TREEasy: An automated workflow to infer gene trees,species trees,and phylogenetic networks from multilocus data

Multilocus genomic data sets can be used to infer a rich set of information about the evolutionary history of a lineage, including gene trees, species trees, and phylogenetic networks. However, user‐friendly tools to run such integrated analyses are lacking, and workflows often require tedious reformatting and handling time to shepherd data through a series of individual programs. Here, we present a tool written in Python—TREEasy—that performs automated sequence alignment (with MAFFT), gene tree inference (with IQ‐Tree), species inference from concatenated data (with IQ‐Tree and RaxML‐NG), species tree inference from gene trees (with ASTRAL, MP‐EST, and STELLS2), and phylogenetic network inference (with SNaQ and PhyloNet). The tool only requires FASTA files and nine parameters as inputs. The tool can be run as command line or through a Graphical User Interface (GUI). As examples, we reproduced a recent analysis of staghorn coral evolution, and performed a new analysis on the evolution of the “WGD clade” of yeast. The latter revealed novel patterns that were not identified by previous analyses. TREEasy represents a reliable and simple tool to accelerate research in systematic biology ( https://github.com/MaoYafei/TREEasy ).     

Overexpression of ER Protein Processing and Apoptosis Regulator Genes in Human Embryonic Kidney 293 Cells Improves Gene Therapy Vectors Production

Gammaretroviral and lentiviral vectors (γ‐RV and LV) are among the most used vectors in gene therapy. Currently, human embryonic kidney (HEK) 293 cells, the manufacture platform of choice for these vectors, provide low transducing particle yields, challenging their therapeutic applications and commercialization. This work studies metabolic pathways, focusing on endoplasmic reticulum (ER) protein processing and anti‐apoptotic mechanisms, influencing vector productivity in HEK 293 cell substrates. To that end, four candidate genes—protein disulfide isomerase family A member 2 gene, heat shock protein family A (Hsp70) member 5 gene, X‐box binding protein 1 gene (ER protein processing), and B‐cell lymphoma 2 protein gene (anti‐apoptotic)—are individually stably expressed in the cells. How their overexpression level influence vector yields is analyzed by establishing cell populations with incremental genomic copies of each. γ‐RV volumetric productivity increases up to 97% when overexpressing ER protein processing genes. LV volumetric production increases 53% when overexpressing the anti‐apoptotic gene. Improvements are associated with higher cell specific productivities and dependent on gene overexpression level, highlighting the importance of fine‐tuning gene expression. Overall, this work discloses gene engineering targets enabling efficient gene therapy product manufacture showing that ER protein processing and anti‐apoptotic pathways are pivotal to producer cell performance.     

TaRPP13-3, a CC-NBS-LRR-like gene located on chr 7D,promotes disease resistance to wheat powdery mildew in Brock

Disease resistance (R) gene, RPP13, plays an important role in the resistance of plants to pathogen infections; its function in resistance of wheat to powdery mildew remains unknown. In this study, a RNA-Seq technique was used to monitor expression of genes in susceptible wheat ‘Jing411’ and resistant near-isogenic line ‘BJ-1’ in response to powdery mildew infection. Overall, 413 differential expression genes were observed and identified as involved in disease resistance. RPP13 homologous gene on wheat chromosome 7D was preliminarily identified using the wheat 660K SNP chip. RPP13 was highly expressed in ‘BJ-1’ and encodes 1,027 amino acids, including CC, NB and LRR domain, termed TaRPP13-3. After inoculation with powdery mildew, expression of TaRPP13-3 in resistant wheat changed with time, but average expression was higher when compared to susceptible variety, thus indicating that TaRPP13-3 is involved in resistance to powdery mildew. Virus-induced gene silencing (VIGS) was used to inhibit expression of TaRPP13-3 in resistant parent ‘Brock’. Results indicated that silencing of TaRPP13-3 led to decreased disease resistance in ‘Brock’. Overall results of this study indicate that TaRPP13-3 gene is involved in the defence response of wheat to powdery mildew and plays a positive role in wheat powdery mildew interactions.     

Improving NK1R-targeted gene delivery of stearyl-antimicrobial peptide CAMEL by conjugating it with substance P

Specificity is a crucial condition that hampers the application of non-viral vectors for cancer gene therapy. In a previous study, we developed an efficient gene vector, stearyl-CAMEL, using N-terminal stearylation of the antimicrobial peptide CAMEL. Substance P (SP), an 11-residue neuropeptide, rapidly enters cells after binding to the neurokinin-1 receptor (NK1R), which is expressed in many cancer cell lines. In this study, the NK1R-targeted gene vector stearyl-CMSP was constructed by conjugating SP to the C-terminus of stearyl-CAMEL. Our results indicated that stearyl-CMSP displayed significant transfection specificity for NK1R-expressing cells compared with that shown by stearyl-CAMEL. Accordingly, the stearyl-CMSP/p53 plasmid complexes had significantly higher antiproliferative activity against HEK293-NK1R cells than they did against HEK293 cells, while the stearyl-CAMEL/p53 plasmid complexes did not show this specificity in antiproliferative activity. Consequently, conjugation of the NK1R-targeted ligand SP is a simple and successful strategy to construct efficient cancer-targeted non-viral gene vectors.     

LCI1, a Chlamydomonas reinhardtii plasma membrane protein,functions in active CO2 uptake under low CO2

In response to high CO₂ environmental variability, green algae, such as Chlamydomonas reinhardtii, have evolved multiple physiological states dictated by external CO₂ concentration. Genetic and physiological studies demonstrated that at least three CO₂ physiological states, a high CO₂ (0.5–5% CO₂), a low CO₂ (0.03–0.4% CO₂) and a very low CO₂ (< 0.02% CO₂) state, exist in Chlamydomonas. To acclimate in the low and very low CO₂ states, Chlamydomonas induces a sophisticated strategy known as a CO₂‐concentrating mechanism (CCM) that enables proliferation and survival in these unfavorable CO₂ environments. Active uptake of C_i from the environment is a fundamental aspect in the Chlamydomonas CCM, and consists of CO₂ and HCO₃^– uptake systems that play distinct roles in low and very low CO₂ acclimation states. LCI1, a putative plasma membrane C_i transporter, has been linked through conditional overexpression to active C_i uptake. However, both the role of LCI1 in various CO₂ acclimation states and the species of C_i, HCO₃^– or CO₂, that LCI1 transports remain obscure. Here we report the impact of an LCI1 loss‐of‐function mutant on growth and photosynthesis in different genetic backgrounds at multiple pH values. These studies show that LCI1 appears to be associated with active CO₂ uptake in low CO₂, especially above air‐level CO₂, and that any LCI1 role in very low CO₂ is minimal.     

The Chimonanthus salicifolius genome provides insight into magnoliid evolution and flavonoid biosynthesis

Chimonanthus salicifolius, a member of the Calycanthaceae of magnoliids, is one of the most famous medicinal plants in Eastern China. Here, we report a chromosome‐level genome assembly of C. salicifolius, comprising 820.1 Mb of genomic sequence with a contig N50 of 2.3 Mb and containing 36 651 annotated protein‐coding genes. Phylogenetic analyses revealed that magnoliids were sister to the eudicots. Two rounds of ancient whole‐genome duplication were inferred in the C. salicifolious genome. One is shared by Calycanthaceae after its divergence with Lauraceae, and the other is in the ancestry of Magnoliales and Laurales. Notably, long genes with > 20 kb in length were much more prevalent in the magnoliid genomes compared with other angiosperms, which could be caused by the length expansion of introns inserted by transposon elements. Homologous genes within the flavonoid pathway for C. salicifolius were identified, and correlation of the gene expression and the contents of flavonoid metabolites revealed potential critical genes involved in flavonoids biosynthesis. This study not only provides an additional whole‐genome sequence from the magnoliids, but also opens the door to functional genomic research and molecular breeding of C. salicifolius.     

Expansion of MIR482/2118 by a class‐II transposable element in cotton

Some plant microRNA (miRNA) families contain multiple members generating identical or highly similar mature miRNA variants. Mechanisms underlying the expansion of miRNA families remain elusive, although tandem and/or segmental duplications have been proposed. In this study of two tetraploid cottons, Gossypium hirsutum and Gossypium barbadense, and their extant diploid progenitors, Gossypium arboreum and Gossypium raimondii, we investigated the gain and loss of members of the miR482/2118 superfamily, which modulates the expression of nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) disease resistance genes. We found significant expansion of MIR482/2118d in G. barbadense, G. hirsutum and G. raimondii, but not in G. arboreum. Several newly expanded MIR482/2118d loci have mutated to produce different miR482/2118 variants with altered target‐gene specificity. Based on detailed analysis of sequences flanking these MIR482/2118 loci, we found that this expansion of MIR482/2118d and its derivatives resulted from an initial capture of an MIR482/2118d by a class‐II DNA transposable element (TE) in G. raimondii prior to the tetraploidization event, followed by transposition to new genomic locations in G. barbadense, G. hirsutum and G. raimondii. The ‘GosTE’ involved in the capture and proliferation of MIR482/2118d and its derivatives belongs to the PIF/Harbinger superfamily, generating a 3‐bp target site duplication upon insertion at new locations. All orthologous MIR482/2118 loci in the two diploids were retained in the two tetraploids, but mutation(s) in miR482/2118 were observed across all four species as well as in different cultivars of both G. barbadense and G. hirsutum, suggesting a dynamic co‐evolution of miR482/2118 and its NBS‐LRR targets. Our results provide fresh insights into the mechanisms contributing to MIRNA proliferation and enrich our knowledge on TEs.