Bioengineering of a Lactococcus lactis subsp. lactis strain enhances nisin production and bioactivity.

Lactococcus lactis subsp. lactis is a food bacterium that has been utilized for decades in food fermentation and the development of high-value industrial goods. Among these, nisin, which is produced by several strains of L. lactis subsp. lactis, plays a crucial role as a food bio-preservative. The gene expression for nisin synthesis was evaluated using qPCR analysis. Additionally, a series of re-transformations of the strain introducing multiple copies of the nisA and nisRK genes related to nisin production were developed. The simultaneous expression of nisA and nisZ genes was used to potentiate the effective inhibition of foodborne pathogens. Furthermore, qPCR analysis indicated that the nisA and nisRK genes were expressed at low levels in wild-type L. lactis subsp. lactis. After several re-transformations of the strain with the nisA and nisRK genes, a high expression of these genes was obtained, contributing to improved nisin production. Also, co-expression of the nisA and nisZ genes resulted in extremely effective antibacterial action. Hence, this study would provide an approach to enhancing nisin production during industrial processes and antimicrobial activity.

New native Bacillus thuringiensis strains induce high insecticidal action against Culex pipiens pallens larvae and adults.

Mosquitoes of many species are key disease vectors, killing millions of people each year. Bacillus thuringiensis-based insecticide formulations are largely recognized as among the most effective, ecologically safe, and long-lasting methods of managing insect pests. New B. thuringiensis strains with high mosquito control effectiveness were isolated, identified, genetically defined, and physiologically characterized. Eight B. thuringiensis strains were identified and shown to carry endotoxin-producing genes. Using a scanning electron microscope, results revealed typical crystal forms of various shapes in B. thuringiensis strains. Fourteen cry and cyt genes were found in the strains examined. Although the genome of the B. thuringiensis A4 strain had twelve cry and cyt genes, not all of them were expressed, and only a few protein profiles were observed. The larvicidal activity of the eight B. thuringiensis strains was found to be positive (LC50: 1.4-28.5 g/ml and LC95: 15.3-130.3 g/ml). Bioassays in a laboratory environment demonstrated that preparations containing B. thuringiensis spores and crystals were particularly active to mosquito larvae and adults. These new findings show that the novel preparation containing B. thuringiensis A4 spores and crystals mixture might be used to control larval and adult mosquitoes in a sustainable and ecologically friendly manner.

Endophytic bacterium Bacillus aryabhattai induces novel transcriptomic changes to stimulate plant growth.

In nature, plants interact with a wide range of microorganisms, and most of these microorganisms could induce growth through the activation of important molecular pathways. The current study evaluated whether the endophytic bacterium Bacillus aryabhattai encourages plant growth and the transcriptional changes that might be implicated in this effect. The endophytic bacterium promotes the growth of Arabidopsis and tobacco plants. The transcriptional changes in Arabidopsis plants treated with the bacterium were also identified, and the results showed that various genes, such as cinnamyl alcohol dehydrogenase, apyrase, thioredoxin H8, benzaldehyde dehydrogenase, indoleacetaldoxime dehydratase, berberine bridge enzyme-like and gibberellin-regulated protein, were highly expressed. Also, endophytic bacterial genes, such as arginine decarboxylase, D-hydantoinase, ATP synthase gamma chain and 2-hydroxyhexa-2,4-dienoate hydratase, were activated during the interaction. These findings demonstrate that the expression of novel plant growth-related genes is induced by interaction with the endophytic bacterium B. aryabhattai and that these changes may promote plant growth in sustainable agriculture.

Effects of nitrogen levels on gene expression and amino acid metabolism in Welsh onion.

BACKGROUND: Welsh onion constitutes an important crop due to its benefits in traditional medicine. Nitrogen is an important nutrient for plant growth and yield; however, little is known about its influence on the mechanisms of Welsh onion regulation genes. In this study, we introduced a gene expression and amino acid analysis of Welsh onion treated with different concentrations of nitrogen (N0, N1, and N2 at 0 kg/ha, 130 kg/ha, and 260 kg/ha, respectively). RESULTS: Approximately 1,665 genes were differentially regulated with different concentrations of nitrogen. Gene ontology enrichment analysis revealed that the genes involved in metabolic processes, protein biosynthesis, and transportation of amino acids were highly represented. KEGG analysis indicated that the pathways were related to amino acid metabolism, cysteine, beta-alanine, arginine, proline, and glutathione. Differential gene expression in response to varying nitrogen concentrations resulted in different amino acid content. A close relationship between gene expression and the content of amino acids was observed. CONCLUSIONS: This work examined the effects of nitrogen on gene expression and amino acid synthesis and provides important evidence on the efficient use of nitrogen in Welsh onion.

Effects of nitrogen application on phytochemical component levels and anticancer and antioxidant activities of Allium fistulosum.

BACKGROUND: Allium fistulosum L. has good nutritional value and is cultivated worldwide as an efficacious traditional medicinal plant. Its biological activities are attributable to its phytochemicals. Nitrogen is an essential nutrient for plant growth and development; however, the effect of nitrogen levels on the level of active components in this species is not well understood. METHODS: In this study, using urea fertilizer, we investigated the effects of different nitrogen levels (N0, N1, and N2 at 0, 130, and 260 kg/ha, respectively) on the phytochemical constituents , and antioxidant and anticancer properties of A. fistulosum. RESULTS: The results suggested that nitrogen fertilizers have a significant effect on the level of total phenols and flavonoids. The analysis of the antioxidant capacity revealed that the lowest IC50 values corresponded to plants treated with the highest nitrogen concentration. Anticancer activity was investigated against cancer cell lines (HeLa and HepG2), and the extracts of A. fistulosum treated with a high nitrogen level showed the highest antiproliferative effect. Collectively, our results suggest that nitrogen fertilizer application enhanced the quality of A. fistulosum, particularly its health benefits.

Endophytic Bacillus altitudinis Strain Uses Different Novelty Molecular Pathways to Enhance Plant Growth.

The identification and use of endophytic bacteria capable of triggering plant growth is an important aim in sustainable agriculture. In nature, plants live in alliance with multiple plant growth-promoting endophytic microorganisms. In the current study, we isolated and identified a new endophytic bacterium from a wild plant species Glyceria chinensis (Keng). The bacterium was designated as a Bacillus altitudinis strain using 16S rDNA sequencing. The endophytic B. altitudinis had a notable influence on plant growth. The results of our assays revealed that the endophytic B. altitudinis raised the growth of different plant species. Remarkably, we found transcriptional changes in plants treated with the bacterium. Genes such as maturase K, tetratricopeptide repeat-like superfamily protein, LOB domain-containing protein, and BTB/POZ/TAZ domain-containing protein were highly expressed. In addition, we identified for the first time an induction in the endophytic bacterium of the major facilitator superfamily transporter and DNA gyrase subunit B genes during interaction with the plant. These new findings show that endophytic B. altitudinis could be used as a favourable candidate source to enhance plant growth in sustainable agriculture.

Heat-killed endophytic bacterium induces robust plant defense responses against important pathogens.

Stress caused by pathogens strongly damages plants. Developing products to control plant disease is an important challenge in sustainable agriculture. In this study, a heat-killed endophytic bacterium (HKEB), Bacillus aryabhattai, is used to induce plant defense against fungal and bacterial pathogens, and the main defense pathways used by the HKEB to activate plant defense are revealed. The HKEB induced high protection against different pathogens through the salicylic and jasmonic acid pathways. We report the presence of gentisic acid in the HKEB for the first time. These results show that HKEBs may be a useful tool for the management of plant diseases.

Phytotoxic Metabolites Produce by Fusarium oxysporum f. sp. cubense Race 2.

Banana is a major tropical fruit crop but banana production worldwide is seriously threatened due to Fusarium wilt. Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt of banana (also referred as Panama disease) is an asexual, soil inhabiting facultative parasite. Foc isolates can be classified into three races that are not defined genetically, but for their pathogenicity to different banana cultivars. Despite mycotoxins being some of the best studied virulence factors of phytopathogenic fungi and these have been useful for the prediction of Foc virulence on banana plants, toxins produced by Foc race 2 strains have not been previously identified. The aim of this contribution was to identify the phytotoxic metabolites closely related to banana wilt caused by a Foc race 2 strain. We used an in vitro bioassay on detached banana leaves to evaluate the specificity of the microbial culture filtrates before a partial purification and further identification of Foc race 2 phytotoxins. A 29-day-old host-specific culture filtrate was obtained but specificity of culture filtrate was unrecovered after partial purification. The non-specific phytotoxins were characterized as fusaric acid, beauvericin, and enniatin A. Whereas some, if not all, of these phytotoxins are important virulence factors, a proteinaceous fraction from the specific 29-day-old culture filtrate protected the leaves of the resistant banana cultivar from damage caused by such phytotoxic metabolites.

Potato virus X-mediated constitutive expression of Plutella xylostella PxSDF2L1 gene in Nicotiana benthamiana confers resistance to Phytophthora parasitica var. nicotianae.

BACKGROUND: The Plutella xylostella PxSDF2L1 gene was previously reported to enhance insect resistance to pathogen at high basal transcription rate. PxSDF2L1 shows similitude with the stromal cell-derived factor 2 (SDF2), an ER stress-induced chaperon protein that is highly conserved throughout animals and plants. The precise biological function of SDF2 is not clear, but its expression is required for innate immunity in plants. Here, we investigate whether a continuous expression of PxSDF2L1 in Nicotiana benthamiana can similarly confer resistance to plant pathogen, particularly, the black shank Phytophthora parasitica var. nicotianae. RESULTS: The N. benthamiana plants were inoculated with agrobacteria transformed with a PVX-based binary vector carrying the PxSDF2L1 gene; similar agroinoculation experiments with a PVX vector carrying the GFP gene were used for controls. In pot trials, agroinfected N. benthamiana plants constitutively expressing PxSDF2L1 showed a significant reduction of stem disease symptoms caused by the inoculation with P. parasitica, compared with controls. CONCLUSIONS: We confirm a role of PxSDF2L1 in resistance to black shank, with a potential application to engineering active resistance against this oomycete in the commercial N. tabacum species and propose its evaluation in other crop families and plant pathogens.

Correction: High nitrogen concentration alter microbial community in Allium fistulosum rhizosphere.

[This corrects the article DOI: 10.1371/journal.pone.0241371.].

High nitrogen concentration alter microbial community in Allium fistulosum rhizosphere.

Welsh onion (Allium fistulosum L.) constitutes an important plant species cultivated in China due the benefits and applications in different areas. Moreover, nitrogen is an essential nutrient during the growth and development of plant. Here, we present the effects of nitrogen on soil microbiome in welsh onion plants. We used High-throughput sequencing analysis to determine the diversity and abundances of microbes associated to soil rhizosphere in welsh onion under the influence of nitrogen application. Nitrogen application significantly influenced in the diversity of fungal community. The relative abundance of Orbiliomycetes increased with the nitrogen concentration. Nitrogen application did not affect the diversity of bacterial community, whereas the relative abundance of Acidobacteria_Gp2, Verrucomicrobiae and Sphingobacteriia decreased with the nitrogen condition. In this work, we introduced evidences of the effect of nitrogen fertilization on microbial community in welsh onion rhizosphere, and the change of microbial community may interfere the growth and development of welsh onion.

Field Resistance to Phakopsora pachyrhizi and Colletotrichum truncatum of Transgenic Soybean Expressing the NmDef02 Plant Defensin Gene.

Fungal diseases lead to significant losses in soybean yields and a decline in seed quality; such is the case of the Asian soybean rust and anthracnose caused by Phakopsora pachyrhizi and Colletotrichum truncatum, respectively. Currently, the development of transgenic plants carrying antifungal defensins offers an alternative for plant protection against pathogens. This paper shows the production of transgenic soybean plants expressing the NmDef02 defensin gene using the biolistic delivery system, in an attempt to improve resistance against diseases and reduce the need for chemicals. Transgenic lines were assessed in field conditions under the natural infections of P. pachyrhizi and C. truncatum. The constitutive expression of the NmDef02 gene in transgenic soybean plants was shown to enhance resistance against these important plant pathogens. The quantification of the P. pachyrhizi biomass in infected soybean leaves revealed significant differences between transgenic lines and the non-transgenic control. In certain transgenic lines there was a strong reduction of fungal biomass, revealing a less severe disease. Integration and expression of the transgenes were confirmed by PCR, Southern blot, and qRT-PCR, where the Def1 line showed a higher relative expression of defensin. It was also found that the expression of the NmDef02 defensin gene in plants of the Def1 line did not have a negative effect on the nodulation induced by Bradyrhizobium japonicum. These results indicate that transgenic soybean plants expressing the NmDef02 defensin gene have a substantially enhanced resistance to economically important diseases, providing a sound environmental approach for decreasing yield losses and lowering the burden of chemicals in agriculture.

Functional expression of a peritrophin A-like SfPER protein is required for larval development in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Peritrophins are associated with structural and functional integrity of peritrophic membranes (PM), structures composed of chitin and proteins. PM lines the insect midgut and has roles in digestion and protection from toxins. We report the full-length cDNA cloning, molecular characterization and functional analysis of SfPER, a novel PM peritrophin A protein, in Spodoptera frugiperda. The predicted amino acid sequence indicated SfPER's domain structure as a CMCMC-type, consisting of a signal peptide and three chitin-binding (C) domains with two intervening mucin-like (M) domains. Phylogenetic analysis determined a close relationship between SfPER and another S. frugiperda PM peritrophin partial sequence. SfPER transcripts were found in larvae and adults but were absent from eggs and pupae. Chitin affinity studies with a recombinant SfPER-C1 peritrophin A-type domain fused to SUMO/His-tag confirmed that SfPER binds to chitin. Western blots of S. frugiperda larval proteins detected different sized variants of SfPER along the PM, with larger variants found towards the posterior PM. In vivo suppression of SfPER expression did not affect susceptibility of larvae to Bacillus thuringiensis toxin, but significantly decreased pupal weight and adult emergence, possibly due to PM structural alterations impairing digestion. Our results suggest SfPER could be a novel target for insect control.

Expression of pathogenesis-related proteins in transplastomic tobacco plants confers resistance to filamentous pathogens under field trials.

Plants are continuously challenged by pathogens, affecting most staple crops compromising food security. They have evolved different mechanisms to counterattack pathogen infection, including the accumulation of pathogenesis-related (PR) proteins. These proteins have been implicated in active defense, and their overexpression has led to enhanced resistance in nuclear transgenic plants, although in many cases constitutive expression resulted in lesion-mimic phenotypes. We decided to evaluate plastid transformation as an alternative to overcome limitations observed for nuclear transgenic technologies. The advantages include the possibilities to express polycistronic RNAs, to obtain higher protein expression levels, and the impeded gene flow due to the maternal inheritance of the plastome. We transformed Nicotiana tabacum plastids to co-express the tobacco PR proteins AP24 and ß-1,3-glucanase. Transplastomic tobacco lines were characterized and subsequently challenged with Rhizoctonia solani, Peronospora hyoscyami f.sp. tabacina and Phytophthora nicotianae. Results showed that transplastomic plants expressing AP24 and ß-1,3-glucanase are resistant to R. solani in greenhouse conditions and, furthermore, they are protected against P.hyoscyami f.sp. tabacina and P. nicotianae in field conditions under high inoculum pressure. Our results suggest that plastid co- expression of PR proteins AP24 and ß-1,3-glucanase resulted in enhanced resistance against filamentous pathogens.

HT-SuperSAGE of the gut tissue of a Vip3Aa-resistant Heliothis virescens (Lepidoptera: Noctuidae) strain provides insights into the basis of resistance.

Multitoxin Bt-crops expressing insecticidal toxins with different modes of action, for example, Cry and Vip, are expected to improve resistance management in target pests. While Cry1A resistance has been relatively well characterized in some insect species, this is not the case for Vip3A, for which no mechanism of resistance has yet been identified. Here we applied HT-SuperSAGE to analyze the transcriptome of the gut tissue of tobacco budworm Heliothis virescens (F.) laboratory-selected for Vip3Aa resistance. From a total of 1 324 252 sequence reads, 5 895 126-bp tags were obtained representing 17 751 nonsingleton unique transcripts (UniTags) from genetically similar Vip3Aa-resistant (Vip-Sel) and susceptible control (Vip-Unsel) strains. Differential expression was significant (≥2.5 fold or ≤0.4; P < 0.05) for 1989 sequences (11.2% of total UniTags), where 420 represented overexpressed (OE) and 1569 underexpressed (UE) genes in Vip-Sel. BLASTN searches mapped 419 UniTags to H. virescens sequence contigs, of which, 416 (106 OE and 310 UE) were unambiguously annotated to proteins in NCBI nonredundant protein databases. Gene Ontology distributed 345 of annotated UniTags in 14 functional categories with metabolism (including serine-type hydrolases) and translation/ribosome biogenesis being the most prevalent. A UniTag homologous to a particular member of the REsponse to PAThogen (REPAT) family was found among most overexpressed, while UniTags related to the putative Vip3Aa-binding ribosomal protein S2 (RpS2) were underexpressed. qRT-PCR of a subset of UniTags validated the HT-SuperSAGE data. This study is the first providing lepidopteran gut transcriptome associated with Vip3Aa resistance and a foundation for future attempts to elucidate the resistance mechanism.

Enhanced anti-proliferative efficacy of epothilone B loaded with Escherichia coli Nissle 1917 bacterial ghosts on the HeLa cells by mitochondrial pathway of apoptosis.

Epothilones constitute a new class of microtubule-stabilizing anti-cancer agents with promising preclinical and clinical activity. However, its systemic application still causes some toxic side effects. To reduce these undesired effects, advanced drug delivery systems based on cell targeting carriers are needed currently. In this study, the high quality bacterial ghosts of the probiotic Escherichia coli Nissle 1917 (EcN) were prepared in a large scale and retained fully intact surface structures for specific attachment to mammalian cells. The EcN ghosts could be efficiently loaded with the low hydrophilic drug Epothilone B (Epo B) and the maximal load efficiency was approximately 2.5% (w/w). Cytotoxicity assays revealed that Epo B-ghosts exhibited enhanced anti-proliferative properties on the HeLa cells. The Epo B associated with EcN ghosts was more cytotoxic at least 10 times than the free Epo B at the same concentrations. Apoptosis assays showed that both Epo B-ghosts and free Epo B induced time course-dependent apoptosis and necrosis in HeLa cells, respectively. While the former induced more apoptosis and necrosis than the latter. Furthermore, the cytochrome C release and the activation of caspase-3 were more remarkable after treatment with the Epo B-ghosts compared to the free Epo B, which implied that Epo B-ghosts might more effectively induce the apoptosis mediated by mitochondrial pathway in HeLa cells. Therefore, the higher anti-proliferative effects of the Epo B-ghosts on the HeLa cells were mediated by mitochondrial pathway of apoptosis. The EcN ghosts may provide a useful drug delivery carrier for drug candidates in cancer therapy.

High-throughput SuperSAGE for gene expression analysis of Nicotiana tabacum-Rhizoctonia solani interaction.

OBJECTIVE: The ubiquitous soil pathogen Rhizoctonia solani causes serious diseases in different plant species. Despite the importance of this disease, little is known regarding the molecular basis of susceptibility. SuperSAGE technology and next-generation sequencing were used to generate transcript libraries during the compatible Nicotiana tabacum-R. solani interaction. Also, we used the post-transcriptional silencing to evaluate the function of a group of important genes. RESULTS: A total of 8960 and 8221 unique Tag sequences identified as differentially up- and down-regulated were obtained. Based on gene ontology classification, several annotated UniTags corresponded to defense response, metabolism and signal transduction. Analysis of the N. tabacum transcriptome during infection identified regulatory genes implicated in a number of hormone pathways. Silencing of an mRNA induced by salicylic acid reduced the susceptibility of N. tabacum to R. solani. We provide evidence that the salicylic acid pathway was involved in disease development. This is important for further development of disease management strategies caused by this pathogen.

'Candidatus Liberibacter asiaticus', Causal Agent of Citrus Huanglongbing, Is Reduced by Treatment with Brassinosteroids.

Huanglongbing (HLB) constitutes the most destructive disease of citrus worldwide, yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, 'Candidatus Liberibacter asiaticus'. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB.