<Emphasis Type="Italic">Muscadinia rotundifolia</Emphasis> ‘Noble’ defense response to <Emphasis Type="Italic">Plasmopara viticola</Emphasis> inoculation by inducing phytohormone-mediated stilbene accumulation

Downy mildew (DM), one of the most devastating grape diseases worldwide, is caused by the biotrophic oomycete Plasmopara viticola (Pv). In general, grapevine responds to Pv infection with the accumulation of phytoalexins as part of the innate immune system, and diverse phytoalexins are induced on grapevines with different DM-resistance levels in response to Pv invasion. However, the regulation of phytoalexin biosynthesis during grapevine against Pv is still unclear. Herein, we detected stilbenes by UPLC-ESI-MS/MS and found that resveratrol was accumulated to higher level and earlier in the DM-immune Muscadinia rotundifolia ‘Noble’ than that in the DM-susceptible Vitis vinifera ‘Thompson Seedless’ after Pv inoculation. Additionally, a considerable amount of pterostilbene and ε-viniferin was found in ‘Noble’, while a little was detected in ‘Thompson Seedless’. Resveratrol was glycosylated into piceid both in ‘Noble’ and ‘Thompson Seedless’ after Pv inoculation. The qPCR analysis of gene expression indicated that the resveratrol-synthesis gene (STS) was induced by Pv inoculation earlier in ‘Noble’ than that in ‘Thompson Seedless’, while the pterostilbene-synthesis gene (ROMT) was induced in ‘Noble’ but not in ‘Thompson Seedless’ at all. The piceid-synthesis gene (GT) was generally up-regulated in both cultivars. Sequence analysis of STS, ROMT, and GT promoters revealed that they contained cis-regulatory elements responsive to phytohormones and pathogens. Following Pv inoculation, the level of SA, MeJA, and ABA was found to be consistently higher in ‘Noble’ than those in ‘Thompson Seedless’. The results of exogenous hormone elicitation further demonstrated that the accumulation of stilbenes was regulated by phytohormones. The earlier and higher accumulation of phytohormones and consequent induction of stilbene synthesis may play an important role in grapevine defense against downy mildew disease.     

Transient silencing of VvCSN5 enhances powdery mildew resistance in grapevine (Vitis vinifera)

As one of the most economically important fruit crops in the world, the grapevine (Vitis vinifera) suffers significant yield losses from various pathogens including powdery mildew caused by Erysiphe necator. In contrast, several wild Chinese grapevines, including Vitis pseudoreticulata accession Baihe-35-1, are highly resistant to powdery mildew pathogens. Here, we identified a grapevine gene CSN5 (COP9 signalosome complex subunit 5), designated VvCSN5, that was differentially expressed between the resistant ‘Baihe-35-1’ and susceptible ‘Thompson Seedless’ during powdery mildew isolate Erysiphe necator NAFU1 infection. Moreover, transient silencing of VvCSN5 in ‘Thompson Seedless’ leaves enhanced resistance to En NAFU1. This resistance manifested in cell wall callose deposition at attempted infection sites and hypersensitive response-like cell death of penetrated epidermal cells. Several defense-related marker genes (VvPR1, VvPR3, VvPAD4, and VvRBOHD) had higher basal expression levels in VvCSN5-silenced leaves. In addition, we found the structure and activity of CSN5 promoters in ‘Thompson Seedless’ and ‘Baihe-35-1’ were different, which may have been behind their different resistances to powdery mildew infection. Taken together, these results implied that grapevine CSN5 plays an important role in the response to powdery mildew infection.

     

Medicarpin confers powdery mildew resistance in Medicago truncatula and activates the salicylic acid signalling pathway

Powdery mildew (PM) caused by the obligate biotrophic fungal pathogen Erysiphe pisi is an economically important disease of legumes. Legumes are rich in isoflavonoids, a class of secondary metabolites whose role in PM resistance is ambiguous. Here we show that the pterocarpan medicarpin accumulates at fungal infection sites, as analysed by fluorescein‐tagged medicarpin, and provides penetration and post‐penetration resistance against E. pisi in Medicago truncatula in part through the activation of the salicylic acid (SA) signalling pathway. Comparative gene expression and metabolite analyses revealed an early induction of isoflavonoid biosynthesis and accumulation of the defence phytohormones SA and jasmonic acid (JA) in the highly resistant M. truncatula genotype A17 but not in moderately susceptible R108 in response to PM infection. Pretreatment of R108 leaves with medicarpin increased SA levels, SA‐associated gene expression, and accumulation of hydrogen peroxide at PM infection sites, and reduced fungal penetration and colony formation. Strong parallels in the levels of medicarpin and SA, but not JA, were observed on medicarpin/SA treatment pre‐ or post‐PM infection. Collectively, our results suggest that medicarpin and SA may act in concert to restrict E. pisi growth, providing new insights into the metabolic and signalling pathways required for PM resistance in legumes.     

Expression pattern, genomic structure, and promoter analysis of the gene encoding stilbene synthase from Chinese wild Vitis pseudoreticulata

The gene encoding stilbene synthase (STS) plays a central role in many biochemical and physiological actions, and its metabolite resveratrol possesses broad-spectrum resistance to pathogens, as well as diverse pharmacological properties, notably an anticancer effect. Here, we report the expression analysis of the gene encoding STS and its promoter function from a powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata, and compare it with two PM-susceptible cultivated grapevines, Vitis vinifera cvs. Carignane and Thompson Seedless. We show an unusual expression pattern of STS in V. pseudoreticulata, which differs markedly from that of the cultivated species. Sequence comparisons reveal that the genomic DNA sequences encoding STS in the three grapevines are highly conserved, but a novel residue mutation within the key motif of STS is solely present in V. pseudoreticulata. Moreover, the STS promoter in V. pseudoreticulata displays a significantly different structure from that found in the two V. vinifera. The three promoter-driven GUS differential expression patterns in transformed tobacco plants induced with Alternaria alternata, methyl jasmonate, and wounding indicated that the structurally different STS promoter of V. pseudoreticulata is responsible for its specific regulatory function. We also demonstrate that the expression of STS genes from their native promoters are functional in transformed tobacco and retain pathogen inducibility. Importantly, the genomic DNA-2 of V. pseudoreticulata under its native promoter shows good induction and the maximum level of resveratrol content. These findings further our understanding of the regulation of STS expression in a resistant grapevine and provide a new pathogen-inducible promoter system for the genetic improvement of plant disease resistance.     

A circulatory system useful both for long-term somatic embryogenesis and genetic transformation in Vitis vinifera L. cv. Thompson Seedless

To establish an efficient regeneration protocol for functional validation and variety resistance improvement, a long-term system that useful for embryogenic culture maintenance and transformation was developed through recurrent cycles of secondary embryogenesis from Vitis vinifera L. cv. Thompson Seedless. Three media and five types of somatic embryo in secondary embryogenesis were evaluated. Somatic embryos (SE) in the torpedo and mid-cotyledonary stages gave the best embryogenic responses with re-induction rates of about 80 %. Embryogenic callus, proembryonic masses and SE produced in the system, could be propagated for over 3 years and all proved competent for Agrobacterium-mediated transformation. Based on this system, different transgenic selection regimes were compared. Addition of kanamycin at 4 weeks after co-cultivation was optimal for embryo recovery. Plant conversion was improved by alternating culture on two media: one containing 0.2 mg l^?1 BA and the other 0.25 mg l^?1 kinetin. To further test the efficiency of the system, a ubiquitin ligase gene (VpPUB23) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. Of the 351 transgenic plants obtained, those overexpressing VpPUB23 exhibited decreased resistance to powdery mildew compared with non-transgenic plants.     

A RAPD Assay for Strain Typing of the Biotrophic Grape Powdery Mildew Fungus Uncinula necator Using DNA Extracted from the Mycelium

Délye, C., Corio-Costet, M.-F., and Laigret, F. 1995. A RAPD assay for strain typing of the biotrophic grape powdery mildew fungus Uncinula necator using DNA extracted from the mycelium. Experimental Mycology 19, 234-237. We describe, for the first time, a RAPD assay using DNA extracted from the mycelium of a powdery mildew fungus, Uncinula necator, a pathogen of grape. No contamination by plant DNA was observed, and the resulting patterns were fully repetitive. RAPD profiles were unchanged when using two different DNA polymerases or three different thermocyclers. Thirteen strains were tested for amplification, using 95 primers. Only 4% of the amplified fragments were polymorphic. Cluster analysis revealed that the strains from the same geographical origin had the higher genetic similarity, suggesting a short-range dissemination of U. necator. This RAPD assay was also successfully applied to the grape downy mildew fungus, Plasmopara viticola, indicating that it can be used for other fungi which cannot be grown on artificial media.     

Physiological Response of Resistant and Susceptible Vitis vinifiera Cultivars to Meloidogyne incognita

The effect of Meloidogyne incognita on growth, general physiological response, and the concentration of reducing and nonreducing sugars at the nematode feeding sites of French Colombard (susceptible) and Thompson Seedless (moderately resistant) Vitis vinifiera cultivars was studied up to 2,100 degree-days (DD-base 10 C). Nematode stress dosage, measured as the product of cumulative number of juveniles and females and their total energy (calories) demand, accounted for up to 15 and 10% of the energy assimilated by French Colombard and Thompson Seedless plants, respectively. Total leaf area, total carbon dioxide fixed, transpiration rate, stomatal conductance, and internal leaf CO₂ concentration were not affected, but energy assimilated into plant tissue and respiration were decreased by nematode infection in both cultivars. Energy consumed by nematodes accounted for most of the difference in total energy assimilated between infected and uninfected plants on French Colombard but not on Thompson Seedless, suggesting that the resistant cultivar may be using more energy to curtail the nematode''s activity. Nematodes did not affect the concentration of reducing sugars, but the concentration of nonreducing sugars increased in French Colombard and decreased in Thompson Seedless. This indicates that there was more translocation of photosynthate to the feeding sites of the susceptible than to those of the resistant cultivar, and may explain why M. incognita causes more damage to French Colombard than to Thompson Seedless.     

LRP5 promotes cancer stem cell traits and chemoresistance in colorectal cancer

The overactivation of canonical Wnt/β‐catenin pathway and the maintenance of cancer stem cells (CSCs) are essential for the onset and malignant progression of most human cancers. However, their regulatory mechanism in colorectal cancer (CRC) has not yet been well demonstrated. Low‐density lipoprotein receptor‐related protein 5 (LRP5) has been identified as an indispensable co‐receptor with frizzled family members for the canonical Wnt/β‐catenin signal transduction. Herein, we show that activation of LRP5 gene promotes CSCs‐like phenotypes, including tumorigenicity and drug resistance in CRC cells, through activating the canonical Wnt/β‐catenin and IL‐6/STAT3 signalling pathways. Clinically, the expression of LRP5 is upregulated in human CRC tissues and closely associated with clinical stages of patients with CRC. Further analysis showed silencing of endogenous LRP5 gene is sufficient to suppress the CSCs‐like phenotypes of CRC through inhibiting these two pathways. In conclusion, our findings not only reveal a regulatory cross‐talk between canonical Wnt/β‐catenin signalling pathway, IL‐6/STAT3 signalling pathway and CD133‐related stemness that promote the malignant behaviour of CRC, but also provide a valuable target for the diagnosis and treatment of CRC.     

Opposing effects of MYZUS PERSICAE‐INDUCED LIPASE 1 and jasmonic acid influence the outcome of Arabidopsis thaliana–Fusarium graminearum interaction

Fusarium graminearum (Fg) is an important fungal pathogen of small grain cereals that can also infect Arabidopsis thaliana. In Arabidopsis, jasmonic acid (JA) signalling involving JASMONATE RESISTANT 1 (JAR1), which synthesizes JA‐isoleucine, a signalling form of JA, promotes susceptibility to Fg. Here we show that Arabidopsis MYZUS PERSICAE‐INDUCED LIPASE 1 (MPL1), via its influence on limiting JA accumulation, restricts Fg infection. MPL1 expression was up‐regulated in response to Fg infection, and MPL1‐OE plants, which overexpress MPL1, exhibited enhanced resistance against Fg. In comparison, disease severity was higher on the mpl1 mutant than the wild type. JA content was lower in MPL1‐OE and higher in mpl1 than in the wild type, indicating that MPL1 limits JA accumulation. Pharmacological experiments confirmed the importance of MPL1‐determined restriction of JA accumulation on curtailment of Fg infection. Methyl‐JA application attenuated the MPL1‐OE‐conferred resistance, while the JA biosynthesis inhibitor ibuprofen enhanced resistance in mpl1. Also, the JA biosynthesis‐defective opr3 mutant was epistatic to mpl1, resulting in enhanced resistance in mpl1 opr3 plants. In comparison, JAR1 was not essential for the mpl1‐conferred susceptibility to Fg. Considering that methyl‐JA promotes Fg growth in culture, we suggest that in part MPL1 curtails disease by limiting the availability of a plant‐derived Fg growth‐promoting factor.     

Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high‐protein feeding to regulate glucose homeostasis in vivo

High‐protein feeding acutely lowers postprandial glucose concentration compared to low‐protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr–PKA–ERK–K_ATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high‐ vs. low‐protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high‐protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.     

PAK4 suppresses motor neuron degeneration in hSOD1G93A‐linked amyotrophic lateral sclerosis cell and rat models

ObjectivesAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons (MN). CREB pathway‐mediated inhibition of apoptosis contributes to neuron protection, and PAK4 activates CREB signalling in diverse cell types. This study aimed to investigate PAK4’s effect and mechanism of action in ALS.MethodsWe analysed RNA levels by qRT‐PCR, protein levels by immunofluorescence and Western blotting, and apoptosis by flow cytometry and TUNEL staining. Cell transfection was performed for in vitro experiment. Mice were injected intraspinally to evaluate PAK4 function in vivo experiment. Rotarod test was performed to measure motor function.ResultsThe expression and activation of PAK4 significantly decreased in the cell and mouse models of ALS as the disease progressed, which was caused by the negative regulation of miR‐9‐5p. Silencing of PAK4 increased the apoptosis of MN by inhibiting CREB‐mediated neuroprotection, whereas overexpression of PAK4 protected MN from hSOD1^G93A‐induced degeneration by activating CREB signalling. The neuroprotective effect of PAK4 was markedly inhibited by CREB inhibitor. In ALS models, the PAK4/CREB pathway was inhibited, and cell apoptosis increased. In vivo experiments revealed that PAK4 overexpression in the spinal neurons of hSOD1^G93A mice suppressed MN degeneration, prolonged survival and promoted the CREB pathway.ConclusionsPAK4 protects MN from degeneration by activating the anti‐apoptotic effects of CREB signalling, suggesting it may be a therapeutic target in ALS.

Schematic representation of the mechanism of PAK4 protecting MN from apoptosis in ALS. PAK4 increases CREB levels and activation, leading to the upregulation of PGC‐1a and Bcl‐2, thereby decreasing cleaved‐caspase3 levels, and inhibiting MN degeneration. miR‐9‐5p is responsible for the decreased expression of PAK4 in ALS.     

METTL3‐m6 A methylase regulates the osteogenic potential of bone marrow mesenchymal stem cells in osteoporotic rats via the Wnt signalling pathway

ObjectivesBone marrow mesenchymal stem cells (BMSCs) hold a high osteogenic differentiation potential, but the mechanisms that control the osteogenic ability of BMSCs from osteoporosis (OP‐BMSCs) need further research. The purpose of this experiment is to discuss the osteogenic effect of Mettl3 on OP‐BMSCs and explore new therapeutic target that can enhance the bone formation ability of OP‐BMSCs.Materials and MethodsThe bilateral ovariectomy (OVX) method was used to establish the SD rat OP model. Dot blots were used to reveal the different methylation levels of BMSCs and OP‐BMSCs. Lentiviral‐mediated overexpression of Mettl3 was applied in OP‐BMSCs. QPCR and WB detected the molecular changes of osteogenic‐related factors and Wnt signalling pathway in vitro experiment. The staining of calcium nodules and alkaline phosphatase detected the osteogenic ability of OP‐BMSCs. Micro‐CT and histological examination evaluated the osteogenesis of Mettl3 in OP rats in vivo.ResultsThe OP rat model was successfully established by OVX. Methylation levels and osteogenic potential of OP‐BMSCs were decreased in OP‐BMSCs. In vitro experiment, overexpression of Mettl3 could upregulate the osteogenic‐related factors and activate the Wnt signalling pathway in OP‐BMSCs. However, osteogenesis of OP‐BMSCs was weakened by treatment with the canonical Wnt inhibitor Dickkopf‐1. Micro‐CT showed that the Mettl3(+) group had an increased amount of new bone formation at 8 weeks. Moreover, the results of histological staining were the same as the micro‐CT results.ConclusionsTaken together, the methylation levels and osteogenic potential of OP‐BMSCs were decreased in OP‐BMSCs. In vitro and in vivo studies, overexpression of Mettl3 could partially rescue the decreased bone formation ability of OP‐BMSCs by the canonical Wnt signalling pathway. Therefore, Mettl3 may be a key targeted gene for bone generation and therapy of bone defects in OP patients.

In this study, the osteoporosis rat model was successfully established by OVX. OP‐BMSCs were successfully isolated and cultured from the femur of OP rat. Lentiviral‐mediated overexpression of Mettl3 could partially rescue the impaired osteogenic ability of OP‐BMSCs by activating the canonical Wnt signalling pathway in vitro and in vivo .