Clustering and evolutionary analysis of small RNAs identify regulatory siRNA clusters induced under drought stress in rice

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were madeMotivation Drought tolerance is an important trait related to growth and yield in crop. Until now, drought related research has focused on coding genes. However, non-coding RNAs also respond significantly to environmental stimuli such as drought stress. Unfortunately, characterizing the role of siRNAs under drought stress is difficult since a large number of heterogenous siRNA species are expressed under drought stress and non-coding RNAs have very weak evolutionary conservation. Thus, to characterize the role of siRNAs, we need a well designed biological and bioinformatics strategy. In this paper, to characterize the function of siRNAs we developed and used a bioinformatics pipeline that includes a genomic-location based clustering technique and an evolutionary conservation tool. Results By comparing the wild type Nipponbare and two drought resistant rice varities, we found that 21 nt and 24 nt siRNAs are significantly expressed in the three rice plants but at different time points under a short-term (0, 1, and 6 hrs) drought treatment. siRNAs were up-regulated in the wild type at an early stage while the up-regulation was delayed in the two drought tolerant plants. Genes targeted by up-regulated siRNAs were related to oxidation reduction and proteolysis, which are well known to be associated with water deficit phenotypes. More interestingly, we found that siRNAs were located in intronic regions as clusters and were of high evolutionary conservation among monocot grass plants. In summary, we show that siRNAs are important respondents to drought stress and regulate genes related to the drought tolerance in water deficit conditions

Analysis of the role of RNA silencing protein 1 (Rsp1) in the biogenesis of ~23-24 nt sRNAs in Tetrahymena thermophila

RNA interference (RNAi) pathways regulate a variety of biological processes, including normal cell growth and development, through the action of protein-RNA complexes containing small RNAs (sRNAs). Our research focused an RNAi pathway in the ciliated unicellular eukaryote Tetrahymena thermophila. This pathway produces ~23-24 nucleotide (nt) sRNAs through the action of RNA-dependent RNA polymerase (RdRP) complexes (complexes termed RdRCs) and their interaction with an RNA nuclease called Dicer 2 (Dcr2). The accumulation of sRNAs also requires a protein called RNA Silencing Protein 1 (Rsp1) which associates with a subset of RdRC proteins. In this study, we first sought to learn more about the potential function and evolutionary conservation of Rsp1 by examining its sequence. Our results indicate Rsp1 may have structural similarity to RNA polymerases, including RdRPs, but lacks the conserved catalytic residues for RNA synthesis. We also identified Rsp1-like predicted proteins in other Tetrahymena species, but no clear homologs in more distantly related organisms. Second, we tested three hypotheses for why Rsp1 is required for sRNA accumulation: 1) Rsp1 stabilizes the precursor RNA transcripts that are later processed into sRNA, 2) Rsp1 is necessary for the accumulation of RdRC proteins, and 3) Rsp1 is necessary for correct assembly of RdRCs. Our experimental results indicate that Rsp1 does not appear to regulate sRNA biogenesis by regulating the levels of sRNA precursors or RdRC proteins levels. Instead, purification of RdRCs revealed that in strains lacking Rsp1, RdRCs cannot be recovered. This suggests that RdRCs are disrupted somehow in the absence of Rsp1

How stress affects rice: a characterization of the rice transcriptome during single and simultaneous abiotic and biotic stresses

2019 Spring.Includes bibliographical references.Environmental stresses, both abiotic and biotic, are large contributors to pre-harvest crop loss. Abiotic stresses, such as drought, salinity, non-optimal temperature and others, are non-living factors in the environment that have a negative effect on plants. Biotic stresses are biological factors that can harm plants, including pathogens, pests and competition from other plants. With climate change increasing the incidence of abiotic stresses and the constant pressures of pests and pathogens, it is critical to world agriculture that varieties of plants broadly tolerant to stresses are developed. For this, it is necessary to understand how plants respond to multiple simultaneous stresses. The goal of this work is to characterize the stress response of the global staple food plant rice. Here, I present the results of two comprehensive transcriptome studies. In the first, I characterize how the rice transcriptome changes in response to simultaneous heat stress and infection by the bacterial pathogen Xanthomonas oryzae (Xo). Xo includes the causal agent for the economically important bacterial blight disease of rice, Xo pathovar oryzae (Xoo). Bacterial blight is more severe during abiotic stresses such as high temperature and drought. Most rice resistance (R) genes that target Xoo lose function at high temperature; however, function of the R-gene Xa7 is enhanced when the host is subjected to abiotic stresses. Understanding why Xa7 is more effective during heat stress gives insight into host processes that are important during combined abiotic and biotic stresses. The major finding of this study was that the abscisic acid (ABA) pathway is a node of cross-talk in the interactions between heat stress and pathogen attack, during both susceptible and resistant interactions. In the second comprehensive study, I characterize how the rice transcriptome is universally regulated by all stresses. Understanding universalities in rice stress response transcriptomes provides insight into how plants endure a wide variety of stresses in the field. To explore the universal rice transcriptome response, I developed a custom workflow to analyze publicly available RNA-Seq data from rice stress response studies, including the abiotic stresses drought, salinity, heat and cold, and the biotic stresses bacterial leaf streak, bacterial blight, rice blast, and two viral diseases. From this study, I concluded that the rice stress response is a robust system with many overlapping features. This core response includes down-regulation of photosynthetic processes and up-regulation of downstream signaling of the hormones ABA, salicylic acid and jasmonic acid. Within this dissertation, I present networks of gene regulation in four major rice responses: (1) response to a susceptible interaction with Xo during high temperature, (2) response to a resistant interaction with Xo during high temperature, (3) core response to abiotic stresses and (4) core response to biotic stresses. Common among all of these pathways are the pathways upstream and downstream of the plant hormone ABA. ABA-related processes are universally up-regulated by abiotic and biotic stresses, and are only repressed during the enhanced Xa7 response at high temperature. Because ABA signaling is critical for stress response, we need a thorough understanding of how genes in the ABA response network interact to most efficiently improve rice to be tolerant to multiple and simultaneous stresses. The gene networks I have characterized can be integrated with genome and transcriptome data from stress-tolerant rice varieties. By having a complete understanding of the rice stress response, we can develop an informed approach for developing new varieties of rice that are resistant to stress

Additional file 2 of Clustering and evolutionary analysis of small RNAs identify regulatory siRNA clusters induced under drought stress in rice

The detected motif sequences in siRNA clusters located in intronic regions. (XLSX 98 kb

Additional file 1 of Clustering and evolutionary analysis of small RNAs identify regulatory siRNA clusters induced under drought stress in rice

The GO (Gene Ontology) enrichment results of genes with siRNA targeting sites. (XLSX 20 kb

Molecular responses of grapevine to environmental stress

In the past decade, advances in sequencing and transcript quantification technology have progressed science through the genomic era into the big data era, yielding thousands of studies examining plant gene expression in response to a vast assortment of conditions. Understanding and characterization of Vitis (grapevine) has been vastly improved with modern technology. Grapevines are a culturally and economically important crop susceptible to abiotic and biotic stresses. The Vitis genus consists of tens of thousands of varieties each belonging on a spectrum of tolerance and susceptibility to different stress conditions. Two genes (VviERF6L1 and NCED3) were previously identified from microarray and RNA-Sequencing experiments and implicated in abiotic stress response but required further investigation and characterization in grapevine. The examination of these genes as hub genes in ABA signaling and stress response employed multi-level analyses of DNA, RNA, protein, and metabolite quantification in diverse Vitis species including Vitis vinifera cv. Cabernet Sauvignon (CS), Vitis champinii cv. Ramsey (RA), Vitis riparia cv. Riparia Gloire (RI), and Vitis vinifera x Vitis girdiana cv. SC2 (SC). First, a bioinformatics approach was used to annotate ABA response elements (ABREs) across all promoter regions in the PN40024 reference genome. ABREs were highly abundant and in the majority of PN40024 promoter regions. Various ABREs were identified in the ERF6L1 and NCED3 promoter regions contributing to the understanding of previous transcriptional changes observed in response to abiotic stresses. Many novel and uncharacterized genes were also identified with high numbers of ABREs in respective promoter regions that may provide valuable targets for future studies to improve grapevine breeding programs and abiotic stress tolerance. Meta-data analysis of publicly available microarray and RNA-Sequencing data identified the VviERF6L clade to transcriptionally respond to numerous stimuli including water deficit, cold, salinity, pathogen infection, wounding, and berry ripening. VviERF6Ls were expressed in many tissues including leaves, roots, and berries. Although VviERF6L1 overexpression vines did not have any obvious quantifiable morphological phenotype, the majority of genes differentially expressed in response to VviERF6L1 overexpression were involved in pathogen response. Cis-acting elements like the WBOXATNPR1 in the VviERF6L1 promoter region further implicated a role of VviERF6L1 in pathogen response. This hypothesized function was also supported by known effects of ERF5 and ERF6 Arabidopsis thaliana orthologs on pathogen susceptibility. To test the functional role of VviERF6L1 in pathogen response, a grapevine-optimized P. syringae infection assay was established. VviERF6L1 was demonstrated to have significantly higher transcript abundance in response to P. syringae infection than mock infection. Additionally, VviERF6L1 overexpression vines had significantly fewer colony-forming units during P. syringae infection and thereby higher resistance to the pathogen than empty vector control vines. NINE-CIS-EPOXYCAROTENOID DIOXYGENASE (NCED3) transcripts, NCED3 protein, and abscisic acid (ABA) concentration were quantified using RNA-Sequencing and RT-qPCR, western blots, and HPLC-MS/MS, respectively, in the leaves and roots of the four Vitis species in response to three different water deficit severities. NCED3 was identified as the only ABA metabolism gene that was a hub gene during water deficit response. NCED3 and ABA metabolism were validated as a major part of the core water deficit responses in the four Vitis species. However, ABA metabolism was highly dependent on species, organ, stress severity, and stress duration during water deficit. Interestingly, NCED3 transcript abundance paralleled ABA concentration, but this similarity was not maintained for NCED3 protein, concentrations of which did not significantly change in response to water deficit. Overall, the Texan grapevine, RA, was found to respond earlier and more sensitively during longer-term moderate and severe water deficits than the other more water deficit sensitive species. Altogether, this work furthered the understanding of two genes involved in stress response in grapevine. VviERF6L1 was identified to have a role in abiotic and biotic stress response, but the mechanism of VviERF6L1 in pathogen response requires further investigation. NCED3 was confirmed as an ABA signaling hub during water deficit with specific expression and downstream ABA concentrations being highly dependent upon species, organ, and stress conditions. However, NCED3 protein abundance response to water deficit requires further examination. These genes provide useful targets for future studies and may have applications in breeding programs to improve grapevine stress tolerance