First worldwide report of Exserohilum rostratum causing leaf spot on strawberry (Fragaria × ananassa Duch.).

During April 2022, leaf spot was observed on strawberry (Fragaria × ananassa Duch.) with a disease incidence of approximately 45% among 100 plants. Strawberry was cultivated in a nursery at Huzhou University (30.87゜N, 120.13゜E), Zhejiang Province, China. In the strawberry greenhouse, the average temperature was 15-18 degrees, 40%-60% humidity. Early symptoms appeared as dark brown or black spotted necrotic lesions, which expanded from 2 to 6 mm in diameter. Dark brown spots with yellow halos occupied half of the leaf area and eventually developed leaf blight with large yellow halos. To isolate the causal agent, 0.5 cm x 0.5 cm fragments were cut from three symptomatic leaves, and were surface sterilized with 75% ethanol for 30 s and then rinsed three times with sterilized water. The airdried leaf fragments were placed on PDA with 50 µg/ml ampicillin and incubated in the dark at 25℃ for two days. Isolates were obtained by transferring hyphal plugs of 1 mm in diameter onto PDA. The colony morphology was circular and dark brown on the upperside and black on the underside, with cottony mycelium and an large amount of gray aerial mycelium. Conidia were large, light olive-brown to dark olive-brown and light olive-black and septate. The typical conidia were oval or rod-shaped, rarely curved, and dark septa defined the basal and apical cells. In the two typical forms of conidia, the average size of oval conidia was approximately 18.77 × 54.92 µm (11.99 to 26.97 × 35.13 to 74.59 µm, n = 20), and the average size of the rod-shaped conidia was approximately 14.80 × 103.24 µm (11.24 to 24.64 × 73.11 to 131.51 µm, n = 20). The morphological characteristics matched well with previous descriptions of Exserohilum rostratum (Sharma et al. 2014; Liu et al. 2021). The identity of C1-L and C1-S from symptomatic tissues was confirmed by means of multi-locus gene sequencing. Genomic DNA was extracted from the mycelium using the CTAB (cetyltrimethylammonium bromide) method (Griffith & Shaw 1998). Molecular identification was conducted by sequencing the internal transcribed spacer (ITS) rDNA region, partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, partial actin (ACT) gene, and partial beta-tubulin 2 (TUB2), using the primers ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT512F/ACT783R (Carbone and Kohn 1999), T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson, 1995). The obtained sequences of C1-L and C1-S were the same. Moreover, the sequences have been deposited in GenBank under accession numbers ON982516 (ITS), ON996915 (GAPDH), ON996916 (ACT), and ON996917 (TUB2). The results of Basic Local Alignment Search Tool (BLAST) analysis revealed that the ITS, GAPDH, and ACT had 100% identity with the sequences of E. rostratum (GenBank Accession No. LT837834, LT883550, and LT837672, respectively), the TUB2 had 99.61% similarity with BLAST sequences of E. rostratum (LT899391). These morphological characteristics and molecular analyses allowed the identification of the pathogen as E. rostratum. Koch's postulates were performed with five healthy detached strawberry leaves with three inoculations per leaf of the 'Akihime' strawberry variety. Surface-sterilized leaves were wounded with an aseptic needle, and inoculated with 2 mm diameter mycelial plugs from 5-day-old cultures of E. rostratum. Control leaves were also wounded with the aseptic needle, and inoculated with a sterile PDA agar plug. The leaves were incubated at 25℃ in Petri plates with petioles wrapped in moist sterile cotton. The diseased symptoms included black spots on the epidermis of the wounded leaves within 5, 10, and 20 days after inoculation. Mock-inoculated controls remained asymptomatic, and three biological repetitions were conducted. The fungus reisolated from the diseased leaves was confirmed as E. rostratum by sequencing. Abundant reports have shown that E. rostratum can infect many economically important crops such as maize, rice, and pineapple (Sun et al. 2021; Kabore et al. 2022; Luo et al. 2012). To the best of our knowledge, this is the first report of E. rostratum on strawberry in China and worldwide.

Functional analysis of a viral promoter from a strawberry vein banding virus isolate from China.

BACKGROUND: Promoters are important factors affecting gene expression in cells. The driven activities of viral promoters were generally assessed to screen available promoters for transgenic and research and biotech industries. In this study, we cloned a full-length promoter from a Chinese isolate of strawberry vein banding virus (SVBV) and produced several deletion mutants for evaluation of applications in production of reporter proteins in stable transgenic plants. METHODS: The full-length promoter of SVBV (SP1) and its three deletion mutants (SP2, SP3, and SP4) were amplified using polymerase chain reaction. The effects of SVBV SP1, SP2, SP3, and SP4 on gene expression were evaluated using ß-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. RESULTS: Transient expression assays showed that the SVBV SP1 promoter and its three deletion mutants all expressed the reporter genes, albeit at very different levels. Interestingly, transcriptional activity driven by the SP1 promoter was much higher than that of the cauliflower mosaic virus (CaMV) 35S promoter. After stable transformation of the GUS gene into Nicotiana tabacum plants, SVBV SP1-driven transgene expression was approximately 2.6-fold higher than CaMV 35S promoter-driven transgene expression. In addition, GUS gene expression levels were enhanced by co-inoculation of the plants with the SP1 promoter-driven vector carrying the GUS gene and the vector expressing SVBV open reading frame (ORF) V or ORF VI. CONCLUSIONS: The SVBV SP1 promoter from the Chinese isolate evaluated in this study could successfully drive transient and stable expression in plants, it was a stronger promoter than the CaMV 35S and FLt-US promoters and may be more useful for the production of stable transgenic plants.

First report of Colletotrichum fructicola causing anthracnose on cherry (Prunus avium) in China.

Cherry (Prunus avium) has become an important economical fruit in China. In October 2020, a leaf spot disease was found on cherry in the orchard of Taizhou Academy of Agriculture Sciences, Zhejiang, China. The symptoms appeared as small, water-soaked spots on the leaves, which later became larger, dark brown, and necrotic lesions of 1 cm to 3 cm in width, 4 cm to 8 cm in length. Disease incidences of approximately 60% of the leaves were observed by sampling five locations. To isolate the causing agent, small fragments from five target symptomatic leaves were surface-sterilized with 1.0% sodium hypochlorite solution for 1 min and then rinsed three times with sterilized water. Afterwards the leaf fragments were air-dried, plated onto potato dextrose agar (PDA) medium, and incubated at 25 ℃ in the dark for 2 days. The pure cultures were obtained by transferring hyphal plug of 2 mm in diameter onto PDA, which followed single spore isolation. The colony morphology showed light to dark gray, cottony mycelium, with the underside of the culture became brownish after 7 days. Conidia (n = 28) were hyaline, smooth-walled, cylindrical, aseptate, broadly rounded ends, and average size around 3.84 × 12.82 µm (2.99 to 4.87 × 10.27 to 15.68 µm). Appressoria (n = 27) were mostly brown, ovoid and slightly irregular in shape, and average size around 8.04 × 9.68 µm (6.29 to 9.67 × 9.32 to 12.06 µm). Perithecia average size is 106.25 µm, textura angularis, thick-walled. Asci 26.35-49.18 × 5.00-12.03 µm (average size 37.44 × 7.80 µm, n = 17), unitunicate, thin-walled, clavate or cymbiform. Ascospores 13.69-20.93 × 3.86-6.69 µm (average size 16.00 × 5.42 µm, n = 30), one-celled, hyaline, one or two large guttulate at the centre, slightly rounded ends. The morphological characteristics matched well with previous descriptions of Colletotrichum species of C. gloeosporioides species complex, including C. fructicola (Prihastuti et al. 2009; Fu et al. 2019). The identity of two representative isolates (cf2-3 and cf4-4) from different leaves was confirmed by means of multi-locus gene sequencing. To this end, genomic DNA was extracted by the Plant Direct PCR kit (Vazyme Biotech Co., Ltd, China). Molecular identification was conducted by sequencing the internal transcribed spacer (ITS) rDNA region, partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, partial actin (ACT) gene, partial beta-tubulin 2 gene (TUB2), and partial chitin synthase gene (CHS). The obtained sequences have been deposited in GenBank under accession numbers MW581851 and MW581852 (ITS), MW590586 and MW590587 (GAPDH), MW616561 and MW616562 (ACT), MW729380 and MW729381 (TUB2), MW729378 and MW729379 (CHS). The results of Basic Local Alignment Search Tool (BLAST) analysis revealed that the ITS, GAPDH, ACT, TUB2 and CHS sequences of both isolates matched with 100% identity to Colletotrichum fructicola culture collection sequences in GenBank database (JX010165, JX009998, JX009491, JX010405, and JX009866 respectively). These morphological characteristics and molecular analyses allowed the identification of the pathogen as C. fructicola. Koch's postulates were performed with healthy detached cherry leaves of cultivar namely 'HongMi' from Taizhou Academy of Agriculture Sciences. Surface-sterilized leaves were inoculated with five-day-old cultures of C. fructicola mycelial discs of 2 mm in diameter after being wounded with a needle or non-wounded. Control leaves were inoculated with discs of same size PDA agar. Treated leaves were incubated at 25 ℃ in the dark at high relative humidity. Anthracnose symptoms appeared within 3 days both on non-wounded and wounded inoculation approaches. Mock-inoculated controls remained asymptomatic. Biological repetitions were carried out three times. The fungus was reisolated from infected leaves and confirmed as C. fructicola following the methods described above. Until recently, it has been found that C. fructicola can infect tea, apple, pear, Pouteria campechiana in China (Fu et al. 2014; Li et al. 2013; Shi et al. 2018; Yang et al. 2020). To the best of our knowledge, this is the first report of C. fructicola on cherry in China.

Comparative analysis of maca (Lepidium meyenii) proteome profiles reveals insights into response mechanisms of herbal plants to high-temperature stress.

BACKGROUND: High-temperature stress (HTS) is one of the main environmental stresses that limit plant growth and crop production in agricultural systems. Maca (Lepidium meyenii) is an important high-altitude herbaceous plant adapted to a wide range of environmental stimuli such as cold, strong wind and UV-B exposure. However, it is an extremely HTS-sensitive plant species. Thus far, there is limited information about gene/protein regulation and signaling pathways related to the heat stress responses in maca. In this study, proteome profiles of maca seedlings exposed to HTS for 12 h were investigated using a tandem mass tag (TMT)-based proteomic approach. RESULTS: In total, 6966 proteins were identified, of which 300 showed significant alterations in expression following HTS. Bioinformatics analyses indicated that protein processing in endoplasmic reticulum was the most significantly up-regulated metabolic pathway following HTS. Quantitative RT-PCR (qRT-PCR) analysis showed that the expression levels of 19 genes encoding proteins mapped to this pathway were significantly up-regulated under HTS. These results show that protein processing in the endoplasmic reticulum may play a crucial role in the responses of maca to HTS. CONCLUSIONS: Our proteomic data can be a good resource for functional proteomics of maca and our results may provide useful insights into the molecular response mechanisms underlying herbal plants to HTS.

[Effects of scaffold microstructure and mechanical properties on regeneration of tubular dentin].

Tubular dentin is of great significance in the process of tooth tissue and tooth regeneration, because it is not only the structural feature of primary dentin, but also can affect the tooth sensory function, affect the differentiation of dental pulp cells and provide strong mechanical support for teeth. Scaffold is one of the three elements of tissue engineering dentin regeneration. Most experiments on dentin regeneration involve the study of the microstructure and mechanical properties of the scaffold. The microstructure and mechanical characteristics of scaffold materials have important effects on the differentiation and adhesion of odontoblast, it can directly affect the tissue structure of regenerated dentin.

Genome-wide identification and expression analysis of the NAC transcription factor family in tomato (Solanum lycopersicum) during aluminum stress.

BACKGROUND: The family of NAC proteins (NAM, ATAF1/2, and CUC2) represent a class of large plant-specific transcription factors. However, identification and functional surveys of NAC genes of tomato (Solanum lycopersicum) remain unstudied, despite the tomato genome being decoded for several years. This study aims to identify the NAC gene family and investigate their potential roles in responding to Al stress. RESULTS: Ninety-three NAC genes were identified and named in accordance with their chromosome location. Phylogenetic analysis found SlNACs are broadly distributed in 5 groups. Gene expression analysis showed that SlNACs had different expression levels in various tissues and at different fruit development stages. Cycloheximide treatment and qRT-PCR analysis indicated that SlNACs may aid regulation of tomato in response to Al stress, 19 of which were significantly up- or down-regulated in roots of tomato following Al stress. CONCLUSION: This work establishes a knowledge base for further studies on biological functions of SlNACs in tomato and will aid in improving agricultural traits of tomato in the future.

Morphine Addiction and Oxidative Stress: The Potential Effects of Thioredoxin-1.

Long-term administration of morphine for the management of chronic pain will result in tolerance to its analgesic effect and could even cause drug dependence. Numerous studies have demonstrated significant redox alteration in morphine dependence and addiction. Thioredoxin-1 (Trx-1) play important roles in controlling the cellular redox balance. In recent years, several recent studies have demonstrated that Trx-1 may be a promising novel therapeutic target for morphine addiction. In this article, we firstly review the redox alteration in morphine addiction. We also summarize the expression and the protective roles of Trx-1 in morphine dependence. We further highlight the protection of geranylgeranylacetone (GGA), a noncytotoxic pharmacological inducer of Trx-1, in morphine-induced conditioned place preference. In conclusion, Trx-1 may be very promising for clinical therapy of morphine addiction in the future.

Low phosphate represses histone deacetylase complex1 to regulate root system architecture remodeling in Arabidopsis.

The mechanisms involved in the regulation of gene expression in response to phosphate (Pi) deficiency have been extensively studied, but their chromatin-level regulation remains poorly understood. We examined the role of histone acetylation in response to Pi deficiency by using the histone deacetylase complex1 (hdc1) mutant. Genes involved in root system architecture (RSA) remodeling were analyzed by quantitative real-time polymerase chain reaction (qPCR) and chromatin immunoprecipitation qPCR. We demonstrate that histone H3 acetylation increased under Pi deficiency, and the hdc1 mutant was hypersensitive to Pi deficiency, with primary root growth inhibition and increases in root hair number. Concomitantly, Pi deficiency repressed HDC1 protein abundances. Under Pi deficiency, hdc1 accumulated higher concentrations of Fe3+ in the root tips and had higher expression of genes involved in RSA remodeling, such as ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (ALMT1), LOW PHOSPHATE ROOT1 (LPR1), and LPR2 compared with wild-type plants. Furthermore, Pi deficiency enriched the histone H3 acetylation of ALMT1 and LPR1. Finally, genetic evidence showed that LPR1/2 was epistatic to HDC1 in regulating RSA remodeling. Our results suggest a chromatin-level control of Pi starvation responses in which HDC1-mediated histone H3 deacetylation represses the transcriptional activation of genes involved in RSA remodeling in Arabidopsis.

Flapless immediate implant placement into fresh molar extraction socket using platelet-rich fibrin: A case report.

BACKGROUND: There are some challenges concerning immediate implant placement in the molar region. Platelet-rich fibrin (PRF), an autologous biomaterial, has been used widely for periodontal intra-bony defects, sinus augmentation, socket preservation, and gingival recession. However, the literature remains scarce for reports on immediate implants with PRF, particularly in the case of fresh molar extraction socket. CASE SUMMARY: The patient was a 43-year-old woman with maxillary molar vertical crown-root fracture. She underwent flapless immediate implant placement into the fresh molar socket with PRF. At the follow-up visit 15 d post procedure, the vascularization of soft tissue was visible. There was no swelling or pain after the surgery. Six months postoperatively, the regeneration of bone and soft tissues was visible. Subsequently, the definitive restoration was placed. The patient was satisfied with the aesthetic outcomes. CONCLUSION: The flapless immediate implant placement into the fresh molar socket with PRF is a feasible procedure. This case report demonstrates that PRF promotes bone and soft tissue regeneration apart from having an enhanced anti-inflammatory ability. Furthermore, the procedure involves a minimally invasive technique, thus reducing the surgical complexity.

Identification and Analysis of Potential Genes Regulated by an Alphasatellite (TYLCCNA) that Contribute to Host Resistance against Tomato Yellow Leaf Curl China Virus and Its Betasatellite (TYLCCNV/TYLCCNB) Infection in Nicotiana benthamiana.

Recently, begomovirus/betasatellite disease complexes were found to be associated with alphasatellites, and their presence modulated disease symptoms and/or viral DNA accumulation in infected plants. However, the biological functions of alphasatellites during begomovirus/betasatellite infections remain unclear. Tomato yellow leaf curl China virus (TYLCCNV) associated with a betasatellite (TYLCCNB) is a widespread monopartite begomovirus in China. In the Yunnan province of China, the TYLCCNV/TYLCCNB disease complex is found in association with an alphasatellite (TYLCCNA). In this study, in order to explain the mechanisms underlying TYLCCNV/TYLCCNB infection and reductions in viral DNA accumulation caused by TYLCCNA, we analyzed the transcriptome profiles of Nicotiana benthamiana seedlings challenged by TYLCCNV/TYLCCNB or TYLCCNV/TYLCCNB/TYLCCNA using RNA sequencing. In total, 2272 and 1207 differentially expressed genes (DEGs) were identified to respond to TYLCCNV/TYLCCNB and TYLCCNV/TYLCCNB/TYLCCNA infections, respectively. Compared with the DEGs in the TYLCCNV/TYLCCNB-infected N. benthamiana seedlings, the number of DEGs in plants co-infected with TYLCCNV/TYLCCNB + TYLCCNA was significantly reduced. Additionally, 36 DEGs were identified to be regulated by TYLCCNA, six of which were further analyzed using the virus-induced gene silencing (VIGS) approach. Silencing of these six TYLCCNA responsive DEGs caused more severe disease symptoms and higher viral DNA accumulation levels, suggesting that TYLCCNA responsive DEGs may attenuate TYLCCNV/TYLCCNB infection.

The role of thioredoxin system in cancer: strategy for cancer therapy.

PURPOSE: Cancer, a major public health problem, exhibits significant redox alteration. Thioredoxin (Trx) system, including Trx and Trx reductase (TrxR), as well as Trx-interacting protein (TXNIP) play important roles in controlling the cellular redox balance in cancer cells. In most cancers, Trx and TrxR are usually overexpressed and TXNIP is underexpressed. In recent years, some agents targeting Trx, TrxR, and TXNIP were used to explore a therapy approach for cancer patients. METHODS: A systematic search of PMC and the PubMed Database was conducted to summarize the potential of Trx system inhibitors for cancer treatment. RESULTS: In this article, we first summarize the functions of Trx, TrxR, and TXNIP in cancers. We also review some small molecule inhibitors of Trx/TrxR and D-allose (TXNIP inducer) and discuss their antitumor mechanisms. We highlight the combined inhibition of Trx system and GSH system in cancer therapy. We expect that a highly specific and selective antitumor agent with no cytotoxicity on human normal cells could be developed in the future. CONCLUSION: In conclusion, Trx system may be very promising for clinical therapy of cancer in the future.

iTRAQ-based analysis of the Arabidopsis proteome reveals insights into the potential mechanisms of anthocyanin accumulation regulation in response to phosphate deficiency.

Phosphate (Pi) deficiency significantly limits plant growth in natural and agricultural systems. Accumulation of anthocyanins in shoots is a common response of Arabidopsis thaliana to Pi deficiency. To elucidate the mechanisms underlying Pi deficiency-induced anthocyanin accumulation, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ) to investigate protein expression profiles of Arabidopsis thaliana seedlings subjected to Pi deficiency for 7 days. In total, 5,106 proteins were identified, of which 156 displayed significant changes in abundance upon Pi deficiency. Bioinformatics analysis indicated that flavonoid biosynthesis was the most significantly elevated metabolic process under Pi deficiency. We further examined the potential role of the flavonoid biosynthetic pathway using a dihydroflavonol 4-reductase (DFR) mutant (tt3) and quantitative RT-PCR (qRT-PCR) analysis, and found that the tt3 mutant was deprived of transcriptional up-regulation of three genes related to anthocyanin biosynthesis, modification and transport under Pi deficiency. These results showed that Pi deficiency probably enhances the anthocyanin accumulation by promoting the flavonoid biosynthesis. The exact functions of these proteins remain to be examined. Nevertheless, our study increases the understanding of the mechanisms implicated in the anthocyanin accumulation induced by Pi deficiency and adaptive responses of plants to Pi starvation.

Transcription factor WRKY22 promotes aluminum tolerance via activation of OsFRDL4 expression and enhancement of citrate secretion in rice (Oryza sativa).

Whilst WRKY transcription factors are known to be involved in diverse plant responses to biotic stresses, their involvement in abiotic stress tolerance is poorly understood. OsFRDL4, encoding a citrate transporter, has been reported to be regulated by ALUMINUM (Al) RESISTANCE TRANSCRIPTION FACTOR 1 (ART1) in rice, but whether it is also regulated by other transcription factors is unknown. We define the role of OsWRKY22 in response to Al stress in rice by using mutation and transgenic complementation assays, and characterize the regulation of OsFRDL4 by OsWRKY22 via yeas one-hybrid, electrophoretic mobility shift assay and ChIP-quantitative PCR. We demonstrate that loss of OsWRKY22 function conferred by the oswrky22 T-DNA insertion allele causes enhanced sensitivity to Al stress, and a reduction in Al-induced citrate secretion. We next show that OsWRKY22 is localized in the nucleus, functions as a transcriptional activator and is able to bind to the promoter of OsFRDL4 via W-box elements. Finally, we find that both OsFRDL4 expression and Al-induced citrate secretion are significantly lower in art1 oswrky22 double mutants than in the respective single mutants. We conclude that OsWRKY22 promotes Al-induced increases in OsFRDL4 expression, thus enhancing Al-induced citrate secretion and Al tolerance in rice.

Mimic Phosphorylation of a ßC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant.

Phosphorylation of the ßC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-ßC1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana However, how phosphorylation of TYLCCNB-ßC1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-ßC1 that are phosphorylated by SnRK1. The effects of TYLCCNB-ßC1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-ßC1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-ßC1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-ßC1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-ßC1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor.IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, ßC1 (TYLCCNB-ßC1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-ßC1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N. benthamiana ASYMMETRIC LEAVES 1. To our knowledge, this is the first report establishing an inhibitory effect of phosphorylation of TYLCCNB-ßC1 on its pathogenic functions as both a VSR and a symptom determinant and to provide a mechanistic explanation of how SNF1-related protein kinase 1 acts as a host defense factor. These findings expand the scope of phosphorylation-mediated defense mechanisms and contribute to further understanding of plant defense mechanisms against geminiviruses.

iTRAQ analysis of the tobacco leaf proteome reveals that RNA-directed DNA methylation (RdDM) has important roles in defense against geminivirus-betasatellite infection.

Geminiviruses have caused serious losses in crop production. To investigate the mechanisms underlying host defenses against geminiviruses, an isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic approach was used to explore the expression profiles of proteins in Nicotiana benthamiana (N. benthamiana) leaves in response to tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB) at an early phase. In total, 4155 proteins were identified and 272 proteins were changed differentially in response to TYLCCNV/TYLCCNB infection. Bioinformatics analysis indicated that S-adenosyl-l-methionine cycle II was the most significantly up-regulated biochemical process during TYLCCNV/TYLCCNB infection. The mRNA levels of three proteins in S-adenosyl-l-methionine cycle II were further analyzed by qPCR, each was found significantly up-regulated in TYLCCNV/TYLCCNB-infected N. benthamiana. This result suggested a strong promotion of the biosynthesis of available methyl groups during geminivirus infections. We further tested the potential role of RdDM in N. benthamiana by virus-induced gene silencing (VIGS) and found that a disruption in RdDM resulted in more severe infectious symptoms and higher accumulation of viral DNA after TYLCCNV/TYLCCNB infection. Although the precise functions of these proteins still need to be determined, our proteomic results enhance the understanding of plant antiviral mechanisms. BIOLOGICAL SIGNIFICANCE: One of the major limitations to crop growth in the worldwide is the prevalence of geminiviruses. They are able to infect food and cash crops and cause serious crop failures and economic losses worldwide, especially in Africa and Asia. Tomato yellow leaf curl China virus (TYLCCNV), which causes severe viral diseases in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). However, the mechanisms underlying the TYLCCNV/TYLCCNB defense in plants are still not fully understood at the molecular level. In this study, the combined proteomic, bioinformatic and VIGS analyses revealed that TYLCCNV/TYLCCNB invasion caused complex proteomic alterations in the leaves of N. benthamiana involving the processes of stress and defense, energy production, photosynthesis, protein homeostasis, metabolism, cell structure, signal transduction, transcription, transportation, and cell growth/division. Promotion of available methyl groups via the S-adenosyl-l-methionine cycle II pathway in N. benthamiana appeared crucial for antiviral responses. These findings enhance our understanding in the proteomic aspects of host antiviral defenses against geminiviruses, and also demonstrate that the combination of proteomics with bioinformatics and VIGS analysis is an effective approach to investigate systemic plant responses to geminiviruses and to shed light on plant-virus interactions.

Role of perivascular adipose tissue in nicotine­induced endothelial cell inflammatory responses.

Smoking is considered to be one of the primary causes of atherosclerosis and vascular injury. Previous studies have shown that nicotine in tobacco can lead to vascular inflammation and endothelial dysfunction. Perivascular adipose tissue (PVAT) is known to secrete various types of adipokines to maintain vascular homeostasis. The present study investigated whether nicotine­induced PVAT malfunction can accelerate endothelial inflammation and eventually lead to endothelial dysfunction. The levels of inflammatory adipokines, including nuclear factor (NF)­κB, interleukin (IL)­1ß, IL­6 and tumor necrosis factor (TNF)­α, the ICAM­1 and VCAM­1 adhesion molecules and secretion of adiponectin were assessed in mature adipocytes and endothelial cells cultured alone or in co­culture under nicotine stimulation. It was found that nicotine reduced the secretion of adiponectin and stimulated secretion of the NF­κB, IL­1ß, IL­6 and TNF­α inflammatory adipokines in mature adipocytes. Although nicotine stimulated endothelial cells to secrete IL­1ß and IL­6, no significant increase in the secretion of TNF­α was observed. The co­culture of mature adipocytes with endothelial cells markedly augmented the expression of the NF­κB, IL­1ß, IL­6 and TNF­α inflammatory adipokines and the ICAM­1 and VCAM­1 adhesion molecules, and significantly lowered the levels of adiponectin. These findings suggested that nicotine induced mature adipocyte dysfunction, which caused the abnormal secretion of adiponectin and inflammatory adipokines, and exacerbated endothelial inflammation. These findings also suggested a mechanism whereby nicotine induced the secretion of adiponectin and inflammatory cytokines by adipocytes. The results of the present study elucidated a novel pathway induced by cigarette smoke, which contributed to atherosclerosis and vascular injury.

Impact of the Action Time of Inducers on Differentiation of 3T3-L1 Cells.

Objective To explore the effect of the action time of inducers on the differentiation of 3T3-L1 cells to adipocytes. Methods According to the "Cocktail" method,3T3-L1 cells were divided into three groups according to the action time of inducers,with the action time being 2,3 or 4 days,respectively. Cell morphology was observed using inverted microscope and adipose content were detected by Oil red "O" staining and detection of triglyceride. The cell viability was identified by trypan blue staining method. Results The proportion of samples (n=12) with differentiation rate above 80% in group A was 66% (12/18),while the differentiation rate of all the samples (n=18)in group B and group C were above 80%. For the Oil red "O",the OD value at 510 nm in group C was 2.59±0.17,which was significantly higher than that in group A (2.12±0.47;F=6.62,P=0.0001)and group B (2.20±0.17;F=5.15,P=0.0001),while no significant difference was found between group A and group B (F=1.14,P=0.74). As for the triglyceride,the value in group C was (1351.04±119.01)ng/ml,which was significantly higher than that in group A[ (1077.88±272.75)ng/ml;F=6.73,P=0.001] and group B [(1089.38±115.39)ng/ml;F=5.78,P=0.001],while no difference was found between group A and group B (F=0.27,P=0.64). The cell viability in group A,B,and C was (98.3±1.2)%,(98.5±1.8)%,and (98.9±2.1)%,respectively,showing no significant difference (F=0.18,P=0.83). Conclusions The modified procedure for the differentiation of 3T3-L1 cells to adipocytes can increase the differentiation rate and thus may be applied for establishing adipocyte models. The recommended action time is three days.

OsTCTP, encoding a translationally controlled tumor protein, plays an important role in mercury tolerance in rice.

BACKGROUND: Mercury (Hg) is not only a threat to public health but also a growth risk factor to plants, as it is readily accumulated by higher plants. Accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development; however, the detoxification and tolerance mechanisms of plants to Hg stress are still not fully understood. Exposure to toxic Hg also occurs in some crops cultivated under anoxic conditions, such as rice (Oryza sativa L.), a model organism and one of the most important cultivated plants worldwide. In this study, we functionally characterized a rice translationally controlled tumor protein gene (Os11g43900, OsTCTP) involved in Hg stress tolerance. RESULTS: OsTCTP was ubiquitously expressed in all examined plant tissues, especially in actively dividing and differentiating tissues, such as roots and nodes. OsTCTP was found to localize both the cytosol and the nucleus. OsTCTP was induced by mercuric chloride, cupric sulfate, abscisic acid, and hydrogen peroxide at the protein level in a time-dependent manner. Overexpression of OsTCTP potentiated the activities of several antioxidant enzymes, reduced the Hg-induced H2O2 levels, and promoted Hg tolerance in rice, whereas knockdown of OsTCTP produced opposite effects. And overexpression of OsTCTP did not prevent Hg absorption and accumulation in rice. We also demonstrated that Asn 48 and Asn 97 of OsTCTP amino acids were not the potential N-glycosylation sites. CONCLUSIONS: Our results suggest that OsTCTP is capable of decreasing the Hg-induced reactive oxygen species (ROS), therefore, reducing the damage of ROS and enhancing the tolerance of rice plants to Hg stress. Thus, OsTCTP is a valuable gene for genetic engineering to improve rice performance under Hg contaminated paddy soils.

[Establishment and functional identification of a stable cell line overexpression heme oxygenase-1].

OBJECTIVE: To establish a stable cell line overexpression heme oxygenase-1 (HO-1) mediated by a modified lentivirus system and identify its function. METHODS: The HO-1 gene was amplified by polymerase chain reaction and cloned into the modified pLentiLox3.7 expression vectors. The recombinant plasmids were transfected into HEK293T cells and the HO-1 was detected by Western blot. The recombinant plasmids were transfected into HEK293T cells to produce the viruses, with the helping plasmids including plp1, plp2, and VSVG. HEK293T cells were infected by the viruses and the cells that can express HO-1 were identified by Western blot. The reactive oxygen species were detected in the HO-1-overexpression HEK293T cells and the normal cells after the adding of hydrogen peroxide. The same experiment was performed with human umbilical vein endothelial cells. RESULTS: The stable cell line that can overexpress HO-1 was established, which was verified by Western blot. The reactive oxygen species in the HO-1-overexpression HEK293T cells and human umbilical vein endothelial cells decreased obviously after exposure to hydrogen peroxide. CONCLUSIONS: The lentivirus-carrying HO-1 was successfully packaged and the stable cell line overexpression HO-1 was established. HO-1 can play a protective role in the course of oxidative damage.

Protective effect of melatonin on cigarette smoke-induced restenosis in rat carotid arteries after balloon injury.

Vascular restenosis after the interventional angioplasty remains the main obstacle to a favorable long-term patency. Many researches suggest cigarette smoking is one of the most important causes of restenosis. This study was designed to investigate whether melatonin could protect against the cigarette smoke-induced restenosis in rat carotid arteries after balloon injury. Three groups of male rats (normal condition, cigarette smoke exposed, cigarette smoke exposed, and melatonin injected) were used in this study. An established balloon-induced carotid artery injury was performed, and the carotid arteries were harvested from these three groups 14 days later. The ratio of intima to media, the infiltration of inflammatory cells, the expression of inflammatory cytokines (NF-κB, IL-1ß, IL-6, TNF-α, MCP-1), adhesion molecules (ICAM-1, VCAM-1), and eNOS were measured. The results showed that cigarette smoke exposure aggravated the stenosis of the lumen, promoted the infiltration of inflammatory cells and induced the expression of the inflammatory cytokines and adhesion molecules after the balloon-induced carotid artery injury. Moreover, cigarette smoke exposure can inhibit the expression of eNOS. Particularly, we surprised that melatonin could minimize this effect caused by cigarette smoke. These results suggested that melatonin could prevent the cigarette smoke-induced restenosis in rat carotid arteries after balloon injury and the mechanism of its protective effect may be the inhibition of the inflammatory reaction. This also implies melatonin has the potential therapeutic applicability in prevention of restenosis after the vascular angioplasty in smokers.