NLRs derepress MED10b- and MED7-mediated repression of jasmonate-dependent transcription to activate immunity.

Plant intracellular nucleotide-binding domain, leucine-rich repeat-containing receptors (NLRs) activate a robust immune response upon detection of pathogen effectors. How NLRs induce downstream immune defense genes remains poorly understood. The Mediator complex plays a central role in transducing signals from gene-specific transcription factors to the transcription machinery for gene transcription/activation. In this study, we demonstrate that MED10b and MED7 of the Mediator complex mediate jasmonate-dependent transcription repression, and coiled-coil NLRs (CNLs) in Solanaceae modulate MED10b/MED7 to activate immunity. Using the tomato CNL Sw-5b, which confers resistance to tospovirus, as a model, we found that the CC domain of Sw-5b directly interacts with MED10b. Knockout/down of MED10b and other subunits including MED7 of the middle module of Mediator activates plant defense against tospovirus. MED10b was found to directly interact with MED7, and MED7 directly interacts with JAZ proteins, which function as transcriptional repressors of jasmonic acid (JA) signaling. MED10b-MED7-JAZ together can strongly repress the expression of JA-responsive genes. The activated Sw-5b CC interferes with the interaction between MED10b and MED7, leading to the activation of JA-dependent defense signaling against tospovirus. Furthermore, we found that CC domains of various other CNLs including helper NLR NRCs from Solanaceae modulate MED10b/MED7 to activate defense against different pathogens. Together, our findings reveal that MED10b/MED7 serve as a previously unknown repressor of jasmonate-dependent transcription repression and are modulated by diverse CNLs in Solanaceae to activate the JA-specific defense pathways.

Exosomes in tumor-stroma crosstalk: Shaping the immune microenvironment in colorectal cancer.

Colorectal cancer (CRC) is a multifaceted disease characterized by a complex interaction between tumor cells and the surrounding microenvironment. Within this intricate landscape, exosomes have emerged as pivotal players in the tumor-stroma crosstalk, influencing the immune microenvironment of CRC. These nano-sized vesicles, secreted by both tumoral and stromal cells, serve as molecular transporters, delivering a heterogeneous mix of biomolecules such as RNAs, proteins, and lipids. In the CRC context, exosomes exert dual roles: they promote tumor growth, metastasis, and immune escape by altering immune cell functions and activating oncogenic signaling pathways and offer potential as biomarkers for early CRC detection and treatment targets. This review delves into the multifunctional roles of exosomes in the CRC immune microenvironment, highlighting their potential implications for future therapeutic strategies and clinical outcomes.

Interspecies/Intergroup Complementation of Orthotospovirus Replication and Movement through Reverse Genetics Systems.

Orthotospoviruses, the plant-infecting bunyaviruses, cause serious diseases in agronomic crops and pose major threats to global food security. The family of Tospoviridae contains more than 30 members that are classified into two geographic groups, American-type and Euro/Asian-type orthotospovirus. However, the genetic interaction between different species and the possibility, during mixed infections, for transcomplementation of gene functions by orthotospoviruses from different geographic groups remains underexplored. In this study, minireplicon-based reverse genetics (RG) systems have been established for Impatiens necrotic spot virus (INSV) (an American-type orthotospovirus) and for Calla lily chlorotic spot virus and Tomato zonate spot virus (CCSV and TZSV) (two representative Euro/Asian orthotospoviruses). Together with the earlier established RG system for Tomato spotted wilt virus (TSWV), a type species of the Orthotospovirus American-clade, viral replicase/movement proteins were exchanged and analyzed on interspecies transcomplementation. Whereas the homologous RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) protein supported the replication of orthotospoviruses from both geographic groups, heterologous combinations of RdRp from one group and N from the other group were unable to support the replication of viruses from both groups. Furthermore, the NSm movement protein (MP), from both geographic groups of orthotospoviruses, was able to transcomplement heterologous orthotospoviruses or a positive-strand Cucumber mosaic virus (CMV) in their movement, albeit with varying efficiency. MP from Rice stripe tenuivirus (RSV), a plant-infecting bunyavirus that is distinct from orthotospoviruses, or MP from CMV also moves orthotospoviruses. Our findings gain insights into the genetic interaction/reassortant potentials for the segmented plant orthotospoviruses. IMPORTANCE Orthotospoviruses are agriculturally important negative-strand RNA viruses and cause severe yield-losses on many crops worldwide. Whereas the emergence of new animal-infecting bunyaviruses is frequently associated with genetic reassortants, this issue remains underexposed with the plant-infecting orthotospovirus. With the development of reverse genetics systems for orthotospoviruses from different geographic regions, the interspecies/intergroup replication/movement complementation between American- and Euro/Asian-type orthotospoviruses were investigated. Genomic RNAs from American orthotospoviruses can be replicated by the RdRp and N from those of Euro/Asia-group orthotospoviruses, and vice versa. However, their genomic RNAs cannot be replicated by a heterologous combination of RdRp from one geographic group and N from another geographic group. Cell-to-cell movement of viral entity is supported by NSm from both geographic groups, with highest efficiency by NSm from viruses belonging to the same group. Our findings provide important insights into the genetic interaction and exchange ability of viral gene functions between different species of orthotospovirus.

Electroacupuncture attenuates neuropathic pain via suppressing BIP-IRE-1α-mediated endoplasmic reticulum stress in the anterior cingulate cortex.

Studies have suggested that endoplasmic reticulum stress (ERS) is involved in neurological dysfunction and that electroacupuncture (EA) attenuates neuropathic pain (NP) via undefined pathways. However, the role of ERS in the anterior cingulate cortex (ACC) in NP and the effect of EA on ERS in the ACC have not yet been investigated. In this study, an NP model was established by chronic constriction injury (CCI) of the left sciatic nerve in rats, and mechanical and cold tests were used to evaluate behavioral hyperalgesia. The protein expression and distribution were evaluated using western blotting and immunofluorescence. The results showed that glucose-regulated protein 78 (BIP) and inositol-requiring enzyme 1α (IRE-1α) were co-localized in neurons in the ACC. After CCI, BIP, IRE-1α, and phosphorylation of IRE-1α were upregulated in the ACC. Intra-ACC administration of 4-PBA and Kira-6 attenuated pain hypersensitivity and downregulated phosphorylation of IRE-1α, while intraperitoneal injection of 4-PBA attenuated hyperalgesia and inhibited the activation of P38 and JNK in ACC. In contrast, ERS activation by intraperitoneal injection of tunicamycin induced behavioral hyperalgesia in naive rats. Furthermore, EA attenuated pain hypersensitivity and inhibited the CCI-induced overexpression of BIP and pIRE-1α. Taken together, these results demonstrate that EA attenuates NP by suppressing BIP- and IRE-1α-mediated ERS in the ACC. Our study presents novel evidence that ERS in the ACC is implicated in the development of NP and provides insights into the molecular mechanisms involved in the analgesic effect of EA.

A randomized-controlled trial of ischemia-free liver transplantation for end-stage liver disease.

BACKGROUND & AIMS: Ischemia-reperfusion injury (IRI) has thus far been considered as an inevitable component of organ transplantation, compromising outcomes, and limiting organ availability. Ischemia-free organ transplantation is a novel approach designed to avoid IRI, with the potential to improve outcomes. METHODS: In this randomized-controlled clinical trial, recipients of livers from donors after brain death were randomly assigned to receive either an ischemia-free or a 'conventional' transplant. The primary endpoint was the incidence of early allograft dysfunction. Secondary endpoints included complications related to graft IRI. RESULTS: Out of 68 randomized patients, 65 underwent transplants and were included in the analysis. 32 patients received ischemia-free liver transplantation (IFLT), and 33 received conventional liver transplantation (CLT). Early allograft dysfunction occurred in two recipients (6%) randomized to IFLT and in eight (24%) randomized to CLT (difference -18%; 95% CI -35% to -1%; p = 0.044). Post-reperfusion syndrome occurred in three recipients (9%) randomized to IFLT and in 21 (64%) randomized to CLT (difference -54%; 95% CI -74% to -35%; p <0.001). Non-anastomotic biliary strictures diagnosed with protocol magnetic resonance cholangiopancreatography at 12 months were observed in two recipients (8%) randomized to IFLT and in nine (36%) randomized to CLT (difference, -28%; 95% CI -50% to -7%; p = 0.014). The comprehensive complication index at 1 year after transplantation was 30.48 (95% CI 23.25-37.71) in the IFLT group vs. 42.14 (95% CI 35.01-49.26) in the CLT group (difference -11.66; 95% CI -21.81 to -1.51; p = 0.025). CONCLUSIONS: Among patients with end-stage liver disease, IFLT significantly reduced complications related to IRI compared to a conventional approach. CLINICAL TRIAL REGISTRATION: chictr.org. ChiCTR1900021158. IMPACT AND IMPLICATIONS: Ischemia-reperfusion injury has thus far been considered as an inevitable event in organ transplantation, compromising outcomes and limiting organ availability. Ischemia-free liver transplantation is a novel approach of transplanting donor livers without interruption of blood supply. We showed that in patients with end-stage liver disease, ischemia-free liver transplantation, compared with a conventional approach, led to reduced complications related to ischemia-reperfusion injury in this randomized trial. This new approach is expected to change the current practice in organ transplantation, improving transplant outcomes, increasing organ utilization, while providing a clinical model to delineate the impact of organ injury on alloimmunity.

NtERF4 promotes the biosynthesis of chlorogenic acid and flavonoids by targeting PAL genes in Nicotiana tabacum.

Chlorogenic acid (CGA) and flavonoids are important secondary metabolites, which modulate plant growth and development, and contribute to plant resistance to various environmental stresses. ERF4 has been shown to be a repressor of anthocyanin accumulation in grape, but its full roles in regulating the biosynthesis of other phenylpropanoid compounds still needs to be further studied. In the present study, two NtERF4 genes were identified from N. tabacum genome. The expression level of NtERF4a was higher than that of NtERF4b in all the tobacco tissues examined. Over-expression of NtERF4a significantly promoted the accumulation of CGA and flavonoids in tobacco leaves, while silencing of NtERF4a significantly repressed the biosynthesis of CGA and flavonoids. RNA-seq analysis of NtERF4a-OE and WT plants revealed 8 phenylpropanoids-related differentially expressed genes (DEGs), including 4 NtPAL genes that encode key enzymes in the phenylpropanoid pathway. Activation of NtERF4a-GR fusion protein in tobacco significantly induced the transcription of NtPAL1 and NtPAL2 in the presence of protein synthesis inhibitor. Chromatin immunoprecipitation and Dual-Luc assays further indicated that NtERF4a could bind to the GCC box presented in the promoters of NtPAL1 and NtPAL2, thereby activating their transcription. Moreover, ectopic expression of NtERF4a induced the transcription of NtGSK1, NtMYC2, and NtJAZ3 genes, and enhanced the resistance of tobacco seedlings to salt and drought stresses, indicating multiple roles of NtERF4a in plants. Our findings revealed new roles of NtERF4a in modulating the accumulation of phenylpropanoid compounds in tobacco, and provided a putative target for improving phenylpropanoids synthesis and stress resistance in plants.

CRISPR/Cas9-mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV-U1 strain in regenerated Nicotiana tabacum.

CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.

High-Performance Ag2Se Film by a Microwave-Assisted Synthesis Method for Flexible Thermoelectric Generators.

Flexible Ag2Se thermoelectric (TE) films are promising for wearable applications near room temperature (RT). Herein, a Ag2Se film on a nylon membrane with high TE performance was fabricated by a facile method. First, Ag2Se powders were prepared by a microwave-assisted synthesis method using Ag nanowires as a template. Second, the Ag2Se powders were deposited onto nylon via vacuum filtration followed by hot pressing. Through modulating the Ag/Se molar ratio for synthesizing the Ag2Se powders, an optimized Ag2Se film demonstrates a high power factor of 1577.1 µW m-1 K-2 and good flexibility at RT. The flexibility of the Ag2Se film is mainly attributed to the flexible nylon membrane. In addition, a six-leg flexible TE generator (f-TEG) fabricated with the optimized Ag2Se film exhibits a maximum power density of 18.4 W m-2 at a temperature difference of 29 K near RT. This work provides a new solution to prepare high-TE-performance flexible Ag2Se films for f-TEGs.

Two TOBAMOVIRUS MULTIPLICATION 2A homologs in tobacco control asymptomatic response to tobacco mosaic virus.

The most common response of a host to pathogens is arguably the asymptomatic response. However, the genetic and molecular mechanisms responsible for asymptomatic responses to pathogens are poorly understood. Here we report on the genetic cloning of two genes controlling the asymptomatic response to tobacco mosaic virus (TMV) in cultivated tobacco (Nicotiana tabacum). These two genes are homologous to tobamovirus multiplication 2A (TOM2A) from Arabidopsis, which was shown to be critical for the accumulation of TMV. Expression analysis indicates that the TOM2A genes might play fundamental roles in plant development or in responses to stresses. Consistent with this hypothesis, a null allele of the TOM2A ortholog in tomato (Solanum lycopersicum) led to the development of bent branches and a high tolerance to both TMV and tomato mosaic virus (ToMV). However, the TOM2A ortholog in Nicotiana glauca did not account for the asymptomatic response to TMV in N. glauca. We showed that TOM2A family is plant-specific and originated from Chlorophyte, and the biological functions of TOM2A orthologs to promote TMV accumulation are highly conserved in the plant kingdom-in both TMV host and nonhost species. In addition, we showed that the interaction between tobacco TOM1 and TOM2A orthologs in plant species is conserved, suggesting a conserved nature of TOM1-TOM2A module in promoting TMV multiplication in plants. The tradeoff between host development, the resistance of hosts to pathogens, and their influence on gene evolution are discussed. Our results shed light on mechanisms that contribute to asymptomatic responses to viruses in plants and provide approaches for developing TMV/ToMV-resistant crops.

Transcriptomic analysis provides insights into the AUXIN RESPONSE FACTOR 6-mediated repression of nicotine biosynthesis in tobacco (Nicotiana tabacum L.).

KEY MESSAGE: NtARF6 overexpression represses nicotine biosynthesis in tobacco. Transcriptome analysis suggests that NtARF6 acts as a regulatory hub that connect different phytohormone signaling pathways to antagonize the jasmonic acid-induced nicotine biosynthesis. Plant specialized metabolic pathways are regulated by a plethora of molecular regulators that form complex networks. In Nicotiana tabacum, nicotine biosynthesis is regulated by transcriptional activators, such as NtMYC2 and the NIC2-locus ERFs. However, the underlying molecular mechanism of the regulatory feedback is largely unknown. Previous research has shown that NbARF1, a nicotine synthesis repressor, reduces nicotine accumulation in N. benthamiana. In this study, we demonstrated that overexpression of NtARF6, an ortholog of NbARF1, was able to reduce pyridine alkaloid accumulation in tobacco. We found that NtARF6 could not directly repress the transcriptional activities of the key nicotine pathway structural gene promoters. Transcriptomic analysis suggested that this NtARF6-induced deactivation of alkaloid biosynthesis might be achieved by the antagonistic effect between jasmonic acid (JA) and other plant hormone signaling pathways, such as ethylene (ETH), salicylic acid (SA), abscisic acid (ABA). The repression of JA biosynthesis is accompanied by the induction of ETH, ABA, and SA signaling and pathogenic infection defensive responses, resulting in counteracting JA-induced metabolic reprogramming and decreasing the expression of nicotine biosynthetic genes in vivo. This study provides transcriptomic evidence for the regulatory mechanism of the NtARF6-mediated repression of alkaloid biosynthesis and indicates that this ARF transcription factor might act as a regulatory hub to connect different hormone signaling pathways in tobacco.

ßC1 protein encoded in geminivirus satellite concertedly targets MKK2 and MPK4 to counter host defense.

Plant viruses have evolved multiple strategies to overcome host defense to establish an infection. Here, we identified two components of a host mitogen-activated protein kinase (MAPK) cascade, MKK2 and MPK4, as bona fide targets of the ßC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNV). ßC1 interacts with the kinase domain of MKK2 and inhibits its activity. In vivo, ßC1 suppresses flagellin-induced MAPK activation and downstream responses by targeting MKK2. Furthermore, ßC1 also interacts with MPK4 and inhibits its kinase activity. TYLCCNV infection induces the activation of the MAPK cascade, mutation in MKK2 or MPK4 renders the plant more susceptible to TYLCCNV, and can complement the lack of ßC1. This work shows for the first time that a plant virus both activates and suppresses a MAPK cascade, and the discovery of the ability of ßC1 to selectively interfere with the host MAPK activation illustrates a novel virulence function and counter-host defense mechanism of geminiviruses.

The Sw-5b NLR nucleotide-binding domain plays a role in oligomerization, and its self-association is important for activation of cell death signaling.

Plant and animal intracellular nucleotide-binding and leucine-rich repeat (NLR) receptors play important roles in sensing pathogens and activating defense signaling. However, the molecular mechanisms underlying the activation of host defense signaling by NLR proteins remain largely unknown. Many studies have determined that the coil-coil (CC) or Toll and interleukin-1 receptor/resistance protein (TIR) domain of NLR proteins and their dimerization/oligomerization are critical for activating downstream defense signaling. In this study, we demonstrated that, in tomato, the nucleotide-binding (NB) domain Sw-5b NLR alone can activate downstream defense signaling, leading to elicitor-independent cell death. Sw-5b NB domains can self-associate, and this self-association is crucial for activating cell death signaling. The self-association was strongly compromised after the introduction of a K568R mutation into the P-loop of the NB domain. Consequently, the NBK568R mutant induced cell death very weakly. The NBCΔ20 mutant lacking the C-terminal 20 amino acids can self-associate but cannot activate cell death signaling. The NBCΔ20 mutant also interfered with wild-type NB domain self-association, leading to compromised cell death induction. By contrast, the NBK568R mutant did not interfere with wild-type NB domain self-association and its ability to induce cell death. Structural modeling of Sw-5b suggests that NB domains associate with one another and likely participate in oligomerization. As Sw-5b-triggered cell death is dependent on helper NLR proteins, we propose that the Sw-5b NB domain acts as a nucleation point for the assembly of an oligomeric resistosome, probably by recruiting downstream helper partners, to trigger defense signaling.

Application of ischaemia-free liver transplantation improves prognosis of patients with steatotic donor livers - a retrospective study.

The use of steatotic livers in liver transplantation (LT) is controversial. Ischaemia-free liver transplantation (IFLT) has obvious advantages for the recovery of allograft function. The aim of this study was to examine the effect of liver grafts with steatosis on outcome and the effect of IFLT with steatotic livers. 360 patients with LT were enrolled in this study. Perioperative characteristics and differences in outcome among different grades of steatotic groups, and between the IFLT and conventional LT (CLT) groups were analysed. Occurrence of early allograft dysfunction (EAD; 50%) and primary nonfunction (PNF; 20%) was significantly higher in the severe steatosis group (P < 0.001 and <0.001, respectively). Survival rate is significantly low in severe steatosis group (3-year: 60%, P = 0.0039). The IFLT group had a significantly lower occurrence of EAD than the CLT group (0% vs. 60%, P = 0.01). The level of postoperative peak AST, GGT and creatine were significantly lower in IFLT group (P = 0.009, 0.032 and 0.024, respectively). In multivariable analysis, IFLT and EAD were independent factors affecting postoperative survival. Severe steatotic livers lead to severe complications and poor outcomes in LT. IFLT has obvious advantages for reducing the rate of EAD in LT with steatotic livers.

Single-center experience of organ transplant practice during the COVID-19 epidemic.

In order to safely carry out organ donation transplants during the outbreak of coronavirus disease 2019 (COVID-19), we have formulated strict procedures in place for organ donation and transplantation. We retrospectively analyzed our transplantation work from January 20 to May 5, 2020, to discuss whether organ transplantation can be carried out safely during the epidemic period. From January 20 to May 5, 43 cases of donation were carried out in our hospital, and the utilization rate of liver, kidney, heart, lung, and pancreas donations was more than 90%. Forty-one cases of liver transplantation and 84 cases of kidney transplantation were performed. No graft loss or recipient death occurred within one month after kidney transplantation, and one patient (2.4%) died after liver transplantation. There was no significant difference in the length of hospital stay compared with that during the same period in the previous three years. More importantly, COVID-19 infection did not occur among healthcare providers, donors, patients, or their accompanying families in our center. Under the premise of correct protection, it is safe and feasible to carry out organ transplantation during the epidemic period. Our experience during the outbreak might provide a clinical reference for countries facing COVID-19 worldwide.

Systematic analysis on multiple Gene Expression Omnibus data sets reveals fierce immune response in hepatitis B virus-related acute liver failure.

Acute liver failure (ALF) caused by hepatitis B virus (HBV) is common type of liver failure in the world, with high morbidity and mortality rates. However, the prevalence, genetic background and factors determining the development of HBV-related ALF are rarely studied. In this study, we examined three Gene Expression Omnibus (GEO) data sets by bioinformatics analysis to identify differentially expressed genes (DEGs), key biological processes and pathways. Immune infiltration analysis showed high immune cells infiltration in HBV-related ALF tissue. We then confirmed natural killer cells and macrophages infiltration in clinical samples by immunohistochemistry assay, implying these cells play a significant role in HBV-ALF. We found 1277 genes were co-up-regulated and that 1082 genes were co-down-regulated in the 3 data sets. Inflammation-related pathways were enriched in the co-up-regulated genes and synthetic metabolic pathways were enriched in the co-down-regulated genes. WGCNA also revealed a key module enriching in immune inflammation response and identified 10 hub genes, differentially expressed in an independent data set. In conclusion, we identified fierce immune inflammatory response to elucidate the immune-driven mechanism of HBV-ALF and 10 hub genes based on gene expression profiles.

Identification of two additional plasmodesmata localization domains in the tobacco mosaic virus cell-to-cell-movement protein.

Despite decades of intensive studies, the failure to identify plasmodesmata (PD) localization sequences has constrained our understanding of Tobacco mosaic virus (TMV) movement. Recently, we identified the first PD localization signal (major PLS) in the TMV movement protein (MP), which encompasses the first 50 amino acid residues of the MP. Although the major PLS is sufficient for PD targeting, the efficiency is lower than the full-length TMV MP. To address this efficiency gap, we identified two additional PLS domains encompassing amino acid residues 61 to 80, and 147 to 170 of the MP and showed that these two domains target to PD, but do not transit to adjacent cells. We also demonstrated that the MP61-80 fragment interacts with Arabidopsis synaptotagmin A, which was also shown to interact with the major TMV MP PLS. Therefore, our findings have provided new insights to more fully understand the mechanism underlying plasmodesmal targeting of TMV MP.

Tomato leaf curl Yunnan virus-encoded C4 induces cell division through enhancing stability of Cyclin D 1.1 via impairing NbSKη -mediated phosphorylation in Nicotiana benthamiana.

The whitefly-transmitted geminiviruses induce severe developmental abnormalities in plants. Geminivirus-encoded C4 protein functions as one of viral symptom determinants that could induce abnormal cell division. However, the molecular mechanism by which C4 contributes to cell division induction remains unclear. Here we report that tomato leaf curl Yunnan virus (TLCYnV) C4 interacts with a glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase, designed NbSKη, in Nicotiana benthamiana. Pro32, Asn34 and Thr35 of TLCYnV C4 are critical for its interaction with NbSKη and required for C4-induced typical symptoms. Interestingly, TLCYnV C4 directs NbSKη to the membrane and reduces the nuclear-accumulation of NbSKη. The relocalization of NbSKη impairs phosphorylation dependent degradation on its substrate-Cyclin D1.1 (NbCycD1;1), thereby increasing the accumulation level of NbCycD1;1 and inducing the cell division. Moreover, NbSKη-RNAi, 35S::NbCycD1;1 transgenic N. benthamiana plants have the similar phenotype as 35S::C4 transgenic N. benthamiana plants on callus-like tissue formation resulted from abnormal cell division induction. Thus, this study provides new insights into mechanism of how a viral protein hijacks NbSKη to induce abnormal cell division in plants.

lncRNA XIST regulates proliferation and migration of hepatocellular carcinoma cells by acting as miR-497-5p molecular sponge and targeting PDCD4.

BACKGROUND: MicroRNAs (miRNAs) play a pivotal role in hepatocellular carcinoma (HCC) progression and have been confirmed to participate in the carcinogenesis and development of HCC. However, the relationship between miR-497-5p and HCC remains unclear. METHODS: Kaplan-Meier curve analysis and the log-rank test were used to investigate the efficacy of miR-497-5p on overall survival (OS) and disease-free survival (DFS) in patients with HCC. According to in vitro experiments, programmed cell death 4 (PDCD4) was a target of miR-497-5p by the dual-luciferase activity assay. The efficacy of PDCD4 on cell proliferation and metastasis in HCC was examined by transwell assays, CCK-8 assays and reverse transcription quantitative PCR (RT-qPCR). Additionally, we conducted a luciferase activity reporter assay to confirm the interaction between lncRNA XIST and miR-49-5p. Then, to evaluate the relationship between lncRNA XIST and miR-497-5p, several mechanistic experiments, including qRT-PCR, Western blotting, transwell assays and tumor xenograft assays, were performed. RESULTS: miR-497-5p was upregulated in HCC tissues, and high expression of miR-497-5p resulted in increases in tumor size and tumor number and a higher tumor-node-metastasis (TNM) stage and Edmondson grade in patients with HCC. Silencing miR-497-5p inhibited the proliferation and migration of HCC cells. PDCD4, which was downregulated in HCC tissues, was shown to be a target of miR-497-5p and was negatively correlated with the expression of miR-497-5p. lncRNA XIST was found to act as a miR-497-5p sponge and to regulate the level of PDCD4, which is targeted by miR-497-5p. lncRNA XIST was observed to be downregulated in the HCC tissues and positively correlated with the expression of PDCD4. CONCLUSIONS: Our findings reveal that the XIST/miR-497-5p/PDCD4 axis participates in HCC development and that XIST could be used as a biomarker of HCC.

A Novel DNA Motif Contributes to Selective Replication of a Geminivirus-Associated Betasatellite by a Helper Virus-Encoded Replication-Related Protein.

UNLABELLED: Rolling-circle replication of single-stranded genomes of plant geminiviruses is initiated by sequence-specific DNA binding of the viral replication-related protein (Rep) to its cognate genome at the replication origin. Monopartite begomovirus-associated betasatellites can be trans replicated by both cognate and some noncognate helper viruses, but the molecular basis of replication promiscuity of betasatellites remains uncharacterized. Earlier studies showed that when tomato yellow leaf curl China virus (TYLCCNV) or tobacco curly shoot virus (TbCSV) is coinoculated with both cognate and noncognate betasatellites, the cognate betasatellite dominates over the noncognate one at the late stages of infection. In this study, we constructed reciprocal chimeric betasatellites between tomato yellow leaf curl China betasatellite and tobacco curly shoot betasatellite and assayed their competitiveness against wild-type betasatellite when coinoculated with TYLCCNV or TbCSV onto plants. We mapped a region immediately upstream of the conserved rolling-circle cruciform structure of betasatellite origin that confers the cognate Rep-mediated replication advantage over the noncognate satellite. DNase I protection and in vitro binding assays further identified a novel sequence element termed Rep-binding motif (RBM), which specifically binds to the cognate Rep protein and to the noncognate Rep, albeit at lower affinity. Furthermore, we showed that RBM-Rep binding affinity is correlated with betasatellite replication efficiency in protoplasts. Our data suggest that although strict specificity of Rep-mediated replication does not exist, betasatellites have adapted to their cognate Reps for efficient replication during coevolution. IMPORTANCE: Begomoviruses are numerous circular DNA viruses that cause devastating diseases of crops worldwide. Monopartite begomoviruses are frequently associated with betasatellites which are essential for induction of typical disease symptoms. Coexistence of two distinct betasatellites with one helper virus is rare in nature. Our previous research showed that begomoviruses can trans replicate cognate betasatellites to higher levels than noncognate ones. However, the molecular mechanisms of betasatellites selective replication remain largely unknown. We investigated the interaction between the begomovirus replication-associated protein and betasatellite DNA. We found that the replication-associated protein specifically binds to a motif in betasatellites, with higher affinity for the cognate motif than the noncognate motif. This preference for cognate motif binding determines the selective replication of betasatellites. We also demonstrated that this motif is essential for betasatellite replication. These findings shed new light on the promiscuous yet selective replication of betasatellites by helper geminiviruses.

Transcriptomic profile of tobacco in response to Tomato zonate spot orthotospovirus infection.

BACKGROUND: Tomato zonate spot virus (TZSV), a dominant species of thrips-transmitted orthotospoviruses in Yunnan and Guangxi provinces in China, causes significant loss of yield in lots of crops and is a major threat to incomes of rural families. However, the detailed molecular mechanism of crop disease caused by TZSV remains obscure. METHODS: Next-generation sequencing (NGS)-based transcriptome analysis (RNA-seq) was performed to investigate and compare the gene expression changes in systemic leaves of tobacco upon infection with TZSV and mock-inoculated plants as a control. RESULTS: De novo assembly and analysis of tobacco transcriptome data by RNA-Seq identified 135,395 unigenes. 2102 differentially expressed genes (DEGs) were obtained in tobacco with TZSV infection, among which 1518 DEGs were induced and 584 were repressed. Gene Ontology enrichment analysis revealed that these DEGs were associated with multiple biological functions, including metabolic process, oxidation-reduction process, photosynthesis process, protein kinase activity. The KEGG pathway analysis of these DEGs indicated that pathogenesis caused by TZSV may affect multiple processes including primary and secondary metabolism, photosynthesis and plant-pathogen interactions. CONCLUSION: Our global survey of transcriptional changes in TZSV infected tobacco provides crucial information into the precise molecular mechanisms underlying pathogenesis and symptom development. This is the first report on the relationships in the TZSV-plant interaction using transcriptome analysis. Findings of present study will significantly help enhance our understanding of the complicated mechanisms of plant responses to orthotospoviral infection.