Vascular Occlusions in Grapevines with Pierce’s Disease Make Disease Symptom Development Worse

Vascular occlusions are common structural modifications made by many plant species in response to pathogen infection. However, the functional role(s) of occlusions in host plant disease resistance/susceptibility remains controversial. This study focuses on vascular occlusions that form in stem secondary xylem of grapevines (Vitis vinifera) infected with Pierce’s disease (PD) and the impact of occlusions on the hosts’ water transport and the systemic spread of the causal bacterium Xylella fastidiosa in infected vines. Tyloses are the predominant type of occlusion that forms in grapevine genotypes with differing PD resistances. Tyloses form throughout PD-susceptible grapevines with over 60% of the vessels in transverse sections of all examined internodes becoming fully blocked. By contrast, tylose development was mainly limited to a few internodes close to the point of inoculation in PD-resistant grapevines, impacting only 20% or less of the vessels. The extensive vessel blockage in PD-susceptible grapevines was correlated to a greater than 90% decrease in stem hydraulic conductivity, compared with an approximately 30% reduction in the stems of PD-resistant vines. Despite the systemic spread of X. fastidiosa in PD-susceptible grapevines, the pathogen colonized only 15% or less of the vessels in any internode and occurred in relatively small numbers, amounts much too small to directly block the vessels. Therefore, we concluded that the extensive formation of vascular occlusions in PD-susceptible grapevines does not prevent the pathogen’s systemic spread in them, but may significantly suppress the vines’ water conduction, contributing to PD symptom development and the vines’ eventual death.Pierce’s disease (PD) of grapevines (Vitis vinifera), currently jeopardizing the wine and table grape industries in the southern United States and California, as well as in many other countries, is a vascular disease caused by the xylem-limited bacterium Xylella fastidiosa (Hopkins, 1989; Varela et al., 2001). The pathogen is transmitted mostly via xylem sap-feeding sharpshooters (e.g. Homalodisca vitripennis; Redak et al., 2004) and inhabits, proliferates, and spreads within the vessel system of a host grapevine (Fry and Milholland, 1990a; Hill and Purcell, 1995). PD symptom development in grapevines depends on the interactions between the pathogen and the host vine’s xylem tissue, through which the pathogen may achieve its systemic spread (Purcell and Hopkins, 1996; Krivanek and Walker, 2005; Pérez-Donoso et al., 2010; Sun et al., 2011). Since the path for this spread is the host’s xylem system, xylem tissue and its vessels have become the major focus for studying potential X. fastidiosa-host vine interactions at the cellular or tissue levels (Fry and Milholland, 1990b; Stevenson et al., 2004a; Sun et al., 2006, 2007; Thorne et al., 2006).One major issue related to this host-pathogen interaction is the relationship of a vine’s xylem anatomy to the X. fastidiosa population’s spread. Sun et al. (2006) did a detailed anatomical analysis of the stem secondary xylem, especially the vessel system. Stevenson et al. (2004b) described xylem connection patterns between a stem and the attached leaves. Other studies reported the presence of open continuous vessels connecting stems and leaves, which represent conduits that might facilitate the pathogen’s stem-to-leaf movement (Thorne et al., 2006; Chatelet et al., 2006, 2011). Chatelet et al. (2011) also suggested that vessel size and ray density were the two xylem features that were most relevant to the restriction of X. fastidiosa’s movement. These studies indicate the importance of understanding the grapevine’s xylem anatomy in order to characterize the grapevine host’s susceptibility or resistance to PD.Another focus of PD-related xylem studies is the tylose, a developmental modification that has important impacts on a vessel’s role in water transport and, potentially, its availability as a path for X. fastidiosa’s systemic spread through a vine. Tyloses are outgrowths into a vessel lumen from living parenchyma cells that are adjacent to the vessel and can transfer solutes into the transpiration stream via vessel-parenchyma (V-P) pit pairs (Esau, 1977). Tylose development involves the expansion of the portions of the parenchyma cell’s wall that are shared with the neighboring vessels, specifically the so-called pit membranes (PMs). Intensive tylose development may eventually block the affected vessel (Sun et al., 2006). Since tyloses occur in the vessel system of PD-infected grapevines (Esau, 1948; Mollenhauer and Hopkins, 1976; Stevenson et al., 2004a; Krivanek et al., 2005) that is also the avenue of X. fastidiosa’s spread and water transport, a great deal of effort has been made to understand tyloses and their possible relations to grapevine PD as well as to diseases caused by other vascular system-localized pathogens. One major aspect is to clarify the process of tylose development itself, in which an open vessel may be gradually sealed (Sun et al., 2006, 2008). Our investigations of the initiation of tylose formation in grapevines have identified ethylene as an important factor (Pérez-Donoso et al., 2007; Sun et al., 2007). In terms of the relationship of tyloses to grapevine PD, studies have so far led to several controversial viewpoints that are discussed below (Mollenhauer and Hopkins, 1976; Fry and Milholland, 1990b; Stevenson et al., 2004a; Krivanek et al., 2005). However, more convincing evidence is still needed to support any of them.Another issue potentially relevant to PD symptom development is the possibility that X. fastidiosa cells and/or their secretions contribute to the blockage of water transport in host vines. The bacteria secrete an exopolysaccharide (Roper et al., 2007a) that contributes to the formation of cellular aggregates. Accumulations of X. fastidiosa cells embedded in an exopolysaccharide matrix (occasionally identified as biofilms, gums, or gels) have been reported in PD-infected grapevines (Mollenhauer and Hopkins, 1974; Fry and Milholland, 1990a; Newman et al., 2003; Stevenson et al., 2004b). However, a more detailed investigation is still needed to clarify if and to what extent these aggregates affect water transport in infected grapevines.The xylem tissue in which X. fastidiosa spreads can be classified as primary xylem or secondary xylem, being derived from procambium or vascular cambium, respectively. Primary xylem is located in and responsible for material transport and structural support in young organs (i.e. leaves, young stems, and roots), while secondary xylem is the conductive and supportive tissue in more mature stems and roots (Esau, 1977). It should be noted that most of the earlier experimental results have been based on examinations of leaves (petioles or veins) or young stems of grapevines, which contain mostly primary xylem with little or no secondary xylem. However, X. fastidiosa’s systemic spread generally occurs after introduction during the insect vector’s feeding from an internode of one shoot. The pathogen then moves upward along that shoot and also downward toward the shoot base. The downward movement allows the bacteria to enter the vine’s other shoots via the shared trunk and then move upward (Stevenson et al., 2004a; Sun et al., 2011). These upward and downward bacterial movements occur through stems that contain significant amounts of secondary xylem but relatively dysfunctional primary xylem. Secondary and primary xylem show some major differences in the structure and arrangement of their cell components (Esau, 1977). In terms of the vessel system that is the path of X. fastidiosa’s spread, the secondary xylem has a large number of much bigger vessels with scalariform (ladder-like) PMs (and pit pairs) as the sole intervessel (I-V) PM type, compared with the primary xylem, which contains only a limited number of smaller vessels with multiple types of I-V PMs (Esau, 1948; Sun et al., 2006). Vessels in secondary xylem are also different from those in primary xylem in forming vessel groups and in the number of parenchyma cells associated with a vessel (as seen in transverse sections of xylem tissue). These features of secondary xylem can affect the initial entry and subsequent I-V movement of the pathogen and the formation of vascular occlusions, respectively, in stems containing significant amounts of secondary xylem. Recently, the X. fastidiosa population size only in stems with secondary xylem was found to correlate with the grapevine’s resistance to PD (Baccari and Lindow, 2011), indicating an important role of stem secondary xylem in determining a host vine’s disease resistance. Despite these facts, little is known about the pathogen-grapevine interactions in the stem secondary xylem and their possible impacts on disease development.This study addresses X. fastidiosa-grapevine interactions in stem secondary xylem and examines the resulting impacts on overall vine physiology, with a primary focus on vine water transport. We have made use of grapevine genotypes displaying different PD resistances and explored whether differences in the pathogen’s induction of vascular occlusions occur among the genotypes and, if so, how the differences impact X. fastidiosa’s systemic spread. Our overall, longer-term aim is to elucidate the functional role of vascular occlusions in PD development, an understanding that we view to be essential for identifying effective approaches for controlling this devastating disease.     

Prohibitin Interacts with Envelope Proteins of White Spot Syndrome Virus and Prevents Infection in the Red Swamp Crayfish,Procambarus clarkii

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses, cell proliferation, and immune regulation. However, the function of PHBs in crustacean immunity remains largely unknown. In the present study, we identified a PHB in Procambarus clarkii red swamp crayfish, which was designated PcPHB1. PcPHB1 was widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge at the mRNA level and the protein level. These observations prompted us to investigate the role of PcPHB1 in the crayfish antiviral response. Recombinant PcPHB1 (rPcPHB1) significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. The quantity of WSSV in PcPHB1 knockdown crayfish was increased compared with that in the controls. The effects of RNA silencing were rescued by rPcPHB1 reinjection. We further confirmed the interaction of PcPHB1 with the WSSV envelope proteins VP28, VP26, and VP24 using pulldown and far-Western overlay assays. Finally, we observed that the colloidal gold-labeled PcPHB1 was located on the outer surface of the WSSV, which suggests that PcPHB1 specifically binds to the envelope proteins of WSSV. VP28, VP26, and VP24 are structural envelope proteins and are essential for attachment and entry into crayfish cells. Therefore, PcPHB1 exerts its anti-WSSV effect by binding to VP28, VP26, and VP24, preventing viral infection. This study is the first report on the antiviral function of PHB in the innate immune system of crustaceans.     

Quantitative and Qualitative Involvement of P3N-PIPO in Overcoming Recessive Resistance against Clover Yellow Vein Virus in Pea Carrying the cyv1 Gene

In pea carrying cyv1, a recessive gene for resistance to Clover yellow vein virus (ClYVV), ClYVV isolate Cl-no30 was restricted to the initially infected cells, whereas isolate 90-1 Br2 overcame this resistance. We mapped the region responsible for breaking of cyv1-mediated resistance by examining infection of cyv1 pea with chimeric viruses constructed from parts of Cl-no30 and 90-1 Br2. The breaking of resistance was attributed to the P3 cistron, which is known to produce two proteins: P3, from the main open reading frame (ORF), and P3N-PIPO, which has the N-terminal part of P3 fused to amino acids encoded by a small open reading frame (ORF) called PIPO in the +2 reading frame. We introduced point mutations that were synonymous with respect to the P3 protein but nonsynonymous with respect to the P3N-PIPO protein, and vice versa, into the chimeric viruses. Infection of plants with these mutant viruses revealed that both P3 and P3N-PIPO were involved in overcoming cyv1-mediated resistance. Moreover, P3N-PIPO quantitatively affected the virulence of Cl-no30 in cyv1 pea. Additional expression in trans of the P3N-PIPO derived from Cl-no30, using White clover mosaic virus as a vector, enabled Cl-no30 to move to systemic leaves in cyv1 pea. Susceptible pea plants infected with chimeric ClYVV possessing the P3 cistron of 90-1 Br2, and which were therefore virulent toward cyv1 pea, accumulated more P3N-PIPO than did those infected with Cl-no30, suggesting that the higher level of P3N-PIPO in infected cells contributed to the breaking of resistance by 90-1 Br2. This is the first report showing that P3N-PIPO is a virulence determinant in plants resistant to a potyvirus.     

Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.     

Anti-inflammatory effects of anethole in lipopolysaccharide-induced acute lung injury in mice

Aims

Anethole, the major component of the essential oil of star anise, has been reported to have antioxidant, antibacterial, antifungal, anti-inflammatory, and anesthetic properties. In this study, we investigated the anti-inflammatory effects of anethole in a mouse model of acute lung injury induced by lipopolysaccharide (LPS).

Main methods

BALB/C mice were intraperitoneally administered anethole (62.5, 125, 250, or 500 mg/kg) 1 h before intratracheal treatment with LPS (1.5 mg/kg) and sacrificed after 4 h. The anti-inflammatory effects of anethole were assessed by measuring total protein and cell levels and inflammatory mediator production and by histological evaluation and Western blot analysis.

Key findings

LPS significantly increased total protein levels; numbers of total cells, including macrophages and neutrophils; and the production of inflammatory mediators such as matrix metalloproteinase 9 (MMP-9), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) in bronchoalveolar lavage fluid. Anethole (250 mg/kg) decreased total protein concentrations; numbers of inflammatory cells, including neutrophils and macrophages; and the inflammatory mediators MMP-9, TNF-α and NO. In addition, pretreatment with anethole decreased LPS-induced histopathological changes. The anti-inflammatory mechanism of anethole in LPS-induced acute lung injury was assessed by investigating the effects of anethole on NF-κB activation. Anethole suppressed the activation of NF-κB by blocking IκB-α degradation.

Significance

These results, showing that anethole prevents LPS-induced acute lung inflammation in mice, suggest that anethole may be therapeutically effective in inflammatory conditions in humans.     

Macrophage migration inhibitory factor in mud crab Scylla paramamosain: Molecular cloning, expression profiles in various tissues and under Vibrio challenge

As one of the first found cytokines, macrophage migration inhibitory factor (MIF) plays an important role in several physiological processes in crabs. In this study, a full-length MIF cDNA (GenBank accession number: JX131610) from mud crab Scylla paramamosain (Sp) was cloned based on a sequence of S. paramamosain cDNA library. The full length of SpMIF was 734 bp consisting of a 363 bp open reading frame encoding the SpMIF, a 120 amino acid peptide chain. The molecular weight of SpMIF was 13.46 kDa with the pI of 6.82. The alignment analysis showed that SpMIF appeared to be closely related to the counterpart from Eriocheir sinensis (68%). Quantitative real-time PCR analysis revealed that SpMIF was highly expressed in hepatopancreas and hemocytes. In addition, the expression level of SpMIF was increased significantly after a 6-h challenge by Vibrio parahaemolyticus (4.00 × 10⁶ CFU/mL), peaked at 8 h, and then declined to the common level in 48 h. This data indicated that SpMIF was cloned successfully, and suggested that it participated in the immune system of mud crabs.     

Correlations and comparisons of quantitative trait loci with family per se and testcross performance for grain yield and related traits in maize

Simultaneous improvement in grain yield and related traits in maize hybrids and their parents (inbred lines) requires a better knowledge of genotypic correlations between family per se performance (FP) and testcross performance (TP). Thus, to understand the genetic basis of yield-related traits in both inbred lines and their testcrosses, two F _2:3 populations (including 230 and 235 families, respectively) were evaluated for both FP and TP of eight yield-related traits in three diverse environments. Genotypic correlations between FP and TP, $ \hat{r}_{\text{g}} $ (FP, TP), were low (0–0.16) for grain yield per plant (GYPP) and kernel number per plant (KNPP) in the two populations, but relatively higher (0.32–0.69) for the other six traits with additive effects as the primary gene action. Similar results were demonstrated by the genotypic correlations between observed and predicted TP values based on quantitative trait loci positions and effects for FP, $ \hat{r}_{\text{g}} $ (M _FP, Y _TP). A total of 88 and 35 QTL were detected with FP and TP, respectively, across all eight traits in the two populations. However, the genotypic variances explained by the QTL detected in the cross-validation analysis were much lower than those in the whole data set for all traits. Several common QTL between FP and TP that accounted for large phenotypic variances were clustered in four genomic regions (bin 1.10, 4.05–4.06, 9.02, and 10.04), which are promising candidate loci for further map-based cloning and improvement in grain yield in maize. Compared with publicly available QTL data, these QTL were also detected in a wide range of genetic backgrounds and environments in maize. These results imply that effective selection based on FP to improve TP could be achieved for traits with prevailing additive effects.     

Adhesion of osteoblast-like cell on silicon-doped TiO2 film prepared by cathodic arc deposition

Silicon-doped TiO₂ (Si–TiO₂) and pure TiO₂ films were deposited on titanium substrates by cathodic arc deposition technique. The surface characteristics of the films, such as surface topography, elemental composition and wettability, were studied. About 4.6 % Si was incorporated into the Si–TiO₂ films with a water contact angle of about 83°. The adhesive behaviors of osteoblast-like MG63 cells on both films were investigated through cell counting assay, immunocytochemistry, real-time PCR and western blotting analysis. Cells cultured on the Si–TiO₂ films had a greater cellular viability, stronger cytoskeleton and focal adhesion, and more cellular spreading than those on the pure TiO₂ films. Moreover, the expression levels of integrin β1 and focal adhesion kinase (FAK) genes, FAK and the phosphorylation of FAK proteins were up-regulated in cells cultured on the Si–TiO₂ films. These results indicated that the Si–TiO₂ films possess significantly enhanced cytocompatibility and provide potential solutions for the surface modification of implants in the future.     

Nanoparticle-tethered NAD+ with in situ cofactor regeneration

A new and simple route for the preparation of immobilized NAD⁺ on carboxyl-activated silica nanoparticles activated by γ-aminpropyltriethoxysilane and glutaric anhydride was developed. In addition, formate dehydrogenase, keto-reductase and the silica nanoparticle-attached NAD⁺ were applied to catalyze the coupled reactions for production of l-lactate with the cofactor regenerated within the reaction cycle. As indicated by thermogravimetric analysis and FT-IR, the silica nanoparticles were successfully activated and the loading of carboxyl groups was 0.53 mmol g^?1 particle. The amount of immobilized NAD⁺ on the support was 73 mg g^?1 particle. With 0.2 M pyruvate and 3 M formate, 0.16 M l-lactate was produced after the coupled reactions. The immobilized system showed excellent efficiency and stabilities in recycling, and it retained 60 % residual activity even after six reuses.