De novo sequencing, assembly, and characterization of Asparagus racemosus transcriptome and analysis of expression profile of genes involved in the flavonoid biosynthesis pathway.

Asparagus racemosus is known for its diverse content of secondary metabolites, i.e., saponins, alkaloids, and a wide range of flavonoids. Flavonoids, including phenols and polyphenols, have a significant role in plant physiology and are synthesized in several tissues. Despite the diverse role of flavonoids, genetic information is limited for flavonoid biosynthesis pathways in A. racemosus. The current study explores full-scale functional genomics information of A. racemosus by de novo transcriptome sequencing using Illumina paired-end sequencing technology to elucidate the genes involved in flavonoid biosynthesis pathways. The de novo assembly of high-quality paired-end reads resulted in ∼2.3 million high-quality reads with a pooled transcript of 45,647 comprising ∼76 Mb transcriptome with a mean length (bp) of 1,674 and N50 of 1,868bp. Furthermore, the coding sequence (CDS) prediction analysis from 45,647 pooled transcripts resulted in 45,444 CDS with a total length and mean length of 76,398,686 and 1,674, respectively. The Gene Ontology (GO) analysis resulted in a high number of CDSs assigned to 25,342 GO terms, which grouped the predicted CDS into three main domains, i.e., Biological Process (19,550), Molecular Function (19,873), and Cellular Component (14,577). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was used to categorize 6,353 CDS into 25 distinct biological pathway categories, in which the majority of mapped CDS were shown to be related to translation (645), followed by signal transduction (532), carbohydrate metabolism (524), folding, sorting, and degradation (522). Among these, only ∼64 and 14 CDSs were found to be involved in the phenylpropanoid and flavonoid biosynthesis pathways, respectively. Quantitative Real-time PCR was used to check the expression profile of fourteen potential flavonoid biosynthesis pathway genes. The qRT-PCR analysis result matches the transcriptome sequence data validating the Illumina sequence results. Moreover, a large number of genes associated with the flavonoids biosynthesis pathway were found to be upregulated under the induction of methyl jasmonate. The present-day study on transcriptome sequence data of A. racemosus can be utilized for characterizing genes involved in flavonoid biosynthesis pathways and for functional genomics analysis in A. racemosus using the reverse genetics approach (CRISPR/Cas9 technology).

Genome-wide comprehensive analysis of miRNAs and their target genes expressed in resistant and susceptible Capsicum annuum genotypes during Phytophthora capsici infection.

Despite extensive works on miRNA's role during plant-oomycete interaction, its role in Capsicum annuum-Phytophthora capsici pathosystem is not fully explored. Therefore, the present study was designed to identify known and novel miRNAs along with their target genes in two contrasting chili peppers genotypes, i.e., GojamMecha_9086 (resistant) and Dabat_80045 (susceptible) under P. capsici infection associated with modulating the defense response during disease pathogenesis. The result demonstrated 79 known miRNAs corresponding to 24 miRNAs families and 477 novel miRNAs along with 22,895 potential targets, including 30 defense-related target genes against P. capsici infection. The expression analysis of 29 known and 157 novel miRNAs in resistant and 30 known and 177 novel miRNAs in susceptible genotypes revealed differential accumulation patterns. qRT-PCR analysis of 8 defense-related miRNAs representing 4 novels (Pz-novel-miR428-1, Pz-novel-miR160-1, Pz-novel-miR1028-1, Pz-novel-miR204-1) and 4 known miRNAs (Pz-known-miR803-1, Pz-known-miR2059-1, Pz-known-miR2560-1, Pz-known-miR1872-1) revealed differential accumulation pattern in both resistant and susceptible genotypes. Additionally, validation of eight target genes of miRNAs using regional amplification quantitative RT-PCR (RA-PCR), a superior technique to 5'-RNA Ligase-Mediated-rapid amplification of cDNA ends (5' RLM-RACE), revealed expression of six target genes positively correlated with their corresponding miRNAs in RC versus RI leaf, while five target genes observed an inverse correlation with their corresponding miRNAs in SC versus SI leaf, suggesting their key role during disease response. The Pz-known-miR1872-PODs pair showed perfect inverse relation in all four samples. The significant findings of the current study provide comprehensive genome-wide information about the repertoire of miRNAs and their target genes expressed in resistant and susceptible chili pepper genotypes, which can serve as a valuable resource for better understanding the post-transcriptional regulatory mechanism during C. annuum-P. capsici pathosystem.

Recent advances and potential applications of cross-kingdom movement of miRNAs in modulating plant's disease response.

In the recent past, cross-kingdom movement of miRNAs, small (20-25 bases), and endogenous regulatory RNA molecules has emerged as one of the major research areas to understand the potential implications in modulating the plant's biotic stress response. The current review discussed the recent developments in the mechanism of cross-kingdom movement (long and short distance) and critical cross-talk between host's miRNAs in regulating gene function in bacteria, fungi, viruses, insects, and nematodes, and vice-versa during host-pathogen interaction and their potential implications in crop protection. Moreover, cross-kingdom movement during symbiotic interaction, the emerging role of plant's miRNAs in modulating animal's gene function, and feasibility of spray-induced gene silencing (SIGS) in combating biotic stresses in plants are also critically evaluated. The current review article analysed the horizontal transfer of miRNAs among plants, animals, and microbes that regulates gene expression in the host or pathogenic organisms, contributing to crop protection. Further, it highlighted the challenges and opportunities to harness the full potential of this emerging approach to mitigate biotic stress efficiently.

Integrative RNA-Seq analysis of Capsicum annuum L.-Phytophthora capsici L. pathosystem reveals molecular cross-talk and activation of host defence response.

Chili pepper (Capsicum annuum L.) is economically one of the most important spice. But, it's productivity is highly affected by the pathogen, Phytophthora capsici L. Our current understanding of the molecular mechanisms associated with the defence response in C. annuum-P. capsici pathosystem is limited. The current study used RNA-seq technology to dissect the genes associated with defence response against P. capsici infection in two contrasting landraces, i.e. GojamMecha_9086 (Resistant) and Dabat_80045 (Susceptible) exposed to P. capsici infection. The transcriptomes from four leaf samples (RC, RI, SC and SI) of chili pepper resulted in a total of 118,879 assembled transcripts along with 52,384 pooled unigenes. The enrichment analysis of the transcripts indicated 23 different KEGG pathways under five main categories. Out of 774 and 484 differentially expressed genes (DEGs) of two landraces (under study), respectively, 57 and 29 DEGs were observed as associated with defence responses against P. capsici infection in RC vs. RI and SC vs. SI leaf samples, respectively. qRT-PCR analysis of six randomly selected genes validated the results of Illumina NextSeq500 sequencing. A total of 58 transcription factor families (bHLH most abundant) and 2095 protein families (Protein kinase most abundant) were observed across all the samples with maximum hits in RI and SI samples. Expression analysis revealed differential regulation of genes associated with defence and signalling response with shared coordination of molecular function, cellular component and biological processing. The results presented here would enhance our present understanding of the defence response in chili pepper against P. capsici infection, which the molecular breeders could utilize to develop resistant chili genotypes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01122-y.