Viroids are the smallest pathogens of angiosperms, consisting of non-coding RNAs that cause severe diseases in agronomic crops. Symptoms associated with viroid infection are linked to developmental alterations due to genetic regulation. To understand the global mechanisms of host viroid response, we implemented network approaches to identify master transcription regulators and their differentially expressed targets in tomato infected with mild and severe variants of PSTVd. Our approach integrates root and leaf transcriptomic data, gene regulatory network analysis, and identification of affected biological processes. Our results reveal that specific bHLH, MYB, and ERF transcription factors regulate genes involved in molecular mechanisms underlying critical signaling pathways. Functional enrichment of regulons shows that bHLH-MTRs are linked to metabolism and plant defense, while MYB-MTRs are involved in signaling and hormone-related processes. Strikingly, a member of the bHLH-TF family has a specific potential role as a microprotein involved in the post-translational regulation of hormone signaling events. We found that ERF-MTRs are characteristic of severe symptoms, while ZNF-TF, tf3a-TF, BZIP-TFs, and NAC-TF act as unique MTRs. Altogether, our results lay a foundation for further research on the PSTVd and host genome interaction, providing evidence for identifying potential key genes that influence symptom development in tomato plants.

Viroids, minimalist plant pathogens, pose significant threats to crops by causing severe diseases. Transcriptome profiling technologies have significantly advanced the analysis of viroid-infected host plants, providing critical insights into gene regulation by these pathogens. Despite these advancements, the presence of numerous genes of unknown function continues to limit a complete understanding of the transcriptome data. Co-expression analysis addresses this issue by clustering genes into modules based on global gene expression levels, with genes in the same cluster likely participating in the same biological pathways. In a previous study, we emphasized the importance of basic helix-loop-helix (bHLH) proteins in transcriptional reprogramming in tomato host in response to different potato spindle tuber viroid (PSTVd) strains. In the current research, we delve into tissue-specific gene modules, particularly in root and leaf tissues, governed by bHLH transcription factors (TFs) during PSTVd infections. Utilizing public datasets that span Control (C), mock-inoculated, PSTVd-mild (M), and PSTVd-severe (S23) strains in time-course infections, we uncovered differentially expressed gene modules. These modules were functionally characterized to identify essential hub genes, notably highlighting the regulatory coordination of bHLH TFs, depicted through the significant bifan motif found in these interactions. Expanding on these findings, we explored bipartite networks, discerning both common and unique bHLH TF regulatory roles. Our findings reveal that bHLH TFs play pivotal roles in regulating processes such as energy metabolism and facilitating rapid membrane repair in infected roots. In leaves, changes in the external layers affected photosynthesis, linking bHLH TFs to distinct metabolic functions. Through this holistic approach, we deepen our understanding of viroid-host interactions and the intricate regulatory mechanisms underpinning them.

Viroids, minimalist plant pathogens, present significant threats to crops by causing severe diseases. The use of high-throughput sequencing technologies for analyzing the transcriptomes of viroid-infected host plants has yielded informative information on gene regulation by these pathogens, however a complete understanding of the transcriptome data suffers from the inclusion of numerous genes of unknown function. Co-expression analysis addresses this by clustering genes into modules based on global gene expression levels. Our previous study emphasized basic helix-loop-helix protein (bHLH) transcriptional reprogramming in tomato in response to different potato spindle tuber viroid (PSTVd) strains. In the current research, we delve into tissue-specific gene modules, particularly in root and leaf tissues, governed by bHLH transcription factors during PSTVd infections. Utilizing public datasets that span Control (C; (mock-inoculated), PSTVd-mild (M), and PSTVd-severe (S23) strains in time-course infections, we uncovered differentially expressed gene modules. These modules were functionally characterized, identifying essential hub genes. We identified the roles of bHLH transcription factors (TFs) in managing processes like photosynthesis and rapid membrane repair in infected roots. In leaves, external layer alterations influenced photosynthesis, linking bHLH TFs to distinct metabolic functions. Expanding on these findings, we explored bipartite networks, discerning both common and unique bHLH TF regulatory roles, notably highlighting the bifan motif’s significance in these interactions. Through this holistic approach, we deepen our understanding of viroid-host interactions and the intricate regulatory mechanisms underpinning them.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, yet the transcriptional hierarchies linking endocrine signaling to tumor progression remain poorly defined. Here, we integrated gene-expression profiles from two independent cohorts (TCGA-THCA and GSE33630) to identify consensus transcriptional master regulators (TMRs) driving PTC. After normalization and differential expression analysis, we reconstructed regulon networks with ARACNe-AP, inferred TMR activity using VIPER, and integrated evidence across datasets via Fisher’s meta-analysis. This cross-cohort strategy yielded 50 shared TMRs, predominantly from the Zinc Finger, Forkhead, ETS, and nuclear receptor families. Network topology highlighted PBX4, GATAD2A, BHLHE40, HEY2, and TEAD4 as upstream regulators controlling other TMRs. Functional enrichment revealed activation of NOTCH, MAPK, PI3K, and TGF-β signaling and enrichment of early and late estrogen-response programs, uncovering a noncanonical role of SMAD9 in TGF-β signaling. Together, these findings delineate the transcriptional and hormonal circuitry underlying thyroid tumorigenesis, providing a regulatory framework for biomarker-driven therapies based on network activity states.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, yet the transcriptional hierarchies linking endocrine signaling to tumor progression remain poorly defined. Here, we integrated gene-expression profiles from two independent cohorts (TCGA-THCA and GSE33630) to identify consensus transcriptional master regulators (TMRs) driving PTC. After normalization and differential expression analysis, we reconstructed regulon networks with ARACNe-AP, inferred TMR activity using VIPER, and integrated evidence across datasets via Fisher’s meta-analysis. This cross-cohort strategy yielded 50 shared TMRs, predominantly from the Zinc Finger, Forkhead, ETS, and nuclear receptor families. Network topology highlighted PBX4, GATAD2A, BHLHE40, HEY2, and TEAD4 as upstream regulators controlling other TMRs. Functional enrichment revealed activation of NOTCH, MAPK, PI3K, and TGF-β signaling and enrichment of early and late estrogen-response programs, uncovering a noncanonical role of SMAD9 in TGF-β signaling. Together, these findings delineate the transcriptional and hormonal circuitry underlying thyroid tumorigenesis, providing a regulatory framework for biomarker-driven therapies based on network activity states.

The evolutionary reconstruction of transcriptional regulatory networks play a crucial role in plant adaptation to biotic stress. In Solanaceae, basic helix-loop-helix (bHLH) transcription factors (TFs) regulate hormone signaling pathways that balance growth and defense responses. In this study, we investigated the phylogenomic evolution of bHLH-mediated hormone networks in response to Potato Spindle Tuber Viroid (PSTVd) infection in tomato ( Solanum lycopersicum ) and its wild relatives. Among them, the JA-associated bHLH Solyc08g076930 , IA3/MYC2 exhibits strong evolutionary conservation across Solanaceae, whereas the auxin regulator Solyc03g113560 miP-bHLH-ARF8 is a tomato-specific gene raising from an ancestral duplication, indicating neofunctionalization. Under PSTVd infection, we observe extensive regulatory rewiring, with bHLH hubs assuming dominant control over target genes typically regulated by multiple TFs in healthy conditions. The JA regulon undergoes the most pronounced shift, with Solyc08g076930 directly regulating 91 genes, compared to shared regulation with over a thousand TFs in healthy plants. This rewiring reflects the adaptive plasticity of bHLH TFs in coordinating stress responses through hormonal crosstalk. These findings reveal key evolutionary pressures shaping Solanaceae stress responses, offering insights for improving viroid tolerance and crop resilience.