Fig 1.
Different stages of disease and RKN development observed in infected tomato roots of susceptible line through acid fuchsin staining.
(A) Invasion of J2s/ initiation of feeding sites (Stage 1). (B) Parasitic J2s/ formation of feeding sites (Stage 2). (C) Feeding J2s and J3s/ expansion of feeding sites (Stage 3). (D) J4s/ maintenance of feeding sites (Stage 4). (E) J4s and females/ maintenance of feeding sites (Stage 5).
Table 1.
Elimination summary showing the abundance of reads obtained from all the libraries of susceptible interaction investigated.
Fig 2.
Chromosomal distribution of predicted miRNA precursors of tomato miRNAs.
The precursors of conserved (MIR), variants of conserved (varMIR) and novel (MIRNA) miRNAs were mapped onto tomato chromosomes using sequenced based map (Mb units) that shows the positions of Kazusa and SolCAP markers on the genome. The loci of pre-miRNAs of conserved and variants of conserved miRNAs are highlighted in light color and the loci of pre-miRNAs of novel miRNAs are highlighted in dark colour.
Fig 3.
Expression analysis of tomato miRNAs through qRT-PCR at four stages of disease development during susceptible response (A-C) and at two stages of disease development during resistance response (D).
To normalize the expression level, 18S rRNA was selected as internal control. All the experiments were conducted using two technical replicates for each of three biological replicates. Fold change was calculated through delta delta Ct method that represents the change in expression level in the infected sample relative to uninfected control sample. Data is the average of three biological replicates ± standard error of the mean. The student’s t-test (P < 0.05) was performed to determine significant difference in miRNA expression between uninfected and infected sample. The significant difference (P < 0.05) obtained for an infected stage is marked with * in the figure. S1-Stage 1, S2-Stage 2, S3-Stage 3, S5-Stage 5, UI-Uninfected sample, I-Infected sample.
Fig 4.
GO analysis of targets of conserved, variants of conserved and novel tomato miRNAs using singular enrichment analysis tool (SEA) of AgriGO toolkit.
(A) The percentage of genes mapped under different GO terms is represented through a bar graph. Blue bars indicate percentage of enriched miRNA target genes in different GO terms and green bars indicate percentage of total annotated tomato genes mapping in different GO terms. (B) Enriched GO terms in molecular function category are represented in hierarchical tree graph.
Fig 5.
Mapping of amplified cDNA cleaved products obtained from 5’RLM-RACE.
Using psRNAtarget Analysis server, conserved miRNAs including, miR164(i), miR171(i), miR159(i), miR394(i), miR156(i), miR482(ii), miR166(i), and miR168(i) were predicted to target genes including NAC, GRAS, GAMYB-like, Peroxiredoxin, SBP, Resistance protein, HB and AGO-1, respectively. A novel miRNA, Sly_miRNA996 was predicted to target MYB-like transcription factor gene. The cleaved products were amplified through 5’RLM-RACE, cloned in pGEMT vector and mapped. Cleavage sites are depicted with arrows and number of positive clones obtained out of total clones is mentioned above the arrows.
Fig 6.
Relative expression analysis of selected conserved and novel tomato miRNAs and their targets through qRT-PCR at four stages of disease development during tomato-RKN susceptible interaction.
The correlation in expression profile was deciphered between conserved miRNAs including, miR156(i), miR164(i), miR159(i), miR168(i) and miR396(i) and their target genes, SBP, NAC, GAMYB-like (MYB33 and MYB65), AGO1 and GRF1, respectively. The correlation in expression profile of novel miRNA, Sly_miRNA996 with its target, MYB-like transcription factor gene was also determined. For qRT-PCR analysis, two technical replicates for each of three biological replicates were used. Fold change was calculated through delta delta Ct method that represents the change in expression level of miRNA and target gene in the infected sample relative to the uninfected control. The fold change of uninfected sample of each stage was taken as 1 and presented with solid line. The data is presented as the mean of three biological replicates ±standard error of the mean. S1-Stage 1, S2-Stage 2, S3-Stage 3, S5-Stage 5, UI-Uninfected sample, I-Infected sample.
Table 2.
GO enrichment analysis of RKN miRNA targets performed with GeneMerge v1.4.