High-quality assembled and annotated genomes of Nicotiana tabacum and Nicotiana benthamiana reveal chromosome evolution and changes in defense arsenals.

Nicotiana tabacum and Nicotiana benthamiana are widely used models in plant biology research. However, genomic studies of these species have lagged. Here we report the chromosome-level reference genome assemblies for N. benthamiana and N. tabacum with an estimated 99.5% and 99.8% completeness, respectively. Sensitive transcription start and termination site sequencing methods were developed and used for accurate gene annotation in N. tabacum. Comparative analyses revealed evidence for the parental origins and chromosome structural changes, leading to hybrid genome formation of each species. Interestingly, the antiviral silencing genes RDR1, RDR6, DCL2, DCL3, and AGO2 were lost from one or both subgenomes in N. benthamiana, while both homeologs were kept in N. tabacum. Furthermore, the N. benthamiana genome encodes fewer immune receptors and signaling components than that of N. tabacum. These findings uncover possible reasons underlying the hypersusceptible nature of N. benthamiana. We developed the user-friendly Nicomics (http://lifenglab.hzau.edu.cn/Nicomics/) web server to facilitate better use of Nicotiana genomic resources as well as gene structure and expression analyses.

A novel effector RipBT contributes to Ralstonia solanacearum virulence on potato.

Ralstonia solanacearum is one of the most destructive plant-pathogenic bacteria, infecting more than 200 plant species, including potato (Solanum tuberosum) and many other solanaceous crops. R. solanacearum has numerous pathogenicity factors, and type III effectors secreted through type III secretion system (T3SS) are key factors to counteract host immunity. Here, we show that RipBT is a novel T3SS-secreted effector by using a cyaA reporter system. Transient expression of RipBT in Nicotiania benthamiana induced strong cell death in a plasma membrane-localization dependent manner. Notably, mutation of RipBT in R. solanacearum showed attenuated virulence on potato, while RipBT transgenic potato plants exhibited enhanced susceptibility to R. solanacearum. Interestingly, transcriptomic analyses suggest that RipBT may interfere with plant reactive oxygen species (ROS) metabolism during the R. solanacearum infection of potato roots. In addition, the expression of RipBT remarkably suppressed the flg22-induced pathogen-associated molecular pattern-triggered immunity responses, such as the ROS burst. Taken together, RipBT acts as a T3SS effector, promoting R. solanacearum infection on potato and presumably disturbing ROS homeostasis.

Heterologous overexpression of StERF3 triggers cell death in Nicotiana benthamiana.

Programmed cell death plays a crucial role in plant development and disease defense. Here, we report that the expression of StERF3, a potato EAR motif-containing transcription factor, promotes Phytophthora infestans colonization in Nicotiana benthamiana. Transient overexpression of StERF3 induces cell death in N. benthamiana leaves. The substitution of two key amino acids (14th and 19th) in its ERF domain (the DNA binding domain) dramatically altered its cell death-inducing ability. In addition, StERF3△EAR EAR motif-deletion or StERF3AAA mutation abolished the cell death-inducing ability. StERF3 interacted with the co-repressors Topless-related protein 1 (StTPL1) and Topless-related protein 3 (StTPL3) via the EAR motif. Moreover, cell death induced by StERF3 was facilitated by co-expression with StTPL1 or StTPL3. Virus-induced gene silencing (VIGS) of NbTPL1 and NbTPL3 in N. benthamiana compromised the cell death-inducing ability of StERF3. Furthermore, StERF3-induced cell death accompanied with ROS bursts and the upregulation of the respiratory burst oxidase homolog (Rboh) genes NbRbohA and NbRbohC. In addition, several cell death regulator genes, including NbCRTD, NbNCBP, and NbBCPL, and a hypersensitive cell death marker gene Hin1 were upregulated. StERF3 may positively regulate cell death through its EAR motif-mediated transcriptional repressor activity by inhibiting the expression of genes potentially coding the repressor of cell death (CD).

Ralstonia solanacearum type III effector RipV2 encoding a novel E3 ubiquitin ligase (NEL) is required for full virulence by suppressing plant PAMP-triggered immunity.

Ralstonia solanacearum causes bacterial wilt disease in a broad range of plants, primarily through type Ⅲ secreted effectors. However, the R. solanacearum effectors promoting susceptibility in host plants remain limited. In this study, we determined that the R. solanacearum effector RipV2 functions as a novel E3 ubiquitin ligase (NEL). RipV2 was observed to be locali in the plasma membrane after translocatio into plant cells. Transient expression of RipV2 in Nicotiana benthamiana could induce cell death and suppress the flg22-induced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, mediating such effects as attenuation of the expression of several PTI-related genes and ROS bursts. Furthermore, we demonstrated that the conserved catalytic residue is highly important for RipV2. Transient expression of the E3 ubiquitin ligase catalytic mutant RipV2 C403A alleviated the PTI suppression ability and cell death induction, indicating that RipV2 requires its E3 ubiquitin ligase activity for its role in plant-microbe interactions. More importantly, mutation of RipV2 in R. solanacearum reduces the virulence of R. solanacearum on potato. In conclusion, we identified a NEL effector that is required for full virulence of R. solanacearum by suppressing plant PTI.

Proteomic Analysis of Potato Responding to the Invasion of Ralstonia solanacearum UW551 and Its Type III Secretion System Mutant.

The infection of potato with Ralstonia solanacearum UW551 gives rise to bacterial wilt disease via colonization of roots. The type III secretion system (T3SS) is a determinant factor for the pathogenicity of R. solanacearum. To fully understand perturbations in potato by R. solanacearum type III effectors(T3Es), we used proteomics to measure differences in potato root protein abundance after inoculation with R. solanacearum UW551 and the T3SS mutant (UW551△HrcV). We identified 21 differentially accumulated proteins. Compared with inoculation with UW551△HrcV, 10 proteins showed significantly lower abundance in potato roots after inoculation with UW551, indicating that those proteins were significantly downregulated by T3Es during the invasion. To identify their functions in immunity, we silenced those genes in Nicotiana benthamiana and tested the resistance of the silenced plants to the pathogen. Results showed that miraculin, HBP2, and TOM20 contribute to immunity to R. solanacearum. In contrast, PP1 contributes to susceptibility. Notably, none of four downregulated proteins (HBP2, PP1, HSP22, and TOM20) were downregulated at the transcriptional level, suggesting that they were significantly downregulated at the posttranscriptional level. We further coexpressed those four proteins with 33 core T3Es. To our surprise, multiple effectors were able to significantly decrease the studied protein abundances. In conclusion, our data showed that T3Es of R. solanacearum could subvert potato root immune-related proteins in a redundant manner.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A systematic screen of conserved Ralstonia solanacearum effectors reveals the role of RipAB, a nuclear-localized effector that suppresses immune responses in potato.

Both Solanum tuberosum and Ralstonia solanacearum phylotype IIB originated in South America and share a long-term co-evolutionary history. However, our knowledge of potato bacterial wilt pathogenesis is scarce as a result of the technical difficulties of potato plant manipulation. Thus, we established a multiple screening system (virulence screen of effector mutants in potato, growth inhibition of yeast and transient expression in Nicotiana benthamiana) of core type III effectors (T3Es) of a major potato pathovar of phylotype IIB, to provide more research perspectives and biological tools. Using this system, we identified four effectors contributing to virulence during potato infection, with two exhibiting multiple phenotypes in two other systems, including RipAB. Further study showed that RipAB is an unknown protein with a nuclear localization signal (NLS). Furthermore, we generated a ripAB complementation strain and transgenic ripAB-expressing potato plants, and subsequent virulence assays confirmed that R. solanacearum requires RipAB for full virulence. Compared with wild-type potato, transcriptomic analysis of transgenic ripAB-expressing potato plants showed a significant down-regulation of Ca2+ signalling-related genes in the enriched Plant-Pathogen Interaction (PPI) gene ontology (GO) term. We further verified that, during infection, RipAB is required for the down-regulation of four Ca2+ sensors, Stcml5, Stcml23, Stcml-cast and Stcdpk2, and a Ca2+ transporter, Stcngc1. Further evidence showed that the immune-associated reactive oxygen species (ROS) burst is attenuated in ripAB transgenic potato plants. In conclusion, a systematic screen of conserved R. solanacearum effectors revealed an important role for RipAB, which interferes with Ca2+ -dependent gene expression to promote disease development in potato.

Fabricating carbon-nanotubes-based porous foam for superoxide electrochemical sensing through one-step hydrothermal process induced by phytic acid.

The detection of superoxide anions (O2•-) is widely considered as a potential way for cancer diagnosis and the development of enzyme-mimic catalysts is the main challenge in the establishment of electrochemical sensors for O2•- sensing in real samples. Here we present a novel enzyme- and metal-free electrochemical catalyst for superoxide (O2•-) sensing based on the widely-used carbon nanotubes (CNT). Through a one-step hydrothermal process induced by phytic acid (PA), CNT-based porous foam (PACNTF) was successfully obtained. Characterizations demonstrated the enhanced defect and disorder degree of PACNTF after PA treatment, which leaded to the increased active sites of PACNTF for electron transfer and the adhesion of O2•- during the electrochemical process. As a result, the PACNTF presented higher conductivity and larger current response toward O2•- sensing when compared with CNT precursor and CNTF without PA treatment. The sensitivity of PACNTF/SPCE was calculated to be 1230 µA cm-2 mM-1 in the linear range of 0-193.6 µM (R2 = 0.965) and 373 µA cm-2 mM-1 in the linear range of 193.6-1153.6 µM (R2 = 0.995) with a limit of detection of 0.16 µM (S/N = 3). Further, the PACNTF/SPCE presented fast response toward cell-released O2•- stimulated by Zymosan A. The above results indicated that the fabricated sensor holds potential usage in biological samples.

Positive effects of treatment of donor cells with aphidicolin on the preimplantation development of somatic cell nuclear transfer embryos in Chinese Bama mini-pig (Sus Scrofa).

To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 µg/mL APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs. 56.25%, 24.55% vs. 17.86%; P < 0.05). The cleavage rate from the EFC was significantly increased by APC treatment compared to serum starvation only (63.36% vs. 57.01%; P < 0.05). In the ooctyes with the CC, the reconstructed embryos could yield high blastocyst formation rate by APC treatment (29.63%; P < 0.05). In the presence of APC, CC gave rise to the highest cleavage and blastocyst formation rates among the three cell types. Therefore, our results suggest that treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs.

[Fast screening ninety-six pesticides in six kinds of agricultural products by high performance liquid chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry].

A high-throughput method for the determination of 96 pesticides in six kinds of agricultural products by liquid chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry was developed. After extraction with 0.1% acetic acid in acetonitrile solution and concentration, dispersive solid-phase extraction was further utilized to reduce the matrix interference. The chromatographic analysis was performed on a C18 column with methanol and 5 mmol/L ammonium acetate solution as the mobile phases with a gradient elution program. The 96 pesticide residues were analyzed in switching positive and negative modes at the same time. With the optimized mass resolution, accurate mass-to-charge ratio extraction of the target pesticide compounds in full scan mode could eliminate matrix interference effectively. Two-stage threshold-triggered full mass scan mode was utilized to further improve the accuracy of qualitative analysis. The linear ranges of all the 96 pesticides were from 1 microg/L to 200 microg/L with correlation coefficients greater than 0.99. By detecting spiked samples, the detection limits were 5 microg/kg for all the residues and the recoveries were in the range of 58% - 105% with the relative standard deviations (RSDs) between 8.8% and 18.3%.

Determination of residues of cyromazine and its metabolite, melamine, in animal-derived food by gas chromatography-mass spectrometry with derivatization.

A gas chromatography-mass spectrometric (GC-MS) method was established for the determination of cyromazine and its metabolite, melamine, in animal-derived food. Chicken and tilapia muscle samples were spiked with (15)N(3)-melamine, extracted with an acidic acetonitrile/water solution, and defatted with dichloromethane. Egg and milk samples were directly extracted with 3% trichloroacetic acid. The extracts were purified using mixed cation-exchange cartridges, derived with N,O-bis(trimethylsilyl)trifluoroacetamide, and detected by GC-MS. Cyromazine and melamine were quantified by external standard methods except for the determination of melamine in animal muscle, which used an internal standard method. Recoveries ranged from 75.0 to 110.0%, and relative standard deviations were <15.0%. In animal muscle the limits of quantification (LOQs) were 20 microg/kg and the limits of detection (LODs) were 10 microg/kg for cyromazine and melamine. In milk and eggs the LOQs were 10 microg/kg and the LODs were 5 microg/kg for both analytes.

[Determination of acrylamide in foods by gas chromatography-mass spectrometry].

A confirmatory method is presented for the determination of acrylamide in different food products by gas chromatography-mass spectrometry (GC-MS). The method is based on the extraction of acrylamide with and methanol, and purification with Carrez I zinc sulfate) and Carrez II (potassium hexacyanoferrate) solution, followed by bromination onto the acrylamide double bond. The derivative was extracted with ethyl acetate/hexane (4: 1, v/v), and converted to the stable 2-bromopropenamide by dehydrobromination using 10% triethylamine, then analyzed by GC-MS, employing 13C3-acrylamide as internal standard. In-house validation data for flour and bread showed good accuracy and precision of the method. The recoveries of acrylamide in the French fries and bread were all in the range from 80% to 110% after correction of analyte loss by the internal standard at three spike levels of 0.02, 0.05 and 0.2 mg/kg, and relative standard deviations (RSDs) no more than 12.7%. The limits of detection for flour and bread were estimated at 5 microg/kg.

The role of the conserved histidine-aspartate pair in the 'base-off' binding of cobalamins.

The conserved cobalamin-binding domain of glutamate mutase exists as a separate dissociable subunit, MutS. The results obtained from BIAcore analysis indicate that MutS alone, in the absence of E component of glutamate mutase (MutE, catalytic subunit), is capable of binding hydroxocobinamide (OHCbi) with a Kd of 15.4+/-1.6 microM, but fails to bind adenosylcobalamin (AdoCbl). The UV-visible spectrum indicates that histidine ligation to the cobalt atom only occurs when both MutE and MutS are present in the solution. MutS mutants, MutS-D14N and MutS-H16G, are also capable of binding OHCbi, but their binding kinetics altered. Our experimental results show that the electrostatic interaction between histidine-aspartate pair is important in the binding of OHCbi or AdoCbl, no matter whether histidine coordinates to the cobalt atom or not. The catalytic subunit is also involved in histidine ligation to the cobalt atom. Meanwhile, mutation of either His16 or Asp14 significantly impairs the enzyme to cleave the cobalt-carbon bond of AdoCbl.

[Dynamic variation of serum and stool level of interleukin-2, interleukin-6 and interferon-alpha in children with rotavirus enteritis and its relation to clinical manifestations].

OBJECTIVE: This study was conducted to observe dynamically the serum and stool level of IL-2, IL-6 and IFN-alpha with RV enteritis, and to analyse the relation of them and their relation to clinical manifestations. METHODS: The double-antibody sandwich ELISA technique was applied. RESULTS: The positive rate of RV in 119 enteritis cases was 63.0% and the peak month was November. These cytokines had different peak phases in the course of disease. The earliest cytokine which had peak phase was IFN-alpha, the last ones were serum and stool IL-2. There was a negative relation between stool IL-2 and IL-6 (P<0.01). There were positive relation between IL-2 and IFN-alpha, stool IL-6 and IFN-alpha, serum IL-2 and IL-6, stool IL-2 and the recovery time of the enteritis (P<0.01 or P<0.05). Serum and stool level of IL-2 and IFN-alpha had positive relation to the number of lymphocyte plus mononuclear cells (P<0.01 or P<0.05). CONCLUSIONS: IL-2, IL-6 and IFN-alpha play important roles of immunoregulation in RV enteritis, and have close relation to clinical manifestations.