Heterologous synthesis of ginsenoside F1 and its precursors in Nicotiana benthamiana.

Ginsenoside F1 has high medicinal values, which is a kind of rare triterpene saponin isolated from Panax plants. The extremely low content of ginsenoside F1 in herbs has limited its research and application in medical field. In this work, we constructed a pathway in tobacco for the biosynthesis of ginsenoside F1 by metabolic engineering. Four enzyme genes (PnDDS, CYP716A47, CYP716S1 and UGT71A56) isolated from Panax notoginseng were introduced into tobacco. Thus, a biosynthetic pathway for ginsenoside F1 synthesis was artificially constructed in tobacco cells; moreover, the four exogenous genes could be expressed in the roots, stems and leaves of transgenic plants. Consequently, ginsenoside F1 and its precursors were successfully synthesized in the transgenic tobacco, compared with Panax plants, the content of ginsenoside F1 in transgenic tobacco was doubled. In addition, accumulation of ginsenoside F1 and its precursors in transgenic tobacco shows organ specificity. Based on these results, a new approach was established to produce rare ginsenoside F1; meanwhile, such strategy could also be employed in plant hosts for the heterologous synthesis of other important or rare natural products.

Proline-rich protein PRPL1 enhances Panax notoginseng defence against Fusarium solani by regulating reactive oxygen species balance and strengthening the cell wall barrier.

The root rot mainly caused by Fusarium solani is a bottleneck in the cultivation of Panax notoginseng. In this study, we reported a gene encoding a plant cell wall structural protein, P. notoginseng proline-rich protein (PnPRPL1), whose transcription was upregulated by F. solani and induced by some hormone signals. The PnPRPL1 recombinant protein significantly inhibited the growth and conidial germination of the root rot pathogens. Downregulation of PnPRPL1 by RNA interference (RNAi) in P. notoginseng leaves increased the susceptibility to F. solani, whereas overexpression of PnPRPL1 in tobacco (Nicotiana tabacum) enhanced the resistance to F. solani. Compared with wild-type tobacco, the PnPRPL1-overexpressing transgenic tobacco had higher reactive oxygen species (ROS)-scavenging enzyme activities, lower ROS levels, and more lignin and callose deposition. The opposite results were obtained for the P. notoginseng expressing PnPRPL1 RNAi fragments. Furthermore, the PnPRPL1 promoter transcription activity was induced by several plant hormones and multiple stress stimuli. In addition, the transcription factor PnWRKY27 activated the expression of PnPRPL1 by directly binding to the promoter region. Thus, PnPRPL1, which is positively regulated by a WRKY transcription factor, encodes an antimicrobial protein that also mediates ROS homoeostasis and callose/lignin deposition during the response to F. solani infection.

Panax notoginseng transcription factor WRKY15 modulates resistance to Fusarium solani by up-regulating osmotin-like protein expression and inducing JA/SA signaling pathways.

BACKGROUND: Panax notoginseng (Burk) F. H. Chen is a valuable traditional Chinese medicinal plant, but its commercial production is seriously affected by root rot caused by some pathogenic fungi, including Fusarium solani. Nevertheless, the genetic breeding for disease resistance of P. notoginseng remains limited. The WRKY transcription factors have been revealed to play important roles in plant defense responses, which might provide an inspiration for resistance improvement in P. notoginseng. RESULTS: In this study, the regulatory mechanism of transcription factor PnWRKY15 on P. notoginseng resistance to F. solani infection was revealed. The suppressed expression of PnWRKY15 via RNA interference increased the sensitivity of P. notoginseng to F. solani and decreased the expression levels of some defense-related genes, including PnOLP1, which encodes an osmotin-like protein that confers resistance to F. solani. Ectopic expression of PnWRKY15 in the model plant tobacco significantly enhanced the resistance to F. solani. Moreover, the transcriptome sequencing analysis discovered that some pathogenesis-related genes were expressed at higher levels in the PnWRKY15-overexpressing tobacco than that in the wild-type tobacco. In addition, the jasmonic acid (JA) and salicylic acid (SA) signaling pathways were evidently induced by PnWRKY15-overexpression, that was evidenced by that the JA and SA contents were significantly higher in the PnWRKY15-overexpressing tobacco than that in the wild-type. Furthermore, PnWRKY15, which was localized in the nucleus, can trans-activate and up-regulate PnOLP1 expression according to the EMSA, yeast one-hybrid and co-expression assays. CONCLUSIONS: PnWRKY15 contributes to P. notoginseng resistance to F. solani by up-regulating the expression of resistance-related gene PnOLP1 and activating JA/SA signaling pathways. These findings will help to further elucidate the transcriptional regulatory mechanism associated with the P. notoginseng defense response to F. solani.

Panax notoginseng WRKY Transcription Factor 9 Is a Positive Regulator in Responding to Root Rot Pathogen Fusarium solani.

Panax notoginseng (Burk) F.H. Chen is a rare and valuable Chinese herb, but root rot mainly caused by Fusarium solani severely affects the yield and quality of P. notoginseng herbal materials. In this study, we isolated 30 P. notoginseng WRKY transcription factors (TFs), which were divided into three groups (I, II, and III) on the basis of a phylogenetic analysis. The expression levels of 10 WRKY genes, including PnWRKY9, in P. notoginseng roots increased in response to a methyl jasmonate (MeJA) treatment and the following F. solani infection. Additionally, PnWRKY9 was functionally characterized. The PnWRKY9 protein was localized to the nucleus. The overexpression of PnWRKY9 in tobacco (Nicotiana tabacum) considerably increased the resistance to F. solani, whereas an RNAi-mediated decrease in the PnWRKY9 expression level in P. notoginseng leaves increased the susceptibility to F. solani. The RNA sequencing and hormone content analyses of PnWRKY9-overexpression tobacco revealed that PnWRKY9 and the jasmonic acid (JA) signaling pathway synergistically enhance disease resistance. The PnWRKY9 recombinant protein was observed to bind specifically to the W-box sequence in the promoter of a JA-responsive and F. solani resistance-related defensin gene (PnDEFL1). A yeast one-hybrid assay indicated that PnWRKY9 can activate the transcription of PnDEFL1. Furthermore, a co-expression assay in tobacco using ß-glucuronidase (GUS) as a reporter further verified that PnWRKY9 positively regulates PnDEFL1 expression. Overall, in this study, we identified P. notoginseng WRKY TFs and demonstrated that PnWRKY9 positively affects plant defenses against the root rot pathogen. The data presented herein provide researchers with fundamental information regarding the regulatory mechanism mediating the coordinated activities of WRKY TFs and the JA signaling pathway in P. notoginseng responses to the root rot pathogen.

Lilium regale Wilson WRKY3 modulates an antimicrobial peptide gene, LrDef1, during response to Fusarium oxysporum.

BACKGROUND: WRKY transcription factors (TFs) play vital roles in plant growth and development, secondary metabolite synthesis, and response to biotic and abiotic stresses. In a previous transcriptome sequencing analysis of Lilium regale Wilson, we identified multiple WRKY TFs that respond to exogenous methyl jasmonate treatment and lily Fusarium wilt (Fusarium oxysporum). RESULTS: In the present study, the WRKY TF LrWRKY3 was further analyzed to reveal its function in defense response to F. oxysporum. The LrWRKY3 protein was localized in the plant cell nucleus, and LrWRKY3 transgenic tobacco lines showed higher resistance to F. oxysporum compared with wild-type (WT) tobacco. In addition, some genes related to jasmonic acid (JA) biosynthesis, salicylic acid (SA) signal transduction, and disease resistance had higher transcriptional levels in the LrWRKY3 transgenic tobacco lines than in the WT. On the contrary, L. regale scales transiently expressing LrWRKY3 RNA interference fragments showed higher sensitivity to F. oxysporum infection. Moreover, a F. oxysporum-induced defensin gene, Def1, was isolated from L. regale, and the recombinant protein LrDef1 isolated and purified from Escherichia coli possessed antifungal activity to several phytopathogens, including F. oxysporum. Furthermore, co-expression of LrWRKY3 and the LrDef1 promoter in tobacco enhanced the LrDef1 promoter-driven expression activity. CONCLUSIONS: These results clearly indicate that LrWRKY3 is an important positive regulator in response to F. oxysporum infection, and one of its targets is the antimicrobial peptide gene LrDef1.

An MYB Transcription Factor Modulates Panax notoginseng Resistance Against the Root Rot Pathogen Fusarium solani by Regulating the Jasmonate Acid Signaling Pathway and Photosynthesis.

Root rot of Panax notoginseng, a precious Chinese medicinal plant, seriously impacts its sustainable production. However, the molecular regulatory mechanisms employed by P. notoginseng against root rot pathogens, including Fusarium solani, are still unclear. In this study, the PnMYB2 gene was isolated, and its expression was affected by independent treatments with four signaling molecules (methyl jasmonate, ethephon, salicylic acid, and hydrogen peroxide) as assessed by quantitative real-time PCR. Moreover, the PnMYB2 expression level was induced by F. solani infection. The PnMYB2 protein localized to the nucleus and may function as a transcription factor. When overexpressed in transgenic tobacco, the PnMYB2 gene conferred resistance to F. solani. Jasmonic acid (JA) metabolism and disease resistance-related genes were induced in the transgenic tobacco, and the JA content significantly increased compared with in the wild type. Additionally, transcriptome sequencing, Kyoto Encyclopedia of Genes and Genomes annotation enrichment, and metabolic pathway analyses of the differentially expressed genes in the transgenic tobacco revealed that JA metabolic, photosynthetic, and defense response-related pathways were activated. In summary, PnMYB2 is an important transcription factor in the defense responses of P. notoginseng against root rot pathogens that acts by regulating JA signaling, photosynthesis, and disease-resistance genes.

Lilium regale Wilson WRKY2 Regulates Chitinase Gene Expression During the Response to the Root Rot Pathogen Fusarium oxysporum.

Root rot, mainly caused by Fusarium oxysporum, is the most destructive disease affecting lily (Lilium spp.) production. The WRKY transcription factors (TFs) have important roles during plant immune responses. To clarify the effects of WRKY TFs on plant defense responses to pathogens, a WRKY gene (LrWRKY2) was isolated from Lilium regale Wilson, which is a wild lily species highly resistant to F. oxysporum. The expression of LrWRKY2, which encodes a nuclear protein, is induced by various hormones (methyl jasmonate, ethephon, salicylic acid, and hydrogen peroxide) and by F. oxysporum infection. In this study, LrWRKY2-overexpressing transgenic tobacco plants were more resistant to F. oxysporum than the wild-type plants. Moreover, the expression levels of jasmonic acid biosynthetic pathway-related genes (NtAOC, NtAOS, NtKAT, NtPACX, NtJMT, NtOPR, and NtLOX), pathogenesis-related genes (NtCHI, NtGlu2, and NtPR-1), and antioxidant stress-related superoxide dismutase genes (NtSOD, NtCu-ZnSOD, and MnSOD) were significantly up-regulated in LrWRKY2 transgenic tobacco lines. Additionally, the transient expression of a hairpin RNA targeting LrWRKY2 increased the susceptibility of L. regale scales to F. oxysporum. Furthermore, an F. oxysporum resistance gene (LrCHI2) encoding a chitinase was isolated from L. regale. An electrophoretic mobility shift assay demonstrated that LrWRKY2 can bind to the LrCHI2 promoter containing the W-box element. Yeast one-hybrid assay results suggested that LrWRKY2 can activate LrCHI2 transcription. An examination of transgenic tobacco transformed with LrWRKY2 and the LrCHI2 promoter revealed that LrWRKY2 activates the LrCHI2 promoter. Therefore, in L. regale, LrWRKY2 is an important positive regulator that contributes to plant defense responses to F. oxysporum by modulating LrCHI2 expression.

WRKY Transcription Factors Actively Respond to Fusarium oxysporum in Lilium regale.

The WRKY transcription factors form a plant-specific superfamily important for regulating plant development, stress responses, and hormone signal transduction. In this study, many WRKY genes (LrWRKY1-35) were identified in Lilium regale, which is a wild lily species highly resistant to Fusarium wilt. These WRKY genes were divided into three classes (I to III) based on a phylogenetic analysis. The Class-II WRKY transcription factors were further divided into five subclasses (IIa, IIb, IIc, IId, and IIe). Moreover, the gene expression patterns based on a quantitative real-time PCR analysis revealed the WRKY genes were differentially expressed in the L. regale roots, stems, leaves, and flowers. Additionally, the expression of the WRKY genes was affected by an infection by Fusarium oxysporum as well as by salicylic acid, methyl jasmonate, ethephon, and hydrogen peroxide treatments. Moreover, the LrWRKY1 protein was localized to the nucleus of onion epidermal cells. The recombinant LrWRKY1 protein purified from Escherichia coli bound specifically to DNA fragments containing the W-box sequence, and a yeast one-hybrid assay indicated that LrWRKY1 can activate transcription. A co-expression assay in tobacco (Nicotiana tabacum) confirmed LrWRKY1 regulates the expression of LrPR10-5. Furthermore, the overexpression of LrWRKY1 in tobacco and the Oriental hybrid 'Siberia' (susceptible to F. oxysporum) increased the resistance of the transgenic plants to F. oxysporum. Overall, LrWRKY1 regulates the expression of the resistance gene LrPR10-5 and is involved in the defense response of L. regale to F. oxysporum. This study provides valuable information regarding the expression and functional characteristics of L. regale WRKY genes.

Env diversity-dependent protection of the attenuated equine infectious anaemia virus vaccine.

Lentiviruses harbour high genetic variability for efficient evasion from host immunity. An attenuated equine infectious anaemia (EIA) vaccine was developed decades ago in China and presented remarkably robust protection against EIA. The vaccine was recently proven to have high genomic diversity, particular in env. However, how and to what extent the high env diversity relates to immune protection remains unclear. In this study, we compared immune protections and responses of three groups of horses stimulated by the high-diversity vaccine EIAV_HD, a single molecular clone of the vaccine EIAV_LD with low env diversity, as well as a constructed vaccine strain EIAV_MD with moderate env diversity. The disparity of virus-host interactions between three env diversity-varied groups (5 horses in each group) was evaluated using clinical manifestation, pathological scores, and env-specific antibody. We found the highest titres of env antibodies (Abs) or neutralizing Abs (nAbs) in the EIAV_HD group, followed by the EIAV_MD group, and the lowest titres in the EIAV_LD group (P<0.05). The occurrence of disease/death was different between EIAV_HD group (1/0), EIAV_MD (2/2), and EIAV_LD group (4/2). A similar env diversity-related linear relationship was observed in the clinical manifestations and pathological changes. This diversity-dependent disparity in changes between the three groups was more distinct after immunosuppression, suggesting that env diversity plays an important role in protection under low host immunocompetence. In summary, inoculation with vaccines with higher genetic diversity could present broader and more efficient protection. Our findings strongly suggest that an abundance of Env antigens are required for efficient protection against lentiviruses.

Osmotin-Like Protein Gene from Panax notoginseng Is Regulated by Jasmonic Acid and Involved in Defense Responses to Fusarium solani.

Osmotin and osmotin-like proteins (OLPs) play important roles in plant defense responses. The full-length cDNA sequence of an OLP gene was cloned from Panax notoginseng using rapid amplification of cDNA-end technology and named PnOLP1. A quantitative reverse transcription-PCR analysis showed that the signaling molecules methyl jasmonate, salicylic acid, ethylene, and hydrogen peroxide induced PnOLP1 expression to different degrees. In addition, the expression level of PnOLP1 rapidly increased within 48 h of inoculating P. notoginseng with the root rot pathogen Fusarium solani. Subcellular localization revealed that PnOLP1 localized to the cell wall. A prokaryotic expression vector containing PnOLP1 was constructed and transformed into Escherichia coli BL21 (DE3), and in vitro antifungal assays were performed using the purified recombinant PnOLP1 protein. The recombinant PnOLP1 protein had strong inhibitory effects on the mycelial growth of F. oxysporum, F. graminearum, and F. solani. A plant PnOLP1-overexpression vector was constructed and transfected into tobacco, and the resistance of T2 transgenic tobacco against F. solani was significantly enhanced compared with wild-type tobacco. Moreover, a PnOLP1 RNAi vector was constructed and transferred to the P. notoginseng leaves for transient expression, and the decrease of PnOLP1 expression level in P. notoginseng leaves increased the susceptibility to F. solani. Thus, PnOLP1 is an important disease resistance gene involved in the defense responses of P. notoginseng to F. solani.

A Pathogenesis-Related Protein-Like Gene Is Involved in the Panax notoginseng Defense Response to the Root Rot Pathogen.

Pathogenesis-related proteins (PRs) are a class of proteins that accumulate in response to biotic and abiotic stresses to protect plants from damage. In this study, a gene encoding a PR-like protein (PnPR-like) was isolated from Panax notoginseng, which is used in traditional Chinese herbal medicines. An analysis of gene expression in P. notoginseng indicated that PnPR-like was responsive to an infection by the root rot pathogen Fusarium solani. The expression of this gene was induced by several signaling molecules, including methyl jasmonate, ethephon, hydrogen peroxide, and salicylic acid. The PnPR-like-GFP fusion gene was transiently expressed in onion (Allium cepa) epidermal cells, which revealed that PnPR-like is a cytoplasmic protein. The purified recombinant PnPR-like protein expressed in Escherichia coli had antifungal effects on F. solani and Colletotrichum gloeosporioides as well as inhibited the spore germination of F. solani. Additionally, the in vitro ribonuclease (RNase) activity of the recombinant PnPR-like protein was revealed. The PnPR-like gene was inserted into tobacco (Nicotiana tabacum) to verify its function. The gene was stably expressed in T2 transgenic tobacco plants, which exhibited more RNase activity and greater disease resistance than the wild-type tobacco. Moreover, the transient expression of hairpin RNA targeting PnPR-like in P. notoginseng leaves increased the susceptibility to F. solani and decreased the PnPR-like expression level. In conclusion, the cytoplasmic protein PnPR-like, which has RNase activity, is involved in the P. notoginseng defense response to F. solani.

[Functional analysis of a chitinase gene PnCHI1 from Panax notoginseng].

Chitinases, a glycosidase enzyme that hydrolyzes chitin to N-acetylglucosamine, are widely found in plant cells, and they are an important part of plant antifungal defense system. The function of a Panax notoginseng chitinase gene PnCHI1 was characterized in this paper. Expression vector of PnCHI1 was constructed and transiently expressed in onion epidermal cells, and laser scanning confocal microscopy demonstrated that PnCHI1 was localized in the cell wall. Prokaryotic expression vector of PnCHI1 was also constructed, and recombinant protein of PnCHI1 was induced and purified. In vitro antibacterial assay showed that recombinant PnCHI1 protein had strong inhibitory activity on the mycelium growth of Fusarium solani, F. oxysporum and F. verticillioide. The function of PnCHI1 was further verified by reverse genetics. PnCHI1 expression vector was transferred into tobacco by Agrobacterium tumefaciens and expression of PnCHI1 was confirmed by qRT-PCR. It was found by leaf inoculation experiment that resistance of transgenic tobacco to F. solani was significantly increased. It is conclnded that: PnCHI1 is a chitinase localized in the cell wall, which inhibits several fungi which cause the root rot disease of P. notoginseng. Overexpression of this chitinase gene in tobacco greatly increased resistance to F. solani. PnCHI1 may be an important resistance gene in P. notoginseng that participates in the defense against root rot disease.

MAPK-mediated auxin signal transduction pathways regulate the malic acid secretion under aluminum stress in wheat (Triticum aestivum L.).

An isobaric tags for relative and absolute quantitative (iTRAQ)-based quantitative proteomic approach was used to screen the differentially expressed proteins during control treatment (CK), aluminum (Al) and Al+ indole-3-acetic acid (IAA) treatment of wheat lines ET8 (Al-tolerant). Further, the the expression levels of auxin response factor (ARF), Aux/IAA, Mitogen activated protein kinase (MAPK) 2c, and MAPK1a were analyzed. Results showed that 16 proteins were determined to be differentially expressed in response to Al and IAA co-treatment compared with Al alone. Among them, MAPK2c and MAPK1a proteins displayed markedly differential expression during the processes. The expression of ARF2 was upregulated and Aux/IAA was downregulated by Al, while both in concentration- and time-dependent manners. Western-blot detection of MAPK2c and MAPK1a indicated that Al upregulated MAPK2c and downregulated MAPK1a in both concentration- and time-dependent manners. Exogenous IAA could promote the expression of MAPK2c, but inhibit the expression of MAPK1a in the presence/absence of Al. These findings indicated that IAA acted as one of the key signaling molecule controls the response mechanism of wheat malic acid efflux to Al stress through the suppression/activation of Aux/IAA and ARFs, and the activity of MAPK2c and MAPK1a were positively or negatively regulated.

Intracellular overexpression of HIV-1 Nef impairs differentiation and maturation of monocytic precursors towards dendritic cells.

Nef functions as an immunosuppressive factor critical for HIV-1 replication, survival and development of AIDS following HIV-1 infection. What effects Nef exerts on differentiation and maturation of monocytes towards dendritic cells (DCs) remains greatly controversial. In this study, we used THP-1 (human monocytic leukemia cell line) as monocytic DC precursors to investigate how overexpression of HIV-1 Nef influences the processes of differentiation and maturation of dendritic cells. In striking contrast to negative controls, our results showed that morphological and phenotypical changes (CD11c, CD14, CD40, CD80, CD83, CD86, and HLA-DR) occurred on recombinant THP-1 expressing HIV-1 Nef (short for Nef) upon co-stimulation of GM-CSF/IL-4 or GM-CSF/IL-4/TNF-α/ionomycin. Moreover, CD4, CCR5, and CXCR4 were also down-regulated on Nef. It might be hypothesized that Nef prevents superinfection and signal transduction in HIV-1 infected monocytes. Collectively, our study demonstrates that long-lasting expression of Nef at high levels indeed retards differentiation and maturation of dendritic cells in terms of phenotype and morphology. We are hopeful that potentially, stable expression of intracellular Nef in vivo may function as a subtle mode to support long-lasting HIV-1 existence.

Expression of infectious pancreatic necrosis virus (IPNV) VP2-VP3 fusion protein in Lactobacillus casei and immunogenicity in rainbow trouts.

Infectious pancreatic necrosis virus (IPNV) infects wild and cultured salmonids, causing high mortality in juvenile trouts and salmons. IPNV VP2-VP3 fusion gene was constructed by splicing overlap extension (SOE) PCR and inserted into Lactobacillus/Escherichia coli shuttle vectors (pPG1and pPG2) followed by transformation of Lactobacillus casei competent cell to yield two recombinant strains: Lc:PG1-VP2-VP3 (surface-displayed) and Lc:PG2-VP2-VP3 (secretory). Subsequently, juvenile rainbow trouts were inoculated with the recombinant strains via orogastric route. Our results demonstrated that Lactobacillus-derived VP2-VP3 fusion protein could induce production of serum IgM specific for IPNV with neutralizing activity in rainbow trouts. Statistical analyses of IgM levels showed that immunogenicity of Lc:PG1-VP2-VP3 was more powerful than that of Lc:PG2-VP2-VP3 (P<0.001) in rainbow trouts. This result has been confirmed by viral loads reduction analyzed by real-time RT-PCR in orogastrically immunized rainbow trouts after virus challenging. Comparing to trouts received Lactobacillus (control), rainbow trouts orogastrically dosed with Lc:PG1-VP2-VP3 resulted in ∼10-fold reduction in viral loads on day 10 post-virus challenging, and ∼4-fold did by Lc:PG2-VP2-VP3. Taken together, Lc:PG1-VP2-VP3 functions as novel mucosal vaccine against IPNV infection in rainbow trouts, which most likely come true.

High-level mucosal and systemic immune responses induced by oral administration with Lactobacillus-expressed porcine epidemic diarrhea virus (PEDV) S1 region combined with Lactobacillus-expressed N protein.

To develop effective mucosal vaccine formulation against porcine epidemic diarrhea virus (PEDV) infection, the DNA fragments encoding spike protein immunodominant region S1 and nucleocapsid N of PEDV were inserted into pPG1 (surface-displayed) or pPG2 (secretory) plasmids followed by electrotransformation into Lactobacillus casei (Lc) to yield four recombinant strains: PG1-S1, PG2-S1, PG1-N, and PG2-N. After intragastric administration, it was observed that live Lc-expressing S1 protein combined with Lc-expressing N protein could elicit much more potent mucosal and systemic immune responses than the former alone (P < 0.001), however slightly inferior to the latter alone (P > 0.05). Furthermore, the surface-displayed mixture (PG1-S1+ PG1-N) revealed stronger immunogenicity than the secretory mixture (PG2-S1+ PG2-N) as well as PEDV-neutralizing potency in vitro (P < 0.001). On 49th day after the last immunization, splenocytes were prepared from mice immunized with surface-displayed mixture, secretory mixture and negative control to be stimulated by purified N and S protein, respectively. The results of ELISA analysis showed that N protein was capable of inducing a higher level of IL-4 (P < 0.001) and IFN-γ (P < 0.001) than S1 protein in the immunized mice. Taken together, Lc-expressed N protein as molecular adjuvant or immunoenhancer was able to effectively facilitate the induction of mucosal and systemic immune responses by Lc-expressing S1 region.