Plants and insects have coexisted for million years and evolved a set of interactions which affect both organisms at different levels. Plants have developed various morphological and biochemical ...adaptations to cope with herbivores attacks. However, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) has become the major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100%.
The aim of this study was to investigate the in vivo effect of a serine proteinase inhibitor (BTI-CMe) and a cysteine proteinase inhibitor (Hv-CPI2) from barley on this insect and to examine the effect their expression has on tomato defensive responses. We found that larvae fed on tomato transgenic plants co-expressing both proteinase inhibitors showed a notable reduction in weight. Moreover, only 56% of these larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Transgenes expression had no harmful effect on Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae), a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We also found that barley cystatin expression promoted plant defense by inducing the expression of the tomato endogenous wound inducible Proteinase inhibitor 2 (Pin2) gene, increasing the production of glandular trichomes and altering the emission of volatile organic compounds.
Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta.
In nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such ...environments. Pseudomonas aeruginosa, an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood.
In this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after 14 years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K
was elicited by high concentration of Na
in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that 78 genes had differential expression between the parental strain and its derivative clones. Sixty-one transcripts were up-regulated, while 17 were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions.
The individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa, a relevant environmental and pathogenic bacterium, survives to long-term salt stress.
The pathogenic bacterium Salmonella enterica serovar Typhimurium elicits a variety of genetic programs to adapt to stress conditions encountered within hostile environments such as host phagocytes ...and preserved food.
In this work, differential display (DD) methodology was used to investigate the effect of one month starvation in a salty microcosm (0.5 M NaCl) on transcript profiling in a Salmonella Typhimurium LT2 strain. cDNA fragments resulting from differentially expressed mRNA were eluted from the gel, re-amplified, cloned, and then sequenced.
A total of 21 differentially expressed bands were detected by DD reverse transcription-polymerase chain reaction (RT-PCR). However, only 12 of them were successfully identified as upregulated genes in stressed cells. Based on the sequencing data and BLAST analysis, these genes were sopA, ssaD, yhhK, gmK, cspC, uspA, ompR, phoP, stcC, fimA, acrA,and yehZ. As a confirmation of the differential expression, RT-PCR was carried out using a set of specific primers. Remarkably, the expression levels of these genes were significantly increased in starved bacteria compared to standard laboratory conditions.
Our results indicate that the starvation of Salmonella Typhimurium over one month in a salty microcosm changes the expression of stress proteins, response regulator in a two-component system, outer membrane proteins, effector proteins translocated by Salmonella pathogenicity island SPI1 and SPI2 type III secretion systems (TTSS), several metabolic enzymes, efflux pumps, and transport proteins. This suggests that the expression of the identified genes is important for the response of this pathogen to starvation in salt.
Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests. We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L.) ...by biolistic bombardment of cultured immature embryos. Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe. BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 1.1% of total extracted protein. No expression was detected in untransformed wheat seeds. Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay. The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance. However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects.
Even with advances in molecular cloning and DNA transformation, new or alternative methods that permit DNA penetration in Salmonella enterica subspecies enterica serovar Typhimurium are required in ...order to use this pathogen in biotechnological or medical applications. In this work, an adapted protocol of bacterial transformation with plasmid DNA based on the “Yoshida effect” was applied and optimized on Salmonella enterica serovar Typhimurium LT2 reference strain. The plasmid transference based on the use of sepiolite as acicular materials to promote cell piercing via friction forces produced by spreading on the surface of a hydrogel. The transforming mixture containing sepiolite nanofibers, bacterial cells to be transformed and plasmid DNA were plated directly on selective medium containing 2% agar. In order to improve the procedure, three variables were tested and the transformation of Salmonella cells was accomplished using plasmids pUC19 and pBR322. Using the optimized protocol on Salmonella LT2 strain, the efficiency was about 105 transformed cells per 109 subjected to transformation with 0.2μg plasmid DNA. In summary, the procedure is fast, offers opportune efficiency and promises to become one of the widely used transformation methods in laboratories.
•A protocol of Salmonella transformation using sepiolite nanofibers was optimized.•The tested variables are: amount of sepiolite, number of cells and spreading time.•Using the general protocol, the efficiency was about 5×105 per 1μg of plasmid.•The method is simple, rapid, and inexpensive and does not require competent cell preparation.
We investigated the association of angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism with preeclampsia (PE) in Tunisian women. ACE I/D genotyping was done by PCR in 342 ...pregnant women with PE and 289 healthy pregnant women. The association between ACE I/D and PE and associated features were also evaluated. Decreased active renin concentration, plasma aldosterone concentration, and placental growth factor (PlGF) were observed in PE cases, while soluble fms-like tyrosine kinase-1 (sFlt-1)/PlGF ratio was significantly higher in the PE group. Distribution of ACE I/D alleles and genotypes were comparable between women with PE and control women. A significant difference in the frequency of the I/I genotype was seen between PE cases and control women according to the recessive model, with a trend towards association in the codominant model. Carriers of the I/I genotype had significantly higher infant birth weights compared to the I/D and the D/D genotype carriers. A dose-dependent relationship was also seen in VEGF and PlGF plasma levels and specific ACE I/D genotypes, with the lowest VEGF levels seen in the I/I genotype carriers compared to the D/D genotype carriers. Similarly, the I/I genotype carriers had the lowest PlGF levels compared to I/D and D/D genotype carriers. Furthermore, when studying the linkage between PE features, we found a positive correlation between PAC and PIGF. Our study suggests a role for ACE I/D polymorphism in the pathogenesis of PE, possibly through modulating VEGF and PlGF levels and infant birth weight, and highlights the relationship between PAC and PlGF.
●Decreased active renin (ARC), aldosterone (PAC), and PlGF levels seen in PE cases.●Higher soluble fms-like tyrosine kinase-1 (sFlt-1)/PlGF ratio seen in PE group.●ACE I/D allele and genotype frequencies comparable between PE cases and controls.●ACE I/I genotype linked with lower PLGF and VEGF levels and high infant birth weight.
Photobacterium damselae subsp. damselae has been isolated from different outbreaks affecting cultured Sparus aurata and Dicentrarchus labrax. The aim of the present study was to characterize the ...phenotype and genotype of 12 P. damselae subsp. damselae strains isolated from these outbreaks. The roles of skin mucus in resistance to the initial infection steps have been studied. All tested strains resisted the bactericidal activity of the mucus and showed an ability to adhere to it, but only those showing hemolytic activity were found to be virulent by intraperitoneal injection. Phenotypic and genetic characterization revealed a considerable degree of variability within the subspecies. We found that RAPD-PCR represents a quick tool to generate information on intraspecific differences in environmental strains. We found, that some biotypes are more pathogenic than others, which by doing correlation between adhesion profile, enzymatic and hemolytic activity.
•During 2012 we had several deaths in the sea bass fish farm.•We isolated a few Photobacterium damselae subsp. damselae strains.•We tried to do a complete characterization of these strains.
The adaptive response of bacteria to stress is variable owing to the effects of various environmental factors and the time of exposure to stress conditions. We have studied the morphological and ...biochemical characters of resuscitated Pseudomonas aeruginosa ATCC 27853 that had been incubated for 14 years in natural sterilized seawater. The resuscitated cells were identified by PCR analysis of the internal transcribed spacer region. The biochemical profile of the resuscitated cells was partially inactive according to the API-20NE system. The cells had lost the ability to reduce nitrate and hydrolyse gelatin but acquired urease activity. In addition, the atomic force micrographs showed a reduction in cell size and an evolution to coccoid shapes. After incubation in a rich medium, the resuscitated cells regained their initial biochemical characters and their original rod shape. The differential display “DDRT-PCR” technique was used to determine the effects of stress on gene expression in this P. aeruginosa strain. The genes algU and iscR were successfully identified in stressed P. aeruginosa ATCC 27853 as two differentially expressed gene fragments. Our results demonstrate that the DDRT-PCR technique can be a useful tool in gene expression analysis studies looking at the stress response in P. aeruginosa.
The development of antibiotic resistance in the opportunistic pathogen
Pseudomonas aeruginosa
is a major cause of the pathogen’s morbidity and is strongly correlated with the biofilm formation. ...Motility and adherence capacity in long-term stressed cells have not been extensively analyzed even though
P. aeruginosa
considered a model organism for the study of biofilm formation. In this investigation,
P. aeruginosa
ATCC 27853 strain has been stored for 12 months in LB broth with 0.5 M NaCl. Several experiments demonstrated that the strain recovery from the salty microcosm had the ability to increase the biofilm formation and to reduce motility comparing with that of the original strain. To identify genes involved in the regulation of biofilm and/or in stress response by the recovered
P. aeruginosa
, differential display “DDRT-PCR” technique was used. The genes
speD
and
ccoN2
, coding, respectively, for an S-adenosylmethionine decarboxylase and Cbb3-type cytochrome oxidase, were identified in recovered strain of
P. aeruginosa
ATCC 27853 as two differentially expressed gene fragments. A comparison of the biofilm produced by the wild-type strain PA14 and the transposon insertion mutant for
speD
gene suggested that spermidine has a potential role in the adaptive response in
P. aeruginosa
incubated in long-term stress conditions.
Current protocols of recombinant DNA research, including gene cloning and complementation, quantification of gene expression and tagging with reporter proteins, are usually limited by the ...availability of effective bacteria transformation tools different from Escherichia coli. This is particularly relevant with respect to the Pseudomonas species due to their biotechnological and sanitary importance. Here, we describe an optimized and efficient plasmid transference protocol based on the Yoshida effect, a method that relies on DNA uptake mediated by friction forces. The main advantages of this method are: (i) no competent cell preparation is needed, (ii) cells in any physiological state can be used, (iii) the procedure is performed directly on agar plates and (iv) the protocol, which is neither time-consuming nor labor-intensive, offers good efficiency. This approach promises to become the gold standard for day to day genetic manipulation in Pseudomonas.
► Efficient plasmid transformation protocol for Pseudomonas sp. ► The method do not require competent cell preparation. ► Cells at any physiological state can be used. ► The procedure is performed directly on agar plates. ► Advantages for high throughput screenings.
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