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.

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.

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.

Expression of a Nicotiana tabacum pathogen-induced gene is involved in the susceptibility to black shank.

Many host genes induced during compatible plant-pathogen interactions constitute targets of pathogen virulence factors that act to suppress host defenses. In order to identify Nicotiana tabacum L. genes for pathogen-induced proteins involved in susceptibility to the oomycete Phytophthora parasitica var. nicotianae, we used SuperSAGE technology combined with next-generation sequencing to identify transcripts that were differentially upregulated during a compatible interaction. We identified a pathogen-induced gene (NtPIP) that was rapidly induced only during the compatible interaction. Virus-induced gene silencing of NtPIP reduced the susceptibility of N. tabacum to P. parasitica var. nicotianae. Additionally, transient expression of NtPIP in the resistant species Nicotiana megalosiphon Van Heurck & Mull. Arg. compromised the resistance to P. parasitica var. nicotianae. This pathogen-induced protein is therefore a positive regulator of the susceptibility response against an oomycete pathogen in tobacco.

NmDef02, a novel antimicrobial gene isolated from Nicotiana megalosiphon confers high-level pathogen resistance under greenhouse and field conditions.

Plant defensins are small cysteine-rich peptides that inhibit the growth of a broad range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a putative defensin isolated from Nicotiana megalosiphon upon inoculation with the tobacco blue mould pathogen Peronospora hyoscyami f.sp. tabacina. NmDef02 was heterologously expressed in the yeast Pichia pastoris, and the purified recombinant protein was found to display antimicrobial activity in vitro against important plant pathogens. Constitutive expression of NmDef02 gene in transgenic tobacco and potato plants enhanced resistance against various plant microbial pathogens, including the oomycete Phytophthora infestans, causal agent of the economically important potato late blight disease, under greenhouse and field conditions.

Over-expression of a protein kinase gene enhances the defense of tobacco against Rhizoctonia solani.

To identify Nicotiana tabacum genes involved in resistance and susceptibility to Rhizoctonia solani, suppression subtractive hybridization was used to generate a cDNA library from transcripts that are differentially expressed during a compatible and incompatible interaction. This allowed the isolation of a protein kinase cDNA that was down-regulated during a compatible and up-regulated during an incompatible interaction. Quantitative RT-PCR analysis of this gene confirmed the differential expression patterns between the compatible and incompatible interactions. Over-expression of this gene in tobacco enhanced the resistance to damping-off produced by an aggressive R. solani strain. Furthermore, silencing of this protein kinase gene reduced the resistance to a non-aggressive R. solani strain. A set of reported tobacco-resistant genes were also evaluated in tobacco plants over-expressing and silencing the protein kinase cDNA. Several genes previously associated with resistance in tobacco, like manganese superoxide dismutase, Hsr203J, chitinases and phenylalanine ammonia-lyase, were up-regulated in tobacco plants over-expressing the protein kinase cDNA. Potentially, the protein kinase gene could be used to engineer resistance to R. solani in tobacco cultivars susceptible to this important pathogen.

Black shank resistant tobacco by silencing of glutathione S-transferase.

A glutathione S-transferase gene was amplified from cDNA of Nicotiana tabacum roots infected with Phytophthora parasitica var. nicotianae. The gene was cloned in sense and anti-sense orientation to an RNAi vector for induced gene silencing, and reduced expression of the gene was detected by RT-PCR. A statistically significant increase in resistance of N. tabacum to infection following gene silencing was found for glutathione S-transferase-silenced plants compared with control plants. Some defense genes were up-regulated in glutathione S-transferase-silenced plants during the interaction with the pathogen. This is the first evidence of the role of glutathione S-transferase as negative regulator of defense response.