Transcriptome sequencing and denovo assembly
The number of raw reads generated by Illumina HiSeq 2000 was 54,699,263 and 52,509,239 for RRL and SRL with respective the number of filtered reads was 41,025,151 and 42,001,812. Quality trimming, adaptor sequence removal and size selection of transcript reads resulted in a total of 10,038 and 4,022 high quality reads for RRL and SRL, respectively. Analyses of two sets of transcripts assigned the transcripts to the 7,849 and 2,899 transcripts for RRL and SRL through BLASTx. There was no homology for 2,189 and 1,123 for RRL and SRL, respectively and they are described as novel genes or hypothetical proteins. The transcripts were submitted in NCBI sequence read archive (SRA) database, with accession numbers SRR2992210 and SRR 2992249 for RRL and SRL respectively.
BLAST homology with other species and annotation
In RRL, the BLAST hits constituted to maximum homology of 34% with Sorghum bicolor followed by 32% with Zea mays and 4% with Oryza sativa. In case of SRL, the respective homologies were 42%, 27% and 5% for Sorghum bicolor, Zea mays and Oryza sativa. In RRL, 1% homology in Triticum urartu, Vitis vinifera, Glycine Max, Hordeum vulgare and Medicago truncatula species were found. In SRL, no homology was found with those species. Saccharum officinarum and Populus trichocarpa had 1% homology in SRL, and no homology was found in RRL. Both RRL and SRL had 1% homology with Saccharum hybrid cultivar (Suppl. Fig. 1a, 1b). In both RRL and SRL, many of the transcripts from BLAST annotation were found to be hypothetical proteins or novel genes. So, to know the functional ontology of the differential transcripts, GO distribution and KEGG-KAAS functional categorization were done.
Gene Ontology (GO) distribution
The high-quality assembled transcripts were annotated with gene ontology (GO) terms. The GO terms were distributed as biological processes, molecular functions and cellular components (Fig. 2). In biological process, transcripts pertaining to aromatic amino acid family biosynthetic process, protein N-linked glycosylation, small GTPase mediated signal transduction, GPI anchor biosynthetic process, IMP biosynthetic process and so on were expressed differentially in RRL. In SRL, transcripts pertaining to sucrose biosynthetic process, negative regulation of peptidase activity and protein deubiquitination were present. In cellular component, transcripts pertaining to RNA polymerase complex, cis-golgi network, anaphase-promoting complex, transcription factor TFIID complex, photosystem I & II were present in RRL. In SRL, a single differential transcript pertaining to cullin-RING ubiquitin ligase complex was present. In molecular function, transcripts pertaining to shikimate kinase activity, cellulose synthase (UDP-forming) activity, P-P-bond-hydrolysis-driven protein transmembrane transporter activity, aspartic-type endopeptidase activity, serine-type endopeptidase activity and mannose-1-phosphate guanylyl transferase (GDP) activity were present in RRL. In SRL, serine-type endopeptidase inhibitor activity and quinone binding - oxidoreductase activity, acting on NADH or NADPH were present. The gene ontology of the 3 GO terms is presented in Table 2.
KEGG-KAAS functional annotation of subtracted transcriptome
High quality reads corresponding to 10,038 for RRL and 4022 for SRL were mapped in KEGG-KAAS database. The transcripts were mapped to 12 categories pertaining to carbohydrate metabolism, energy metabolism, lipid metabolism, nucleotide metabolism, amino acid metabolism, glycan metabolism and biosynthesis, metabolism of cofactors and vitamins, metabolism of terpenoids and polyketides, biosynthesis of other secondary metabolites, genetic information processing, environment information processing and plant pathogen interaction. Of the total transcripts mapped, 42% were found to be present in both the interactions or unchanged during C. falcatum pathogenesis, 47% of the transcripts were upregulated in RRL and 11% of the transcripts were found to be upregulated in SRL. In all the categories several transcripts were mapped in common i.e., those transcripts were present in both the responses or unchanged during the interaction (Fig 3). The representative transcripts are listed in Table 3.
Differential transcripts from Glomerella graminicola from BLAST homology search
From the BLAST homology search, a total number of 17 transcripts homologous to Glomerella graminicola were present only in SRL. The transcripts represented pathogenic determinants of G. tucumanensis, the perfect stage of C. falcatum. The transcripts were found to be involved in fungal morphogenesis (alanine glyoxylate aminotransferase), intra cellular signal transduction (Ras), translation (ribosomal proteins), glycolysis (hexokinase), RNA splicing and the E3 Ub liagase of the Ub-26S proteasome pathway (Table 4). The expression of transcripts corresponding to the pathogen even after subtraction of cDNA suggested that the C. falcatum could colonize the host tissues in compatible interaction whereas transcripts related to C. falcatum colonization was not found in the incompatible interaction.
Validation of gene expression through qRT-PCR
The gene expression of the following transcripts CEBiP, MAPKKK1, MAPKK1, DRPRPM1, DRPRPS5, CBPCML, JAAS and ABAREBF showed a gradual increase in their expression at 12h and 36h and a decline at 72h post C. falcatum inoculation in RR. Whereas, in SR, CEBiP, MAPKKK1 and CBPCML exhibited an inconsistency in their expression at all the time intervals. MAPKK1 and JAAS showed gradual decrease from 12h to 72h. DRPRPM1 showed a similar response as RR but the transcript level was less than 2-fold. ABAREBF showed gradual raise from 12 to 72h and reached 2.5-fold at 72h in SR which is higher than the RR. The defense gene chitinase showed a gradual raise in both RR and SR from 12 to 72h. However, the transcript accumulation was found to be higher in RR and reached a maximum of 4-fold at 72h post C. falcatum inoculation. The transcripts CNGC and CDPK showed a gradual increase in RR and a gradual decrease in SR from 12 to 72h post C. falcatum inoculation. The transcripts 14-3-3 PE and SOD Cu Zn showed an increase in expression from 12 to 36h and a decline at 72h in the SR. In RR, there was an unstable expression. 5-fold expression was noticed in RRL for SOD Cu-Zn at 12h post C. falcatum inoculation. For, VTP ATPase, the expression in RR was unstable and SR showed a constant 6-fold expression at 36 and 72h post C. falcatum inoculation. BRSK and ER showed an inconsistent expression in both the responses. At 12h, BRSK showed more than 5-fold expression in RR. At 36h ER showed more than 10-fold expression in SR. Overall, incompatible interaction revealed higher expression of different transcripts associated with host resistance to defense upon C. falcatum inoculation whereas in the compatible interaction, except for a few transcripts where the gene expression was not prominent.