Expression of Human FGF18 by Fusion with Oleosin in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Seeds

Expression of recombinant human fibroblast growth factor 18 (hFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, but they are difficult, expensive. The expression of recombinant proteins fused to oleosin protein have distinct advantages, such as safety, ease, low cost. So we have expressed hFGF18 fused to oleosin protein in the oil bodies of Arabidopsis thaliana (A. thaliana) and screen the proliferation effect of NIH3T3 cells. The vector of oleosinhFGF18 fusion gene was constructed and transformed into wild A. thaliana. Transformed A. thaliana lines were obtained by the floral dip method and confirmed using polymerase chain reaction (PCR). The PCR results indicated that the oleosin-hFGF18 fusion gene was integrated into the A. thaliana genome. The oil bodies expression of oleosin-hFGF18 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biological activity showed that oil bodies expressing oleosin-hFGF18 could stimulate the proliferation of NIH3T3 cells.     

Antifungal peptidic compound from the deep-sea bacterium <Emphasis Type="Italic">Aneurinibacillus</Emphasis> sp. YR247

Aneurinibacillus: sp. YR247 was newly isolated from the deep-sea sediment inside the Calyptogena community at a depth of 1171 m in Sagami Bay. The strain exhibited antifungal activity against the filamentous fungus Aspergillus brasiliensis NBRC9455. A crude extract prepared from the YR247 cells by ethanol extraction exhibited broad antimicrobial activities. The antifungal compound is stable at 4–70?°C and pH 2.0–12.0. After treatment with proteinase K, the antifungal activity was not detected, indicating that the antifungal compound of strain YR247 is a peptidic compound. Electrospray ionization mass spectrometry of the purified antifungal compound indicated that the peptidic compound has an average molecular weight of 1167.9. The molecular weight of the antifungal compound from strain YR247 is different from those of antimicrobial peptides produced by the related Aneurinibacillus and Bacillus bacteria. The antifungal peptidic compound from the deep-sea bacterium Aneurinibacillus sp. YR247 may be useful as a biocontrol agent.     

Evaluation of antifungal,phosphate solubilisation,and siderophore and chitinase release activities of endophytic fungi from <Emphasis Type="Italic">Pistacia vera</Emphasis>

Fungal endophytes use different strategies to protect host plants from abiotic and biotic stress. In this study, we isolated endophytic fungi from Pistacia vera and characterised their antifungal activity against Aspergillus flavus, Rhizoctonia solani and Sclerotinia sclerotiorum, and their release of some factors that can alter plant growth capability. Trichoderma harzianum TH 5-1-2, T. harzianum TH 10-2-2 and T. atroviride TA 2-2-1 exhibited the highest growth inhibition percentages in dual culture assays against A. flavus, R. solani and S. sclerotiorum, respectively. Among the fungal endophyte cultures, ethyl acetate extracts of T. harzianum TH 10-2-2, T. harzianum TH 5-1-2 and T. atroviride TA 2-2-1 exhibited the highest growth inhibition of S. sclerotiorum, R. solani and A. flavus, respectively. Phosphate solubilisation was induced by Byssochlamys nivea BN 1-1-1 in culture. Large amounts of siderophore production were observed with Quambalaria cyanescens QC 11-3-2 and Epicoccum nigrum EN1, but Trichoderma spp. also produced siderophore in lower amounts. Trichoderma harzianum TH 5-1-2 produced the highest chitinase activity (2.92 U/mL). In general, among the endophytes isolated, Trichoderma spp. appear to have the most promise for promoting healthy growth of P. vera.     

Molecular characterization of <Emphasis Type="Italic">bmyC</Emphasis> gene of the mosquito pupicidal bacteria, <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> (VCRC B483) and in silico analysis of bacillomycin D synthetase C protein

A strain of Bacillus amyloliquefaciens (VCRC B483) exhibiting mosquito pupicidal, keratinase and antimicrobial activities was isolated from mangrove forest ecosystem of Andaman and Nicobar Islands. Molecular characterization of the strain showed the presence of lipopeptide encoding bmyC gene. Phylogenetic tree based on protein sequence of this gene exhibited homology with mycosubtilin synthetase of Bacilus atropheus and Iturin synthetase of Bacillus subtilis and B. amyloliquefaciens. This is the first report on the evolutionary conservation of amino acids concerned with the function and structure of bmyC protein of B. amyloliquefaciens. The presence of valine at the 1197th position in our strain was found to be unique and different from the existing strains of B. subtilis and B. amyloliquefaciens. Molecular modelling studies revealed significant changes in the structure of epimerization domain of the bmyC protein with A1197V variation. Crude metabolite of this strain exhibited antifungal activity against Fusarium sp. and Carvularia sp.     

Increase in Salt Tolerance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by <Emphasis Type="Italic">TaDi19</Emphasis>

The gene expression profile chip of salt-resistant wheat mutant RH8706-49 under salt stress was investigated. The overall length of the cDNA sequence of the probe was obtained using electronic cloning and RT-PCR. An unknown gene induced by salt was obtained, cloned, and named TaDi19 (Triticum aestivum drought-induced protein). No related report or research on the protein is available. qPCR analysis showed that gene expression was induced by many stresses, such as salt. Arabidopsis thaliana was genetically transferred using the overexpressing gene, which increased its salt tolerance. After salt stress, the transgenic plant demonstrated better physiological indicators (higher Ca²⁺ and lower Na⁺) than those of the wild-type plant. Results of non-invasive micro-test technology indicate that TaDi19-overexpressing A. thaliana significantly effluxed Na⁺ after salt treatment, whereas the wild-type plant influxed Na⁺. Chelating extracellular Ca²⁺ resulted in insignificant differences in salt tolerance between overexpressing and wild-type A. thaliana. Subcellular localization showed that the gene encoding protein was mainly located in the cell membrane and nucleus. TaDi19 was overexpressed in wild-type A. thaliana, and the transgenic lines were more salt-tolerant than the control A. thaliana. Thus, the wheat gene TaDi19 could increase the salt tolerance of A. thaliana.     

Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

Induction of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Resistance to Pathogenic Bacteria by Lipopolysaccharide and Salicylic Acid

The effects of combined treatment with an elicitor (lipopolysaccharide) and a signaling molecule (salicylic acid) on the disease resistance of wild-type (Col-0) and mutant Arabidopsis thaliana L. plants have been compared. The mutant lines used were jin1 (with impaired jasmonate signaling), npr1 (lacking expression of pathogen-dependent PR genes), and NahG (expressing an active bacterial salicylate hydroxylase transgene). The lipopolysaccharide was isolated from a saprophytic strain (8614) of Pseudomonas aeruginosa bacteria. Treatment of A. thaliana seeds with a composite preparation (lipopolysaccharide and salicylic acid–SA) increased the resistance of seedlings to a subsequent infection by the pathogenic 9096 strain of P. aeruginosa bacteria. The protective effect was more pronounced in jin1 mutant seedlings, which was indicative of the possible compensation of jasmonate signaling impairment due to activation of the SA-dependent signaling pathway. We concluded that a preparation composed of an elicitor and a signaling molecule could affect regulatory mechanism functioning in a plant cell and, in particular, compensate for the absence of a certain signaling pathway by activating another.     

Macromolecular Toxins Secreted by <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Induce Programmed Cell Death in Arabidopsis Leaves

Botrytis cinerea causes grey mold disease in crops and horticultural plants. It is suspected to kill plant cells via secreted toxins and to derive nutrients from dead or dying cells. However, whether macromolecular phytotoxins (MPs) secreted by B. cinerea induce necrosis or also trigger a programmed cell death (PCD) remains to be determined. We have previously partially characterized MPs secreted by B. cinerea. Here we isolated MPs from B. cinerea culture and applied them to leaf cells, assessing PCD over the following 120 h. Cell death was assessed by propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) staining. Catalase (CAT), peroxidase (POD) activity and the cytochrome c/a ratio were assessed by spectrophotometer. POD isomers were measured using the benzidine acetate method. In Arabidopsis thaliana (L.) Heynh. exposed to B. cinerea MPs, we observed chromatin condensation and marginalization, nuclear substance leakage and accumulation of autofluorescent materials in the cell wall. Furthermore, B. cinerea MPs induced release of cytochrome c from the mitochondria into the cytosol. Moreover, CAT and POD activity was upregulated and the POD isoenzyme pattern was altered. In conclusion, A. thaliana exposed to B. cinerea MPs exhibits multiple hallmarks of PCD, suggesting that B. cinerea induces PCD in host cells through secreted macromolecules.     

Molecular cloning and characterization of a PR-5 like protein gene from <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>

Downy mildew caused by Hyaloperonospora parasitica is a serious fungal disease in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). Pathogenesis-related 5 (PR-5) genes play an important role in plant resistance to disease invasion. In this study, a gene encoding pathogenesis-related 5-like (PR-5L) protein, named BcPR-5L, was successfully cloned from non-heading Chinese cabbage. The cDNA sequence of BcPR-5L was 747 bp in length. It encoded a protein of molecular mass of 25.78 kDa, an isoelectric point of 4.42, and containing 248 amino acids. Multiple sequence alignment indicated that BcPR-5L protein was highly homologous to other PR-5L proteins identified in 13 different species, with the highest homology to Brassica rapa. We analyzed the subcellular localization of BcPR-5L protein by using onion epidermal cells and found that it was localized in the membrane. Real time quantitative PCR analyses revealed that the expression of BcPR-5L gene was significantly upregulated after H. parasitica infection, and the expression in the resistant cultivar was higher than that in the susceptible cultivar. In summary, our data suggest that BcPR-5L gene may play an important role in the resistance of non-heading Chinese cabbage to H. parasitica infection.     

Modulation of proline metabolic gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> under water-stressed conditions by a drought-mitigating <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strain

Although amelioration of drought stress in plants by plant growth promoting rhizobacteria (PGPR) is a well reported phenomenon, the molecular mechanisms governing it are not well understood. We have investigated the role of a drought ameliorating PGPR strain, Pseudomonas putida GAP-P45 on the regulation of proline metabolic gene expression in Arabidopsis thaliana under water-stressed conditions. Indeed, we found that Pseudomonas putida GAP-P45 alleviates the effects of water-stress in A. thaliana by drastic changes in proline metabolic gene expression profile at different time points post stress induction. Quantitative real-time expression analysis of proline metabolic genes in inoculated plants under water-stressed conditions showed a delayed but prolonged up-regulation of the expression of genes involved in proline biosynthesis, i.e., ornithine-Δ-aminotransferase (OAT), Δ ¹ -pyrroline-5-carboxylate synthetase1 (P5CS1), Δ ¹ -pyrroline-5-carboxylate reductase (P5CR), as well as proline catabolism, i.e., proline dehydrogenase1 (PDH1) and Δ ¹ -pyrroline-5-carboxylate dehydrogenase (P5CDH). These observations were positively correlated with morpho-physiological evidences of water-stress mitigation in the plants inoculated with Pseudomonas putida GAP-P45 that showed better growth, increased fresh weight, enhanced plant water content, reduction in primary root length, enhanced chlorophyll content in leaves, and increased accumulation of endogenous proline. Our observations point towards PGPR-mediated enhanced proline turnover rate in A. thaliana under dehydration conditions.     

Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

In vitro antifungal efficacy of <Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9642 chitosan-AgNPs composite against post-harvest disease of citrus fruits

Green and blue mold postharvest diseases are the most vital negative components influencing the local market of citrus fruits. Citrus fruits were collected, and fungi were isolated. Among the fungal isolates identified, Penicillium digitatum and Penicillium italicum recorded the highest occurrence of 39.5 and 25.6%, respectively. In this work, we extracted chitosan from Aspergillum niger ATCC 9642. Fourier transform infrared spectroscopy was utilized to confirm the functional groups of the obtained compound, which exhibited the main characteristic bands of O–H stretching at 3302 cm^-1, and C–O–C band at 1125 cm^-1. A. niger ATCC 9642 chitosan had the degree of deacetylation of 88.5%, a molecular weight of 1.8 × 10⁵ Da, and viscosity of 7.3 centipoises; these values were comparable to those for standard shrimp chitosan. Ultraviolet-visible light spectra revealed the presence of A. niger ATCC 9642 chitosan-AgNPs composite. Using antifungal and spore germination assays, it was found that this composite exhibited effective antifungal action against P. digitatum and P. italicum compared with a chitosan standard. In a comet assay, the percentage of tail DNA was considered as a parameter that indicated DNA damage. The comet parameter increased significantly (P < 0.05) with A. niger ATCC 9642 chitosan–AgNPs composite, and the increase was dose-dependent. The increase in the DNA damage positively correlated with the inhibition performance of the A. niger ATCC 9642 chitosan–AgNPs composite.     

Assessment of the antifungal activity of <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Pediococcus</Emphasis> spp. for use as bioprotective cultures in dairy products

Fungi are commonly involved in dairy product spoilage and the use of bioprotective cultures can be a complementary approach to reduce food waste and economic losses. In this study, the antifungal activity of 89 Lactobacillus and 23 Pediococcus spp. isolates against three spoilage species, e.g., Yarrowia lipolytica, Rhodotorula mucilaginosa and Penicillium brevicompactum, was first evaluated in milk agar. None of the tested pediococci showed antifungal activity while 3, 23 and 43 lactobacilli isolates showed strong antifungal activity or total inhibition against Y. lipolytica, R. mucilaginosa and P. brevicompactum, respectively. Then, the three most promising strains, Lactobacillus paracasei SYR90, Lactobacillus plantarum O_VI9 and Lactobacillus rhamnosus BIO_III28 at initial concentrations of 10⁵ and 10⁷ CFU/ml were tested as bioprotective cultures against the same fungal targets in a yogurt model during a 5-week storage period at 10 °C. While limited effects were observed at 10⁵ CFU/ml inoculum level, L. paracasei SYR90 and L. rhamnosus BIO_III28 at 10⁷ CFU/ml respectively retarded the growth of R. mucilaginosa and P. brevicompactum as compared to a control without selected cultures. In contrast, growth of Y. lipolytica was only slightly affected. In conclusion, these selected strains may be good candidates for bioprotection of fermented dairy products.     

Fungal load in <Emphasis Type="Italic">Bradysia agrestis</Emphasis>, a phytopathogen-transmitting insect vector

Larvae of Bradysia agrestis, a phytopathogen-transmitting insect vector in East Asia, were sampled from geographically (ecologically) segregated regions to identify their intestinal fungal flora. A total of 24 fungal strains were isolated from the insect vectors and selected based on morphological differences. In addition, 38 fungal strains were isolated from the ulcerated parts of invaded host plants by the same method, revealing the impact of vector fungal flora on their host plants. For molecular identification of the fungi, internal transcribed spacer (ITS) regions were amplified and sequenced. Their sequences were compared with sequences of other fungal strains obtained from NCBI GenBank, and their phylogeny was determined. The dominant fungal genera in the insect vector were Penicillium (25%), Aspergillus (21%), and Cladosporium (13%). In plant scar lesions, most fungal isolates belonged to the genera Fusarium (31.6%), Phoma (7.8%), Didymella (7.8%), and Epicoccum (7.8%). Fungal genera in vectors or host plant lesions differed by study site. Furthermore, diversity indices by study site showed clear differences based on Margalef’s richness (2.06, 2.40, 3.04), and Menhinick’s (1.89, 2.12, 2.53), and Simpson’s indices (0.14, 0.07, 0.07). In addition, common fungal strains in insect vectors were found to be closely related to members of the genera Cladosporium, Penicillium, or Aspergillus. Among these strains, those showing the highest homology with Aspergillus terreus, which regarded as beneficial fungal genera could be considered ideal paratransgenesis candidates. Some other fungal strains from vectors or ulcerated plant parts from each study site after B. agrestis invasion may be harmful in terms of plant disease or agrifood safety. This study provides information on the fungal microbiota of B. agrestis, an emerging problem in East Asia, and proposes paratransgenesis candidates to control this insect vector. Furthermore, potential transferable pathogens or commensal fungi were revealed by comparing the fungal biota between the insect gut and the ulcerated parts of the invaded host plants.