The Role of PnTCP2 in the Lobed Leaf Formation of Phoebe neurantha var. lobophylla.

A lobed leaf is a common trait in plants, but it is very rare in Lauraceae plants, including species of Phoebe. In the study of germplasm resources of Phoebe neurantha, we found lobed leaf variant seedlings, and the variation could be inherited stably. Studying the lobed leaf mechanism of P. neurantha var. lobophylla can offer insight into the leaf development mechanism of woody plants. RNA-seq and small RNA-seq analysis results showed that a total of 8091 differentially expressed genes (DEGs) and 16 differentially expressed miRNAs were identified in P. neurantha var. lobophylla. Considering previous research results, a leaf margin morphological development related miRNA, pne-miRNA319a, was primary identified as a candidate miRNA. Target gene prediction showed that a total of 2070 genes were predicted to be the target genes of differentially expressed miRNAs. GO enrichment analysis of differentially expressed target genes suggested that PnTCP2 is related to lobed leaf formation. The TRV-VIGS gene silencing of PnTCP2 led to lobed leaves in P. neurantha seedlings. The downregulation of PnTCP2 led to lobed leaves. The yeast two-hybrid test and bimolecular fluorescence complementation test confirmed that the PnTCP2 protein interacted with the PnLBD41 protein. Based on the expression analysis of gene-silenced leaves and RNA-seq and small RNA-seq analysis results, pne- miRNA319a and PnLBD41 might also play important roles in this process. In conclusion, PnTCP2 plays an important and vital role in the formation of the lobed leaves of P. neurantha var. lobophylla.

Comparative Transcriptome Analysis Revealing the Potential Mechanism of Low-Temperature Stress in Machilus microcarpa.

Machilus microcarpa is a rare national tree species in China and possesses important ornamental and ecological value. M. microcarpa can be planted in low-temperature areas, depending on whether its seedlings can withstand the harm. To face this problem, the annual seedlings of M. microcarpa were subjected to five temperature treatments, and eight physiological indicators were measured. Furthermore, comparative transcriptome analysis was performed between M. microcarpa leaves treated at 25°C and -2.8°C. A total of 9,385 differentially expressed genes (DEGs) were involved in low-temperature stress in M. microcarpa. An upregulated (cobA) and five downregulated (HEM, CHLM, CRD, CLH, and PORA) genes associated with the porphyrin and chlorophyll metabolism pathway may reduce chlorophyll synthesis under low-temperature stress. Upregulation of six DEGs (two GAPDHs, PFK, PGAM, PDC, and PK) involved in the glycolysis/gluconeogenesis pathway provided energy for M. microcarpa under adverse cold conditions. Thirteen upregulated and seven downregulated genes related to antioxidant enzymes were also observed under low-temperature stress. Candidate transcription factors (TFs) played key roles in signal transduction under low-temperature stress in M. microcarpa, and quantitative real-time PCR (qRT-PCR) analysis validated the RNA-seq data. The results provide valuable information for further studies on the cold response mechanisms for low-temperature stress in M. microcarpa.

Immunization with adenovirus LIGHT-engineered dendritic cells induces potent T cell responses and therapeutic immunity in HBV transgenic mice.

LIGHT, a TNF superfamily member (TNFSF14), is a type II transmembrane protein expressed on activated T cells and immature dendritic cells (DCs). However, the expression of LIGHT on mature DCs is down-regulated. Recent studies demonstrated that LIGHT provides potent costimulatory activity for T cells, enhancing proliferation and the production of Th1 cytokines independently of the B7-CD28 pathway. Here, we evaluated the effectiveness of peptide-pulsed DC-mediated antiviral immunity in HBV transgenic mice and the immunoadjuvant effect of LIGHT. The bone marrow-derived DCs were modified in vitro with an adenovirus (Ad) vector expressing mouse LIGHT (Ad-LIGHT), the expression of costimulatory molecules was up-regulated and the secretion of cytokines IL-12 and IFN-γ increased. LIGHT-modified DCs enhanced allostimulation for T cells in mixed lymphocyte reaction (MLR). HBV peptide-pulsed DCs elicited HBV specific CD8+ T cell response and reduced the level of HBsAg and HBV DNA in sera of HBV transgenic mice. Importantly, LIGHT-modified DCs could induce stronger antiviral immunity. These results support the concept that genetic modification of DCs with a recombinant LIGHT adenovirus vector may be a useful strategy for antiviral immunotherapy.

Improvement in efficacy of DNA vaccine encoding HIV-1 Vif by LIGHT gene adjuvant.

DNA vaccine can induce the prolonged immune responses against the encoded antigen with the appropriate adjuvant. To study the immunogenicity of the HIV-1 vif DNA vaccine in inducing the humoral and cellular immune responses and the immunoadjuvant effect of LIGHT, which is a member of TNF superfamily and can stimulate the proliferation of naïve T cells as a co-stimulatory molecule, DNA vaccine plasmid pcDNA-Vif was constructed by inserting HIV-1 vif gene into the downstream of CMV promoter in eukaryotic expression vector pcDNA3.1(+). In vitro expression of HIV-1 Vif in pcDNA-Vif-transfected HeLa cells was confirmed in transcriptional and protein level by RT-PCR and Western blot, respectively. After BALB/c mice were injected muscularly with DNA vaccines for three times, the specific immune responses were analyzed. The data showed that anti-Vif antibody response, Vif-specific T cell proliferation, and CTL activities were induced in the mice that were inoculated with HIV-1 vif DNA vaccine plasmid. Interestingly, stronger humoral and cellular immune responses were detected in mice that were immunized with plasmid pcDNA-Vif and pcDNA-LIGHT together compared to the single immunization with plasmid pcDNA-Vif alone. Together, the results of the study suggest that candidate HIV-1 DNA vaccine can elicit HIV-1 Vif-specific immune responses in mice and that LIGHT plays the role of immunoadjuvant in co-immunization with DNA vaccine.