Fig 1.
Pipeline for the network approaches.
(A) We acquired publicly available microarray expression data encompassing both control and infected leaves and roots from the NCBI. (B) With the obtained expression data, we constructed an expression matrix of each tissue (roots, and leaf separately) and curated additional pertinent files, collectively serving as the foundational input for our subsequent analysis as follows: functional annotation, expression matrix, interest genes interactions, and sample phenotypes (the required input files are depicted in pink). (C) Then, we performed a co-expression modular network analysis using CEMiTool, a specialized tool for inferring co-expression modules. (D) Following the completion of the network analysis, we delved into Gene Set Enrichment Analysis (GSEA) performed by CEMiTool to unveil significant associations, and functional enrichment analysis was executed on the modules. Furthermore, it identifies hub genes residing within the network clusters. (E) Additionally, we constructed bipartite networks delineating the bHLH-regulon interactions. (G) Finally, we identified the most representative motifs within each network module.
Fig 2.
Deciphering Root-Specific Regulatory Patterns.
Bipartite network analysis unveils connections between bHLH (right) TFs and target genes (left). Connections-colors represent the number of bHLH TFs for each target gene as follows: blue for one, pink for two, green for three, and yellow for four. While functional enrichment analysis highlights specific pathways, including chloroplast, Photosystem I and Photosystem II, and KEGG 00592 Alpha-linoleic acid metabolism.
Fig 3.
Deciphering Leaf-Specific Regulatory Patterns: bipartite network analysis unveils connections between bHLH (right) TFs and target genes (left).
Connection color represents the number of bHLH regulation of each target gene as follows: blue for one, pink for two, green for three, and yellow for four. While functional enrichment analysis highlights specific pathways, including cutin, suberine, and wax biosynthesis, endopeptidase activity, and ornithine metabolic processes, the leaf-specific network showcases the exclusive presence of genes regulated by more than four TFs.
Fig 4.
Dynamics of Co-Expression Modules in Root Tissue.
GSEA was employed to unveil the module activity across distinct conditions, including control, PSTVd M, and PSTVd S23 strains, throughout the time course analysis of root tissue. Notably, the M5, M4, M2, M9, and M8 modules emerged as highly significant in this context, showcasing distinctive regulatory dynamics and potential functional implications across the different experimental conditions.
Fig 5.
Co-expression interaction network and functional enrichment of modules firstly repressed and then overexpressed in root.
Each node on the network represents a gene, size is proportional to the number of interactions (node degree). Hub genes are listed with their names, in red if they only interact, and in blue if they are co-expressed with their targets. Next to each network, biological functions of their genes are shown. Length of the bar represents significance, and color gradient number of genes implicated in each GO Term.Interaction network and biological functions for A) M2; B) M4; and C) M5.
Fig 6.
Co-expression network and functional enrichment of modules at early symptoms and repressed at recovery in root-tissue.
Each node on the network represents a gene, size is proportional to the number of interactions (node degree). Hub genes are listed with their names, in red if they only interact, and in blue if they are co-expressed with their targets. Next to each network, biological functions of their genes are shown. Length of the bar represents significance, and colour gradient number of genes implicated in each Gene Ontology Term. (A) Interaction network and biological functions for M8; (B) Interaction network and biological functions for M9.
Fig 7.
Dynamics of Co-Expression Modules in Leaf Tissue.
GSEA for the significant co-expression modules (M1, M2, M3, M5, and M6) within leaf tissue. It visually represents the module activity under control, PSTVd M, and PSTVd S23 strains during the time course conditions. A visual representation captures the regulatory dynamics of the five key co-expression modules (M5, M1, M3, M6, and M2) within leaf tissue. The figure illustrates the contrasting behavior of these modules in control plants and those infected with PSTVd-S.
Fig 8.
Co-expression network and functional enrichment of modules repressed at recovery with PSTVd-S23 in leaf.
Each node on the network represents a gene, size is proportional to the number of interactions (node degree). Hub genes are listed with their names, in red if they only interact, and in blue if they are co-expressed with their targets. Next to each network, biological functions of their genes are shown. Length of the bar represents significance, and color gradient number of genes implicated in each GO Term. (A) Interaction network and biological functions for M1; (B) Interaction network and biological functions for M3; (C) Interaction network and biological functions for M5; (D) Interaction network and biological functions for M6.
Fig 9.
Co-expression network and functional enrichment of module 2 in leaf.
Each node on the network represents a gene, size is proportional to the number of interactions (node degree). Hub genes are listed with their names, in red if they only interact, and in blue if they are co-expressed with their targets. Next to each network, biological functions of their genes are shown. Length of the bar represents significance, and color gradient number of genes implicated in each Gene Ontology Term. Interaction network and biological functions for M2.
Fig 10.
Bifan Motif and its functionality in gene regulation.
(A) Graphic illustration of the Bifan motif: two different bHLH regulating the expression of two different target genes; arrows depict regulation; (B) Examples of Bifan motif activity in gene regulation. Legend for the diagram is shown at the top. Four different regulation types are shown: Cooperative, Competitive, Redundant, and Combinatorial. The color gradient indicates level of regulation: the darker, the stronger.