Interference Efficiency and Effects of Bacterium-mediated RNAi in the Fall Armyworm (Lepidoptera: Noctuidae).

RNAi is an effective tool for gene function analysis and a promising strategy to provide environmentally friendly control approaches for pathogens and pests. Recent studies support the utility of bacterium-mediated RNAi as a cost-effective method for gene function study and a suitable externally applied delivery mechanism for pest control. Here, we developed a bacterium-mediated RNAi system in Spodoptera frugiperda based on four target genes, specifically, Chitinase (Sf-CHI), Chitin synthase B (Sf-CHSB), Sugar transporter SWEET1 (Sf-ST), and Hemolin (Sf-HEM). RNAi conducted by feeding larvae with bacteria expressing dsRNAs of target genes or injecting pupae and adults with bacterially synthesized dsRNA induced silencing of target genes and resulted in significant negative effects on growth and survival of S. frugiperda. However, RNAi efficiency and effects were variable among different target genes and dsRNA delivery methods. Injection of pupae with dsCHI and dsCHSB induced a significant increase in wing malformation in adults, suggesting that precise regulation of chitin digestion and synthesis is crucial during wing formation. Injection of female moths with dsHEM resulted in lower mating, fecundity, and egg hatching, signifying a critical role of Sf-HEM in the process of egg production and/or embryo development. Our collective results demonstrate that bacterium-mediated RNAi presents an alternative technique for gene function study in S. frugiperda and a potentially effective strategy for control of this pest, and that Sf-CHI, Sf-CHSB, Sf-ST, and Sf-HEM encoding genes can be potent targets.

Antifungal activity and expression patterns of extracellular chitinase and ß-1,3-glucanase in Wickerhamomyces anomalus EG2 treated with chitin and glucan.

In this study, the expression patterns of extracellular chitinase and ß-1,3-glucanase from cultured Wickerhamomyces anomalus EG2 treated with chitin, glucan, and chemical chitinase inhibitors (kinetin, caffeine, and acetazolamide) were investigated using SDS-PAGE. Relationship between enzyme expression and antifungal activity from yeast plays a very important role for biocontrol of phytopathoges. To determine antifungal activity against phytopathogens, W. anomalus EG2 was shown to strongly inhibit hyphal growth of Fusarium oxysporum KACC 40032 and Rhizoctonia solani KACC 40111. Slight chitinase activity was observed 12 h after incubation in both PDB and YPD medium without colloidal chitin. The molecular weight of chitinase was approximately 124 kDa ß-1,3-Glucanase isoenzyme (GN1 and GN2) was observed distinctly on SDS-PAGE gels when laminarin was used as a substrate. ß-1,3-Glucanase isoenzyme was not observed when using glucan-containing high polymer complex (GHPC) as a substrate. Production of chitinase from W. anomalus EG2 was inhibited slightly by acetazolamide. Abnormal and cluster-shaped cells of W. anomalus EG2 were observed in both PDB and YPD medium treated with colloidal chitin. These results indicated that W. anomalus EG2 could be applied commercially as a biological control agent of phytopathogens and as a bioinhibitor of yeast cell growth.

The roles of three Serratia marcescens chitinases in chitin conversion are reflected in different thermodynamic signatures of allosamidin binding.

Binding of allosamidin to the three family 18 chitinases of Serratia marcescens has been studied using isothermal titration calorimetry (ITC). Interestingly, the thermodynamic signatures of allosamidin binding were different for all three chitinases. At pH 6.0, chitinase A (ChiA) binds allosamidin with a K(d) value of 0.17 +/- 0.06 microM where the main part of the driving force is due to enthalpic change (DeltaH(r) degrees = -6.2 +/- 0.2 kcal/mol) and less to entropic change (-TDeltaS(r) degrees = -3.2 kcal/mol). A large part of DeltaH is due to allosamidin stacking with Trp(167) in the -3 subsite. Binding of allosamidin to both chitinase B (ChiB) (K(d) = 0.16 +/- 0.04 microM) and chitinase C (ChiC) (K(d) = 2.0 +/- 0.2 microM) is driven by entropy (DeltaH(r) degrees = 3.8 +/- 0.2 kcal/mol and -TDeltaS(r) degrees = -13.2 kcal/mol for ChiB and DeltaH(r) degrees = -0.6 +/- 0.1 and -TDeltaS(r) degrees = -7.3 kcal/mol for ChiC). For ChiC, the entropic term is dominated by changes in solvation entropy (DeltaS(conf) = 1 cal/K.mol and DeltaS(solv) = 31 cal/K.mol), while, for ChiB, changes in conformational entropy dominate (DeltaS(conf) = 37 cal/K x mol and DeltaS(solv) = 15 cal/K x mol). Corresponding values for ChiA are DeltaS(conf) = 4 cal/K x mol and DeltaS(solv) = 15 cal/K x mol. These remarkable differences in binding parameters reflect the different architectures of the catalytic centers in these enzymes that are adapted to different types of actions: ChiA and ChiB are processive enzymes that move in opposite directions, meaning that allosamidin binds in to "product" subsites in ChiB, while it binds to polymer-binding subsites in ChiA. The values for ChiC are compatible with this enzyme being a nonprocessive endochitinase with a much more open and solvated substrate-binding-site cleft.

Chitin stimulates expression of acidic mammalian chitinase and eotaxin-3 by human sinonasal epithelial cells in vitro.

BACKGROUND: Sinonasal epithelial cells participate in host defense by initiating innate immune mechanisms against potential pathogens. Antimicrobial innate mechanisms have been shown to involve Th1-like inflammatory responses. Although epithelial cells can also be induced by Th2 cytokines to express proeosinophilic mediators, no environmental agents have been identified that promote this effect. METHODS: Human sinonasal epithelial cells from patients with chronic rhinosinusitis with nasal polyps (CRSwNPs) and controls were harvested and grown in primary culture. Cell cultures were exposed to a range of concentrations of chitin for 24 hours, and mRNA for acidic mammalian chitinase (AMCase), eotaxin-3, and thymic stromal-derived lymphopoietin (TSLP) were assessed. Other cultures were exposed to interleukin 4 (IL- 4) alone and in combination with dust-mite antigen (DMA) for 36 hours. Extracted mRNA and cell culture supernatant were analyzed for expression of AMCase and eotaxin-3. RESULTS: Chitin induced a dose-dependent expression of AMCase and eotaxin-3 mRNA but not TSLP. Patients with recalcitrant CRSwNPs showed lower baseline expression of AMCase when compared with treatment-responsive CRSwNP and less induction of AMCase expression by chitin. DMA did not directly induce expression of AMCase or eotaxin-3. Expression of eotaxin-3 was stimulated by IL-4 and further enhanced with the addition of DMA. Levels of AMCase were not significantly affected by either IL-4 or DMA exposure. In some cases, the combination of IL-4 and DMA was able to induce AMCase expression in cell cultures not producing AMCase at baseline. CONCLUSION: The abundant biopolymer chitin appears to be recognized by a yet uncharacterized receptor on sinonasal epithelial cells. Chitin stimulates production of AMCase and eotaxin-3, two pro-Th2 effector proteins. This finding suggests the existence of a novel innate immune pathway for local defense against chitin-containing organisms in the sinonasal tract. Dysregulation of this function could precipitate or exacerbate Th2 inflammation, potentially acting as an underlying factor in recalcitrant CRSwNP.

Dexamethasone alters bronchoalveolar lavage fluid proteome in a mouse asthma model.

BACKGROUND: Glucocorticoid is the most effective anti-inflammatory agent for asthma. The spectrum of protein targets that can be regulated by glucocorticoid in asthma is not fully understood. The present study tried to identify novel protein targets of dexamethasone in allergic airway inflammation by analyzing the proteome of mouse bronchoalveolar lavage (BAL) fluid. METHODS: BALB/c mice sensitized and challenged with ovalbumin (OVA) showed increased pulmonary inflammatory cell infiltration, airway mucus production and serum OVA-specific IgE level. Dexamethasone inhibited all these allergic airway inflammation endpoints. BAL fluid proteins were resolved by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: The levels of 26 BAL fluid proteins were found to be markedly altered by dexamethasone. A family of chitinases (Ym1, Ym2 and acidic mammalian chitinase, AMCase), lungkine, gob-5, surfactant protein D and polymeric immunoglobulin receptor have been found for the first time to be downregulated by dexamethasone in allergic airways. The downregulatory effects were confirmed by immunoblotting and RT-PCR analyses. Dexamethasone was also shown to significantly inhibit lavage fluid chitinase bioactivity. In addition, dexamethasone promoted airway expression of vitamin D-binding protein, heptoglobin and alpha(1)-antitrypsin. CONCLUSIONS: Among all these newly identified protein targets of dexamethasone, AMCase and gob-5 have been shown to be pro-inflammatory in asthma. Downregulation of AMCase and gob-5 may be considered as two novel anti-inflammatory actions of glucocorticoid in asthma.

Elicitor-induced activation of transcription via W box-related cis-acting elements from a basic chitinase gene by WRKY transcription factors in tobacco.

A putative elicitor responsive element with two W boxes (CTGACC/T) has been identified in the region between -125 and -69 of a tobacco class I basic chitinase gene CHN48. We generated transgenic tobacco calli that contained the -125/-69 region fused to a luciferase reporter gene. The expression of the reporter gene was induced upon treatment with an elicitor, xylanase from Trichoderma viride (TvX). This induction required protein kinase activity. We isolated three cDNA clones encoding DNA-binding proteins, designated as NtWRKY1, NtWRKY2, and NtWRKY4, from tobacco cultured cells. Gel mobility shift assays showed that in vitro translation products of NtWRKY1, NtWRKY2 and NtWRKY4 bound to W box of CHN48 gene. These NtWRKY proteins stimulated W box-mediated transcription of a luciferase reporter gene in the transient assay. In addition, the transactivation of W box-mediated transcription by NtWRKY1 and NtWRKY4 was enhanced in response to elicitor treatment, suggesting elicitor-induced posttranscriptional activation of these NtWRKYs. Northern blot analyses showed that mRNAs for NtWRKY1 and NtWRKY2 increased after treatment with the elicitor, whereas mRNAs for NtWRKY4 were expressed constitutively at a low level. These results suggested possible involvement of NtWRKYs in elicitor-responsive transcription of defense genes in tobacco.

Systemic acquired resistance in sunflower (Helianthus annuus L.).

Systemic acquired resistance (SAR) to infection by Botrytis cinerea in the leaves of sunflower (Helianthus annuus L.) plants was induced following cotyledon inoculation with B. cinerea or treatment with abiotic inducers. Salicylic acid (SA), benzo-(1,2,3)-thiadiazole-7-carbothioic S-methyl ester (BTH), 2,6-dichloroisonicotinic acid (INA) or EDTA protected sunflower plants against Botrytis infection, that was revealed by a reduction in the number and area of the necrotic lesions in upper leaves after challenge inoculation with the pathogen. SA and BTH were more potent inducers than INA, EDTA or pre-inoculation with the fungus. In addition to resistance to B. cinerea, the upper leaves have also developed resistance to maceration by a mixture of cell wall-degrading enzymes. Calcium nitrate inhibited both the protective effect and the resistance of leaf discs to cell-wall degrading enzymes. All the tested chemicals increased the synthesis and excretion of sunflower phytoalexins--coumarins scopoletin and ayapin and induced the PR-proteins chitinase and 1,3-beta-glucanase, being the inducer effect of each activator correlated with the level of protection against B. cinerea (BTH > SA > INA > EDTA). Thus, SAR induction is mediated by general increase of plant defence responses. This is the first report on SAR in sunflower.

A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407.

Chitinases are produced throughout the growth process of fungi and are thought to play important roles in morphogenesis. Aspergillus fumigatus, is an important pathogen of immunocompromised individuals in which it causes pneumonia and invasive disseminated disease with high mortality; it is also known to produce chitinase. We have induced an exceptionally stable extracellular chitinase in A. fumigatus YJ-407, which could be isolated readily in a homogeneous form by using ammonium sulfate precipitation followed by DEAE-cellulose chromatography and preparative PAGE. The molecular mass of this chitinase was estimated to be 46 000 by SDS/PAGE, and its isoelectric point was pH 5.6. The enzyme was most active at pH 5.0 and 60 degrees C, and was inhibited strongly by Hg2+, Pb2+, Ag+, Fe2+, Mn2+ and Zn2+. The enzyme was stable over a broad pH range 4-8 and below 45 degrees C. Tryptophan and carboxyl groups were found to be essential for the enzyme activity. The Michaelis constants for swollen chitin and chitosan were 1.12 mg.mL-1 and 1.84 mg.mL-1, respectively. The enzyme showed maximum activity towards glycol chitin and partially deacetylated chitosan, and lower activity towards colloidal chitin. Analysis of the hydrolysis product showed that the enzyme has both endo- and exo-hydrolytic activities. In addition, a transglycosyl activity was also observed.

Elicitor action via cell membrane of a cultured rice cell demonstrated by the single-cell transient assay.

In order to analyze intracellular signal transduction, we investigated the mechanism of chemical elicitor action by single-cell transient assay using green fluorescent protein (GFP) as a reporter gene. When the elicitor was applied from outside the cell into which the chitinase promoter and GFP reporter were introduced beforehand, fluorescence emission of GFP was observed. In contrast, when the elicitor was introduced in the cell to let the elicitor act from inside, no emission was observed. Addition of further elicitor from outside, however, did cause GFP emission. Therefore, it is clear that the elicitor does not act after entering the cell but that its signal is transduced into the cell via the cell membrane.