Biology subjects

Tian, E.

Publications and source records attributed to Tian, E..

8 recordsLinked to original sources

bioRxiv2026

A RecBCD-like enzyme regulates expression of bacterial defense operons in response to DNA damage

Bacteria must quickly respond to stress signals including bacteriophage (phage) infection, invasion by mobile genetic elements, and other threats. DNA damage is a stress-signaling nexus in bacteria, initiating global responses such as the SOS response and triggering expression of specific defense and immune pathways through DNA damage-sensing transcriptional regulators. Here we identify a three-protein pathway termed Cerberus that senses dsDNA ends that become available during DNA damage or inhibition of the key DNA repair complex RecBCD, and controls expression of an associated defense/immune operon. The central player in Cerberus is a large peptidase-helicase-nuclease fusion protein, CerC, that is architecturally similar to the RecBC complex itself. Upon DNA damage or RecBCD inhibition, CerC binds and unwinds free DNA ends to allosterically activate its fused HExxH metallopeptidase domain. CerC specifically cleaves the adjacently encoded anti-sigma factor CerB to release its cognate sigma factor CerA, allowing CerA to mediate transcription of a defense/immune operon encoded nearby. Cerberus therefore represents a pathway that has co-opted a key DNA repair machine for DNA damage sensing, and linked that activity to transcriptional control through anti-sigma factor cleavage.

biochemistry↗

bioRxiv2025

pGGTOX: a versatile plasmid platform for recombination-based genome editing across Enterobacteriaceae

Precise genome editing in Enterobacteriaceae is essential for studying gene function, pathogenesis, and antimicrobial resistance, yet many current systems face host-specific and efficiency limitations. We developed pGGTOX, a modular plasmid platform that enables efficient homologous recombination-mediated genome editing across diverse Enterobacteriaceae, including Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, and Enterobacter intestinihominis. The system integrates a rhamnose-inducible toxin (MqsR) for stringent counterselection, a sfGFP reporter for visual tracking of recombination events, Golden Gate cloning for rapid assembly of homologous arms, an FRT-flanked resistance cassette for marker removal, and an oriT sequence for conjugative transfer. Together with the companion plasmid pCP20-oriT, pGGTOX supports precise, marker-free genomic modification. Using pGGTOX, we achieved targeted deletions of dapA in E. coli and mrkCD in carbapenem-resistant K. pneumoniae, both with 100% efficiency. The dapA mutant exhibited diaminopimelate auxotrophy, while mrkCD deletion markedly reduced biofilm formation, consistent with the loss of function associated with these genes. pGGTOX also enabled deletion of a 43.1-kb type IV secretion gene cluster (tra) from an IncN/FII plasmid in E. intestinihominis and insertion of a 10-kb CRISPR-Cas9 plasmid-curing module (pCasCure) into an S. enterica IncX1 plasmid. Deletion of the tra gene cluster resulted in a substantial reduction in plasmid conjugation efficiency. Conjugative transfer of the engineered IncX1-pCasCure plasmid into K. pneumoniae facilitated CRISPR-mediated curing of blaKPC, sensitizing carbapenem resistance to susceptibility. In summary, pGGTOX provides a versatile, efficient, and broadly applicable platform for genome engineering and CRISPR delivery in Enterobacteriaceae, expanding the toolkit for bacterial genetics and translational antimicrobial research. ImportancePrecise genetic manipulation in Enterobacteriaceae remains a major technical challenge, particularly for non-model or multidrug-resistant strains. We developed pGGTOX, a versatile and broadly applicable plasmid platform that enables efficient, marker-free genome editing through homologous recombination. By integrating stringent counterselection, visual screening, modular cloning, and conjugative transfer, pGGTOX simplifies construction and streamlines editing across multiple clinically relevant species. We demonstrate its utility in deleting chromosomal and plasmid-borne loci, inserting large genetic modules, and delivering CRISPR-Cas9 systems for targeted elimination of antibiotic resistance genes. This platform expands the molecular toolkit for functional genomics and provides a powerful new strategy for dissecting bacterial virulence, resistance, and plasmid biology.

microbiology↗

bioRxiv2024

Molecular regulation of Bna1205ams1 required for male fertility and development of a recessive genic male-sterility system in Brassica napus

Through the comprehensive use of two pollination control systems of cytoplasmic male sterility (CMS) and genic male sterility (GMS), the rapeseed yield was increased by more than 20%. However, more hybrid production systems and detailed mechanisms underlying male sterility are required. Here, we reported a novel two-line hybrid production system of 1205A for GMS and also investigated the underlying mechanism for male sterility. Five co-segregated kompetitive allele specific PCR (KASP) markers were developed and validated, which could be used for transferring the male sterility trait into external B. napus breeding lines and developing further two-line hybrid production systems of GMS. Inheritance studies detected one gene locus of Bna1205ams1 for regulating the male sterility of 1205A. As a potential candidate gene of Bna1205ams1, BnaC03g27700D was fine mapped and narrowed down to a 181.47 kb region on chrC03 and validated by functional analysis. The mutation of BnaC03g27700D in 1205A resulted in large metabolic fluctuations, most of which were involved in aborted tapetal PCD, which could lead to reduced pollen fertility with abnormal pollen exine. The developed new GMS line of 1205AB provided us with the opportunity to identify a new male sterility gene of BnaC03g27700D in B. napus. The study of BnaC03g27700D aims to renew the annotation of the gene and provide new resources for basic research on the genetic control of male sterility.

molecular biology↗

bioRxiv2024

Disease resistance of Brassica juncea to Sclerotinia sclerotiorum is established through the induction of indole glucosinolate biosynthesis

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is the main disease threat to oilseeds in Brassiceae, causing significant yield losses and reduction in oil content and quality. The studies on S. sclerotiorum require a great focus and extensive research on B. juncea compared to those on B. napus and B. oleracea. Transcriptome analysis revealed a large number of defense-related genes and response processes in B. napus and B. oleracea. However, similarities and differences in the defense responses to S. sclerotiorum on B. juncea are rarely reported. In the present study, we reported a B. juncea breeding line of H83 with high S. sclerotiorum resistance, which was used for transcriptome analysis compared to L36 with low resistance. A novel regulatory network was proposed to defend against S. sclerotiorum invasion in B. juncea. Upon infection of S. sclerotiorum, a series of auxin and MAPK signaling pathways were initiated within 12 h, and then defenses were activated to restrict the development and spread of S. sclerotiorum by inducing the massive synthesis of indole glucosinolates after 24 h. Twelve hub genes involved in the network were identified by the weighted gene co-expression network (WGCNA), which are involved in plant-pathogen interaction, signaling pathway genes, indole glucosinolate biosynthesis and cell wall formation. The hub genes were further validated by qRT-PCR. The research revealed a new resistant line of H83 against S. sclerotiorum and a different regulatory network in B. juncea, which would be beneficial for the future effective breeding of Sclerotinia-resistant varieties.

plant biology↗

bioRxiv2021

Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a unique furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation. Here, we show that O-glycosylation near the furin cleavage site is mediated by specific members of the GALNT enzyme family and is dependent on the novel proline at position 681 (P681). We further demonstrate that O-glycosylation of S decreases furin cleavage. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the potential role of P681 mutations in the recently identified, highly transmissible B.1.1.7 variant.

biochemistry↗

bioRxiv2019

Development of a multiparent advanced generation intercross (MAGIC) population for genetic exploitation of complex traits in Brassica juncea: glucosinolate content as an example

Multiparent advanced generation intercross (MAGIC) populations have recently been developed to allow the high-resolution mapping of complex quantitative traits. This article describes the development of one MAGIC population and verifies its potential application for mapping quantitative trait loci (QTLs) in B. juncea. The population was developed from eight founders with diverse traits and composed of 408 F6 recombinant inbred lines (RILs). To develop one rapid and simplified way for using the MAGIC population, a subset of 133 RILs as the primary mapping population were genotyped using 346 intron-length polymorphism (ILP) polymorphic markers. The population lacks significant signatures of population structure that are suitable for the analysis of complex traits. Genome-wide association mapping (GWAS) identified three major glucosinolate (GSL) QTLs of QGsl.ig01.1 on J01 for indole GSL (IG), QGsl.atg09.1 on J09 and QGsl.atg11.1 on J11 for aliphatic GSL (AG) and total GSL (TG). The candidate genes for QGsl.ig01.1, QGsl.atg09.1 and QGsl.atg11.1 are GSH1, GSL-ALK and MYB28, which are involved in converting glutamate and cysteine to{gamma} -EC, the accumulation of glucoraphanin, and the whole process of AG metabolism, respectively. One effective method for association mapping of quantitative traits in the B. juncea MAGIC population is also suggested by utilization of the remaining 275 RILs and incorporation of the novel kompetitive allele specific PCR (KASP) technique. In addition to its QTL mapping purpose, the MAGIC population could also be potentially utilized in variety development by breeders.

genetics↗

bioRxiv2019

Exploring the basis of 2-propenyl and 3-butenyl glucosinolate synthesis by QTL mapping and RNA-sequencing in Brassica juncea

Brassica juncea is used as a condiment, as vegetables and as an oilseed crop, especially in semiarid areas. In the present study, we constructed a genetic map using one recombinant inbred line (RIL) of B. juncea. A total of 304 ILP (intron length polymorphism) markers were mapped to 18 linkage groups designated LG01-LG18 in B. juncea. The constructed map covered a total genetic length of 1671.13 cM with an average marker interval of 5.50 cM. The QTLs for 2-propenyl glucosinolates (GSLs) colocalized with the QTLs for 3-butenyl GSLs between At1g26180 and BnapPIP1580 on LG08 in the field experiments of 2016 and 2017. These QTLs accounted for an average of 42.3% and 42.6% phenotypic variation for 2-propenyl and 3-butenyl GSLs, respectively. Furthermore, the Illumina RNA-sequencing technique was used to excavate the genes responsible for the synthesis of GSLs in the siliques of the parental lines of the RIL mapping population, because the bulk of the seed GSLs might originate from the siliques. Comparative analysis and annotation by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) revealed that 324 genes were involved in GSL metabolism, among which only 24 transcripts were differentially expressed genes (DEGs). Among those DEGs, 15 genes were involved in the biosynthesis and transport of aliphatic GSLs, and their expression patterns were further validated by qRT-PCR analysis. These RNA-Seq results will be helpful for further fine mapping, gene cloning and genetic mechanisms of 2-propenyl and 3-butenyl GSLs in B. juncea.

molecular biology↗

bioRxiv2019

Microhomology-mediated end joining drives complex rearrangements and over expression of MYC and PVT1 in multiple myeloma

MYC is a widely acting transcription factor and its deregulation is a crucial event in many human cancers. MYC is important biologically and clinically in multiple myeloma, but the mechanisms underlying its dysregulation are poorly understood. We show that MYC rearrangements are present in 36.0% of newly diagnosed myeloma patients, as detected in the largest set of next generation sequencing data to date (n=1267). Rearrangements were complex and associated with increased expression of MYC and PVT1, but not other genes at 8q24. The highest effect on gene expression was detected in cases where the MYC locus is juxtaposed next to super-enhancers associated with genes such as IGH, IGK, IGL, TXNDC5/BMP6, FAM46C and FOXO3. We identified three hotspots of recombination at 8q24, one of which is enriched for IGH-MYC translocations. Breakpoint analysis indicates primary myeloma rearrangements involving the IGH locus occur through non-homologous end joining, whereas secondary MYC rearrangements occur through microhomology-mediated end joining. This mechanism is different to lymphomas, where non-homologous end joining generates MYC rearrangements. Rearrangements resulted in over-expression of key genes and ChIP-seq identified that HK2, a member of the glucose metabolism pathway, is directly over-expressed through binding of MYC at its promoter.

cancer biology↗