Corrigendum: Expression of Pumpkin CmbHLH87 Gene Improves Powdery Mildew Resistance in Tobacco.

[This corrects the article DOI: 10.3389/fpls.2020.00163.].

Expression of Pumpkin CmbHLH87 Gene Improves Powdery Mildew Resistance in Tobacco.

Powdery mildew (PM), caused by Podosphaera xanthii, is a major threat to the global cucurbit yield. The molecular mechanisms underlying the PM resistance of pumpkin (Cucurbita moschata Duch.) are largely unknown. A homolog of the basic helix-loop-helix (bHLH) transcription factor was previously identified through a transcriptomic analysis of a PM-resistant pumpkin. In this study, this bHLH homolog in pumpkin has been functionally characterized. CmbHLH87 is present in the nucleus. CmbHLH87 expression in the PM-resistant material was considerably downregulated by PM; and abscisic acid, methyl jasmonate, ethephon, and NaCl treatments induced CmbHLH87 expression. Ectopic expression of CmbHLH87 in tobacco plants alleviated the PM symptoms on the leaves, accelerated cell necrosis, and enhanced H2O2 accumulation. The expression levels of PR1a, PR5, and NPR1 were higher in the PM-infected transgenic plants than in PM-infected wild-type plants. Additionally, the chlorosis and yellowing of plant materials were less extensive and the concentration of bacteria at infection sites was lower in the transgenic tobacco plants than in the wild-type plants in response to bacterial wilt and scab pathogens. CmbHLH87 may be useful for genetic engineering of novel pumpkin cultivars in the future.

Improved Powdery Mildew Resistance of Transgenic Nicotiana benthamiana Overexpressing the Cucurbita moschata CmSGT1 Gene.

Powdery mildew (PM), which is mainly caused by Podosphaera xanthii, is a serious biotrophic pathogen disease affecting field-grown and greenhouse-grown cucurbit crops worldwide. Because fungicides poorly control PM, the development and cultivation of PM-resistant varieties is critical. A homolog of SGT1 (suppressor of the G2 allele of skp1), which encodes a key component of the plant disease-associated signal transduction pathway, was previously identified through a transcriptomic analysis of a PM-resistant pumpkin (Cucurbita moschata) inbred line infected with PM. In this study, we have characterized this SGT1 homolog in C. moschata, and investigated its effects on biotic stress resistance. Subcellular localization results revealed that CmSGT1 is present in the nucleus. Additionally, CmSGT1 expression levels in the PM-resistant material was strongly induced by PM, salicylic acid (SA) and hydrogen peroxide (H2O2). In contrast, SA and H2O2 downregulated CmSGT1 expression in the PM-susceptible material. The ethephon (Eth) and methyl jasmonate (MeJA) treatments upregulated CmSGT1 expression in both plant materials. The constitutive overexpression of CmSGT1 in Nicotiana benthamiana (N. benthamiana) minimized the PM symptoms on the leaves of PM-infected seedlings, accelerated the onset of cell necrosis, and enhanced the accumulation of H2O2. Furthermore, the expression levels of PR1a and PR5, which are SA signaling transduction markers, were higher in the transgenic plants than in wild-type plants. Thus, the transgenic N. benthamiana plants were significantly more resistant to Erysiphe cichoracearum than the wild-type plants. This increased resistance was correlated with cell death, H2O2 accumulation, and upregulated expression of SA-dependent defense genes. However, the chlorosis and yellowing of plant materials and the concentration of bacteria at infection sites were greater in the transgenic N. benthamiana plants than in the wild-type plants in response to infections by the pathogens responsible for bacterial wilt and scab. Therefore, CmSGT1-overexpressing N. benthamiana plants were hypersensitive to these two diseases. The results of this study may represent valuable genetic information for the breeding of disease-resistant pumpkin varieties, and may also help to reveal the molecular mechanism underlying CmSGT1 functions.

Transcriptome profiling of pumpkin (Cucurbita moschata Duch.) leaves infected with powdery mildew.

Cucurbit powdery mildew (PM) is one of the most severe fungal diseases, but the molecular mechanisms underlying PM resistance remain largely unknown, especially in pumpkin (Cucurbita moschata Duch.). The goal of this study was to identify gene expression differences in PM-treated plants (harvested at 24 h and 48 h after inoculation) and untreated (control) plants of inbred line "112-2" using RNA sequencing (RNA-Seq). The inbred line "112-2" has been purified over 8 consecutive generations of self-pollination and shows high resistance to PM. More than 7600 transcripts were examined in pumpkin leaves, and 3129 and 3080 differentially expressed genes (DEGs) were identified in inbred line "112-2" at 24 and 48 hours post inoculation (hpi), respectively. Based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database and GO (Gene Ontology) database, a complex regulatory network for PM resistance that may involve hormone signal transduction pathways, transcription factors and defense responses was revealed at the transcription level. In addition, the expression profiles of 16 selected genes were analyzed using quantitative RT-PCR. Among these genes, the transcript levels of 6 DEGs, including bHLH87 (Basic Helix-loop-helix transcription factor), ERF014 (Ethylene response factor), WRKY21 (WRKY domain), HSF (heat stress transcription factor A), MLO3 (Mildew Locus O), and SGT1 (Suppressor of G-Two Allele of Skp1), in PM-resistant "112-2" were found to be significantly up- or down-regulated both before 9 hpi and at 24 hpi or 48 hpi; this behavior differed from that observed in the PM-susceptible material (cultivar "Jiujiangjiaoding"). The transcriptome data provide novel insights into the response of Cucurbita moschata to PM stress and are expected to be highly useful for dissecting PM defense mechanisms in this major vegetable and for improving pumpkin breeding with enhanced resistance to PM.

Characterization and expression profile of CaNAC2 pepper gene.

The plant-specific NAC (NAM, ATAF, and CUC) transcription factors have diverse role in development and stress regulation. A new transcript encoding NAC protein, homologous to nam-like protein 4 from Petunia was identified from an ABA-regulated subtractive cDNA library of Capsicum annuum seedling. Here, this homolog (named CaNAC2) from C. annuum was characterized and investigated its role in abiotic stress tolerance. Our results indicated that a plant-specific and conserved NAC domain was located in the N-terminus domain of CaNAC2 which was predicted to encode a polypeptide of 410 amino acids. Phylogenetic analysis showed that CaNAC2 belonged to the NAC2 subgroup of the orthologous group 4d. The protein CaNAC2 was subcellularly localized in the nucleus and it had transcriptional activity in yeast cell. CaNAC2 was expressed mainly in seed and root. The transcription expression of CaNAC2 was strongly induced by cold, salt and ABA treatment and inhibited by osmotic stress and SA treatment. Silence of CaNAC2 in virus-induced gene silenced pepper seedlings resulted in the increased susceptibility to cold stress and delayed the salt-induced leaf chlorophyll degradation. These results indicated that this novel CaNAC2 gene might be involved in pepper response to abiotic stress tolerance.

[Advances in the research of application of clinical decision support system in fluid resuscitation following severe burn].

Although guidelines and formulas have been developed through clinical practice to define infusion rate and volume, over- and under-resuscitation are still common, followed by increasing morbidity and mortality. In order to establish an effective management for early fluid resuscitation, the clinical decision support system (CDSS) has been established. The CDSS, by utilizing information systems coupled with decision support technology, could provide recommendations for the amount of fluid to be infused based on measured biological response. The results showed that patients treated with CDSS had a significantly lower mortality, increased ventilator-free days, and ICU-free days as compared with those treated with traditional fluid management. This article reviews the concepts as well as the result of recent clinical studies of CDSS for burn patients.

[Study on lead absorption in pumpkin by atomic absorption spectrophotometry].

A study was carried out on the characteristic of lead absorption in pumpkin via atomic absorption spectrophotometer. The results showed that lead absorption amount in pumpkin increased with time, but the absorption rate decreased with time; And the lead absorption amount reached the peak in pH 7. Lead and cadmium have similar characteristic of absorption in pumpkin.

[The effects of ezrin on the proliferation and the invasiveness of cells of different hepatocellular carcinoma cell lines].

OBJECTIVE: To investigate the different expressions of cytoskeletal organizer ezrin and cytoskeleton protein beta- and gamma-actin in hepatocellular carcinoma (HCC) cell lines with different metastatic potentials and to explore the role of ezrin in cell growth and metastasis in HCC cell lines SF7721 and MHCC97-H. METHODS: Immunofluorescence, RT-PCR and Western blot were used to detect the gene and protein expressions of ezrin and actin in hepatocellular carcinoma cell lines with different metastatic potentials. RNA interference (RNAi) was applied to down-regulate the ezrin expression in SF7721 and MHCC97-H. Changes of the cell growth and metastasis potentials after the RNAi treatment were studied. MTT assay was used to detect cell proliferation changes and Transwell assay was applied to observe the changes of cell motility and invasiveness. RESULTS: Both ezrin and cytoskeleton protein were demonstrated in the cytoplasma of the cells at the same time. The expression of them in cell lines with high metastatic potential, such as SF7721, MHCC-1 and MHCC97-H was obviously higher than in those with low metastatic potentials, such as SMMC-7721, Hep3B and HepG2 (chi2= 13.277, P = 0.010; chi2= 21.815). The mRNA and ezrin and cytoskeleton protein gamma-actin were over-expressed in HCC cell lines with high metastatic potentials. The expressions of beta-actin of cell lines with different metastatic potentials showed no differences. Ezrin protein was successfully down-regulated and the proliferation and the invasiveness of the cells decreased with low ezrin protein level in SF7721 and MHCC97-H. CONCLUSION: Over-expression of ezrin and cytoskeleton protein gamma-actin are associated with the process of metastasis of hepatocellular carcinoma cells. The growth and invasiveness of SF7721 and MHCC97-H cells can be inhibited by down-regulating ezrin expression.

[The influence of downregulation of ezrin expression by RNA interference on the growth and metastasis of hepatocellular carcinoma: experiment in vitro].

OBJECTIVE: To investigate the effect of membrane-cytoskeleton linker ezrin on the growth and metastasis of human hepatocellular carcinoma cell (HCC) lines. METHODS: Human HCC cells of the lines SF/SMMC7721, MHCC97-H, MHCC-1, and HepG2 were cultured. Four pairs of small interfering RNA (siRNA) targeting erzin were designed and transfected into the HCC cells. 48 h after transfection the cell total RNA was extracted and 72 h later the total cell protein was extracted. RT-PCR and Western blotting were used to detect the transfection rates so as to screen the most effective siRNA to be transfected into the HCC cells. HCC cells were collected every day for 7 days to extract the total RNA and protein. Real-time PCR and Western blotting were used to detect the downregulation rate of erzin at different times. MTT method was used to detect the proliferation of the cells. Flow cytometry was used to detect the cell cycle and apoptosis. Scanning electron microscopy was used to observe the cell pseudopods. Transwell test was used to detect the invasion ability of the transfected HCC cells. RESULTS: Real-time PCR and western-blotting revealed that ezrin siRNA notably down-regulated ezrin expression at both mRNA and protein levels. Down-regulation of ezrin expression distinctly decreased the proliferation rates of these 4 kinds of HCC line. After RNAi treatment the cell proportion in G(2)-M phase decreased from 28.07% to 21.53% in the SF/SMMC7721 cells, from 24.94% to 13.92% in the MHCC97-H cells, from 19.30% to 13.2% in the MHCC-1cells, and from 7.73% to 4.24% in the HepG2 cells. After RNAi treatment, the number of pseudopods decreased from 20.8 +/- 3.0 to 13.2 +/- 2.4 in the SF/SMMC7721: cells (P < 0.05), from 18.4 +/- 2.7 to 14.0 +/- 2.9 in the MHCC97-H cells (P < 0.01), from 22.6 +/- 3.5 to 13.3 +/- 1.9 in the MHCC-1: cells (P < 0.01), and from 31.0 +/- 2.9 to 17.8 +/- 2.3 in the HepG2 cells (P < 0.01); and the motility and invasiveness decreased from 49.9 +/- 7.7 to 31.9 +/- 5.2 in the SF/SMMC7721 cells (P < 0.05), from 58.5 +/- 4.2 to 33.0 +/- 3.3 in the MHCC97-H cells (P < 0.01), from 57.6 +/- 6.1 to 28.3 +/- 3.4 in the MHCC-1 cells (P < 0.01), and from 37.3 +/- 3.0 to 25.3 +/- 2.3 in the HepG2 cells (P < 0.01). The pseudopods of the HCC cells remarkably shortened and decreased in number (for the SF/SMMC7721 cells: t = 4.95, P < 0.05, for the MHCC97-H cells: t = 5.88, P < 0.01, for the MHCC-1 cells: t = 5.56, P < 0.01, and for the HepG2 cells: t = 5.71, P < 0.01) after siRNA interference. CONCLUSION: Ezrin is necessary for HCC proliferation and invasion. It is probably an important factor to inhibit tumor reoccurrence and metastasis.

[Influence of the inhibitor of c-Met on the growth and motility of hepatocellular carcinoma cells].

OBJECTIVES: To explore the influence of c-Met inhibitor by synthetic c-Met antisense oligonucleotide, constructive c-Met antisense plasmid and the complex plasmid of U1SnRNA/ ribozyme/anti-Met on the growth and metastasis of hepatocellular carcinoma cells. METHODS: Gene transfection was operated by Lipofectin on SF7721 cells. The difference of the cells before and after transfection was compared by MTT, growth curves and transwell test in vitro. In vivo, the cells before and after transfection were implanted subcutaneously into nude mice respectively to observe tumor growth and metastasis. RESULTS: C-Met antisense oligonucleotide could inhibit the growth of hepatocellular carcinoma SF7721 cells (t=3.58, P<0.05). After transfection, the expression of c-Met protein decreased. Growth curves showed that the cells after transfection proliferated more slowly, about 50% of control cells (F=4.87, P<0.05), and their motility and invasiveness decreased, compared with those before transfected. In vivo experiment, tumors originated from c-Met antisense oligonucleotide treated cells and the antisense/ribozyme/U1SnRNA treated cells grew more slowly (about 54.5% of those from the control cells), and the latent prolonged. After 35 days, the average weight of tumors in the two group nude mice were lighter than that in the control group nude mice (F=5.17, P<0.05). CONCLUSION: Inhibition of c-Met expression by c-Met antisense oligonucleotide and the complex of antisense/ribozyme/U1SnRNA can inhibit the growth and metastasis of SF7721 hepatocarcinoma cells in vitro and in vivo.