Virus-derived circular RNAs populate hepatitis C virus-infected cells.

It is known that pre-mRNAs in eukaryotic cells can be processed to circular RNAs by a backsplicing mechanism. Circular RNAs have great stability and can sequester proteins or small RNAs to exert functions on cellular pathways. Because viruses often exploit host pathways, we explored whether the RNA genome of the cytoplasmic hepatitis C virus is processed to yield virus-derived circRNAs (vcircRNAs). Computational analyses of RNA-seq experiments predicted that the viral RNA genome is fragmented to generate hundreds of vcircRNAs. More than a dozen of them were experimentally verified by rolling-circle amplification. VcircRNAs that contained the viral internal ribosome entry site were found to be translated into proteins that displayed proviral functions. Furthermore, two highly abundant, nontranslated vcircRNAs were shown to enhance viral RNA abundance. These findings argue that novel vcircRNA molecules modulate viral amplification in cells infected by a cytoplasmic RNA virus.

Participation of shrimp pva-miR-166 in hemocyte homeostasis by modulating apoptosis-related gene PvProsaposin during white spot syndrome virus infection.

Tiny controllers referred to as microRNAs (miRNAs) impede the expression of genes to modulate biological processes. In invertebrates, particularly in shrimp as a model organism, it has been demonstrated that miRNAs play a crucial role in modulating innate immune responses against viral infection. By analyzing small RNAs, we identified 60 differentially expressed miRNAs (DEMs) in Penaues vannamei hemocytes following infection with white spot syndrome virus (WSSV). We predicted the target genes of WSSV-responsive miRNAs, shedding light on their participation in diverse biological pathways. We are particularly intrigued by pva-miR-166, which is the most notably elevated miRNA among 60 DEMs. At 24 h post-infection (hpi), the negative correlation between the expression of pva-miR-166 and its target gene, PvProsaposin, was evident and their interaction was confirmed by a reduction in luciferase activity in vitro. Suppression of PvProsaposin in unchallenged shrimp led to decreased survival rates, reduced total hemocyte count (THC), and increased caspase 3/7 activity, suggesting its significant role in maintaining hemocyte homeostasis. In WSSV-infected shrimp, a lower number of hemocytes corresponded to a lower WSSV load, but higher shrimp mortality was observed when PvProsaposin was suppressed. Conformingly, the introduction of the pva-miR-166 mimic to WSSV-infected shrimp resulted in decreased levels of PvProsaposin transcripts, a significant loss of THC, and an increase in the hemocyte apoptosis. Taken together, we propose that pva-miR-166 modulates hemocyte homeostasis during WSSV infection by suppressing the PvProsaposin, an anti-apoptotic gene. PvProsaposin inhibition disrupts hemocyte homeostasis, rendering the shrimp's inability to withstand WSSV invasion.IMPORTANCEGene regulation by microRNAs (miRNAs) has been reported during viral infection. Furthermore, hemocytes serve a dual role, not only producing various immune-related molecules to combat viral infections but also acting as a viral replication site. Maintaining hemocyte homeostasis is pivotal for the shrimp's survival during infection. The upregulated miRNA pva-miR-166 could repress PvProsaposin expression in shrimp hemocytes infected with WSSV. The significance of PvProsaposin in maintaining hemocyte homeostasis via apoptosis led to reduced survival rate, decreased total hemocyte numbers, and elevated caspase 3/7 activity in PvProsaposin-silenced shrimp. Additionally, the inhibitory ability of pva-miR-166-mimic and dsRNA-PvProsaposin on the expression of PvProsaposin also lowered the THC, increases the hemocyte apoptosis, resulting in a lower WSSV copy number. Ultimately, the dysregulation of the anti-apoptotic gene PvProsaposin by pva-miR-166 during WSSV infection disrupts hemocyte homeostasis, leading to an immunocompromised state in shrimp, rendering them incapable of surviving WSSV invasion.

Identification of immune-responsive circular RNAs in shrimp (Litopenaeus vannamei) upon yellow head virus infection.

Circular RNAs (circRNAs) are a subclass of non-coding RNAs (ncRNAs) formed through a process known as back-splicing. They play a crucial role in the genetic regulation of various biological processes. Currently, circRNAs have been identified as participants in the antiviral response within mammalian cells. However, circRNAs in shrimp infected with the yellow head virus (YHV) remain largely unexplored. Therefore, this study aims to identify circRNAs in the hemocytes of Litopenaeus vannamei during YHV infection. We discovered 358 differentially expressed circRNAs (DECs), with 177 of them being up-regulated and 181 down-regulated. Subsequently, eight DECs, including circ_alpha-1-inhibitor 3, circ_CDC42 small effector protein 2, circ_hemicentin 2, circ_integrin alpha V, circ_kazal-type proteinase inhibitor, circ_phenoloxidase 3, circ_related protein rab-8B, and circ_protein toll-like, were randomly selected for analysis of their expression patterns during YHV infection using qRT-PCR. Furthermore, the circRNAs' characteristics were confirmed through PCR, RNase R treatment, and Sanger sequencing, all of which were consistent with the features of circRNAs. These findings contribute to a better understanding of circRNAs' involvement in the antiviral response in shrimp.

Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor.

Acute hepatopancreatic necrosis disease (AHPND) caused by PirABVP-producing strain of Vibrio parahaemolyticus, VPAHPND, has seriously impacted the shrimp production. Although the VPAHPND toxin is known as the VPAHPND virulence factor, a receptor that mediates its action has not been identified. An in-house transcriptome of Litopenaeus vannamei hemocytes allows us to identify two proteins from the aminopeptidase N family, LvAPN1 and LvAPN2, the proteins of which in insect are known to be receptors for Cry toxin. The membrane-bound APN, LvAPN1, was characterized to determine if it was a VPAHPND toxin receptor. The increased expression of LvAPN1 was found in hemocytes, stomach, and hepatopancreas after the shrimp were challenged with either VPAHPND or the partially purified VPAHPND toxin. LvAPN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the stomach after VPAHPND toxin challenge. In addition, LvAPN1 silencing prevented the toxin from causing severe damage to the hemocytes and sustained both the total hemocyte count (THC) and the percentage of living hemocytes. We found that the rLvAPN1 directly bound to both rPirAVP and rPirBVP toxins, supporting the notion that silencing of LvAPN1 prevented the VPAHPND toxin from passing through the cell membrane of hemocytes. We concluded that the LvAPN1 was involved in AHPND pathogenesis and acted as a VPAHPND toxin receptor mediating the toxin penetration into hemocytes. Besides, this was the first report on the toxic effect of VPAHPND toxin on hemocytes other than the known target tissues, hepatopancreas and stomach.

Transcriptome-based insights into the regulatory role of immune-responsive circular RNAs in Litopanaeus vannamei upon WSSV infection.

Circular RNAs (circRNAs) are non-coding RNAs (ncRNAs) originating from a post-transcriptional modification process called back-splicing. Despite circRNAs being traditionally considered by-products rather than independently functional, circRNAs play many vital roles, such as in host immunity during viral infection. However, in shrimp, these remain largely unexplored. Therefore, this study aims to identify circRNAs in Litopenaeus vannamei in the context of WSSV infection, one of the most eradicative pathogens threatening shrimp populations worldwide. We identified 290 differentially expressed circRNAs (DECs) in L. vannamei upon WSSV infection. Eight DECs were expressed from their parental genes, including alpha-1-inhibitor-3, calpain-B, integrin-V, hemicentin-2, hemocytin, mucin-17, proPO2, and rab11-FIP4. These were examined quantitatively by qRT-PCR, which revealed the relevant expression profiles to those obtained from circRNA-Seq. Furthermore, the structural and chemical validation of the DECs conformed to the characteristics of circRNAs. One of the functional properties of circRNAs as a miRNA sponge was examined via the interaction network between DECs and WSSV-responsive miRNAs, which highlighted the targets of miRNA sponges. Our discovery could provide insight into the participation of these ncRNAs in shrimp antiviral responses.

Host-derived circular RNAs display proviral activities in Hepatitis C virus-infected cells.

Viruses subvert macromolecular pathways in infected host cells to aid in viral gene amplification or to counteract innate immune responses. Roles for host-encoded, noncoding RNAs, including microRNAs, have been found to provide pro- and anti-viral functions. Recently, circular RNAs (circRNAs), that are generated by a nuclear back-splicing mechanism of pre-mRNAs, have been implicated to have roles in DNA virus-infected cells. This study examines the circular RNA landscape in uninfected and hepatitis C virus (HCV)-infected liver cells. Results showed that the abundances of distinct classes of circRNAs were up-regulated or down-regulated in infected cells. Identified circRNAs displayed pro-viral effects. One particular up-regulated circRNA, circPSD3, displayed a very pronounced effect on viral RNA abundances in both hepatitis C virus- and Dengue virus-infected cells. Though circPSD3 has been shown to bind factor eIF4A3 that modulates the cellular nonsense-mediated decay (NMD) pathway, circPSD3 regulates RNA amplification in a pro-viral manner at a post-translational step, while eIF4A3 exhibits the anti-viral property of the NMD pathway. Findings from the global analyses of the circular RNA landscape argue that pro-, and likely, anti-viral functions are executed by circRNAs that modulate viral gene expression as well as host pathways. Because of their long half-lives, circRNAs likely play hitherto unknown, important roles in viral pathogenesis.

Shrimp Vago5 activates an innate immune defense upon bacterial infection.

Acute hepatopancreatic necrosis disease, AHPND, caused by a specific Vibrio parahaemolyticus (VPAHPND) strain, results in a great loss of global shrimp production. This study performed suppression subtractive hybridization (SSH) to identify differentially expressed genes from white shrimp Penaeus vannamei hemocyte upon VPAHPND infection. Among the immune-related genes identified, Vago5, kunitz, secretory leukocyte proteinase inhibitor, and profilin are the most abundant genes classified as the up-regulated genes in the SSH library. The qRT-PCR results show that only Vago5 was highly up-regulated at 3 and 6 h post-VPAHPND challenge, whereas kunitz, secretory leukocyte proteinase inhibitor, and profilin were highly up-regulated at 48 h post-VPAHPND challenge. As an early VPAHPND infection-responsive gene, Vago5 was further functional characterized by RNA interference. Knockdown of Vago5 gene resulted in the significantly rapid increase of shrimp mortality and the number of bacteria in the stomach and hepatopancreas upon VPAHPND infection. Moreover, downstream genes of Toll, IMD, and JAK/STAT pathways and phenoloxidase system were analyzed for the expression in the VPAHPND-infected shrimp hemocyte after dsVago5 treatment. Vago5 gene knockdown resulted in a significant decrease in transcript levels of PEN4, TNF, and PO2 genes as well as PO activity in the hemolymph, suggesting that Vago5 might modulate antibacterial infection through activation of the genes in the NF-κB mediated pathways, JAK/STAT pathway, and phenoloxidase system.

Transcriptome profiling reveals the novel immunometabolism-related genes against WSSV infection from Fenneropenaeus merguiensis.

The white spot syndrome virus (WSSV) has been considered a serious threat to shrimp aquaculture. Besides, the activation of cell metabolism as an immune reaction to the virus is now recognized as a piece of the pivotal puzzle of the antiviral responses. Hence, this study explores the relationship between metabolic gene expression and antiviral responses in shrimp using transcriptome analysis. The RNA-seq libraries of Fenneropenaeus merguensis hemocytes after WSSV challenge at early (6 hpi) and late (24 hpi) stages of infection were analyzed to identify differentially expressed genes (DEGs) that the WSSV subverted the expression. One-hundred-thirty-three DEGs that were expressed in response to WSSV infection at both stages were identified. Based on the GO annotation, they were related to innate immunity and metabolic pathway. The expression correlation between "full term" (NGS) and qRT-PCR of 16 representative DEGs is shown. Noticeably, the expression profiles of all the selected metabolic genes involved in glucose metabolism, lipid metabolism, amino acid metabolism, and nucleotide metabolism showed a specific correlation between NGS and qRT-PCR upon WSSV infection. Of these, we further characterized the function related to the WSSV response of glutamine: fructose-6-phosphate aminotransferase (FmGFAT), the rate-limiting enzyme of the hexosamine biosynthesis pathway, which was found to be up-regulated at the late stage of WSSV infection. Suppression of FmGFAT by RNA interference resulted in postponing the death of WSSV-infected shrimp and reduction of viral copy number. These results suggested that the FmGFAT is linked between metabolic change and WSSV responses in shrimp, where the virus-induced metabolic rewiring hijack biological compounds and/or energy sources to benefit the viral replication process.

The interferon-like proteins, Vagos, in Fenneropenaeus merguiensis elicit antimicrobial responses against WSSV and VPAHPND infection.

The Vago interferon-like protein participates in the interplay between interferon regulatory factors and the expression of immune-responsive genes. Vago was initially perceived to participate only in the antiviral activation through JAK/STAT pathway. However, certain isoforms of Vago can stimulate antimicrobial responses. Here we identify Vago isoforms in Fenneropenaeus merguiensis (FmVagos) and how they function in antiviral and antibacterial responses against highly invasive pathogens, including white spot syndrome virus (WSSV) and Vibrio parahaemolyticus (VPAHPND). Three isoforms of FmVagos were identified: FmVago4, FmVago5a, and FmVago5b, and expressed throughout tissues of the shrimp. During infection, FmVago4, FmVago5a, and FmVago5b, were up-regulated after WSSV and VPAHPND challenges at certain time points. Pre-injection of purified recombinant FmVago4 (rVago4), FmVago5a (rVago5a), and FmVago5b (rVago5b) proteins could significantly reduce the mortality of shrimp upon WSSV infection, while the increase of survival rate of VPAHPND-infected shrimp was observed only in rVago4 treatment. The immunity routes that FmVagos might instigate in response to the pathogens were examined by qRT-PCR, revealing that the JAK/STAT pathway was activated after introducing rVago4, rVago5a, and rVago5b, while the Toll/IMD pathway and proPO system, combined with PO activity, were provoked only in the rVago4-treated shrimp. Our finding suggests cross-talk between Vago's antiviral and antimicrobial responses in shrimp immunity. These findings complement previous studies in which Vago and its specific isoform could promote viral and bacterial clearance in shrimp.

Differentially expressed genes in hemocytes of Litopenaeus vannamei challenged with Vibrio parahaemolyticus AHPND (VPAHPND) and VPAHPND toxin.

While toxin-harboring Vibrio parahaemolyticus has been previously established as the causative agent of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, information on the mechanistic processes that happen in the host during infection is still lacking. Here, we examined the expression responses of the shrimp hemocyte transcriptome to V. parahaemolyticus AHPND (VPAHPND) by RNA sequencing (RNA-seq). Using libraries (SRA accession number SRP137285) prepared from shrimp hemocytes under experimental conditions, a reference library was de novo assembled for gene expression analysis of VPAHPND-challenged samples at 0, 3/6, and 48 h post infection (hpi). Using the library from 0-hpi as the control, 359 transcripts were found to be differentially expressed in the 3/6-hpi library, while 429 were differentially expressed in the 48-hpi library. The expression patterns reported in the RNA-seq of 9 representative genes such as anti-lipopolysaccharide factor (LvALF), crustin p (CRU), serpin 3 (SER), C-type lectin 3 (CTL), clottable protein 2 (CLO), mitogen-activated protein kinase kinase 4 (MKK4), P38 mitogen-activated protein kinase (P38), protein kinase A regulatory subunit 1 (PKA) and DNAJ homolog subfamily C member 1-like (DNJ) were validated by qRT-PCR. The expression of these genes was also analyzed in shrimp that were injected with the partially purified VPAHPND toxin. A VPAHPND toxin-responsive gene, LvALF was identified, and its function was characterized by RNA interference. LvALF knockdown resulted in significantly rapid increase of shrimp mortality caused by toxin injection. Protein-protein interaction analysis by molecular docking suggested that LvALF possibly neutralizes VPAHPND toxin through its LPS-binding domain. The data generated in this study provide preliminary insights into the differences in the immune response of shrimp to the bacterial and toxic aspect of VPAHPND as a disease.

The impact of hot-air oven drying combined with Bacillus subtilis KC3 inoculation on quality characteristics and microbial profiles of salted shrimp paste.

This study used hot-air oven drying with Bacillus subtilis KC3 inoculation to improve shrimp paste production. The fermentation rate, quality characteristics, as well as microbial profiles, were compared to those produced using sun-drying with/without inoculation. B. subtilis inoculation increased the degree of hydrolysis of shrimp paste (22.3-32.1 %) during fermentation, compared to those without inoculation (12.7-25.4 %), regardless of different drying methods (p < 0.05). The result corresponded to the faster development of shrimp paste characteristics, particularly color and browning intensity of inoculated samples when fermented for the same duration. More abundant halophilic, proteolytic, and lipolytic bacteria (p < 0.05) were also obtained in inoculated samples, confirming accelerated fermentation. Interestingly, there was no difference in proximate composition, pH, and aw among samples (p > 0.05), which were still in the range regulated by the product's standard. However, the protein and lipid degradation products such as nitrogen contents, 5'-nucleotides, free fatty acids or TBARS values, varied among samples, potentially influencing the release of desirable flavor precursors to a certain extent. The inoculation increased microbial richness and evenness/uniformity, according to next-generation sequencing analysis on microbiota profiles. Pearson's correlation also revealed that these microbiota profiles were correlated with several desirable quality characteristics to varying degrees. Thus, combining the inoculation with B. subtilis KC3 can enhance shrimp paste fermentation and quality when produced using an alternative hot-air oven while maintaining quality characteristics. The findings suggested the possibility of achieving a more efficient and consistent production process for shrimp paste.

Transcriptomics and starch biosynthesis analysis in leaves and developing seeds of mung bean provide a basis for genetic engineering of starch composition and seed quality.

Mung bean starch is distinguished by its exceptional high amylose content and regulation of starch biosynthesis in leaves and storage tissues, such as seeds, share considerable similarities. Genetic engineering of starch composition and content, requires detailed knowledge of starch biosynthetic gene expression and enzymatic regulation. In this study we applied detailed transcriptomic analyses to unravel the global differential gene expression patterns in mung bean leaves and in seeds during various stages of development. The objective was to identify candidate genes and regulatory mechanisms that may enable generation of desirable seed qualities through the use of genetic engineering. Notable differences in gene expression, in particular low expression of the Protein Targeting to Starch (PTST), starch synthase (SS) 3, and starch branching enzyme1 (SBE1) encoding genes in developing seeds as compared to leaves were evident. These differences were related to starch molecular structures and granule morphologies. Specifically, the starch molecular size distribution at different stages of seed development correlated with the starch biosynthesis gene expression of the SBE1, SS1, granule-bound starch synthases (GBSS) and isoamylase 1 (ISA1) encoding genes. Furthermore, putative hormonal and redox controlled regulation were observed, which may be explained by abscisic acid (ABA) and indole-3-acetic acid (IAA) induced signal transduction, and redox regulation of ferredoxins and thioredoxins, respectively. The morphology of starch granules in leaves and developing seeds were clearly distinguishable and could be correlated to differential expression of SS1. Here, we present a first comprehensive transcriptomic dataset of developing mung bean seeds, and combined these findings may enable generation of genetic engineering strategies of for example starch biosynthetic genes for increasing starch levels in seeds and constitute a valuable toolkit for improving mung bean seed quality.

Exploring the therapeutic potential of Thai medicinal plants: in vitro screening and in silico docking of phytoconstituents for novel anti-SARS-CoV-2 agents.

BACKGROUND: The high virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), has triggered global health and economic concerns. The absence of specific antiviral treatments and the side effects of repurposed drugs present persistent challenges. This study explored a promising antiviral herbal extract against SARS-CoV-2 from selected Thai medicinal plants based on in vitro efficacy and evaluated its antiviral lead compounds by molecular docking. METHODS: Twenty-two different ethanolic-aqueous crude extracts (CEs) were rapidly screened for their potential activity against porcine epidemic diarrhea virus (PEDV) as a surrogate using a plaque reduction assay. Extracts achieving ≥ 70% anti-PEDV efficacy proceeded to the anti-SARS-CoV-2 activity test using a 50% tissue culture infectious dose method in Vero E6 cells. Molnupiravir and extract-free media served as positive and negative controls, respectively. Potent CEs underwent water/ethyl acetate fractionation to enhance antiviral efficacy, and the fractions were tested for anti-SARS-CoV-2 performance. The fraction with the highest antiviral potency was identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Molecular docking analyses of these compounds against the main protease (Mpro) of SARS-CoV-2 (6LU7) were performed to identify antiviral lead molecules. The top three hits were further evaluated for their conformational stability in the docked complex using molecular dynamics (MD) simulation. RESULTS: The water fraction of mulberry (Morus alba Linn.) leaf CE (WF-MLCE) exhibited the most potent anti-SARS-CoV-2 efficacy with low cytotoxicity profile (CC50 of ~ 0.7 mg/mL), achieving 99.92% in pre-entry mode and 99.88% in postinfection treatment mode at 0.25 mg/mL. Flavonoids and conjugates were the predominant compounds identified in WF-MLCE. Molecular docking scores of several flavonoids against SARS-CoV-2 Mpro demonstrated their superior antiviral potency compared to molnupiravir. Remarkably, myricetin-3-O-ß-D-galactopyranoside, maragrol B, and quercetin 3-O-robinobioside exhibited binding energies of ~ - 9 kcal/mol. The stability of each ligand-protein complex of these compounds with the Mpro system showed stability during MD simulation. These three molecules were pronounced as antiviral leads of WF-MLCE. Given the low cytotoxicity and high antiviral potency of WF-MLCE, it holds promise as a candidate for future therapeutic development for COVID-19 treatment, especially considering its economic and pharmacological advantages.

CopG1, a Novel Transcriptional Regulator Affecting Symbiosis in Bradyrhizobium sp. SUTN9-2.

The symbiotic interaction between leguminous and Bradyrhizobium sp. SUTN9-2 mainly relies on the nodulation process through Nod factors (NFs), while the type IV secretion system (T4SS) acts as an alternative pathway in this symbiosis. Two copies of T4SS (T4SS1 and T4SS2) are located on the chromosome of SUTN9-2. ΔT4SS1 reduces both nodule number and nitrogenase activity in all SUTN9-2 nodulating legumes. The functions of three selected genes (copG1, traG1, and virD21) within the region of T4SS1 were examined. We generated deleted mutants and tested them in Vigna radiata cv. SUT4. ΔtraG1 and ΔvirD21 exhibited lower invasion efficiency at the early stages of root infection but could be recently restored. In contrast, ΔcopG1 completely hindered nodule organogenesis and nitrogenase activity in all tested legumes. ΔcopG1 showed low expression of the nodulation gene and ttsI but exhibited high expression levels of the T4SS genes, traG1 and trbE1. The secreted proteins from ΔT4SS1 were down-regulated compared to the wild-type. Although ΔcopG1 secreted several proteins after flavonoid induction, T3SS (nopP and nopX) and the C4-dicarboxylate transporter (dct) were not detected. These results confirm the crucial role of the copG1 gene as a novel key regulator in the symbiotic relationship between SUTN9-2 and legumes.

Insights into Antifungal Mechanisms of Bacillus velezensis S141 against Cercospora Leaf Spot in Mungbean (V. radiata).

Cercospora leaf spot (CLS) is caused by Cercospora canescens and is one of the most important diseases of mungbean (Vigna radiata). Cercospora leaf spot may result in economic loss in production areas. The present study investigated the potential of Bacillus velezensis S141 as a biocontrol agent for C. canescens PAK1 growth on culture plates. Cell-free secretions from a dual culture of S141+PAK1 inhibited fungal growth more than those from a single culture of S141. The biocontrol efficiency of S141 against Cercospora leaf spot on mungbean was then evaluated by spraying. The disease severity of Cercospora leaf spot was significantly reduced in plants treated with S141, with a control efficiency of 83% after 2 days of infection. Comparative transcriptomics and qRT-PCR ana-lyses of S141 during C. canescens inhibition were performed to elucidate the antifungal mechanisms underlying its antifungal activity against Cercospora leaf spot. According to the differentially expressed genes, most up-regulated genes involved in the biosynthetic genes encoding enzymatic hydrolases, including protease, ß-glucanase, and N-acyl glucosaminase, were detected in strain S141 following its interaction. Moreover, genes related to secondary metabolites (surfactin, bacilysin, and bacillomycin D) were up-regulated. Collectively, these results suggest that S141 exhibited strong antifungal activity against C. canescens due to multiple enzymatic hydrolases and secondary metabolites. Therefore, the present study provides insights into the biological network responsible for the antifungal activity of B. velezensis S141 against C. canescens.

Effect of fed dietary yeast (Rhodotorula paludigena CM33) on shrimp growth, gene expression, intestinal microbial, disease resistance, and meat composition of Litopenaeus vannamei.

Yeast is a health-promoting and bio-therapeutic probiotic that is commonly used in aquaculture. Rhodotorula paludigena CM33 can accumulate amounts of intracellular carotenoids and lipid, which are regarded as nutritionally beneficial compounds in various aspects. The aim of this study was to evaluate the impact of different levels of R. paludigena CM33 (RD) incorporated in a dietary composition at 0% (control), 1% (1% RD), 2% (2% RD), and 5% (5% RD) on the growth of shrimp (Litopenaeus vannamei), their immune-related gene expression, intestinal health, resistance to Vibrio parahaemolyticus (VPAHPND) infection, and meat composition. The results showed significant improvements in the specific growth rate, weight gain, and survival of shrimp fed with 1% RD, 2% RD, and 5% RD, which were higher than the control group after 4 weeks of administration. The administration of 5% RD group resulted in a decrease in cumulative mortality upon VPAHPND challenge when compared to the control group. Furthermore, the expression levels of immune-responsive genes, including proPO system (prophenoloxidase-2: PO2), antioxidant enzyme (superoxide dismutase: SOD, glutathione peroxidase: GPX, and catalase: CAT), JAK/STAT pathway (signal transducer and activator of transcription: STAT, gamma interferon inducible lysosomal thiol reductase: GILT), IMD pathway (inhibitor of nuclear factor kappa-B kinase subunit beta and epsilon: IKKb and IKKe), and Toll pathway (Lysozyme) genes, were up-regulated in the 5% RD group. In the context of microbiota, microbiome analysis revealed that the main phyla in shrimp intestines were Proteobacteria, Firmicutes, Bacteroidota, Campilobacterota, Actinobacteriota, and Verrucomicrobiota. At the genus level, Vibrio was found to be reduced in the 5% RD group, whereas the abundance of potentially beneficial bacteria Bifidobacterium was increased. The 5% RD group showed a significant increase in the levels of crude protein and crude lipid, both of which are essential nutritious components. Our results show the capability of R. paludigena CM33 as a probiotic supplement in shrimp feed in improving growth, antimicrobial responses against VPAHPND, and meat quality by increasing protein and lipid content in shrimp.

Symbiosis Contribution of Non-nodulating Bradyrhizobium cosmicum S23321 after Transferal of the Symbiotic Plasmid pDOA9.

The symbiotic properties of rhizobial bacteria are driven by the horizontal gene transfer of symbiotic genes, which are located in symbiosis islands or on plasmids. The symbiotic megaplasmid pDOA9 of Bradyrhizobium sp. DOA9, carrying the nod, nif, fix, and type three secretion system (T3SS) genes, has been conjugatively transferred to different Bradyrhizobium strains. In the present study, non-nodulating B. cosmicum S23321, which shows a close phylogenetic relationship with Bradyrhizobium sp. DOA9, but lacks symbiotic properties, was used to carry pDOA9 (annotated as chimeric S2:pDOA9). The results obtained showed that pDOA9 conferred symbiotic properties on S23321; however, nodulation phenotypes varied among the DOA9, chimeric ORS278:pDOA9, and S2:pDOA9 strains even though they all carried symbiotic pDOA9 plasmid. S23321 appeared to gain symbiotic nodulation from pDOA9 by processing nodulation genes and broadening the host range. The present results also showed the successful formation of active nodules in Arachis hypogaea (Dalbergoid) and Vigna radiata (Millitoid) by chimeric S2:pDOA9, while Crotalaria juncea (Genistoid) and Macroptilium atropurpureum (Millitoid) formed nodule-like structures. The formation of nodules and nodule-like structures occurred in a nod factor-dependent manner because the nod factor-lacking strain (S2:pDOA9ΩnodB) completely abolished nodulation in all legumes tested. Moreover, T3SS carried by S2:pDOA9 exerted negative effects on symbiosis with Crotalaria juncea, which was consistent with the results obtained on DOA9. T3SS exhibited symbiotic compatibility with V. radiata when nodulated by S23321. These outcomes implied that pDOA9 underwent changes during legume evolution that broadened host specificity and the compatibility of nodulation in a manner that was dependent on the chromosomal background of the recipient as well as legume host restrictions.

Regulation of shrimp prophenoloxidase activating system by lva-miR-4850 during bacterial infection.

MicroRNAs (miRNAs) suppress gene expression and regulate biological processes. Following small RNA sequencing, shrimp hemocytes miRNAs differentially expressed in response to acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus (VPAHPND) were discovered and some were confirmed by qRT-PCR. VPAHPND-responsive miRNAs were predicted to target several genes in various immune pathways. Among them, lva-miR-4850 is of interest because its predicted target mRNAs are two important genes of the proPO system; proPO2 (PO2) and proPO activating factor 2 (PPAF2). The expression of lva-miR-4850 was significantly decreased after VPAHPND infection, whereas those of the target mRNAs, PO2 and PPAF2, and PO activity were significantly upregulated. Introducing the lva-miR-4850 mimic into VPAHPND-infected shrimps caused a reduction in the PO2 and PPAF2 transcript levels and the PO activity, but significantly increased the number of bacteria in the VPAHPND targeted tissues. This result inferred that lva-miR-4850 plays a crucial role in regulating the proPO system via suppressing expression of PPAF2 and PO2. To fight against VPAHPND infection, shrimp downregulated lva-miR-4850 expression resulted in proPO activation.

The Bradyrhizobium diazoefficiens type III effector NopE modulates the regulation of plant hormones towards nodulation in Vigna radiata.

Host-specific legume-rhizobium symbiosis is strictly controlled by rhizobial type III effectors (T3Es) in some cases. Here, we demonstrated that the symbiosis of Vigna radiata (mung bean) with Bradyrhizobium diazoefficiens USDA110 is determined by NopE, and this symbiosis is highly dependent on host genotype. NopE specifically triggered incompatibility with V. radiata cv. KPS2, but it promoted nodulation in other varieties of V. radiata, including KPS1. Interestingly, NopE1 and its paralogue NopE2, which exhibits calcium-dependent autocleavage, yield similar results in modulating KPS1 nodulation. Furthermore, NopE is required for early infection and nodule organogenesis in compatible plants. Evolutionary analysis revealed that NopE is highly conserved among bradyrhizobia and plant-associated endophytic and pathogenic bacteria. Our findings suggest that V. radiata and B. diazoefficiens USDA110 may use NopE to optimize their symbiotic interactions by reducing phytohormone-mediated ETI-type (PmETI) responses via salicylic acid (SA) biosynthesis suppression.

MicroRNA and mRNA interactions coordinate the immune response in non-lethal heat stressed Litopenaeus vannamei against AHPND-causing Vibrio parahaemolyticus.

While Vibrio parahaemolyticus (VPAHPND) has been identified as the cause of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, mechanisms of host response remain unknown. Understanding these processes is important to improve farming practices because this understanding will help to develop methods to enhance shrimp immunity. Pre-treatment of shrimp with 5-minute chronic non-lethal heat stress (NLHS) for 7 days was found to significantly increase Litopenaeus vannamei survival against VPAHPND infection. To elucidate the mechanism involved, mRNA and miRNA expression profiles from the hemocyte of L. vannamei challenged with VPAHPND after NLHS with corresponding control conditions were determined by RNA-Seq. A total of 2,664 mRNAs and 41 miRNAs were differentially expressed after the NLHS treatment and VPAHPND challenge. A miRNA-mRNA regulatory network of differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) was subsequently constructed and the interactions of DEMs in regulating the NLHS-induced immune-related pathways were identified. Transcriptomic data revealed that miRNA and mRNA interactions contribute to the modulation of NLHS-induced immune responses, such as the prophenoloxidase-activating system, hemocyte homeostasis, and antimicrobial peptide production, and these responses enhance VPAHPND resistance in L. vannamei.