How Rice Plants Response to Abiotic Stresses.

With the combustion of fossil fuels, unequal and unsustainable energy and land use, and irrational human activities, greenhouse gas emissions remain high, which leads to global warming [...].

RGA1 Negatively Regulates Thermo-tolerance by Affecting Carbohydrate Metabolism and the Energy Supply in Rice.

BACKGROUND: Signal transduction mediated by heterotrimeric G proteins, which comprise the α, ß, and γ subunits, is one of the most important signaling pathways in rice plants. RGA1, which encodes the Gα subunit of the G protein, plays an important role in the response to various types of abiotic stress, including salt, drought, and cold stress. However, the role of RGA1 in the response to heat stress remains unclear. RESULTS: The heat-resistant mutant ett1 (enhanced thermo-tolerance 1) with a new allele of the RGA1 gene was derived from an ethane methyl sulfonate-induced Zhonghua11 mutant. After 45 °C heat stress treatment for 36 h and recovery for 7 d, the survival rate of the ett1 mutants was significantly higher than that of wild-type (WT) plants. The malondialdehyde content was lower, and the maximum fluorescence quantum yield of photosystem II, peroxidase activity, and hsp expression were higher in ett1 mutants than in WT plants after 12 h of exposure to 45 °C. The RNA-sequencing results revealed that the expression of genes involved in the metabolism of carbohydrate, nicotinamide adenine dinucleotide, and energy was up-regulated in ett1 under heat stress. The carbohydrate content and the relative expression of genes involved in sucrose metabolism indicated that carbohydrate metabolism was accelerated in ett1 under heat stress. Energy parameters, including the adenosine triphosphate (ATP) content and the energy charge, were significantly higher in the ett1 mutants than in WT plants under heat stress. Importantly, exogenous glucose can alleviate the damages on rice seedling plants caused by heat stress. CONCLUSION: RGA1 negatively regulates the thermo-tolerance in rice seedling plants through affecting carbohydrate and energy metabolism.

RGA1 alleviates low-light-repressed pollen tube elongation by improving the metabolism and allocation of sugars and energy.

Low-light stress compromises photosynthetic and energy efficiency and leads to spikelet sterility; however, the effect of low-light stress on pollen tube elongation in the pistil remains poorly understood. The gene RGA1, which encodes a Gα-subunit of the heterotrimeric G-protein, enhanced low-light tolerance at anthesis by preventing the cessation of pollen tube elongation in the pistil of rice plants. In this process, marked increases in the activities of acid invertase (INV), sucrose synthase (SUS) and mitochondrial respiratory electron transport chain complexes, as well as the relative expression levels of SUTs (sucrose transporter), SWEETs (sugars will eventually be exported transporters), SUSs, INVs, CINs (cell-wall INV 1), SnRK1A (sucrose-nonfermenting 1-related kinase 1) and SnRK1B, were observed in OE-1 plants. Accordingly, notable increases in contents of ATP and ATPase were presented in OE-1 plants under low-light conditions, while they were decreased in d1 plants. Importantly, INV and ATPase activators (sucrose and Na2 SO3 , respectively) increased spikelet fertility by improving the energy status in the pistil under low-light conditions, and the ATPase inhibitor Na2 VO4 induced spikelet sterility and decreased ATPase activity. These results suggest that RGA1 could alleviate the low-light stress-induced impairment of pollen tube elongation to increase spikelet fertility by promoting sucrose unloading in the pistil and improving the metabolism and allocation of energy.

Abscisic Acid Improves Rice Thermo-Tolerance by Affecting Trehalose Metabolism.

Heat stress that occurs during the flowering stage severely decreases the rice (Oryza sativa L.) seed-setting rate. This damage can be reversed by abscisic acid (ABA), through effects on reactive oxygen species, carbohydrate metabolism, and heat shock proteins, but the exact role of trehalose and ATP in this process remains unclear. Two rice genotypes, namely, Zhefu802 (heat-resistant plant, a recurrent parent) and its near-isogenic line (faded green leaf, Fgl, heat-sensitive plant), were subjected to 38 °C heat stress after being sprayed with ABA or its biosynthetic inhibitor, fluridone (Flu), at the flowering stage. The results showed that exogenous ABA significantly increased the seed-setting rate of rice under heat stress, by 14.31 and 22.40% in Zhefu802 and Fgl, respectively, when compared with the H2O treatment. Similarly, exogenous ABA increased trehalose content, key enzyme activities of trehalose metabolism, ATP content, and F1Fo-ATPase activity. Importantly, the opposite results were observed in plants treated with Flu. Therefore, ABA may improve rice thermo-tolerance by affecting trehalose metabolism and ATP consumption.

The Evolution and Functional Roles of miR408 and Its Targets in Plants.

MicroRNA408 (miR408) is an ancient and highly conserved miRNA, which is involved in the regulation of plant growth, development and stress response. However, previous research results on the evolution and functional roles of miR408 and its targets are relatively scattered, and there is a lack of a systematic comparison and comprehensive summary of the detailed evolutionary pathways and regulatory mechanisms of miR408 and its targets in plants. Here, we analyzed the evolutionary pathway of miR408 in plants, and summarized the functions of miR408 and its targets in regulating plant growth and development and plant responses to various abiotic and biotic stresses. The evolutionary analysis shows that miR408 is an ancient and highly conserved microRNA, which is widely distributed in different plants. miR408 regulates the growth and development of different plants by down-regulating its targets, encoding blue copper (Cu) proteins, and by transporting Cu to plastocyanin (PC), which affects photosynthesis and ultimately promotes grain yield. In addition, miR408 improves tolerance to stress by down-regulating target genes and enhancing cellular antioxidants, thereby increasing the antioxidant capacity of plants. This review expands and promotes an in-depth understanding of the evolutionary and regulatory roles of miR408 and its targets in plants.

Oligomeric Proanthocyanidins Confer Cold Tolerance in Rice through Maintaining Energy Homeostasis.

Oligomeric proanthocyanidins (OPCs) are abundant polyphenols found in foods and botanicals that benefit human health, but our understanding of the functions of OPCs in rice plants is limited, particularly under cold stress. Two rice genotypes, named Zhongzao39 (ZZ39) and its recombinant inbred line RIL82, were subjected to cold stress. More damage was caused to RIL82 by cold stress than to ZZ39 plants. Transcriptome analysis suggested that OPCs were involved in regulating cold tolerance in the two genotypes. A greater increase in OPCs content was detected in ZZ39 than in RIL82 plants under cold stress compared to their respective controls. Exogenous OPCs alleviated cold damage of rice plants by increasing antioxidant capacity. ATPase activity was higher and poly (ADP-ribose) polymerase (PARP) activity was lower under cold stress in ZZ39 than in RIL82 plants. Importantly, improvements in cold tolerance were observed in plants treated with the OPCs and 3-aminobenzamide (PARP inhibitor, 3ab) combination compared to the seedling plants treated with H2O, OPCs, or 3ab alone. Therefore, OPCs increased ATPase activity and inhibited PARP activity to provide sufficient energy for rice seedling plants to develop antioxidant capacity against cold stress.

Increased ATPase activity promotes heat-resistance, high-yield, and high-quality traits in rice by improving energy status.

Heat stress during the reproductive stage results in major losses in yield and quality, which might be mainly caused by an energy imbalance. However, how energy status affected heat response, yield and quality remains unclear. No relationships were observed among the heat resistance, yield, and quality of the forty-nine early rice cultivars under normal temperature conditions. However, two cultivars, Zhuliangyou30 (ZLY30) and Luliangyou35 (LLY35), differing in heat resistance, yield, and quality were detected. The yield was higher and the chalkiness degree was lower in ZLY30 than in LLY35. Decreases in yields and increases in the chalkiness degree with temperatures were more pronounced in LLY35 than in ZLY30. The accumulation and allocation (ratio of the panicle to the whole plant) of dry matter weight and non-structural carbohydrates were higher in ZLY30 than in LLY35 across all sowing times and temperatures. The accumulation and allocation of dry matter weight and non-structural carbohydrates in panicles were higher in ZLY30 than in LLY35. Similar patterns were observed in the relative expression levels of sucrose unloading related genes SUT1 and SUT2 in grains. The ATP content was higher in the grains of LLY35 than in ZLY30, whereas the ATPase activity, which determined the energy status, was significantly lower in the former than in the latter. Thus, increased ATPase activity, which improved the energy status of rice, was the factor mediating the balance among heat-resistance, high-yield, and high-quality traits in rice.

Respiration, Rather Than Photosynthesis, Determines Rice Yield Loss Under Moderate High-Temperature Conditions.

Photosynthesis is an important biophysical and biochemical reaction that provides food and oxygen to maintain aerobic life on earth. Recently, increasing photosynthesis has been revisited as an approach for reducing rice yield losses caused by high temperatures. We found that moderate high temperature causes less damage to photosynthesis but significantly increases respiration. In this case, the energy production efficiency is enhanced, but most of this energy is allocated to maintenance respiration, resulting in an overall decrease in the energy utilization efficiency. In this perspective, respiration, rather than photosynthesis, may be the primary contributor to yield losses in a high-temperature climate. Indeed, the dry matter weight and yield could be enhanced if the energy was mainly allocated to the growth respiration. Therefore, we proposed that engineering smart rice cultivars with a highly efficient system of energy production, allocation, and utilization could effectively solve the world food crisis under high-temperature conditions.

ATP Hydrolysis Determines Cold Tolerance by Regulating Available Energy for Glutathione Synthesis in Rice Seedling Plants.

BACKGROUND: Glutathione (GSH) is important for plants to resist abiotic stress, and a large amount of energy is required in the process. However, it is not clear how the energy status affects the accumulation of GSH in plants under cold stress. RESULTS: Two rice pure lines, Zhongzao39 (ZZ39) and its recombinant inbred line 82 (RIL82) were subjected to cold stress for 48 h. Under cold stress, RIL82 suffered more damages than ZZ39 plants, in which higher increases in APX activity and GSH content were showed in the latter than the former compared with their respective controls. This indicated that GSH was mainly responsible for the different cold tolerance between these two rice plants. Interestingly, under cold stress, greater increases in contents of carbohydrate, NAD(H), NADP(H) and ATP as well as the expression levels of GSH1 and GSH2 were showed in RIL82 than ZZ39 plants. In contrast, ATPase content in RIL82 plants was adversely inhibited by cold stress while it increased significantly in ZZ39 plants. This indicated that cold stress reduced the accumulation of GSH in RIL82 plants mainly due to the inhibition on ATP hydrolysis rather than energy deficit. CONCLUSION: We inferred that the energy status determined by ATP hydrolysis involved in regulating the cold tolerance of plants by controlling GSH synthesis.

Proteomic analysis of salicylic acid regulation of grain filling of two near-isogenic rice (Oryza sativa L.) varieties under soil drying condition.

Grain filling is the final determinant of yield, and this process is susceptible to abiotic stresses. Salicylic acid (SA) regulates grain filling in rice plants. A comparative proteomic study was conducted to understand how SA mediates grain filling under soil drying (SD) condition. Zhefu802 and its near-isogenic line (NIL) were planted in pots in an artificial chamber. SA (100 mg L-1) was applied, followed by SD treatment (with a water potential of -30 to -35 kPa) at anthesis. The results showed that the grain yield and grain weight significantly decreased under SD in Zhefu802, but not in its NIL variety. SD also decreased expression of photosynthesis-related proteins in grains of Zhefu802, which resulted in its poorer drought resistance. Furthermore, the decreased grain filling rate rather than the grain size explained the observed decreased grain weight and grain yield under SD. Interestingly, these reductions were reversed by SA. Expression of proteins involved in glycolysis/TCA circle, starch and sucrose metabolism, antioxidation and detoxication, oxidative phosphorylation, transcription, translation, and signal transduction, were significantly down-regulated under SD and were significantly up-regulated in response to SA. The expression of these proteins was examined at transcriptional level and similar results were obtained. Inhibited expression of these proteins and related pathways contributed to the observed decrease in the grain filling rate of Zhefu802, and application of SA up-regulated expression of these proteins to improve grain weight. The findings of this study provide new insights into grain filling regulation by SA, and offer the scientific foundation for cultivation practice.

Non-Photochemical Quenching Involved in the Regulation of Photosynthesis of Rice Leaves under High Nitrogen Conditions.

Excess and deficient nitrogen (N) inhibit photosynthesis in the leaves of rice plants, but the underlying mechanism is still unclear. N can improve the chlorophyll content and thus affect photon absorption, but the photosynthetic rate does not increase accordingly. To investigate this mechanism, three concentrations of N treatments were applied to two rice varieties, Zhefu802 and Fgl. The results indicated increased chlorophyll content of leaves with an increased N supply. Little discrepancy was detected in Rubisco enzyme activity and Non-photochemical quenching (NPQ) in the high nitrogen (HN) and moderate nitrogen (MN) treatments. The model that photoinhibition occurs in Zhefu802 due to a lack of balance of light absorption and utilization is supported by the higher malondialdehyde (MDA) content, higher H2O2 content, and photoinhibitory quenching (qI) in HN treatment compared with MN treatment. A lower proportion of N in leaf was used to synthesize chlorophyll for Fgl compared with Zhefu802, reducing the likelihood of photoinhibition under HN treatment. In conclusion, HN supply does not allow ideal photosynthetic rate and increases the likelihood of photoinhibition because it does not sustain the balance of light absorption and utilization. Apart from Rubisco enzyme activity, NPQ mainly contributes to the unbalance. These results of this study will provide reference for the effective N management of rice.

Abscisic Acid Negatively Modulates Heat Tolerance in Rolled Leaf Rice by Increasing Leaf Temperature and Regulating Energy Homeostasis.

BACKGROUND: Abscisic acid (ABA) acts as a signaling hormone in plants against abiotic stress, but its function in energy homeostasis under heat stress is unclear. RESULTS: Two rice genotypes, Nipponbare (wild-type, WT) with flat leaves and its mutant high temperature susceptibility (hts) plant with semi-rolled leaves, were subjected to heat stress. We found significantly higher tissue temperature, respiration rate, and ABA and H2O2 contents in leaves as well as a lower transpiration rate and stomatal conductance in hts than WT plants. Additionally, increased expression of HSP71.1 and HSP24.1 as well as greater increases in carbohydrate content, ATP, NAD (H), and dry matter weight, were detected in WT than hts plants under heat stress. More importantly, exogenous ABA significantly decreased heat tolerance of hts plants, but clearly enhanced heat resistance of WT plants. The increases in carbohydrates, ATP, NAD (H), and heat shock proteins in WT plants were enhanced by ABA under heat stress, whereas these increases were reduced in hts plants. CONCLUSION: It was concluded that ABA is a negative regulator of heat tolerance in hts plants with semi-rolled leaves by modulating energy homeostasis.

Acid invertase confers heat tolerance in rice plants by maintaining energy homoeostasis of spikelets.

Heat stress impairs both pollen germination and pollen tube elongation, resulting in pollination failure caused by energy imbalance. Invertase plays a critical role in the maintenance of energy homoeostasis; however, few studies investigated this during heat stress. Two rice cultivars with different heat tolerance, namely, TLY83 (heat tolerant) and LLY722 (heat susceptible), were subjected to heat stress. At anthesis, heat stress significantly decreased spikelet fertility, accompanied by notable reductions in pollen germination on stigma and pollen tube elongation in ovule, especially in LLY722. Acid invertase (INV), rather than sucrose synthase, contributed to sucrose metabolism, which explains the different tolerances of both cultivars. Under heat stress, larger enhancements in NAD(H), ATP, and antioxidant capacity were found in TLY83 compared with LLY722, whereas a sharp reduction in poly(ADP-ribose) polymerase (PARP) activity was found in the former compared with the latter. Importantly, exogenous INV, 3-aminobenzamide (a PARP inhibitor), sucrose, glucose, and fructose significantly increased spikelet fertility under heat stress, where INV activity was enhanced and PARP activity was inhibited. Therefore, INV can balance the energy production and consumption to provide sufficient energy for pollen germination and pollen tube growth under heat stress.

Epidemiological investigation of feline infectious peritonitis in cats living in Harbin, Northeast China from 2017 to 2019 using a combination of an EvaGreen-based real-time RT-PCR and serum chemistry assays.

Feline infectious peritonitis (FIP) is caused by the FIP virus (FIPV), a highly virulent mutant form of feline coronavirus (FCoV). This disease is one of the most important infectious diseases in cats, and it is associated with high mortality, particularly among younger cats. In this study, we isolated a wild-type FIPV HRB-17 epidemic strain from the blood sample of household pet cat exhibiting the characteristic wet-form FIP symptoms, which has been confirmed further by animal infection. Further, we developed an EvaGreen-based real-time RT-PCR assay for the accurate detection of FCoV based on the amplification of the highly conserved FIPV N gene. Then, using a combination of the real-time RT-PCR approach and a serum chemistry assay, we performed an epidemiological survey of FIPV infection in cats living in Harbin City, Northeast China. The results indicated that the EvaGreen-based real-time RT-PCR assay can be used for screening FCoV infection in the affected cats at an analytical detection limit of 8.2 × 101 viral genome copies/µL, but could not effectively distinguish FIPVs from FECVs. Additionally, the results of the epidemiological survey investigating feline blood samples (n = 1523) collected between July 2017 to July 2019 revealed an FIPV prevalence of approximately 12% (189/1523). Maybe, the prevalence would be less than 12% due to the real-time RT-PCR assay could not accurately differentiate FIPV and FECV. Nevertheless, it still highlighted the severity of the FIP epidemic in cats and reiterated the urgent need to develop effective anti-FIP therapeutic agents and anti-FIPV vaccines. As pet cats are household animals, risk communication and continuous region-extended surveillance cat programs are recommended.

Abscisic acid synergizes with sucrose to enhance grain yield and quality of rice by improving the source-sink relationship.

BACKGROUND: Abscisic acid (ABA) and sucrose act as molecular signals in response to abiotic stress. However, how their synergy regulates the source-sink relationship has rarely been studied. This study aimed to reveal the mechanism underlying the synergy between ABA and sucrose on assimilates allocation to improve grain yield and quality of rice. The early indica rice cultivar Zhefu802 was selected and planted in an artificial climate chamber at 32/24 °C (day/night) under natural sunlight conditions. Sucrose and ABA were exogenously sprayed (either alone or in combination) onto rice plants at flowering and 10 days after flowering. RESULTS: ABA plus sucrose significantly improved both the grain yield and quality of rice, which was mainly a result of the higher proportion of dry matter accumulation and non-structural carbohydrates in panicles. These results were mainly ascribed to the large improvement in sucrose transport in the sheath-stems in response to the ABA plus sucrose treatment. In this process, ABA plus sucrose significantly enhanced the contents of starch, gibberellic acids, and zeatin ribosides as well as the activities and gene expression of enzymes involved in starch synthesis in grains. Additionally, remarkable increases in trehalose content and expression levels of trehalose-6-phosphate synthase1, trehalose-6-phosphate phosphatase7, and sucrose non-fermenting related protein kinase 1A were also found in grains treated with ABA plus sucrose. CONCLUSION: The synergy between ABA and sucrose increased grain yield and quality by improving the source-sink relationship through sucrose and trehalose metabolism in grains.

Dual priming oligonucleotide (DPO)-based real-time RT-PCR assay for accurate differentiation of four major viruses causing porcine viral diarrhea.

Currently in China, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are the major causes of porcine viral diarrhea, and mixed infections in clinics are common, resulting in significant economic losses in pig industry. Here, a dual priming oligonucleotide (DPO)-based multiplex real-time SYBR Green RT-PCR assay were developed for accurately differentiating PEDV, TGEV, PoRV, and PDCoV in clinical specimens targeting the N gene of TGEV, PEDV, and PDCoV, and the VP7 gene of PoRV. Results showed that the DPO primer allowed a wider annealing temperature range (40-65 °C) and had a higher priming specificity compared to conventional primer, in which more than 3 nucleotides in the 3'- or 5'-segment of DPO primer mismatched with DNA template, PCR amplification efficiency would decrease substantially or extension would not proceed. DPO-based multiplex real-time RT-PCR method had analytical detection limit of 8.63 × 102 copies/µL, 1.92 × 102 copies/µL, 1.74 × 102 copies/µL, and 1.76 × 102 copies/µL for PEDV, TGEV, PoRV, and PDCoV in clinical specimens, respectively. A total of 672 clinical specimens of piglets with diarrheal symptoms were collected in Northeastern China from 2017 to 2018 followed by analysis using the assay, and epidemiological investigation results showed that PEDV, TGEV, PoRV, and PDCoV prevalence was 19.05%, 5.21%, 4.32%, and 3.87%, respectively. The assay developed in this study showed higher detection accuracy than conventional RT-PCR method, suggesting a useful tool for the accurate differentiation of the four major viruses causing porcine viral diarrhea in practice.

An EGFP-marked recombinant lactobacillus oral tetravalent vaccine constitutively expressing α, ε, ß1, and ß2 toxoids for Clostridium perfringens elicits effective anti-toxins protective immunity.

Clostridium perfringens is a common opportunistic pathogen endangering livestock and poultry breeds. Here, using enhanced green fluorescent protein as screening marker, a recombinant lactobacillus tetravalent vaccine constitutively expressing α, ε, ß1, and ß2 toxoids of C. perfringens was developed, and its immunogenicity in mice was investigated via oral administration. This probiotic vaccine could effectively induce antigen-specific secretory IgA (sIgA)-based mucosal and IgG-based humoral immune responses, and significantly high levels (p< 0.05) of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and IFN-γ were produced in immunized mice. Moreover, lymphoproliferation and percentage of CD4+ and CD8+ T cells significantly increased in mice of the probiotic vaccine group. Challenge experiments were performed in mice with C. perfringens toxinotypes A, C, and D crude toxins to evaluate protection efficiency of the probiotic vaccine, using a commercial inactivated C. perfringens vaccine made by C. perfringens toxinotypes A, C, and D as vaccine control. We observed 80% protection rate in the probiotic vaccine group, which was higher than commercial vaccine group, whereas all mice in control groups died and obvious histopathological changes were observed in liver, spleen, kidney, and intestines of mice. Significantly, we compared the immunogenicity and protection efficiency of lactobacillus constitutive expression system and lactobacillus inducible expression system, and results showed that lactobacillus constitutive expression system has obvious advantages. Our study clearly demonstrated that the probiotics vaccine could effectively induce mucosal, humoral, and cellular immunity, and provide effective protection against C. perfringens toxins, suggesting a promising strategy for the development of oral vaccine against C. perfringens.

Dendritic Cell Targeting of Bovine Viral Diarrhea Virus E2 Protein Expressed by Lactobacillus casei Effectively Induces Antigen-Specific Immune Responses via Oral Vaccination.

Bovine viral diarrhea caused by bovine viral diarrhea virus (BVDV) is an important disease in cattle, resulting in significant economic losses to the cattle industry worldwide. In order to develop an effective vaccine against BVDV infection, we constructed a dendritic cell (DC)-targeting oral probiotic vaccine (pPG-E2-DCpep/LC W56) using Lactobacillus casei as antigen delivery carrier to express BVDV glycoprotein E2 fused with DC-targeting peptide, and the immunogenicity of orally administered probiotic vaccine was evaluated in mice model. Our results showed that after immunization with the probiotic vaccine, significantly levels of antigen-specific sera IgG and mucosal sIgA antibodies (p < 0.05) with BVDV-neutralizing activity were induced in vivo. Challenge experiment showed that pPG-E2-DCpep/LC W56 can provide effective immune protection against BVDV, and BVDV could be effectively cleared from the intestine of immunized mice post-challenge. Moreover, the pPG-E2-DCpep/LC W56 could efficiently activate DCs in the intestinal Peyer's patches, and significantly levels of lymphoproliferative responses, Th1-associated IFN-γ, and Th2-associated IL-4 were observed in mice immunized with pPG-E2-DCpep/LC W56 (p < 0.01). Our results clearly demonstrate that the probiotic vaccine could efficiently induce anti-BVDV mucosal, humoral, and cellular immune responses via oral immunization, indicating a promising strategy for the development of oral vaccine against BVDV.

N-linked glycosylation sites in G protein of infectious hematopoietic necrosis virus (IHNV) affect its virulence and immunogenicity in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis in salmonid fish, resulting in substantial economic losses to the aquaculture industry worldwide. The G protein, which harbors the major antigenic determinants of IHNV, is an envelope glycoprotein that plays an important role in both pathogenicity and immunogenicity of IHNV. Previous studies have demonstrated that changes to viral glycosylation sites may affect replication and immunogenicity, but little is known about the specific contributions of G protein glycosylation to IHNV replication and pathogenicity. In this study, we predicted four N-linked glycosylation sites at position 56, 379, 401, and 438 Asp (N) in G protein, and using a reverse genetics system developed in our laboratory, constructed nine recombinant viruses with single, triple, or quadruple glycosylation site disruptions using alanine substitutions in the following combinations: rIHNV-N56A, rIHNV-N379A, rIHNV-N401A, rIHNV-N438A, rIHNV-N56A-N379A-N401A, rIHNV-N56A-N379A-N438A, rIHNV-N56A-N401A-N438A, rIHNV-N379A-N401A-N438A, and rIHNV-N56A-N379A-N401A-N438A. Our results confirmed that all four asparagines are sites of N-linked glycosylation, and Western blot confirmed that mutation of each predicted N-glycosylation sited impaired glycosylation. Among the nine recombinant IHNVs, replication levels decreased significantly in vitro and in vivo in the triple and quadruple mutants that combined mutation of asparagines 401 and 438, indicating the importance of glycosylation at these sites for efficient replication. Moreover, juvenile rainbow trout mortality after challenge by each of the nine mutants showed that, while eight mutants suffered almost 100% cumulative mortality over 30 days, the mutant with a single alanine substitution at position 438 resulted in cumulative mortality of less than 50% over 30 days. This mutant also elicited specific anti-IHNV IgM production earlier than other mutants, suggesting that glycosylation of asparagine 438 may be important for viral immune escape. In conclusion, our study reveals the effect of G protein glycosylation on the pathogenicity and immunogenicity of IHNV and provides a foundation for developing a live-attenuated vaccine.

Oral immunization of mice with a probiotic Lactobacillus casei constitutively expressing the α-toxoid induces protective immunity against Clostridium perfringens α-toxin.

Clostridium perfringens α-toxin is one of the major virulence factors during C. perfringens infection, causing hemolysis of erythrocytes in various species. Here, genetically engineered Lactobacillus casei (pPG-α/L. casei 393) constitutively expressing the toxoid of C. perfringens α-toxin was generated and its immunogenicity in mice for induction of protective immunity against the α-toxin was evaluated via oral immunization. The α-toxoid was constitutively expressed by pPG-α/L. casei 393 without a specific inducer, as confirmed by western blotting, laser confocal microscopy, and flow cytometry. In an experiment on BALB/c mice to evaluate the oral immunogenicity of pPG-α/L. casei 393, significant levels of a specific secretory IgA (sIgA) antibody in the intestinal mucus and feces and an IgG antibody in the serum of the probiotic vaccine group were detected after booster immunization (p < 0.05) as compared with the pPG/L. casei 393 and PBS control groups. These antibodies effectively neutralized C. perfringens natural α-toxin. Moreover, significantly higher levels of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and interferon (IFN) γ in the serum and increased proliferation of spleen lymphocytes obtained from mice orally immunized with pPG-α/L. casei 393 were detected. With a commercial C. perfringens type A inactivated vaccine as a control, immune protection provided by the probiotic vaccine against C. perfringens α-toxin was evaluated, and 90% and 80% protection rates were observed, respectively. Therefore, strain pPG-α/L. casei 393 effectively elicited mucosal, humoral, and cellular immunity, suggesting that pPG-α/L. casei 393 is a promising candidate for development of a vaccine against C. perfringens α-toxin.