DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate.

BACKGROUND: Quantification of gene expression such as RNA-Seq is a popular approach to study various biological phenomena. Despite the development of RNA-Seq library preparation methods and sequencing platforms in the last decade, RNA extraction remains the most laborious and costly step in RNA-Seq of tissue samples of various organisms. Thus, it is still difficult to examine gene expression in thousands of samples. RESULTS: Here, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. The DTT concentration in Direct-RT buffer prevented RNA degradation but not RT in the lysates of several plant tissues, yeast, and zebrafish larvae. Direct reverse transcription on these lysates in Direct-RT buffer produced comparable amounts of cDNA to those synthesized from purified RNA. To maximize the advantage of the Direct-RT buffer, we integrated Direct-RT and targeted RNA-Seq to develop a cost-effective, high-throughput quantification method for the expressions of hundreds of genes: DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq). The DeLTa-Seq method could drastically improve the efficiency and accuracy of gene expression analysis. DeLTa-Seq analysis of 1056 samples revealed the temperature-dependent effects of jasmonic acid and salicylic acid in Arabidopsis thaliana. CONCLUSIONS: The DeLTa-Seq method can realize large-scale studies using thousands of animal, plant, and microorganism samples, such as chemical screening, field experiments, and studies focusing on individual variability. In addition, Direct-RT is also beneficial for gene expression analysis in small tissues from which it is difficult to purify enough RNA for the experiments.

Fillable and unfillable gaps in plant transcriptome under field and controlled environments.

The differences between plants grown in field and in controlled environments have long been recognized. However, few studies have addressed the underlying molecular mechanisms. To evaluate plant responses to fluctuating environments using laboratory equipment, we developed SmartGC, a high-performance growth chamber that reproduces the fluctuating irradiance, temperature and humidity of field environments. We analysed massive transcriptome data of rice plants grown under field and SmartGC conditions to clarify the differences in plant responses to field and controlled environments. Rice transcriptome dynamics in SmartGC mimicked those in the field, particularly during the morning and evening but those in conventional growth chamber conditions did not. Further analysis revealed that fluctuation of irradiance affects transcriptome dynamics in the morning and evening, while fluctuation of temperature affects transcriptome dynamics only in the morning. We found upregulation of genes related to biotic and abiotic stress, and their expression was affected by environmental factors that cannot be mimicked by SmartGC. Our results reveal fillable and unfillable gaps in the transcriptomes of rice grown in field and controlled environments and can accelerate the understanding of plant responses to field environments for both basic biology and agricultural applications.

H3K27me3 demethylases alter HSP22 and HSP17.6C expression in response to recurring heat in Arabidopsis.

Acclimation to high temperature increases plants' tolerance of subsequent lethal high temperatures. Although epigenetic regulation of plant gene expression is well studied, how plants maintain a memory of environmental changes over time remains unclear. Here, we show that JUMONJI (JMJ) proteins, demethylases involved in histone H3 lysine 27 trimethylation (H3K27me3), are necessary for Arabidopsis thaliana heat acclimation. Acclimation induces sustained H3K27me3 demethylation at HEAT SHOCK PROTEIN22 (HSP22) and HSP17.6C loci by JMJs, poising the HSP genes for subsequent activation. Upon sensing heat after a 3-day interval, JMJs directly reactivate these HSP genes. Finally, jmj mutants fail to maintain heat memory under fluctuating field temperature conditions. Our findings of an epigenetic memory mechanism involving histone demethylases may have implications for environmental adaptation of field plants.

Pepper vein yellows virus 9: a novel polerovirus isolated from chili pepper in Indonesia.

In 2017, a leaf sample from a single chili pepper (Capsicum annuum) plant exhibiting yellowing was collected from Aceh province, Indonesia. Total RNA was extracted from this sample, and RNA-Seq analysis was conducted. Putative infecting viruses were detected by mapping the obtained reads to the full-length viral genome sequences available in the GenBank database (7457 sequences) and the de novo-assembled contigs. RNA-Seq analysis detected polerovirus, begomovirus, and amalgavirus sequences, and the polerovirus-like sequences showed strong similarity to those of previously reported pepper vein yellows viruses (PeVYVs). The complete viral genome sequence obtained by RT-PCR had a length of 6023 nt, had the typical genome organization of a polerovirus and showed a high degree of sequence similarity to PeVYV-2 from Israel. Moreover, the predicted amino acid sequence of the P0 protein of the Indonesian isolate was 85.1% to 88.8% identical to those of other PeVYVs. In accordance with the polerovirus species demarcation criteria, this isolate should be assigned to a new polerovirus species, and we propose the name "pepper vein yellows virus 9" (PeVYV-9) for this virus.

Complete genome sequence of a novel partitivirus from a wild brassicaceous plant, Arabidopsis halleri.

Two contigs with high similarity to partitivirus sequences were identified by de novo assembly of sequences obtained by RNA-Seq from a wild brassicaceous plant, Arabidopsis halleri subsp. gemmifera. Here, we report the complete genome sequence of a putative novel partitivirus. Excluding the poly-A tail, it consists of two RNA genome segments of 1912 and 1769 bp, which are predicted to encode a 585-amino-acid-long putative RNA-dependent RNA polymerase (RdRp) and a 487-amino-acid-long putative capsid protein (CP), respectively. Phylogenetically, this virus belongs to the genus Alphapartitivirus and is most closely related to Raphanus sativus partitivirus 1 from radish. We propose the name "Arabidopsis halleri partitivirus 1" (AhPV1) for this novel virus.

Seasonality of interactions between a plant virus and its host during persistent infection in a natural environment.

Persistent infection, wherein a pathogen is continually present in a host individual, is widespread in virus-host systems. However, little is known regarding how seasonal environments alter virus-host interaction during such metastability. We observed a lineage-to-lineage infection of the host plant Arabidopsis halleri with Turnip mosaic virus for 3 years without severe damage. Virus dynamics and virus-host interactions within hosts were highly season dependent. Virus accumulation in the newly formed leaves was temperature dependent and was suppressed during winter. Transcriptome analyses suggested that distinct defence mechanisms, i.e. salicylic acid (SA)-dependent resistance and RNA silencing, were predominant during spring and autumn, respectively. Transcriptomic difference between infected and uninfected plants other than defence genes appeared transiently only during autumn in upper leaves. However, the virus preserved in the lower leaves is transferred to the clonal offspring of the host plants during spring. In the linage-to-linage infection of the A. halleri-TuMV system, both host clonal reproduction and virus transmission into new clonal rosettes are secured during the winter-spring transition. How virus and host overwinter turned out to be critical for understanding a long-term virus-host interaction within hosts under temperate climates, and more generally, understanding seasonality provides new insight into ecology of plant viruses.

Lasy-Seq: a high-throughput library preparation method for RNA-Seq and its application in the analysis of plant responses to fluctuating temperatures.

RNA-Seq is a whole-transcriptome analysis method used to research biological mechanisms and functions but its use in large-scale experiments is limited by its high cost and labour requirements. In this study, we have established a high-throughput and cost-effective RNA-Seq library preparation method that does not require mRNA enrichment. The method adds unique index sequences to samples during reverse transcription (RT) that is conducted at a higher temperature (≥62 °C) to suppress RT of A-rich sequences in rRNA, and then pools all samples into a single tube. Both single-read and paired-end sequencing of libraries is enabled. We found that the pooled RT products contained large amounts of RNA, mainly rRNA, causing over-estimations of the quantity of DNA and unstable tagmentation results. Degradation of RNA before tagmentation was found to be necessary for the stable preparation of libraries. We named this protocol low-cost and easy RNA-Seq (Lasy-Seq) and used it to investigate temperature responses in Arabidopsis thaliana. We analysed how sub-ambient temperatures (10-30 °C) affected the plant transcriptomes using time-courses of RNA-Seq from plants grown in randomly fluctuating temperature conditions. Our results suggest that there are diverse mechanisms behind plant temperature responses at different time scales.

A Survey on Plant Viruses in Natural Brassicaceae Communities Using RNA-Seq.

Studies on plant viruses are biased towards crop diseases and little is known about viruses in natural vegetation. We conducted extensive surveys of plant viruses in wild Brassicaceae plants occurring in three local plant communities in central Japan. We applied RNA-Seq with selective depletion of rRNA, which allowed us to detect infections of all genome-reported viruses simultaneously. Infections of Turnip mosaic virus (TuMV), Cucumber mosaic virus (CMV), Brassica yellows virus, Pelargonium zonate spot virus, and Arabidopsis halleri partitivirus 1 were detected from the two perennial species, Arabidopsis halleri subsp. gemmifera and Rorippa indica. De novo assembly further detected partial sequences of a putative novel virus in Arabis fragellosa. Virus species composition and infection rate differed depending on site and plant species. Viruses were most frequently detected from the perennial clonal plant, A. halleri, in which a high clonal transmission rate of viruses across multiple years was confirmed. Phylogenetic analysis of TuMV and CMV showed that virus strains from wild Brassicaceae were included as a major clade of these viruses with other reported strains from crop plants, suggesting that viruses were shared among wild plants and crops. Our studies indicated that distribution of viruses in natural plant populations are determined by the combinations of life histories of viruses and hosts. Revealing viral distribution in the natural plant communities improves our knowledge on the ecology of plant viruses.

Broad distribution spectrum from Gaussian to power law appears in stochastic variations in RNA-seq data.

Gene expression levels exhibit stochastic variations among genetically identical organisms under the same environmental conditions. In many recent transcriptome analyses based on RNA sequencing (RNA-seq), variations in gene expression levels among replicates were assumed to follow a negative binomial distribution, although the physiological basis of this assumption remains unclear. In this study, RNA-seq data were obtained from Arabidopsis thaliana under eight conditions (21-27 replicates), and the characteristics of gene-dependent empirical probability density function (ePDF) profiles of gene expression levels were analyzed. For A. thaliana and Saccharomyces cerevisiae, various types of ePDF of gene expression levels were obtained that were classified as Gaussian, power law-like containing a long tail, or intermediate. These ePDF profiles were well fitted with a Gauss-power mixing distribution function derived from a simple model of a stochastic transcriptional network containing a feedback loop. The fitting function suggested that gene expression levels with long-tailed ePDFs would be strongly influenced by feedback regulation. Furthermore, the features of gene expression levels are correlated with their functions, with the levels of essential genes tending to follow a Gaussian-like ePDF while those of genes encoding nucleic acid-binding proteins and transcription factors exhibit long-tailed ePDF.

RNA-Seq reveals virus-virus and virus-plant interactions in nature.

As research on plant viruses has focused mainly on crop diseases, little is known about these viruses in natural environments. To understand the ecology of viruses in natural systems, comprehensive information on virus-virus and virus-host interactions is required. We applied RNA-Seq to plants from a natural population of Arabidopsis halleri subsp. gemmifera to simultaneously determine the presence/absence of all sequence-reported viruses, identify novel viruses and quantify the host transcriptome. By introducing the criteria of read number and genome coverage, we detected infections by Turnip mosaic virus (TuMV), Cucumber mosaic virus and Brassica yellows virus Active TuMV replication was observed by ultramicroscopy. De novo assembly further identified a novel partitivirus, Arabidopsis halleri partitivirus 1 Interestingly, virus reads reached a maximum level that was equivalent to that of the host's total mRNA, although asymptomatic infection was common. AhgAGO2, a key gene in host defence systems, was upregulated in TuMV-infected plants. Multiple infection was frequent in TuMV-infected leaves, suggesting that TuMV facilitates multiple infection, probably by suppressing host RNA silencing. Revealing hidden plant-virus interactions in nature can enhance our understanding of biological interactions and may have agricultural applications.