Fig 1.
The working flowchart of the integrated bioinformatics analyses approaches to select the best candidates for DCL, AGO and RDR genes and their associated regulatory elements in C. sinensis.
Fig 2.
The multiple sequence alignment profile of PIWI domain of the amino acids sequences of C. sinensis and Arabidopsis AGO proteins by Clustal-W program in MEGA5.
The downward yellow arrows indicate the position of conserved DDH triad of PIWI domain and the conserved H798 positions are surrounded by red box.
Table 1.
Basic information about C. sinensis Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families.
Table 2.
Comparison of the Argonaute proteins with missing catalytic residue(s) in PIWI domains between C. sinensis and A. thaliana.
Fig 3.
Phylogenetic tree for the (A) Dicer-like (DCL) proteins (B) Argonaute (AGO) proteins and (C) RDR proteins from C. sinensis and A. thaliana. All the phylogenetic trees were constructed by neighbour-joining method considering significant bootstrap values. The accession number and the abbreviations of proteins from A. thaliana are given below while others are tabulated in (Table 1): (A) Four DCL proteins, AtDCL1 (At1g01040), AtDCL2 (At3g03300), AtDCL3 (At3g43920) and AtDCL4 (At5g20320) were used in DCL analysis. (B) AtAGO1 (At1g48410), AtAGO4 (At2g27040), AtAGO5 (At2g27880), AtAGO6 (At2g32940), AtAGO7 (At1g69440) and AtAGO10 (At5g43810) were considered for AGO analysis. (C) Among six AtRDR proteins, the phylogenetic tree exhibited only four major classes with CsRDR proteins. The RDR proteins from A. thaliana were AtRDR1 (At1g14790), AtRDR2 (At4g11130), AtRDR3 (At2g19910) and AtRDR6 (At3g49500). The three different gene families from A. thaliana are indicated by different colours in the constructed tree adjacent to the designation.
Table 3.
Domain analysis of the DCLs, AGOs and RDRs proteins of the predicted gene mapping on C. sinensis with Pfam, SMART and NCBI-CDD.
Fig 4.
The conserved domains of the predicted proteins were drawn by using Pfam database.
Fig 5.
Conserved motifs of the proteins of different gene families are drawn by MEME-suite (maximum 20 motifs are displayed).
Different colour represented various motifs distributed in the domains of the proteins.
Fig 6.
Gene structure of the predicted CsDCLs, CsAGOs and CsRDRs genes in C. sinensis with A. thaliana using Gene Structure Display Server (GSDS 2.0, http://gsds.cbi.pku.edu.cn/index.php) [77].
Fig 7.
The genomic location of the reported CsDCL, CsAGO and CsRDR genes.
The chromosomal length indicating scale is provided on the left. The ChrUn means the unknown chromosome.
Fig 8.
The heatmap for the predicted GO terms corresponding to the reported RNAi genes are presented for (A) biological process (B) cellular components (C) molecular function whether the genes are related (Present) or not (Absent). The p-value corresponding to the GO terms are showed in histogram adjacent to the heatmap, using -log10 (p-value). The Ven diagrams are drawn to observe the shared GO terms by three gene families considering the (D) biological process (E) cellular components (F) molecular functions.
Fig 9.
Sub-cellular localization analysis for (A) CsDCL (B) CsRDR and (C) CsAGO proteins. (D) The percentage of protein appears in different cellular components. Here cytosol (cytos), endoplasmic reticulum (ER), extracellular (extra), golgi apparatus (golgi), membrane (membr), mitochondria (mito), nuclear (nucl), peroxisome (pero), plastid (plast) and vacuole (vacu). Overall report is tabulated in (S2 Table).
Fig 10.
(A) The regulatory network among the TFs and the predicted RNAi genes. The nodes of the network were coloured based on RNAi genes and TFs. DCL, AGO and RDR genes were represented by blue, red and green node colour, respectively, and the TFs were represented by yellow node colour. Different node symbols were used for different families of TFs. Magenta node level was used for the hub TFs. (B) The map representing the associated number of TFs with the CsRNAi genes.
Fig 11.
RNAi gene mediated sub-network for (A) ERF, (B) NAC, (C) bZIP, and (D) WRKY TF family. (E) Sub-network among the hub TFs those regulate more than five RNAi genes.
Fig 12.
The cis-regulatory element in the promoter region of the identified C. sinenis DCLs, AGOs and RDRs genes, respectively.
The deep color represents the presence of that element with the corresponding genes.
Fig 13.
The in silico Expressed Sequence Tag (EST) analysis of the identified RNAi genes in C. sinensis plant.
The green color represents the existence of expression and off color stands for absent of expression.