150 research outputs found
Molecular Defense Response of Pine Trees (Pinus spp.) to the Parasitic Nematode Bursaphelenchus xylophilus
ReviewPine wilt disease (PWD) is a severe environmental problem in Eastern Asia andWestern
Europe, devastating large forest areas and causing significant economic losses. This disease is caused
by the pine wood nematode (PWN), Bursaphelenchus xylophilus, a parasitic migratory nematode
that infects the stem of conifer trees. Here we review what is currently known about the molecular
defense response in pine trees after infection with PWN, focusing on common responses in different
species. By giving particular emphasis to resistance mechanisms reported for selected varieties
and families, we identified shared genes and pathways associated with resistance, including the
activation of oxidative stress response, cell wall lignification, and biosynthesis of terpenoids and
phenylpropanoids. The role of post-transcriptional regulation by small RNAs in pine response
to PWN infection is also discussed, as well as the possible implementation of innovative RNAinterference
technologies, with a focus on trans-kingdom small RNAs. Finally, the defense response
induced by elicitors applied to pine plants before PWN infection to prompt resistance is reviewed.
Perspectives about the impact of these findings and future research approaches are discussedinfo:eu-repo/semantics/publishedVersio
SNP Detection in Pinus pinaster Transcriptome and Association with Resistance to Pinewood Nematode
Pinewood nematode (PWN, Bursaphelenchus xylophilus) is the causal agent of pine wilt
disease (PWD), which severely affects Pinus pinaster stands in southwestern Europe. Despite the high
susceptibility of P. pinaster, individuals of selected half-sib families have shown genetic variability in
survival after PWN inoculation, indicating that breeding for resistance can be a valuable strategy
to control PWD. In this work, RNA-seq data from susceptible and resistant plants inoculated with
PWN were used for SNP discovery and analysis. A total of 186,506 SNPs were identified, of which
31 were highly differentiated between resistant and susceptible plants, including SNPs in genes
involved in cell wall lignification, a process previously linked to PWN resistance. Fifteen of these
SNPs were selected for validation through Sanger sequencing and 14 were validated. To evaluate
SNP-phenotype associations, 40 half-sib plants were genotyped for six validated SNPs. Associations
with phenotype after PWN inoculation were found for two SNPs in two different genes (MEE12
and PCMP-E91), as well as two haplotypes of HIPP41, although significance was not maintained
following Bonferroni correction. SNPs here detected may be useful for the development of molecular
markers for PWD resistance and should be further investigated in future association studiesinfo:eu-repo/semantics/publishedVersio
Antimicrobial resistance patterns inEnterobacteriaceae isolated froman urbanwastewater treatment plant
Over 18 months, enterobacteria were isolated from the raw (189 isolates) and
treated (156 isolates) wastewater of a municipal treatment plant. The isolates were
identified as members of the genera Escherichia (76%), Shigella (7%), Klebsiella
(12%) and Acinetobacter (4%). Antimicrobial susceptibility phenotypes were
determined using the agar diffusion method for the antibiotics amoxicillin,
gentamicin, ciprofloxacin, sulfamethoxazole/trimethoprim, tetracycline and cephalothin,
the disinfectants hydrogen peroxide, sodium hypochlorite, quaternary
ammonium/formaldehyde and iodine, and the heavy metals nickel, cadmium,
chromium, mercury and zinc. Class 1 integrons were detected by PCR amplification
using the primers CS5 and CS3. Compared with the raw influent, the treated
wastewater presented higher relative proportions of Escherichia spp. isolates
resistant to ciprofloxacin and cephalothin (Po0.0001 and Po0.05, respectively).
Except for mercury, which showed a positive correlation with tetracycline and
sulfamethoxazole/trimethoprim, no significant positive correlations were observed
between antibiotic, disinfectant and heavy metal resistance. The variable regions of
class 1 integrons, detected in c. 10% of the Escherichia spp. isolates, contained
predominantly the gene cassettes aadA1/dhfrI
NEP-TC a rRNA methyltransferase involved on somatic embryogenesis of tamarillo (Solanum betaceum cav.)
Somatic embryogenesis (SE) is an important biotechnological tool for large-scale clonal propagation and for embryogenesis research. Moreover, genetic transformation and cryopreservation procedures in many species rely on efficient SE protocols. We have been studying different aspects related to SE induction and somatic embryo development in tamarillo (Solanum betaceum Cav.), a small tree from the Solanaceae family. Previous proteomic analyses identified a protein (NEP-TC, 26.5 kDa) consistently present in non-embryogenic calluses of tamarillo, but absent in the embryogenic ones. In this work, the role of NEP-TC during SE was assessed by gene expression analysis and immunolocalization. The results obtained demonstrated that NEP-TC is a putative member of the SpoU rRNA methylase family. This protein, present in the cytoplasm and nucleus, is expressed in non-embryogenic cells and not expressed in embryogenic cells. Slightly enhanced SE induction levels in tamarillo plants with NEP-TC down-regulated levels also supports the role of this protein on SE induction. Heterologous expression was used to confirm NEP-TC rRNA methyltransferase activity, with enhanced activity levels when rRNA was used as a substrate. These data relate a putative member of the SpoU methylase family with plant morphogenesis, in particular with SE induction.publishe
Star-Shaped Gold Nanoparticles as Friendly Interfaces for Protein Electrochemistry: the Case Study of Cytochrome c
UID/QUI/50006/2019
POCI‐01‐0145‐FEDER‐007265
UID/Multi/04378/2019
POCI‐01‐0145‐FEDER‐007728
PTDC/NAN‐MAT/30589/2017
grant NORTE‐01‐0145‐FEDER‐000011
SFRH/BD/132057/2017Gold nanostars with an average tip-to-tip length of 52±6 nm were functionalized with different capping agents and used as electrode modification materials for protein electrochemistry. Direct electron transfer between cytochrome c and nanostar-coated pyrolytic graphite electrodes was observed with the protein in solution. The electrochemical response was improved at nanostars functionalized with a 1 : 1 mixture of 11-mercaptoundecanoic acid and 4-mercaptobenzoic acid in comparison with gold nanospheres coated with a similar functionalization. Further immobilization of cytochrome c on pyrolytic graphite while conjugated with the same nanostars guaranteed the maintenance of the protein's native properties, whereas direct adsorption on the bare or nanostar-modified electrodes resulted in an altered conformational state. The pseudo-peroxidase activity of the altered cytochrome c was enhanced in the presence of the nanostars.publishe
Susceptibility of embryogenic and organogenic tissues of maritime pine (Pinus pinaster) to antibiotics used in Agrobacterium-mediated genetic transformation
The effects of antibiotics commonly
used in Agrobacterium-mediated transformation
were studied on Pinus pinaster tissues. Embryogenic
tissue growth from three embryogenic lines
and adventitious bud induction from cotyledons
from three open-pollinated seed families were
analysed. Cefotaxizme, carbenicillin and timentin
commonly used for Agrobacterium elimination, at
concentrations of 200–400 mg l –1 did not inhibit
the embryogenic tissue growth on filter paper nor
as clumps. Adventitious bud induction and bud
number were significantly reduced for one of the
tested families when using 400 mg l–1 cefotaxime
or timentin. The selection agent kanamycin
significantly inhibited growth of embryogenic
tissue on filter paper in all the embryogenic
lines and concentrations tested (20–50 mg l–1).
Kanamycin also inhibited growth of embryogenic
clumps after two subcultures at 5–50 mg l–1.
In cotyledons, kanamycin inhibited adventitious
bud formation in the three seed families used,
regardless of the concentrations tested (5–
25 mg l–1). There was a significant effect of the
seed family on the bud induction and the number
of adventitious buds produced. From the results
obtained, we propose the use of timentin to
eliminate Agrobacterium in transformation
experiments, at concentrations of 400 mg l–1 for
embryogenic tissues and of 300 mg l–1 for cotyledons.
For selection of transformed tissues carrying
the kanamycin resistance gene, kanamycin
should be used at 20 mg l–1 for embryogenic tissues
on filter paper, at 5 mg l–1 when clumps are
in direct contact with the selection medium, and
bellow 5 mg l–1 for adventitious bud induction
Penicillium crustosum as a potential OTA producer - new insights from whole - genome sequencing of strain MUM 16.125
Ochratoxin A (OTA) is a well-studied mycotoxin that poses severe health risks. OTA is mainly
produced by Aspergillus and Penicillium species associated with food spoilage and it is present
in a wide diversity of food and feed products. Recent studies have reported the presence of
OTA in food matrices where known OTA producers are not present1,2. For that reason, other
species such as P. crustosum are now being considered. A recent study using comparative
genomic analysis3 clarified the OTA biosynthetic gene cluster composition.
In order to gain insight into the secondary metabolism of P. crustosum, this study aimed to
sequence and explore the complete genome of strain MUM 16.125. This strain was isolated
from cheese rind sample contaminated with OTA in which no known OTA producers were
present1.
The genome assembly comprises 199 contigs with a total length of 30.95 Mb and contains
10975 predicted protein-coding genes. In total, 109 gene clusters potentially related with
secondary metabolism were identified, including putative gene clusters for penitrem, clavaric
acid or naphthopyrones biosynthesis. Nevertheless, no evidence of an OTA biosynthetic gene
cluster was found. A total of 83 complete and 49 partial protein sequences from published OTA
biosynthetic genes from 11 Aspergillus and 3 Penicillium species were queried against the
predicted P. crustosum proteins. Only 3 strong matches were found (to a short partial P.
verrucosum PKS and 2 P. thymicola chloroperoxidases) but matches to complete key genes
were absent.
Considering these findings, it appears that strain MUM 16.125 lacks the most common genetic
pathway to produce OTA, providing important information relevant to understand the role of P.
crustosum as putative OTA producer. Nevertheless, the additional secondary metabolism gene
clusters found (such as penitrem, clavaric acid or naphthopyrones) highlight the potential of
this strain for metabolite production, including other mycotoxins or compounds with antioxidant,
anticancer or antibiotic properties.This study was supported by the Portuguese Foundation for Science and Technology
(FCT) under the scope of the strategic funding of CEB (UID/BIO/04469/2019) and iBiMED (UIDB/04501/2020)
units; and by CANCYL (POCI-01-0145-FEDER-031849) and GenomePT (POCI-01-0145-FEDER-022184)
projectsinfo:eu-repo/semantics/publishedVersio
Enhancing legume ecosystem services through an understanding of plant–pollinator interplay
Legumes are bee-pollinated, but to a different extent. The importance of the plant– pollinator interplay (PPI), in flowering crops such as legumes lies in a combination of the importance of pollination for the production service and breeding strategies, plus the increasing urgency in mitigating the decline of pollinators through the development and implementation of conservation measures. To realize the full potential of the PPI, a multidisciplinary approach is required. This article assembles an international team of genebank managers, geneticists, plant breeders, experts on environmental governance and agro-ecology, and comprises several sections. The contributions in these sections outline both the state of the art of knowledge in the field and the novel aspects under development, and encompass a range of reviews, opinions and perspectives. The first three sections explore the role of PPI in legume breeding strategies. PPI based approaches to crop improvement can make it possible to adapt and re-design breeding strategies to meet both goals of: (1) optimal productivity, based on an efficient use of pollinators, and (2) biodiversity conservation. The next section deals with entomological aspects and focuses on the protection of the “pest control service” and pollinators in legume crops. The final section addresses general approaches to encourage the synergybetweenfoodproductionandpollinationservicesatfarmerfieldlevel.Twobasic approaches are proposed: (a) Farming with Alternative Pollinators and (b) Crop Design System
Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis
In order to determine the suitability of reference
or housekeeping genes as internal controls in
real-time reverse transcriptase PCR (RT-PCR) assays
for quantification of target mRNAs, we studied the
levels of expression of four candidate reference genes
in maritime pine by real-time RT-PCR. The expression
levels obtained for glyceraldehyde-3-phosphate-dehydrogenase,
18S ribosomal RNA, eukaryotic translation
initiation factor eIF4AII and ubiquitin in nine stages
of embryo development revealed that none of the
genes tested proved to be suitable as an internal control.
Copy number quantification of the four transcripts
showed an average relative variation of seven
fold. We propose that the combination of a precise
method for RNA quantification, internal controls for
monitoring RT reaction and PCR efficiency and a
robust external standard curve can guarantee a reliable
absolute quantification of mRNA transcripts in real
time RT-PCR. This approach may avoid the controversy
in the use of housekeeping genes and may assume
special significance in tissues undergoing
developmental changes
Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)
Somatic embryogenesis (SE) is a propagation
tool of particular interest for accelerating the deployment
of new high-performance planting stock in multivarietal
forestry. However, genetic conformity in in vitro propagated
plants should be assessed as early as possible,
especially in long-living trees such as conifers. The main
objective of this work was to study such conformity based
on genetic stability at simple sequence repeat (SSR) loci
during somatic embryogenesis in maritime pine (Pinus
pinaster Ait.). Embryogenic cell lines (ECLs) subjected to
tissue proliferation during 6, 14 or 22 months, as well as
emblings regenerated from several ECLs, were analyzed.
Genetic variation at seven SSR loci was detected in ECLs
under proliferation conditions for all time points, and in 5
out of 52 emblings recovered from somatic embryos. Three
of these five emblings showed an abnormal phenotype
consisting mainly of plagiotropism and loss of apical
dominance. Despite the variation found in somatic
embryogenesis-derived plant material, no correlation was
established between genetic stability at the analyzed loci
and abnormal embling phenotype, present in 64% of the
emblings. The use of microsatellites in this work was
efficient for monitoring mutation events during the somatic
embryogenesis in P. pinaster. These molecular markers
should be useful in the implementation of new breeding
and deployment strategies for improved trees using SE
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