Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.

The hepatitis B virus pre-core protein p22 suppresses TNFα-induced apoptosis by regulating the NF-κB pathway.

OBJECTIVE: Cell apoptosis is strongly associated with hepatocellular carcinoma (HCC) progress. Thus, gaining a comprehensive understanding of the virus interfering with the apoptotic process is important for the development of effective anti-tumor therapies. The objective of this study is to explore the potential involvement of HBeAg-p22 (HBV-p22) in TNFα-induced apoptosis. METHODS: Protein expression was detected using western blot. Cell viability and apoptosis were assessed by employing Cell Counting Kit-8 (CCK8) and flow cytometry, respectively. Evaluation of protein-protein interactions was accomplished through co-immunoprecipitation and glutathione-S-transferase (GST) pull-down assays. RESULTS: In this study, it was shown that HBV-p22 inhibited apoptosis of human hepatoma cell lines after tumor necrosis factor-alpha (TNF-α) stimulation. Mechanistically, HBV-p22 suppressed Jun N-terminal kinases (JNK) signaling and enhanced nuclear factor kappa-B (NF-κB) signaling. Moreover, HBV-p22 interacted with I-kappa B kinase α (IKKα) and increased its phosphorylation. CONCLUSIONS: Collectively, HBV-p22, whereby the mechanism contributing to anti-apoptotic effect was regulation of the NF-κB pathway via enhancing the phosphorylation of IKKα.

Arabidopsis Protein Phosphatase PIA1 Impairs Plant Drought Tolerance by Serving as a Common Negative Regulator in ABA Signaling Pathway.

Reversible phosphorylation of proteins is a ubiquitous regulatory mechanism in vivo that can respond to external changes, and plays an extremely important role in cell signal transduction. Protein phosphatase 2C is the largest protein phosphatase family in higher plants. Recently, it has been found that some clade A members can negatively regulate ABA signaling pathways. However, the functions of several subgroups of Arabidopsis PP2C other than clade A have not been reported, and whether other members of the PP2C family also participate in the regulation of ABA signaling pathways remains to be studied. In this study, based on the previous screening and identification work of PP2C involved in the ABA pathway, the clade F member PIA1 encoding a gene of the PP2C family, which was down-regulated after ABA treatment during the screening, was selected as the target. Overexpression of PIA1 significantly down-regulated the expression of ABA marker gene RD29A in Arabidopsis protoplasts, and ABA-responsive elements have been found in the cis-regulatory elements of PIA1 by promoter analysis. When compared to Col-0, transgenic plants overexpressing PIA1 were less sensitive to ABA, whereas pia1 showed the opposite trait in seed germination, root growth, and stomatal opening experiments. Under drought stress, SOD, POD, CAT, and APX activities of PIA1 overexpression lines were lower than Col-0 and pia1, while the content of H2O2 was higher, leading to its lowest survival rate in test plants, which were consistent with the significant inhibition of the expression of ABA-dependent stress-responsive genes RD29B, ABI5, ABF3, and ABF4 in the PIA1 transgenic background after ABA treatment. Using yeast two-hybrid and luciferase complementation assays, PIA1 was found to interact with multiple ABA key signaling elements, including 2 RCARs and 6 SnRK2s. Our results indicate that PIA1 may reduce plant drought tolerance by functioning as a common negative regulator involved in ABA signaling pathway.

[Hepatitis B virus P22(e) inhibit hepatocyte apoptosis via nuclear factor kappa B].

OBJECTIVE: To test whether nuclear factor kappa B plays an important role in the apoptosis-inhibitory effect of hepatitis B virus (HBV) P22(e) protein. METHODS: HepG2 cells were transfected with recombination plasmid pEGFP-HBVP22(e). The Act-D and TNF alpha were used to induce apoptosis. NF-kappa B inhibitor ALLN were used to inhibit the signaling pathway. The activation of NF-kappa B was EMSA, and the nulear translocation of NF-kappa B was determined by immuno-staining. RESULTS: Laser scanning confocal microscopy and EMSA indicated that HBV P22(e) protein enhanced the nuclear translocation of NF-kappa B after apoptosis induction. ALLN treatment inhibited the nuclear translocation of NF-kappa B, and blocked the apoptosis-inhibiting effect of HBV P22(e) protein. CONCLUSION: This study indicates that HBV P22(e) protein inhibits apoptosis of hepatocyte via the NF-kappa B signaling pathway.

[The influence of HBV/P22 protein on the apoptosis of HepG2 cells: an experimental study].

OBJECTIVE: To study the influence of HBV/P22 protein on the induced apoptosis of HepG2 cells. METHODS: In vitro, two HepG2 strains were transfected with pcDNA3.1+ and pcDNA3.1+HBV/P22 respectively and the cells were exposed to Act D and TNF alpha for 6h and then the induced apoptosis was detected by flow cytometry (FCM) and TUNEL technique. Supernatant HBeAg was detected by Abbott reagent. The intracellular expression of HBV/P22 protein was measured by Western blot and immunochemistry. In vivo, three cell groups were inoculated into nude mice by subcutaneous injections. After two weeks, Act D and TNF alpha were injected into the tumors and then the induced apoptosis in the tissues was detected by TUNEL technique. The expression of HBV/P22 protein in the tumor tissues was detected by immunochemistry. RESULTS: In vitro, in HepG2- pcDNA3.1+HBV/P22 cells, supernatant HBeAg was positive and intracellular HBV/P22 protein was positively expressed. The apoptosis proportion of HepG2-pCDNA3.1+HBV/P22 cells was markedly lower than HepG2 and HepG2-pcDNA3.1+ cells (P < 0.05). In vivo, HBV/P22 protein was expressed in the tumor tissues, and the apoptosis proportion in the group injected with HepG2-pcDNA3.1+HBV/P22 cells was markedly lower than those injected with HepG2 and HepG2-pcDNA3.1+cells (P < 0.05). CONCLUSION: HBV/P22 protein could inhibit the induced apoptosis of HepG2 cells both in vitro and in vivo.

[Hepatitis B virus P22e protein inhibits human hepatocellular carcinoma HepG2 cell apoptosis in vitro].

OBJECTIVE: To investigate the effects of the hepatitis B virus (HBV) P22e protein on the apoptosis of human hepatocellular carcinoma HepG2 cells. METHODS: HepG2 cells were transfected with recombinant plasmid pEGFP-HBVP22e and exposed to Act-D and tumor necrosis factor alpha (TNFalpha) treatment to induce cell apoptosis. Flow cytometry was performed to determine the proportion of cells containing sub-G1 DNA to represent the number of apoptotic cells. Laser scanning confocal microscopy was used to observe the nuclear alterations in the apoptotic cells. RESULTS: HepG2EGFP-C2HBVP22e cell strain showed a much delayed apoptosis as well as obviously lowered apoptotic rate in comparison with the HepG2 strain (P<0.01). CONCLUSION: The introduction and expression of extraneous gene HBVP22e significantly inhibits the apoptosis of HepG2 cells.

[Stable expression of HBV C gene mutants in immortalized human B-cell lines].

OBJECTIVE: To provide an cell model of immortalized lymphoblstoid B-cell lines for studying the biological characteristics of full-length hepatitis B virus (HBV) genome carrying the hot-spot mutations V60, G87, and L97. METHODS: V60, G87, and L97 mutation points were introduced into HBV p3.8 II plasmid containing 1.2 copy of HBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8 II and p3.8 II-V60, G87, L97 plasmid, and the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into immortalized human lymphoblasts with lipofectamine 2000 and selected with hygromycin. Steady expression of the target genes was determined by RT-PCR, Western blotting and microparticle enzyme immunoassay. RESULTS: DNA sequence analysis indicated that the desired mutation was introduced into wild-type HBV DNA. HBsAg, HBeAg and HBcAg could be detected in EBO-HBV-transfected cell lysate or culture supernatant. CONCLUSION: Transfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in vitro.

[Establishment of immortalized B lymphoblast cell line from patients with chronic hepatitis B].

AIM: To establish immortalized B lymphoblast cell line (BLCL) from patients with chronic hepatitis B (CHB) in vitro. METHODS: The peripheral blood mononuclear cells (PBMC) were isolated from the patients with CHB by routine method and incubated with EB virus (EBV) in the presence of CpG DNA motifs and cyclosporin A (CysA) for about 28 days. Morphological characteristic of the established immortalized BLCL was observed by microscope and the expression of CD19 and CD23 on cellular surface was determined by flow cytometry. RESULTS: BLCL was successfully established and could be cultured and propagated for a long time in vitro. CONCLUSION: EBV-immortalized BLCL provides a resource of target cells for further research on the cellular immunity of patients with CHB.

[Investigation of single nucleotide polymorphisms in the promoter region of mannan-binding lectin gene in a Han population from Guangdong].

OBJECTIVE: To investigate the single nucleotide polymorphisms (SNP) in the promoter region of mannan-binding lectin (MBL) gene in a Han population in Guangdong Province. METHODS: A total of 167 blood samples were obtained from this Han population to isolate the genomic DNA from the leucocytes. The polymorphism alleles -550(G/C, named H/L alleles), -220(G/C, X/Y alleles) and +4(C/Tr, P/Q alleles) in the promoter region of MBL gene were detected by PCR with sequence-specific primers and molecular beacon real-time fluorescent PCR, and the frequencies of haplotypes and genotypes were analyzed. RESULTS: The frequencies of several genotypes in the 167 samples were: LYP/LYP, 10(5.9%); HYP/LYQ, 7(4.2%); LYP/LYQ, 94(56.3%); LXP/LXP, 6(3.6%); LYQ/LYQ, 4(2.4%); LXP/LYQ, 29(17.4%); HYP/LYP, 3(1.8%); HYP/LXP, 2(1.2%); HYP/HYP, 12(7.2%). CONCLUSION: The polymorphism genotypes in the promoter region of MBL gene in this chosen population are mostly LYP/LYQ and LXP/LYQ.