An improved bacterial mRNA enrichment strategy in dual RNA sequencing to unveil the dynamics of plant-bacterial interactions.

BACKGROUND: Dual RNA sequencing is a powerful tool that enables a comprehensive understanding of the molecular dynamics underlying plant-microbe interactions. RNA sequencing (RNA-seq) poses technical hurdles in the transcriptional analysis of plant-bacterial interactions, especially in bacterial transcriptomics, owing to the presence of abundant ribosomal RNA (rRNA), which potentially limits the coverage of essential transcripts. Therefore, to achieve cost-effective and comprehensive sequencing of the bacterial transcriptome, it is imperative to devise efficient methods for eliminating rRNA and enhancing the proportion of bacterial mRNA. In this study, we modified a strand-specific dual RNA-seq method with the goal of enriching the proportion of bacterial mRNA in the bacteria-infected plant samples. The enriched method involved the sequential separation of plant mRNA by poly A selection and rRNA removal for bacterial mRNA enrichment followed by strand specific RNA-seq library preparation steps. We assessed the efficiency of the enriched method in comparison to the conventional method by employing various plant-bacterial interactions, including both host and non-host resistance interactions with pathogenic bacteria, as well as an interaction with a beneficial rhizosphere associated bacteria using pepper and tomato plants respectively. RESULTS: In all cases of plant-bacterial interactions examined, an increase in mapping efficiency was observed with the enriched method although it produced a lower read count. Especially in the compatible interaction with Xanthmonas campestris pv. Vesicatoria race 3 (Xcv3), the enriched method enhanced the mapping ratio of Xcv3-infected pepper samples to its own genome (15.09%; 1.45-fold increase) and the CDS (8.92%; 1.49-fold increase). The enriched method consistently displayed a greater number of differentially expressed genes (DEGs) than the conventional RNA-seq method at all fold change threshold levels investigated, notably during the early stages of Xcv3 infection in peppers. The Gene Ontology (GO) enrichment analysis revealed that the DEGs were predominantly enriched in proteolysis, kinase, serine type endopeptidase and heme binding activities. CONCLUSION: The enriched method demonstrated in this study will serve as a suitable alternative to the existing RNA-seq method to enrich bacterial mRNA and provide novel insights into the intricate transcriptomic alterations within the plant-bacterial interplay.

Beyond NGS data sharing for plant ecological resilience and improvement of agronomic traits.

Decoding complex plant omics is essential for advancing our understanding of plant biology, evolution, and breeding as well as for practical applications in agriculture, conservation, and biotechnology. The advent of Next-Generation Sequencing (NGS) has revolutionized global plant genomic research, offering high-throughput, cost-effective, and accurate methods for generating genomic data. However, challenges still exist that suggest an entirely unresolved genome characterized by high heterozygosity, extensive repetitive sequences, and complex ploidy features. In addition, individual investigation of genomic information from various genetic resources is essential for omics research, as there are differences in traits within a single breed beyond a species due to the uniqueness of sequence variation. This article provides high-quality genomic and transcriptomic insights targeted at the agronomical background.

The landscape of abiotic and biotic stress-responsive splice variants with deep RNA-seq datasets in hot pepper.

Alternative splicing (AS) is a widely observed phenomenon in eukaryotes that plays a critical role in development and stress responses. In plants, the large number of RNA-seq datasets in response to different environmental stressors can provide clues for identification of condition-specific and/or common AS variants for preferred agronomic traits. We report RNA-seq datasets (350.7 Gb) from Capsicum annuum inoculated with one of three bacteria, one virus, or one oomycete and obtained additional existing transcriptome datasets. In this study, we investigated the landscape of AS in response to environmental stressors, signaling molecules, and tissues from 425 total samples comprising 841.49 Gb. In addition, we identified genes that undergo AS under specific and shared stress conditions to obtain potential genes that may be involved in enhancing tolerance to stressors. We uncovered 1,642,007 AS events and identified 4,354 differential alternative splicing genes related to environmental stressors, tissues, and signaling molecules. This information and approach provide useful data for basic-research focused on enhancing tolerance to environmental stressors in hot pepper or establishing breeding programs.

Global co-expression network for key factor selection on environmental stress RNA-seq dataset in Capsicum annuum.

Environmental stresses significantly affect plant growth, development, and productivity. Therefore, a deeper understanding of the underlying stress responses at the molecular level is needed. In this study, to identify critical genetic factors associated with environmental stress responses, the entire 737.3 Gb clean RNA-seq dataset across abiotic, biotic stress, and phytohormone conditions in Capsicum annuum was used to perform individual differentially expressed gene analysis and to construct gene co-expression networks for each stress condition. Subsequently, gene networks were reconstructed around transcription factors to identify critical factors involved in the stress responses, including the NLR gene family, previously implicated in resistance. The abiotic and biotic stress networks comprise 233 and 597 hubs respectively, with 10 and 89 NLRs. Each gene within the NLR groups in the network exhibited substantial expression to particular stresses. The integrated analysis strategy of the transcriptome network revealed potential key genes for complex environmental conditions. Together, this could provide important clues to uncover novel key factors using high-throughput transcriptome data in other species as well as plants.

Universal gene co-expression network reveals receptor-like protein genes involved in broad-spectrum resistance in pepper (Capsicum annuum L.).

Receptor-like proteins (RLPs) on plant cells have been implicated in immune responses and developmental processes. Although hundreds of RLP genes have been identified in plants, only a few RLPs have been functionally characterized in a limited number of plant species. Here, we identified RLPs in the pepper (Capsicum annuum) genome and performed comparative transcriptomics coupled with the analysis of conserved gene co-expression networks (GCNs) to reveal the role of core RLP regulators in pepper-pathogen interactions. A total of 102 RNA-seq datasets of pepper plants infected with four pathogens were used to construct CaRLP-targeted GCNs (CaRLP-GCNs). Resistance-responsive CaRLP-GCNs were merged to construct a universal GCN. Fourteen hub CaRLPs, tightly connected with defense-related gene clusters, were identified in eight modules. Based on the CaRLP-GCNs, we evaluated whether hub CaRLPs in the universal GCN are involved in the biotic stress response. Of the nine hub CaRLPs tested by virus-induced gene silencing, three genes (CaRLP264, CaRLP277, and CaRLP351) showed defense suppression with less hypersensitive response-like cell death in race-specific and non-host resistance response to viruses and bacteria, respectively, and consistently enhanced susceptibility to Ralstonia solanacearum and/or Phytophthora capsici. These data suggest that key CaRLPs are involved in the defense response to multiple biotic stresses and can be used to engineer a plant with broad-spectrum resistance. Together, our data show that generating a universal GCN using comprehensive transcriptome datasets can provide important clues to uncover genes involved in various biological processes.

Tissue-Specific RNA-Seq Analysis and Identification of Receptor-Like Proteins Related to Plant Growth in Capsicum annuum.

Receptor-like proteins (RLPs) are a gene family of cell surface receptors that are involved in plant growth, development, and disease resistance. In a recent study, 438 pepper RLP genes were identified in the Capsicum annuum genome (CaRLPs) and determined to be present in response to multiple biotic stresses. To further understand the role of CaRLPs in plant growth and development, we analyzed expression patterns of all CaRLPs from various pepper tissues and developmental stages using RNA-seq. Ten CaRLP genes were selected for further analysis according to transcript levels with hierarchical clustering. The selected CaRLP genes displayed similarity of motifs within the same groups and structures typical of RLPs. To examine RLP function in growth and development, we performed loss-of-function analysis using a virus-induced gene silencing system. Three of the ten tested CaRLPs (CaRLP238, 253, and 360) in silenced plants exhibited phenotypic alteration with growth retardation compared to controls. All three gene-silenced peppers showed significant differences in root dry weight. Only CaRLP238 had significant differences in both root and shoot dry weight. Our results suggest that CaRLPs may play important roles in regulation of plant growth and development as well as function in defense responses to biotic stresses in the RLP gene family.

Comprehensive transcriptome resource for response to phytohormone-induced signaling in Capsicum annuum L.

OBJECTIVES: Phytohormones are small signaling molecules with crucial roles in plant growth, development, and environmental adaptation to biotic and abiotic stress responses. Despite several previously published molecular studies focused on plant hormones, our understanding of the transcriptome induced by phytohormones remains unclear, especially in major crops. Here, we aimed to provide transcriptome dataset using RNA sequencing for phytohormone-induced signaling in plant. DATA DESCRIPTION: We used high-throughput RNA sequencing profiling to investigate the pepper plant response to treatment with four major phytohormones (salicylic acid, jasmonic acid, ethylene, and abscisic acid). This dataset yielded 78 samples containing three biological replicates per six different time points for each treatment and the control, constituting 187.8 Gb of transcriptome data (2.4 Gb of each sample). This comprehensive parallel transcriptome data provides valuable information for understanding the relationships and molecular networks that regulate the expression of phytohormone-related genes involved in plant developments and environmental stress adaptation.

Transcriptome profiling of abiotic responses to heat, cold, salt, and osmotic stress of Capsicum annuum L.

Peppers (Capsicum annuum L.), belonging to the Solanaceae family, are one of the most economically important crops globally. Like other crops, peppers are threatened by diverse environmental conditions due to different pathogens and abiotic stresses. High-quality reference genomes with massive datasets of transcriptomes from various conditions can provide clues to preferred agronomic traits for breeding. However, few global gene expression profiling datasets have been published to examine the environmental stress-resistant mechanisms in peppers. In this study, we report the RNA-seq analyses of peppers treated with heat, cold, salinity, and osmotic stress at six different time points. RNA-seq libraries from 78 RNA samples containing three biological replicates per time point for each of the abiotic stresses and a mock control were constructed. A total of 204.68 Gb of transcriptome data were verified by differentially expressed genes and gene ontology enrichment analysis. Analyses of the transcriptome data in this study will provide useful information for basic studies of various stimuli to facilitate the development of stress-resistant pepper cultivars.