Generation of low-cadmium rice germplasms via knockout of OsLCD using CRISPR/Cas9.

The OsLCD gene, which has been implicated in cadmium (Cd) accumulation in rice, might be a useful target for CRISPR/Cas9 editing. However, the effects of OsLCD gene editing on Cd accumulation, plant growth, and yield traits remain unknown. Here, we used CRISPR/Cas9 to generate oslcd single mutants from indica and japonica rice cultivars. We also generated osnramp5 single mutants and oslcd osnramp5 double mutants in the indica background. When grown in Cd-contaminated paddy soils, all oslcd single mutants accumulated less Cd than the wild types (WTs). Consistent with this, oslcd single mutants grown in Cd-contaminated hydroponic culture accumulated significantly less Cd in the shoots as compared to WTs. This decrease in accumulation probably resulted from the reduction of Cd translocation under Cd stress. Oxidative damage also decreased, and plant growth increased in all oslcd single mutant seedlings as compared to WTs in the presence of Cd. Plant growth and most yield traits, as well essential element concentrations in rice seedling shoots, brown rice, and rice straw, were similar between oslcd single mutants and WTs. In the presence of Cd, Cd concentrations in the brown rice and shoots of oslcd osnramp5 double mutants were significantly decreased compared with WTs as well as osnramp single mutants. Our results suggested that OsLCD knockout may reduce Cd accumulation alone or in combination with other knockout mutations in a variety of rice genotypes; unlike OsNramp5 mutations, OsLCD knockout did not reduce essential element contents. Therefore, OsLCD knockout might be used to generate low-Cd rice germplasms.

LACpG10-HL Functions Effectively in Antibiotic-Free and Healthy Husbandry by Improving the Innate Immunity.

Antibiotics are broadly restricted in modern husbandry farming, necessitating the need for efficient and low-cost immunomodulatory preparations in antibiotic-free and healthful farming. As is known to all, CpG oligonucleotides (CpG-ODNs, an effective innate immunostimulatory agent) recognized by TLR9 in mammals (while TLR21 in avians) could collaborate with some united agent to induce stronger immune responses, but the cost is prohibitively expensive for farmers. Here, considering the coordination between TLR2 and TLR9/TLR21, we firstly proposed the idea that the well-fermented Lactococcus lactis could be utilized as a CpG-plasmid carrier (LACpG10) to enhance the host's innate immunity against pathogenic invasion. In the present study, after obtaining LACpG10-HL from homogenized and lyophilized recombinant strain LACpG10, we treated primary chicken lymphocytes, two cell lines (HD11 and IPEC-J2), and chickens with LACpG10-HL, CpG plasmids (pNZ8148-CpG10), and other stimulants, and respectively confirmed the effects by conducting qRT-PCR, bacterial infection assays, and a zoological experiment. Our data showed that LACpG10-HL could induce excellent innate immunity by regulating autophagy reactions, cytokine expression, and motivating PRRs. Interestingly, despite having no direct antiseptic effect, LACpG10-HL improved the antibacterial capacities of lymphocytes and enterocytes at the first line of defense. Most importantly, water-supplied LACpG10-HL treatment reduced the average adverse event rates, demonstrating that LACpG10-HL maintained its excellent immunostimulatory and protective properties under farming conditions. Our research not only contributes to revealing the satisfactory effects of LACpG10-HL but also sheds new light on a cost-effective solution with optimal immune effects in green, antibiotic-free, and healthful husbandry farming.

Comparative transcriptomics provide new insights into the mechanisms by which foliar silicon alleviates the effects of cadmium exposure in rice.

Silicon (Si) has been shown to alleviate Cd stress in rice. Here, we investigated the beneficial effects of foliar Si in an indica rice Huanghuazhan (HHZ). Our results showed that foliar Si increases the dry weight and decreases Cd translocation in Cd-exposed rice at the grain-filling stage only, implying that the filling stage is critical for foliar Si to reduce Cd accumulation. We also investigated the transcriptomics in flag leaves (FLs), spikelets (SPs), and node Is (NIs) of Cd-exposed HHZ after foliar Si application at the filling stage. Importantly, the gene expression profiles associated with the Si-mediated alleviation of Cd stress were tissue specific, while shared pathways were mediated by Si in Cd-exposed rice tissues. Furthermore, after the Si treatment of Cd-exposed rice, the ATP-binding cassette (ABC)-transporters were mostly upregulated in FL and SP, while the bivalent cation transporters were mostly downregulated in FL and NI, possibly helping to reduce Cd accumulation. The genes associated with essential nutrient transporters, carbohydrate and secondary metabolite biosynthesis, and cytochrome oxidase activity were mostly upregulated in Cd-exposed FL and SP, which may help to alleviate oxidative stress and improve plant growth under Cd exposure. Interestingly, genes responsible for signal transduction were negatively regulated in FL, but positively regulated in SP, by foliar Si. Our results provide transcriptomic evidence that foliar Si plays an active role in alleviating the effects of Cd exposure in rice. In particular, foliar Si may alter the expression pattern of genes associated with transport, biosynthesis and metabolism, and oxidation reduction.

Comparative physiological and transcriptomic analyses illuminate common mechanisms by which silicon alleviates cadmium and arsenic toxicity in rice seedlings.

The inessential heavy metal/loids cadmium (Cd) and arsenic (As), which often co-occur in polluted paddy soils, are toxic to rice. Silicon (Si) treatment is known to reduce Cd and As toxicity in rice plants. To better understand the shared mechanisms by which Si alleviates Cd and As stress, rice seedlings were hydroponically exposed to Cd or As, then treated with Si. The addition of Si significantly ameliorated the inhibitory effects of Cd and As on rice seedling growth. Si supplementation decreased Cd and As translocation from roots to shoots, and significantly reduced Cd- and As-induced reactive oxygen species generation in rice seedlings. Transcriptomics analyses were conducted to elucidate molecular mechanisms underlying the Si-mediated response to Cd or As stress in rice. The expression patterns of the differentially expressed genes in Cd- or As-stressed rice roots with and without Si application were compared. The transcriptomes of the Cd- and As-stressed rice roots were similarly and profoundly reshaped by Si application, suggesting that Si may play a fundamental, active role in plant defense against heavy metal/loid stresses by modulating whole genome expression. We also identified two novel genes, Os01g0524500 and Os06g0514800, encoding a myeloblastosis (MYB) transcription factor and a thionin, respectively, which may be candidate targets for Si to alleviate Cd and As stress in rice, as well as for the generation of Cd- and/or As-resistant plants. This study provides valuable resources for further clarification of the shared molecular mechanisms underlying the Si-mediated alleviation of Cd and As toxicity in rice.

YBX has functional roles in CpG-ODN against cold stress and bacterial infection of Misgurnus anguillicaudatus.

Misgurnus anguillicaudatus (M. anguillicaudatus) is a widely cultivated fish. However, in M. anguillicaudatus breeding, the frequent cold stress during daily breeding could induce immune suppression and increase the risk of infection, causing serious economic loss. Based on existing findings, CpG Oligonucleotides (CpG-ODNs) may be an ideal protective agent for low temperature fish breeding, performing anti-infective when faced with cold stress with cold shock proteins Y box binding proteins (YBX). Although YBX has pleiotropic functions, its roles in CpG-ODNs-mediated immunity (especially under cold situations) remain largely unexplored. To clarify the relationship among them, we identified the YBX1/YBX2 in M. anguillicaudatus and analyzed using a series of bioinformatics methods. After that, we immunized the fish with 3 types of CpG-ODNs and challenged with Aeromonas hydrophila (A. hydrophila). Here we showed that the best anti-bacterial effect of CpG-B was accompanied by the significant upregulation of YBX1. And the detection of the YBX1 downstream effectors confirmed that CpG-B induced the YBX1-mediated Th1 oriented responses to A. hydrophila by regulation of the NLRP3 (Caspase-A/-B), IL-1ß, IL-12 and IFN-γ. Afterwards, we found that under cold stress, CpG-B can activate the NLRP3 and NF-κB pathways through YBX1, a key mediator of anti-A. hydrophila in CpG-B immunization. In this study, we demonstrated CpG-B protection against infection in low temperature, and its interaction with YBX1, expanded the research of CpG-ODN under cold stress, and provided a new CpG-ODN application for low temperature fish farming.

Overexpression of pEGF improved the gut protective function of Clostridium butyricum partly through STAT3 signal pathway.

Clostridium butyricum (C. butyricum) is a probiotic that could promote animal growth and protect gut health. So far, current studies mainly keep up with the basic biological functions of C. butyricum, missing the effective strategy to further improve its protective efficiency. A recent report about C. butyricum alleviating intestinal injury through epidermal growth factor receptor (EGFR) inspired us to bridge this gap by porcine epidermal growth factor (EGF) overexpression. Lacking a secretory overexpression system, we constructed the recombinant strains overexpressing pEGF in C. butyricum for the first time and obtained 4 recombinant strains for highly efficient secretion of pEGF (BC/pPD1, BC/pSPP, BC/pGHF, and BC/pDBD). Compared to the wild-type strain, we confirmed that the expression level ranges of the intestinal development-related genes (Claudin-1, GLUT-2, SUC, GLP2R, and EGFR) and anti-inflammation-related gene (IL-10) in IPECs were upregulated under recombinant strain stimulation, and the growth of Staphylococcus aureus and Salmonella typhimurium was significantly inhibited as well. Furthermore, a particular inhibitor (stattic) was used to block STAT3 tyrosine phosphorylation, resulting in the downregulation on antibacterial effect of recombinant strains. This study demonstrated that the secretory overexpression of pEGF in C. butyricum could upregulate the expression level of EGFR, consequently improving the intestinal protective functions of C. butyricum partly following STAT3 signal activation in IPECs and making it a positive loop. These findings on the overexpression strains pointed out a new direction for further development and utilization of C. butyricum. KEY POINTS: • By 12 signal peptide screening in silico, 4 pEGF overexpression strains of C. butyricum/pMTL82151-pEGF for highly efficient secretion of pEGF were generated for the first time. • The secretory overexpression of pEGF promoted the intestinal development, antimicrobial action, and anti-inflammatory function of C. butyricum. • The overexpressed pEGF upregulated the expression level of EGFR and further magnified the gut protective function of recombinant strains which in turn partly depended on STAT3 signal pathway in IPECs.

Intestinal guard: Human CXCL17 modulates protective response against mycotoxins and CXCL17-mimetic peptides development.

Mycotoxin contamination is an ongoing and growing issue that can create health risks and even cause death. Unfortunately, there is currently a lack of specific therapy against mycotoxins with few side effects. On the other hand, the strategic expression of CXCL17 in mucosal tissues suggests that it may be involved in immune response when exposed to mycotoxins, but the exact role of CXCL17 remains largely unknown. Using Caco-2 as a cell model of the intestinal epithelial barrier (the first line of defense against mycotoxins), we showed that a strong production of ROS-dependent CXCL17 was triggered by mycotoxins via p38 and JNK pathways. Under the mycotoxins stress, CXCL17 modulated enhanced immuno-protective response with a remission of inflammation and apoptosis through PI3K/AKT/mTOR. Based on our observed feedback of CXCL17 to the mycotoxins, we developed the CXCL17-mimetic peptides in silico (CX1 and CX2) that possessed the safety and the capability to ameliorate mycotoxins-inducible inflammation and apoptosis. In this study, the identification of detoxifying feature of CXCL17 is a prominent addition to the chemokine field, pointing out a new direction for curing the mycotoxins-caused damage.

A transcriptomic (RNA-seq) analysis of genes responsive to both cadmium and arsenic stress in rice root.

Cadmium (Cd) and arsenic (As) are nonessential and toxic elements in rice that often occur together in contaminated paddy field soils. To understand whether rice has a common molecular response mechanism against Cd and As toxicity, 30-day seedlings (Oryza sativa L. indica) were exposed separately to Cd and As3+ in hydroponic cultures for up to 7 days. Root transcriptomic analysis of plants exposed to Cd and As for 3 days revealed that a total of 2224 genes in rice roots responded to Cd stress, while 1503 genes responded to As stress. Of these, 841 genes responded to both stressors. The genes in common to Cd and As stress were associated with redox control, stress response, transcriptional regulation, transmembrane transport, signal transduction, as well as biosynthesis and metabolism of macromolecules and sulfur compounds. In plants exposed to Cd and As separately or in combination for 3 and 7 days, qRT-PCR verification revealed that the glutathione metabolism associated gene Os09g0367700 was significantly up-regulated with respect to unexposed controls and had a positive synergistic effect under combined Cd and As stress. In addition, the redox control related genes Os06g0216000, Os07g0638300 and Os01g0294500, the glutathione metabolism related gene Os01g0530900, the cell wall biogenesis related genes Os05g0247800, Os11g0592000 and Os03g0416200, the expression regulation related genes Os07g0597200 and Os02g0168200, and the transmembrane transport related genes Os04g0524500, also varied significantly with respect to an unexposed control and displayed synergistic effects after 7 days of simultaneous exposure to Cd and As. Our identification of a novel set of genes in rice which responded to both Cd and As3+ stress may be of value in mitigating the toxicity of co-contaminated soils. These results also provide a deeper understanding of the molecular mechanisms involved in response to multi-metal/loids stress, and may be used in the genetic improvement of rice varieties.

The feasibility of a novel bioreactor for vaccine production of classical swine fever virus.

The performance of a new type of tide mode culture system was investigated in this study. This novel bioreactor provides two separated stages, liquid and gas, for cell growth requirements. The immobilized cells absorbed the nutrient from medium during the liquid stage and subsequently were exposed directly to fresh air to absorb oxygen during the gas stage. Operating with PK15 cells under optimal conditions, we obtained 2.3×10(9) cells in 500ml reactor. It is 30 times higher than the initial inoculum and about 11 times higher than the production by roller bottle. For the vaccine production of classical swine fever (CSFV), a high virus titer of 2.1×10(9) median tissue culture infective dose (TCID(50)) was yielded which provided exceed 300 doses per milliliter of CSFV solution. Therefore, this new cultural system performed well not only for cell production but also for virus yield. It should be a highly efficient production for the CSFV vaccine and have practical potential in other animal cell culture vaccine.