/PYL of ABA signalling, ERTF1/2 of ET signalling and BKI1 of BR signalling were significantly down-regulated in SU vs SI indicating their role in conferring DM signalling in DPO-14 (Susceptible genotype).
Genes involved in phenylpropanoid biosynthetic pathway
Phenylpropanoid biosynthetic pathway is one of the important secondary metabolic pathways which plays important role in plant defense [39]. There were 14 genes involved in phenylpropanoid biosynthetic pathway [39-40]. KEGG annotation of the phenylpropanoid biosynthetic pathway in response to PP infection is presented in Fig. 9. In the present study, CDS were detected for 13 of the 14 genes involved in phenylpropanoid biosynthetic pathway in the leaf transcriptome indicating their role in DM resistance (Additional Table 10 and 11). We didn’t find the CDS for UGT72E encoding Coniferyl-alcohol glucosyltransferase involved in phenylpropanoid biosynthetic pathway. The genes CCR and HCT were significantly up-regulated while the genes CAD and REF1 were significantly down-regulated in RU vs RI indicating their role in plant defense up on infection in DPO-185 (resistant genotype). The genes 4CL and CSE were significantly down-regulated in SU vs SI showing their role in conferring the plant defense up on infection in DPO-14 (susceptible genotype).
Enriched R-Genes in response to DM infection
According to the gene-for-gene hypothesis, resistance (R) proteins recognize and interact with effectors and trigger ETI [7]. Resistance (R) genes are the members of a large multigene family genes encoding disease resistance proteins in plants. More than 70 different R genes showing resistance to major plant pathogens have been isolated and characterized in plants [41]. R proteins posses multi-domain structure (Nucleotide-binding site (NBS)-leucine-rich repeat (LRR) (NL), coiled-coil (CC)-NBS-LRR (CNL), Toll Interleukin-1 Receptor (TIR)-NBS-LRR (TNL), nucleotide-binding adaptor shared by apoptotic protease activating factor 1 (NB-ARC), Receptor-like kinases (RLK) and others) which allows simultaneously recognition of Avr proteins of the pathogen and trigger plant defense reactions [42]. In the present study, CDS were found for 58 candidate R genes in the leaf transcriptome indicating their role in conferring DM resistance (Additional Table 12). Of these, 25 encode CNL proteins, 10 encode other, six encode NL, three encode TNL, two encode RLK, and one encode NB-ARC domain containing proteins. Remaining 11 encode unclassified R proteins. The most represented group of R genes in plants cloned to date are CNL proteins characterized by nucleotide-binding site (NBS) and leucine rich repeat (LRR) domains as well as variable amino- and carboxy-terminal domains. However, only 43 of these genes were differentially expressed (Table 4) in Isabgol transcriptome. There were 10 (Alanine--glyoxylate aminotransferase 2 (At2), NBS type disease resistance protein, NBS-LRR class resistance protein Fy12-Ry1, NBS-LRR class resistance protein Fy1-Ry1, Disease resistance protein RPP13, Late blight resistance protein homolog R1A-10, Late blight resistance protein homolog R1A-3, Late blight resistance protein homolog R1A-4, Late blight resistance protein homolog R1B-16 and Late blight resistance protein homolog R1B-8) R genes significantly up-regulated and 12 (Restricted Tev Movement 2 (RTM2), General transcription factor IIA subunit 2 (TFIIAy), Mildew Locus O (MLO) gene, Disease resistance family protein/LRR family protein, Disease resistance protein RGA2, Late blight resistance protein R1-A, Disease resistance protein At1g58602, Enhanced disease resistance 2, Disease resistance protein At3g14460, Disease resistance protein At3g14460, Disease resistance protein RGA4 and Late blight resistance protein homolog R1C-3) R genes down-regulated in RU vs RI indicating their expression up on DM infection in the resistant parent DPO-185. Similarly, there were seven (Resistance to Fusarium oxysporum 1 (RFO-1), General transcription factor IIA subunit 2 (TFIIAy), IAA-alanine resistance protein 1, Disease resistance protein At4g33300, Enhanced disease resistance 2, Disease resistance protein RGA1 and Late blight resistance protein homolog R1B-16) genes significantly up-regulated and 12 (PEP1 Receptor 1(PEPR1), Late blight resistance protein homolog R1B-14 (Sw-5), NBS-LRR class resistance protein Fy8-Ry8, Disease resistance protein RPP13, Disease resistance RPP8, Disease resistance protein RGA3, Putative disease resistance RPP13, Late blight resistance protein homolog R1B-12, Late blight resistance protein homolog R1B-14, Late blight resistance protein homolog R1B-8 and Late blight resistance protein homolog R1C-3) genes down-regulated in SU vs SI showing their expression in the susceptible genotype DPO-14 indicating their role in conferring resistance to DM disease. Interestingly, the Late blight resistance protein homolog R1B-14 (Sw-5), R1B-12 and R1B-8 identified as CNL (CC-NBS-LRR) type of R protein were down-regulated 6.32, 6.00 and 8.20 -fold respectively in SU vs SI, indicating a crucial role in the DM defense response.
Table 4. Differentially expressed R-genes identified in leaf transcriptome of Isabgol (Plantago ovata)
S.No.
|
R-genes (cloned)
|
Gene Id
|
Log2 fold change values
|
RU vs RI
|
SU vs SI
|
1
|
Alternaria stem canker resistance protein (ASC)
|
XP_011077707
|
#N/A
|
#N/A
|
2
|
Restricted Tev Movement 2 (RTM2)
|
XP_011091969
|
-2.305439972
|
-0.193337627
|
3
|
PEP1 Receptor 1(PEPR1)
|
XP_011080517
|
-0.617383978
|
-2.012695073
|
4
|
Resistance to Fusarium oxysporum 1 (RFO-1)
|
XP_012835527
|
0.33191121
|
2.461446713
|
5
|
General transcription factor IIA subunit 2 (TFIIAy )
|
XP_012856602
|
-1.220048481
|
1.745157888
|
6
|
Serine--glyoxylate aminotransferase (At1)
|
XP_011090165
|
0.020550403
|
-0.303689105
|
7
|
Alanine--glyoxylate aminotransferase 2 (At2)
|
XP_011080767
|
1.040641984
|
-0.024767905
|
8
|
ABC transporter G family member 35 (Lr34)
|
XP_011080481
|
-0.535611608
|
0.33695378
|
9
|
MLO (Mildew Locus O) gene
|
XP_011098016
|
-3.151787363
|
0.335446369
|
10
|
NB-LRR type disease resistance protein (Rps1-k-1)
|
KRH65532
|
#N/A
|
#N/A
|
11
|
Late blight resistance protein homolog R1B-14 (Sw-5)
|
XP_011072124
|
0.914259704
|
-6.322692958
|
|
R-Genes (Others)
|
|
|
|
12
|
Disease resistance family protein / LRR family protein
|
XP_007038696
|
-2.68055
|
#N/A
|
13
|
Disease resistance protein [Glycine soja]
|
KHN30606
|
#N/A
|
#N/A
|
14
|
Disease resistance protein RPM1
|
XP_011089079
|
0.930459
|
-0.43654
|
15
|
NB-ARC domain-containing disease resistance protein
|
XP_007012768
|
#N/A
|
#N/A
|
16
|
NBS resistance protein RGA43
|
AKC03708
|
#N/A
|
#N/A
|
17
|
NBS type disease resistance protein
|
ABF81447
|
2.325131
|
0.63691
|
18
|
NBS-coding resistance gene analog
|
ACE79511
|
#N/A
|
#N/A
|
19
|
NBS-LRR class resistance protein Fy12-Ry1
|
AGX27506
|
4.137504
|
#N/A
|
20
|
NBS-LRR class resistance protein Fy12-Ry12
|
AGW28126
|
#N/A
|
#N/A
|
21
|
NBS-LRR class resistance protein Fy1-Ry1
|
AGX27499
|
4.137504
|
#N/A
|
22
|
NBS-LRR class resistance protein Fy8-Ry8
|
AGX27504
|
#N/A
|
-4.417085328
|
23
|
Disease resistance protein RGA2
|
XP_011072600
|
-1.66985
|
0.596377
|
24
|
Disease resistance protein RPM1
|
XP_012849603
|
-0.12553
|
0.132773
|
25
|
Disease resistance protein RPS2
|
XP_002279295
|
#N/A
|
#N/A
|
26
|
Disease resistance protein RPS6
|
XP_012829386
|
#N/A
|
#N/A
|
27
|
Disease resistance protein RPP13
|
XP_012851388
|
2.137504
|
-1.36724
|
28
|
Disease resistance RPP8
|
XP_011072174
|
0.485427
|
-1.96189
|
29
|
IAA-alanine resistance protein 1
|
XP_011070517
|
0.485427
|
1.208624
|
30
|
Late blight resistance protein R1-A
|
XP_011097122
|
-2.12928
|
0.888602
|
31
|
Disease resistance protein At1g58602
|
XP_011091324
|
-2.51355
|
-0.20903
|
31
|
Disease resistance protein At4g33300
|
XP_011083793
|
0.525741
|
1.451477
|
32
|
Enhanced disease resistance 2 |
XP_011089926
|
-3.83404
|
3.290901
|
33
|
Disease resistance protein At1g50180
|
XP_011073815
|
0.904013
|
0.381976
|
34
|
Disease resistance protein At3g14460
|
XP_011089052
|
-1.35755
|
-0.32865
|
35
|
Disease resistance protein At5g63020
|
XP_010645051
|
#N/A
|
#N/A
|
36
|
Disease resistance RPP8
|
XP_011072174
|
0.485427
|
-1.96189
|
37
|
Enhanced disease resistance 2
|
XP_011078691
|
0.412816
|
-0.13334
|
38
|
Disease resistance protein At1g50180
|
XP_011073815
|
0.904013
|
0.381976
|
39
|
Disease resistance protein At1g58400
|
XP_012829221
|
#N/A
|
#N/A
|
40
|
Disease resistance protein At3g14460
|
XP_011089052
|
-1.35755
|
-0.32865
|
41
|
Disease resistance protein RGA1
|
XP_011097211
|
-0.66985
|
2.893527
|
42
|
Disease resistance protein RGA3
|
XP_012833088
|
0.067114
|
-1.10319
|
43
|
Disease resistance protein RGA4
|
XP_012854094
|
-3.6504
|
#N/A
|
44
|
Putative disease resistance RPP13
|
XP_011091694
|
#N/A
|
-3.18827
|
45
|
Late blight resistance protein homolog R1A-10
|
XP_011071970
|
4.055041
|
#N/A
|
46
|
Late blight resistance protein homolog R1A-3
|
XP_011072560
|
4.076103
|
#N/A
|
47
|
Late blight resistance protein homolog R1A-4
|
XP_011072135
|
4.722466
|
#N/A
|
48
|
Late blight resistance protein homolog R1B-11
|
XP_009785700
|
#N/A
|
#N/A
|
49
|
Late blight resistance protein homolog R1B-12
|
XP_012856432
|
#N/A
|
-6.00685
|
50
|
Late blight resistance protein homolog R1B-13
|
XP_012853219
|
#N/A
|
#N/A
|
51
|
Late blight resistance protein homolog R1B-16
|
XP_011088087
|
3.544679
|
2.665655
|
52
|
Late blight resistance protein homolog R1B-17
|
XP_012853777
|
#N/A
|
#N/A
|
53
|
Late blight resistance protein homolog R1B-19
|
XP_011080908
|
-0.16916
|
#N/A
|
54
|
Late blight resistance protein homolog R1B-8
|
XP_012833866
|
2.353232
|
-8.2017
|
55
|
Late blight resistance protein homolog R1C-3
|
XP_012853759
|
-3.53492
|
-1.5659
|
56
|
Disease resistance protein RGA3
|
EMT15286
|
#N/A
|
#N/A
|
57
|
TIR-NBS-LRR type disease resistance protein
|
AEL30371
|
#N/A
|
#N/A
|
58
|
TMV resistance protein N
|
KHN11124
|
#N/A
|
#N/A
|
Susceptible infected (SI), Susceptible uninfected (SU), Resistant infected (RI) and Resistant uninfected (RU). No change (#N/A), (-): down regulated.
Realtime Gene expression analysis
To validate the gene expression profiles from the high throughput RNA sequencing, the transcript levels of 11 DEGs were selected to profile their expression upon DM infection in resistant (DPO-185) and susceptible (DPO-14) genotypes (Table 5). Differential expression of transcripts in RI (infection level: slight (S), medium (M) and high (H)) and SI (infection level: slight (S), medium (M) and high (H)) compared to their respective controls (uninfected DM) were found significant. The genes BXL1, TT4, ERF1, ERF, R1B-14 (XP_011072119) and R1B-14 (XP_012833868) in RI and the AUX1 in SI were upregulated in S, M, H infection levels (Additional file S5; Fig 10). Further, BXL1, ERF1 and ERF genes were down regulated in S, M, H infection levels in SI. The genes TT4, R1B-14 (XP_011088007) showed upregulation in S, down regulation in M and upregulation in H in SI. In contrast, the genes PR1 in both RI and SI, and R1B-14 (XP_011070688) in SI were down regulated in S, upregulated in M and down regulated in H. The genes AUX1 in RI and R1B-14 (XP_012833868) in SI were upregulated in S and M, and downregulated in H infection levels. Whereas the genes CHS, R1B-14 (XP_011070688) and R1B-14 (XP_011088007) were down regulated in infection level S and upregulated in M and H infection levels. Although, RNA-Seq and RT-qPCR methods uses different algorithms to quantify gene expression levels and vast variation of transcript abundance between infected and uninfected as well as between two DPO-14 and DPO-185 exist for some selected genes, some of the tested DEGs showed the consistency in gene expression patterns using qRT-PCR.
Table 5. List of gene specific primers used in RT-qPCR
Gene
|
Name
|
Forward (5→3)
|
Reverse (5→3)
|
Tm
|
Expected product size (bp)
|
Observed product size (bp)
|
Pathway
|
Auxin transporter protein 1 (AUX1)
|
XDPOAUX1
|
TTTGCATGCACACCACTGTA
|
AGGGGAAGATAATGGCCAAG
|
59.8
|
133
|
130
|
Phytohormone signaling
|
Beta-D-xylosidase 1 (BXL1)
|
XDPOBXL1
|
GTCACAAACAAGAGCCTTCTCA
|
GCTAGGATCACAGGCGAAAG
|
59.7
|
100
|
100
|
Secondary metabolism
|
Chalcone and stilbene synthase (TT4)
|
XDPOTT4
|
GTGCGAGAAATCAACGATCA
|
CCACCACAATGTCCTGTCTG
|
59.8
|
122
|
130
|
Secondary metabolism
|
Ethylene responsive factor (ERF1)
|
XDPOERF1
|
CGGTGAGATCTGGTTTGGTT
|
GCAGAAGTGGTCTTGGAAGC
|
59.97
|
148
|
150
|
Phytohormone signaling
|
Chalcone and stilbene synthase (CHS/ TT4)
|
XDPOCHS/ TT4
|
TGGTTGAGGTCCCAAAACTC
|
AGGAGCTTGGTGAGCTGGTA
|
59.94
|
148
|
150
|
Secondary metabolism
|
Pathogen related protein 1 (PR1)
|
XDPOPR1
|
AACTCTTGCGTTGGAGGAGA
|
TGGGCCGTAACTGCATATAA
|
59.99
|
126
|
120
|
Phytohormone signaling
|
Ethylene response transcription factor 1 (ETR1)
|
XDPOERF
|
TGACAAGCACAGATCCAAGC
|
CTTCAGGAAAGGGTCACGAA
|
59.99
|
169
|
170
|
Phytohormone signaling
|
Late blight resistance protein homolog R1B-14 (XP_011070688)
|
Xdaposw5m2
|
AATTCCGCCGAGAGAGATTT
|
AAAGGCCTGTCATCAACTGG
|
60.00
|
212
|
200
|
Resistance gene
|
Late blight resistance protein homolog R1B-14 (XP_011072119)
|
Xdaposw5m4
|
GTGAGGTTATGGAGGGCTGA
|
CCTTCCTCAACGGATACGAA
|
60.00
|
241
|
240
|
Resistance gene
|
Late blight resistance protein homolog R1B-14 (XP_012833868)
|
Xdaposw5-5770
|
TGAGTGTGGCTTCTTATATAGGTTCA
|
CAATCGCTAAAGGCAGTCCT
|
60.00
|
200
|
200
|
Resistance gene
|
Late blight resistance protein homolog R1B-14 (XP_011088007)
|
Xdaposw5-5970
|
TCACAGGTTTGGATGGGTCT
|
CAATCGCTAAAGGCAGTCCT
|
60.00
|
200
|
200
|
Resistance gene
|