FASTdRNA: a workflow for the analysis of ONT direct RNA sequencing.

Motivation: Direct RNA-seq (dRNA-seq) using Oxford Nanopore Technology (ONT) has revolutionized transcript mapping by offering enhanced precision due to its long-read length. Unlike traditional techniques, dRNA-seq eliminates the need for PCR amplification, reducing the impact of GC bias, and preserving valuable base physical information, such as RNA modification and poly(A) length estimation. However, the rapid advancement of ONT devices has set higher standards for analytical software, resulting in potential challenges of software incompatibility and reduced efficiency. Results: We present a novel workflow, called FASTdRNA, to manipulate dRNA-seq data efficiently. This workflow comprises two modules: a data preprocessing module and a data analysis module. The preprocessing data module, dRNAmain, encompasses basecalling, mapping, and transcript counting, which are essential for subsequent analyses. The data analysis module consists of a range of downstream analyses that facilitate the estimation of poly(A) length, prediction of RNA modifications, and assessment of alternative splicing events across different conditions with duplication. The FASTdRNA workflow is designed for the Snakemake framework and can be efficiently executed locally or in the cloud. Comparative experiments have demonstrated its superior performance compared to previous methods. This innovative workflow enhances the research capabilities of dRNA-seq data analysis pipelines by optimizing existing processes and expanding the scope of analysis. Availability and implementation: The workflow is freely available at https://github.com/Tomcxf/FASTdRNA under an MIT license. Detailed install and usage guidance can be found in the GitHub repository.

Bacterium-enabled transient gene activation by artificial transcription factors for resolving gene regulation in maize.

Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.

Citrinin Is a Potential Quorum Sensing Inhibitor against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that infects patients by regulating virulence factors and biofilms through a quorum sensing (QS) system to protect itself from antibiotics and environmental stress. Therefore, the development of quorum sensing inhibitors (QSIs) is expected to become a new strategy for studying drug resistance to P. aeruginosa infections. Marine fungi are valuable resources for screening QSIs. A marine fungus, Penicillium sp. JH1, with anti-QS activity was isolated from the offshore waters of Qingdao (China), and citrinin, a novel QSI, was purified from secondary metabolites of this fungus. Citrinin could significantly inhibit the production of violacein in Chromobacterium violaceum CV12472 and the production of three virulence factors (elastase, rhamnolipid and pyocyanin) in P. aeruginosa PAO1. It could also inhibit the biofilm formation and motility of PAO1. In addition, citrinin downregulated the transcript levels of nine genes (lasI, rhlI, pqsA, lasR, rhlR, pqsR, lasB, rhlA and phzH) associated with QS. Molecular docking results showed that citrinin bound to PqsR and LasR with better affinity than the natural ligands. This study laid a foundation for the further study of the structure optimization and structure-activity relationship of citrinin.

A R2R3-MYB Transcription Factor Gene, BpMYB123, Regulates BpLEA14 to Improve Drought Tolerance in Betula platyphylla.

Drought stress causes various negative impacts on plant growth and crop production. R2R3-MYB transcription factors (TFs) play crucial roles in the response to abiotic stress. However, their functions in Betula platyphylla haven't been fully investigated. In this study, a R2R3 MYB transcription factor gene, BpMYB123, was identified from Betula platyphylla and reveals its significant role in drought stress. Overexpression of BpMYB123 enhances tolerance to drought stress in contrast to repression of BpMYB123 by RNA interference (RNAi) in transgenic experiment. The overexpression lines increased peroxidase (POD) and superoxide dismatase (SOD) activities, while decreased hydrogen peroxide (H2O2), superoxide radicals (O2 -), electrolyte leakage (EL) and malondialdehyde (MDA) contents. Our study showed that overexpression of BpMYB123 increased BpLEA14 gene expression up to 20-fold due to BpMYB123 directly binding to the MYB1AT element of BpLEA14 promoter. These results indicate that BpMYB123 acts as a regulator via regulating BpLEA14 to improve drought tolerance in birch.

Growth-regulating factor 5 (GRF5)-mediated gene regulatory network promotes leaf growth and expansion in poplar.

Although polyploid plants have larger leaves than their diploid counterparts, the molecular mechanisms underlying this difference (or trait) remain elusive. Differentially expressed genes (DEGs) between triploid and full-sib diploid poplar trees were identified from two transcriptomic data sets followed by a gene association study among DEGs to identify key leaf growth regulators. Yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay were employed to substantiate that PpnGRF5-1 directly regulated PpnCKX1. The interactions between PpnGRF5-1 and growth-regulating factor (GRF)-interacting factors (GIFs) were experimentally validated and a multilayered hierarchical regulatory network (ML-hGRN)-mediated by PpnGRF5-1 was constructed with top-down graphic Gaussian model (GGM) algorithm by combining RNA-sequencing data from its overexpression lines and DAP-sequencing data. PpnGRF5-1 is a negative regulator of PpnCKX1. Overexpression of PpnGRF5-1 in diploid transgenic lines resulted in larger leaves resembling those of triploids, and significantly increased zeatin and isopentenyladenine in the apical buds and third leaves. PpnGRF5-1 also interacted with GIFs to increase its regulatory diversity and capacity. An ML-hGRN-mediated by PpnGRF5-1 was obtained and could largely elucidate larger leaves. PpnGRF5-1 and the ML-hGRN-mediated by PpnGRF5-1 were underlying the leaf growth and development.

PuHox52-mediated hierarchical multilayered gene regulatory network promotes adventitious root formation in Populus ussuriensis.

Adventitious root (AR) formation is critically important in vegetative propagation through cuttings in some plants, especially woody species. However, the underlying molecular mechanisms remain elusive. Here, we report the identification of a poplar homeobox gene, PuHox52, which was induced rapidly (within 15 min) at the basal ends of stems upon cutting and played a key regulatory role in adventitious rooting. We demonstrated that overexpression of PuHox52 significantly increased the number of ARs while suppression of PuHox52 had the opposite effect. A multilayered hierarchical gene regulatory network (ML-hGRN) mediated by PuHox52 was reverse-engineered and demonstrated to govern AR formation. PuHox52 regulated AR formation through upregulation of nine hub regulators, including a jasmonate signaling pathway gene, PuMYC2, and an auxin signaling pathway gene, PuAGL12. We also identified coherent type 4 feed-forward loops within this ML-hGRN; PuHox52 repressed PuHDA9, which encodes a histone deacetylase, and led to an increase in acetylation and presumably expression of three hub regulators, PuWRKY51, PuLBD21 and PuIAA7. Our results indicate that the ML-hGRN mediated by PuHox52 governs AR formation at the basal ends of stem cuttings from poplar trees.

Overexpression of an AP2/ERF family gene, BpERF13, in birch enhances cold tolerance through upregulating CBF genes and mitigating reactive oxygen species.

The AP2/ERF (APETALA2/ethylene-responsive factor) family of transcription factors (TF) is involved in regulating biotic and abiotic stress responses in plants. To explore the role of AP2/ERFs in cold tolerance in woody plants, BpERF13 was cloned and characterized in Betula platyphylla (white birch), a species primarily found in Asia in temperate and boreal climates. Based on phylogenetic analysis, BpERF13 is a member of the IXb subfamily of ERFs. Using qRT-PCR, we found that BpERF13 was differentially expressed in different tissues, and its expression could be induced by cold treatment (4 °C). BpERF13 protein, fused with GFP, was exclusively localized to nuclei. To further assess the role of BpERF13 in cold tolerance, BpERF13 overexpression (OE) transgenic lines were generated in B. platyphylla and used for cold stress treatment and biochemical/physiological studies. BpERF13 overexpression lines had significantly increased tolerance to subfreezing treatment and reduced reactive oxygen species. Using a TF-centered yeast one-hybrid (Y1H) experimental system, we showed that BpERF13 could bind to LTRECOREATCOR15 and MYBCORE cis-elements to activate a reporter gene. ChIP-seq and ChIP-PCR experiments further demonstrated that BpERF13 bound to these cis-elements when present in the 5' proximal regions of superoxide dismutase (SOD), peroxidase (POD), and C-repeat-binding factor (CBF) genes. qRT-PCR was employed to examine the expression levels of these genes in response to cold stress; SOD, POD, and CBF genes were significantly upregulated in BpERF13 transgenic lines compared to wild-type plants in response to cold stress. These results indicate that the transcription factor BpERF13 regulates physiological processes underlying cold tolerance in woody plants.

Functional characterization of poplar WRKY75 in salt and osmotic tolerance.

The WRKY transcription factor family is one of the most important families in plants, playing a significant role in plant growth and development, as well as in stress responses. However, functional studies on the family in response to abiotic stresses are limited in poplar. In the present study, we cloned a WRKY transcription factor gene PagWRKY75, which was down-regulated during early stages of salt and osmotic stresses. The PagWRKY75 protein belongs to the WRKY IIc subfamily. It is located in the nucleus and can bind to the W box. We obtained transgenic poplar lines with PagWRKY75 overexpression or inhibited expression by RNA interference. Stress treatment experiments indicated that the transgenic poplar lines overexpressing PagWRKY75 were more sensitive to salt and osmotic stresses, compared to wild type. The transgenic lines with PagWRKY75 inhibition displayed opposite effects. Furthermore, our results showed that PagWRKY75 can reduce the ability of reactive oxygen species scavenging and the accumulation of proline under stresses, and positively regulate the water loss rate of leaves. These results indicate that the transcription factor PagWRKY75 can negatively regulate salt and osmotic tolerance by modulating various physiological processes.