Yun-Ru Chen

Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening

Ripening of tomato fruits is triggered by the plant hormone ethylene, but its effect is restricted by an unknown developmental cue to mature fruits containing viable seeds. To determine whether this cue involves epigenetic remodeling, we expose tomatoes to the methyltransferase inhibitor 5-azacytidine and find that they ripen prematurely. We performed whole-genome bisulfite sequencing on fruit in four stages of development, from immature to ripe. We identified 52,095 differentially methylated regions (representing 1% of the genome) in the 90% of the genome covered by our analysis. Furthermore, binding sites for RIN, one of the main ripening transcription factors, are frequently localized in the demethylated regions of the promoters of numerous ripening genes, and binding occurs in concert with demethylation. Our data show that the epigenome is not static during development and may have been selected to ensure the fidelity of developmental processes such as ripening. Crop-improvement strategies could benefit by taking into account not only DNA sequence variation among plant lines, but also the information encoded in the epigenome.

High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation

Silin Zhong,1,2,5 Je-Gun Joung,1 Yi Zheng,1 Yun-ru Chen,1 Bao Liu,2 Ying Shao,3 Jenny Z. Xiang,3 Zhangjun Fei,1,4,5 and James J. Giovannoni1,4,5 Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853, USA Institute of Genetics and Cytology, Northeast Normal University, Changchun, 130024, China Weill Medical College, Cornell University, New York, NY 10021, USA U.S. Department of Agriculture/Agriculture Research Service, Plant, Soil, and Nutrition Laboratory, Ithaca, NY 14853, USA

The Transcriptome of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus in Trichoplusia ni Cells

ABSTRACT Baculoviruses are important insect pathogens that have been developed as protein expression vectors in insect cells and as transduction vectors for mammalian cells. They have large double-stranded DNA genomes containing approximately 156 tightly spaced genes, and they present significant challenges for transcriptome analysis. In this study, we report the first comprehensive analysis of AcMNPV transcription over the course of infection in Trichoplusia ni cells, by a combination of strand-specific RNA sequencing (RNA-Seq) and deep sequencing of 5′ capped transcription start sites and 3′ polyadenylation sites. We identified four clusters of genes associated with distinctive patterns of mRNA accumulation through the AcMNPV infection cycle. A total of 218 transcription start sites (TSS) and 120 polyadenylation sites (PAS) were mapped. Only 29 TSS were associated with a canonical TATA box, and 14 initiated within or near the previously identified CAGT initiator motif. The majority of viral transcripts (126) initiated within the baculovirus late promoter motif (TAAG), and late transcripts initiated precisely at the second position of the motif. Analysis of 3′ ends showed that 92 (77%) of the 3′ PAS were located within 30 nucleotides (nt) downstream of a consensus termination signal (AAUAAA or AUUAAA). A conserved U-rich region was found approximately 2 to 10 nt downstream of the PAS for 58 transcripts. Twelve splicing events and an unexpectedly large number of antisense RNAs were identified, revealing new details of possible regulatory mechanisms controlling AcMNPV gene expression. Combined, these data provide an emerging global picture of the organization and regulation of AcMNPV transcription through the infection cycle.

Mutation of a major CG methylase in rice causes genome-wide hypomethylation, dysregulated genome expression, and seedling lethality

Significance CG cytosine methylation (mCG) is an important epigenetic marker present in most eukaryotic genomes that is maintained by an evolutionarily conserved DNA methyltransferase dubbed DNMT1 in mammals and MET1 in plants. Null mutation of DNMT1 or MET1 results in global loss of mCG and leads to embryonic death in mouse, inviability in human cancer cells, and wide-ranging developmental abnormality in Arabidopsis thaliana. This study characterizes global effects of null mutation of a MET1 gene in rice, a model plant for monocotyledons, through methylome, transcriptome, and small RNAome analyses. The findings of this study have implications for improving our understanding of the biological roles of cytosine methylation in monocots and, from an applied point of view, in epigenetic manipulation of cereal crops. Cytosine methylation at CG sites (mCG) plays critical roles in development, epigenetic inheritance, and genome stability in mammals and plants. In the dicot model plant Arabidopsis thaliana, methyltransferase 1 (MET1), a principal CG methylase, functions to maintain mCG during DNA replication, with its null mutation resulting in global hypomethylation and pleiotropic developmental defects. Null mutation of a critical CG methylase has not been characterized at a whole-genome level in other higher eukaryotes, leaving the generality of the Arabidopsis findings largely speculative. Rice is a model plant of monocots, to which many of our important crops belong. Here we have characterized a null mutant of OsMet1-2, the major CG methylase in rice. We found that seeds homozygous for OsMet1-2 gene mutation (OsMET1-2−/−), which directly segregated from normal heterozygote plants (OsMET1-2+/−), were seriously maldeveloped, and all germinated seedlings underwent swift necrotic death. Compared with wild type, genome-wide loss of mCG occurred in the mutant methylome, which was accompanied by a plethora of quantitative molecular phenotypes including dysregulated expression of diverse protein-coding genes, activation and repression of transposable elements, and altered small RNA profiles. Our results have revealed conservation but also distinct functional differences in CG methylases between rice and Arabidopsis.

Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta.

Serine protease (SP) and serine protease homolog (SPH) genes in insects encode a large family of proteins involved in digestion, development, immunity, and other processes. While 68 digestive SPs and their close homologs are reported in a companion paper (Kuwar et al., in preparation), we have identified 125 other SPs/SPHs in Manduca sexta and studied their structure, evolution, and expression. Fifty-two of them contain cystine-stabilized structures for molecular recognition, including clip, LDLa, Sushi, Wonton, TSP, CUB, Frizzle, and SR domains. There are nineteen groups of genes evolved from relatively recent gene duplication and sequence divergence. Thirty-five SPs and seven SPHs contain 1, 2 or 5 clip domains. Multiple sequence alignment and molecular modeling of the 54 clip domains have revealed structural diversity of these regulatory modules. Sequence comparison with their homologs in Drosophila melanogaster, Anopheles gambiae and Tribolium castaneum allows us to classify them into five subfamilies: A are SPHs with 1 or 5 group-3 clip domains, B are SPs with 1 or 2 group-2 clip domains, C, D1 and D2 are SPs with a single clip domain in group-1a, 1b and 1c, respectively. We have classified into six categories the 125 expression profiles of SP-related proteins in fat body, brain, midgut, Malpighian tubule, testis, and ovary at different stages, suggesting that they participate in various physiological processes. Through RNA-Seq-based gene annotation and expression profiling, as well as intragenomic sequence comparisons, we have established a framework of information for future biochemical research of nondigestive SPs and SPHs in this model species.

A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

BackgroundDeep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available.ResultsWe demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum) using the T4 RNA ligase 1 adenylated adapter.ConclusionWe provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around

Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin-binding domains in insects

20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.

Overview of chitin metabolism enzymes in Manduca sexta: Identification, domain organization, phylogenetic analysis and gene expression.

In insects, chitin is a major structural component of the cuticle and the peritrophic membrane (PM). In nature, chitin is always associated with proteins among which chitin-binding proteins (CBPs) are the most important for forming, maintaining and regulating the functions of these extracellular structures. In this study, a genome-wide search for genes encoding proteins with ChtBD2-type (peritrophin A-type) chitin-binding domains (CBDs) was conducted. A total of 53 genes encoding 56 CBPs were identified, including 15 CPAP1s (cuticular proteins analogous to peritrophins with 1 CBD), 11 CPAP3s (CPAPs with 3 CBDs) and 17 PMPs (PM proteins) with a variable number of CBDs, which are structural components of cuticle or of the PM. CBDs were also identified in enzymes of chitin metabolism including 6 chitinases and 7 chitin deacetylases encoded by 6 and 5 genes, respectively. RNA-seq analysis confirmed that PMP and CPAP genes have differential spatial expression patterns. The expression of PMP genes is midgut-specific, while CPAP genes are widely expressed in different cuticle forming tissues. Phylogenetic analysis of CBDs of proteins in insects belonging to different orders revealed that CPAP1s from different species constitute a separate family with 16 different groups, including 6 new groups identified in this study. The CPAP3s are clustered into a separate family of 7 groups present in all insect orders. Altogether, they reveal that duplication events of CBDs in CPAP1s and CPAP3s occurred prior to the evolutionary radiation of insect species. In contrast to the CPAPs, all CBDs from individual PMPs are generally clustered and distinct from other PMPs in the same species in phylogenetic analyses, indicating that the duplication of CBDs in each of these PMPs occurred after divergence of insect species. Phylogenetic analysis of these three CBP families showed that the CBDs in CPAP1s form a clearly separate family, while those found in PMPs and CPAP3s were clustered together in the phylogenetic tree. For chitinases and chitin deacetylases, most of phylogenetic analysis performed with the CBD sequences resulted in similar clustering to the one obtained by using catalytic domain sequences alone, suggesting that CBDs were incorporated into these enzymes and evolved in tandem with the catalytic domains before the diversification of different insect orders. Based on these results, the evolution of CBDs in insect CBPs is discussed to provide a new insight into the CBD sequence structure and diversity, and their evolution and expression in insects.

Structural features, evolutionary relationships, and transcriptional regulation of C-type lectin-domain proteins in Manduca sexta

Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes.

Complete Dosage Compensation and Sex-Biased Gene Expression in the Moth Manduca sexta

C-type lectins (CTLs) are a large family of Ca(2+)-dependent carbohydrate-binding proteins recognizing various glycoconjugates and functioning primarily in immunity and cell adhesion. We have identified 34 CTLDP (for CTL-domain protein) genes in the Manduca sexta genome, which encode proteins with one to three CTL domains. CTL-S1 through S9 (S for simple) have one or three CTL domains; immulectin-1 through 19 have two CTL domains; CTL-X1 through X6 (X for complex) have one or two CTL domains along with other structural modules. Nine simple CTLs and seventeen immulectins have a signal peptide and are likely extracellular. Five complex CTLs have both an N-terminal signal peptide and a C-terminal transmembrane region, indicating that they are membrane anchored. Immulectins exist broadly in Lepidoptera and lineage-specific gene duplications have generated three clusters of fourteen genes in the M. sexta genome, thirteen of which have similar expression patterns. In contrast to the family expansion, CTL-S1∼S6, S8, and X1∼X6 have 1:1 orthologs in at least four lepidopteran/dipteran/coleopteran species, suggestive of conserved functions in a wide range of holometabolous insects. Structural modeling suggests the key residues for Ca(2+)-dependent or independent binding of certain carbohydrates by CTL domains. Promoter analysis identified putative κB motifs in eighteen of the CTL genes, which did not have a strong correlation with immune inducibility in the mRNA or protein levels. Together, the gene identification, sequence comparisons, structure modeling, phylogenetic analysis, and expression profiling establish a solid foundation for future studies of M. sexta CTL-domain proteins.