Selection of endogenous genes for gene expression studies in Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using RT-qPCR

<p>Abstract</p> <p>Background</p> <p>Rust caused by <it>Puccinia psidii </it>Winter has been limiting for the establishment of new <it>Eucalyptus </it>plantations, as well as for resprouting of susceptible genetic materials. Identifying host genes involved in defense responses is important to elucidate resistance mechanisms. Reverse transcription-quantitative PCR is the most common method of mRNA quantitation for gene expression analysis. This method generally employs a reference gene as an internal control to normalize results. A good endogenous control transcript shows minimal variation due to experimental conditions.</p> <p>Findings</p> <p>We analyzed the expression of 13 genes to identify transcripts with minimal variation in leaves of 60-day-old clonal seedlings of two <it>Eucalyptus </it>clones (rust-resistant and susceptible) subjected to biotic (<it>P. psidii</it>) and abiotic (acibenzolar-S-methyl, ASM) stresses.</p> <p>Conclusions</p> <p>For tissue samples of clones that did not receive any stimulus, a combination of the <it>eEF2 </it>and <it>EglDH </it>genes was the best control for normalization. When pathogen-inoculated and uninoculated plant samples were compared, <it>eEF2 </it>and <it>UBQ </it>together were more appropriate as normalizers. In ASM-treated and untreated leaves of both clones, transcripts of the <it>CYP </it>and <it>elF4B </it>genes combined were the ones with minimal variation. Finally, when comparing expression in both clones for ASM-treated leaves, <it>P. psidii</it>-inoculated leaves, ASM-treated plus <it>P. psidii</it>-inoculated leaves, and their respective controls, the genes with the most stable expression were <it>EgIDH </it>and <it>UBQ</it>. The chitinase gene, which is highly expressed in studies on plant resistance to phytopathogens, was used to confirm variation in gene expression due to the treatments.</p

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

<p>Abstract</p> <p>Background</p> <p>Citrus canker is a disease caused by the phytopathogens <it>Xanthomonas citri </it>subsp. <it>citri</it>, <it>Xanthomonas fuscans </it>subsp. <it>aurantifolli </it>and <it>Xanthomonas alfalfae </it>subsp. <it>citrumelonis</it>. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.</p> <p>Results</p> <p>Through transposon insertion mutagenesis, 10,000 mutants of <it>Xanthomonas citri </it>subsp. <it>citri </it>strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (<it>Citrus limonia</it>) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with <it>Xanthomonas </it>pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the <it>hrpB4 </it>and <it>hrpX </it>genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the <it>htrA </it>gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. Finally, comparative genomic analysis revealed that 5 mutated ORFs are in new putative pathogenicity islands.</p> <p>Conclusion</p> <p>The identification of these new genes related with Xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker.</p

TOIMINNANOHJAUSJÄRJESTELMÄN KÄYTTÖÖNOTON VAIKUTUS

Tässä tutkielmassa tutkitaan kuinka toiminnanohjausjärjestelmän käyttöönotto on vaikuttanut yrityksen tulokseen ja suorituskykyyn suomalaisissa yrityksissä 1999-2015. Tavoitteena on testata empiirisesti aiheuttaako toiminnanohjausjärjestelmän käyttöönotto merkittäviä säästöjä, yrityk-sen toiminnan tehokkuuden parannuksia ja muutoksia yrityksen kannattavuudessa. Toiminnanohjaus- eli ERP-järjestelmät integroivat yrityksen eri toimintoja esimerkiksi logistiikkaa, tuotantoa, kirjanpitoa, varastointia ja laskutusta yhteen järjestelmään ja näin ollen tehostavat yrityksen toimintaa useiden eri järjestelmien käytön sijaan. Onkin tärkeää selvittää kuinka toiminnanohjausjärjestelmien käyttöönotto on vaikuttanut yritysten kannattavuuteen ja voidaanko investointi nähdä tuottavana. Aikaisempien tutkimusten perusteella tulokset ovat ristiriitaisia; tutkimukset osoittavat, että vaikka ERP- järjestelmien käyttöönotto tutkitusti vaikuttaa ei-rahamääräisiin mittareihin, vaikutusta rahamääräisiin mittareihin ei aina pystytä todistamaan. Yritysten kannalta mielenkiintoista onkin ovatko kalliit ERP-järjestelmät, sekä niiden implementointiprosessit todella kannattavia investointeja. Tutkimuksen empiirisessä osiossa selvitettiin toiminnanohjausjärjestelmien käyttöönoton vaiku-tuksia suorituskykyyn suomalaisissa yhtiöissä, jotka ovat implementoineet toiminnanohjausjärjestelmän vuosina 1999-2015. Aineistona on käytetty julkisuudessa ilmoitettuja järjestelmähank-keita sekä tilinpäätöstietoja implementointiprosessin jälkeisiltä sekä edeltäviltä vuosilta. Suorituskyvyn muutosta on seurattu vertailemalla suorituskyvyn ja kannattavuuden muutosta implementoinnin jälkeen. Tutkimus on suoritettu määrällisenä tutkimuksena ja tilastollisina menetelminä on käytetty toistomittausten varianssianalyysiä sekä t-testiä parivertailulle. Tutkimuksessa havaittiin, että näyttäisi siltä, ettei toiminnanohjausjärjestelmien käyttöönotolla ei ole ollut vaikutusta kohdeyhtiöiden rahamääräiseen suorituskykyyn.fi=Opinnäytetyö kokotekstinä PDF-muodossa.|en=Thesis fulltext in PDF format.|sv=Lärdomsprov tillgängligt som fulltext i PDF-format

Xylella fastidiosa : An in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis

Abstract Xylella fastidiosa inhabits the plant xylem, a nutrient-poor environment, so that mechanisms to sense and respond to adverse environmental conditions are extremely important for bacterial survival in the plant host. Although the complete genome sequences of different Xylella strains have been determined, little is known about stress responses and gene regulation in these organisms. In this work, a DNA microarray was constructed containing 2,600 ORFs identified in the genome sequencing project of Xylella fastidiosa 9a5c strain, and used to check global gene expression differences in the bacteria when it is infecting a symptomatic and a tolerant citrus tree. Different patterns of expression were found in each variety, suggesting that bacteria are responding differentially according to each plant xylem environment. The global gene expression profile was determined and several genes related to bacterial survival in stressed conditions were found to be differentially expressed between varieties, suggesting the involvement of different strategies for adaptation to the environment. The expression pattern of some genes related to the heat shock response, toxin and detoxification processes, adaptation to atypical conditions, repair systems as well as some regulatory genes are discussed in this paper. DNA microarray proved to be a powerful technique for global transcriptome analyses. This is one of the first studies of Xylella fastidiosa gene expression in vivo which helped to increase insight into stress responses and possible bacterial survival mechanisms in the nutrient-poor environment of xylem vessels

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii

Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento CientIfico e Tecnologico (CNPq)Coordenacao para Aperfeicoamento de Pessoal de Ensino Superior (CAPES)Fundo de Defesa da Citricultura (FUNDECITRUS

Global disparities in surgeons’ workloads, academic engagement and rest periods: the on-calL shIft fOr geNEral SurgeonS (LIONESS) study

: The workload of general surgeons is multifaceted, encompassing not only surgical procedures but also a myriad of other responsibilities. From April to May 2023, we conducted a CHERRIES-compliant internet-based survey analyzing clinical practice, academic engagement, and post-on-call rest. The questionnaire featured six sections with 35 questions. Statistical analysis used Chi-square tests, ANOVA, and logistic regression (SPSS® v. 28). The survey received a total of 1.046 responses (65.4%). Over 78.0% of responders came from Europe, 65.1% came from a general surgery unit; 92.8% of European and 87.5% of North American respondents were involved in research, compared to 71.7% in Africa. Europe led in publishing research studies (6.6 ± 8.6 yearly). Teaching involvement was high in North America (100%) and Africa (91.7%). Surgeons reported an average of 6.7 ± 4.9 on-call shifts per month, with European and North American surgeons experiencing 6.5 ± 4.9 and 7.8 ± 4.1 on-calls monthly, respectively. African surgeons had the highest on-call frequency (8.7 ± 6.1). Post-on-call, only 35.1% of respondents received a day off. Europeans were most likely (40%) to have a day off, while African surgeons were least likely (6.7%). On the adjusted multivariable analysis HDI (Human Development Index) (aOR 1.993) hospital capacity &gt; 400 beds (aOR 2.423), working in a specialty surgery unit (aOR 2.087), and making the on-call in-house (aOR 5.446), significantly predicted the likelihood of having a day off after an on-call shift. Our study revealed critical insights into the disparities in workload, access to research, and professional opportunities for surgeons across different continents, underscored by the HDI

Xylella fastidiosa: An in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis

Xylella fastidiosa inhabits the plant xylem, a nutrient-poor environment, so that mechanisms to sense and respond to adverse environmental conditions are extremely important for bacterial survival in the plant host. Although the complete genome sequences of different Xylella strains have been determined, little is known about stress responses and gene regulation in these organisms. In this work, a DNA microarray was constructed containing 2,600 ORFs identified in the genome sequencing project of Xylella fastidiosa 9a5c strain, and used to check global gene expression differences in the bacteria when it is infecting a symptomatic and a tolerant citrus tree. Different patterns of expression were found in each variety, suggesting that bacteria are responding differentially according to each plant xylem environment. The global gene expression profile was determined and several genes related to bacterial survival in stressed conditions were found to be differentially expressed between varieties, suggesting the involvement of different strategies for adaptation to the environment. The expression pattern of some genes related to the heat shock response, toxin and detoxification processes, adaptation to atypical conditions, repair systems as well as some regulatory genes are discussed in this paper. DNA microarray proved to be a powerful technique for global transcriptome analyses. This is one of the first studies of Xylella fastidiosa gene expression in vivo which helped to increase insight into stress responses and possible bacterial survival mechanisms in the nutrient-poor environment of xylem vessels.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

Background. From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings. The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions. Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas. © 2010 Moreira et al; licensee BioMed Central Ltd