siqRNA-seq is a spike-in-independent technique for quantitative mapping of mRNA landscape.

BACKGROUND: RNA sequencing (RNA-seq) is widely used for gene expression profiling and quantification. Quantitative RNA sequencing usually requires cell counting and spike-in, which is not always applicable to many samples. Here, we present a novel quantitative RNA sequencing method independent of spike-ins or cell counting, named siqRNA-seq, which can be used to quantitatively profile gene expression by utilizing gDNA as an internal control. Single-stranded library preparation used in siqRNA-seq profiles gDNA and cDNA with equal efficiency. RESULTS: To quantify mRNA expression levels, siqRNA-seq constructs libraries for total nucleic acid to establish a model for expression quantification. Compared to Relative Quantification RNA-seq, siqRNA-seq is technically reliable and reproducible for expression profiling but also can sequence reads from gDNA which can be used as an internal reference for accurate expression quantification. Applying siqRNA-seq to investigate the effects of actinomycin D on gene expression in HEK293T cells, we show the advantages of siqRNA-seq in accurately identifying differentially expressed genes between samples with distinct global mRNA levels. Furthermore, we analyzed factors influencing the downward trend of gene expression regulated by ActD using siqRNA-seq and found that mRNA with m6A modification exhibited a faster decay rate compared to mRNA without m6A modification. Additionally, applying this technique to the quantitative analysis of seven tumor cell lines revealed a high degree of diversity in total mRNA expression among tumor cell lines. CONCLUSIONS: Collectively, siqRNA-seq is a spike-in independent quantitative RNA sequencing method, which creatively uses gDNA as an internal reference to absolutely quantify gene expression. We consider that siqRNA-seq provides a convenient and versatile method to quantitatively profile the mRNA landscape in various samples.

Plasma membrane-nucleo-cytoplasmic coordination of a receptor-like cytoplasmic kinase promotes EDS1-dependent plant immunity.

Plants use cell-surface immune receptors to recognize pathogen-specific patterns to evoke basal immunity. ENHANCED DISEASE SUSCEPTIBILITY (EDS1) is known to be crucial for plant basal immunity, whereas its activation mechanism by pattern recognition remains enigmatic. Here, we show that the fungal pattern chitin induced the plasma membrane-anchored receptor-like cytoplasmic kinase PBS1-LIKE 19 (PBL19) to undergo nuclear translocation in Arabidopsis. The palmitoylation-deficient PBL19C3A variant constantly resided in the nucleus, triggering transcriptional self-amplification mainly through WRKY8 and EDS1-dependent constitutive immunity. Unexpectedly, the metacaspase-cleaved PBL19 lacking the N-terminal nuclear localization sequence specifically interacted with and phosphorylated EDS1 in the cytoplasm. Phosphodeficient EDS1 attenuated PBL19C3A-induced constitutive immunity, while phosphomimetic EDS1 complemented the loss of PBL19 for fungal resistance. Collectively, these findings reveal a compelling model wherein the plasma membrane, nuclear and cytoplasmic pools of PBL19 temporally coordinate distinct roles of immune signal receiver, amplifier and effector to boost plant antifungal immunity via EDS1.

Plant elicitor peptide signalling confers rice resistance to piercing-sucking insect herbivores and pathogens.

Rice is a staple food crop worldwide, and its production is severely threatened by phloem-feeding insect herbivores, particularly the brown planthopper (BPH, Nilaparvata lugens), and destructive pathogens. Despite the identification of many BPH resistance genes, the molecular basis of rice resistance to BPH remains largely unclear. Here, we report that the plant elicitor peptide (Pep) signalling confers rice resistance to BPH. Both rice PEP RECEPTORs (PEPRs) and PRECURSORs of PEP (PROPEPs), particularly OsPROPEP3, were transcriptionally induced in leaf sheaths upon BPH infestation. Knockout of OsPEPRs impaired rice resistance to BPH, whereas exogenous application of OsPep3 improved the resistance. Hormone measurement and co-profiling of transcriptomics and metabolomics in OsPep3-treated rice leaf sheaths suggested potential contributions of jasmonic acid biosynthesis, lipid metabolism and phenylpropanoid metabolism to OsPep3-induced rice immunity. Moreover, OsPep3 elicitation also strengthened rice resistance to the fungal pathogen Magnaporthe oryzae and bacterial pathogen Xanthamonas oryzae pv. oryzae and provoked immune responses in wheat. Collectively, this work demonstrates a previously unappreciated importance of the Pep signalling in plants for combating piercing-sucking insect herbivores and promises exogenous application of OsPep3 as an eco-friendly immune stimulator in agriculture for crop protection against a broad spectrum of insect pests and pathogens.