scholarly journals Ontogeny of OPN4, OPN5, GnRH and GnIH mRNA Expression in the Posthatch Male and Female Pekin Duck (Anas platyrhynchos domesticus) Suggests OPN4 May Have Additional Functions beyond Reproduction

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1121
Author(s):  
Brooke Van Wyk ◽  
Gregory Fraley
Keyword(s):  
Gene Expression ◽  
Repeated Measures ◽  
Time Course ◽  
Gonadal Steroids ◽  
Pekin Duck ◽  
Mrna Levels ◽  
Qrt Pcr ◽  
Circulating Levels ◽  
Deep Brain ◽  
Hatch Period

The hypothalamic–pituitary–gonadal axis (HPG) is known to be regulated by daylength through the deep brain photoreceptor (DBP) system. The post-hatch ontogeny is not known for any of the DBPs. We set out to determine the ontogeny of OPN4 and OPN5 gene expression relative to GnRH and GnIH using qRT-PCR. Brains and serum were collected from five drakes and five hens on the day of hatching (Day 0) and again at 2, 4, 6, 10, 14, 19, 25 and 31 weeks of age and analyzed by qRT-PCR. Hen and drake serum was assayed for circulating levels of estradiol and testosterone, respectively. Data were analyzed between sexes over time using a repeated measures two-way ANOVA. Interestingly, the results show that on the day of hatching (Day 0), ducks showed adult-like levels of relative OPN4, but not OPN5, gene expression. During week 10, DBP levels increased, achieving highest relative expression levels at week 19 that maintained through week 31, typically peak fertility in ducks. GnRH mRNA levels increased following the DBP expression at the onset of puberty, and gonadal steroids increased after GnRH at week 14 while estradiol preceded testosterone. GnIH mRNA levels did not appreciably change during the time course of this experiment. These observations suggest that OPN4 may be active during the peri-hatch period and may have physiological roles beyond puberty and fertility.

scholarly journals Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 331-340
Author(s):  
WE Kaminski ◽  
E Jendraschak ◽  
K Baumann ◽  
R Kiefl ◽  
S Fischer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Time Course ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Constitutive Expression ◽  
Ionophore A23187 ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Human Mononuclear Cells

Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

scholarly journals Comparative transcriptome and histomorphology analysis of testis tissues from mulard and Pekin ducks

2020 ◽  
Vol 63 (2) ◽  
pp. 303-313
Author(s):  
Li Li ◽  
Linli Zhang ◽  
Zhenghong Zhang ◽  
Nemat O. Keyhani ◽  
Qingwu Xin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differential Expression ◽  
Candidate Genes ◽  
Transcriptome Sequencing ◽  
Differentially Expressed ◽  
Pekin Duck ◽  
Comparative Transcriptome ◽  
Qrt Pcr ◽  
Pekin Ducks ◽  
Testis Tissue

Abstract. Testicular transcriptomes were analyzed to characterize the differentially expressed genes between mulard and Pekin ducks, which will help establish gene expression datasets to assist in further determination of the mechanisms of genetic sterility in mulard ducks. Paraffin sections were made to compare the developmental differences in testis tissue between mulard and Pekin ducks. Comparative transcriptome sequencing of testis tissues was performed, and the expression of candidate genes was verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In mulard ducks, spermatogonia and spermatocytes were arranged in a disordered manner, and no mature sperm were observed in the testis tissue. However, different stages of development of sperm were observed in seminiferous tubules in the testis tissue of Pekin ducks. A total of 43.84 Gb of clean reads were assembled into 193 535 UniGenes. Of these, 2131 transcripts exhibited differential expression (false discover rate <0.001 and fold change ≥2), including 997 upregulated and 1134 downregulated transcripts in mulard ducks as compared to those in Pekin duck testis tissues. Several upregulated genes were related to reproductive functions, including ryanodine receptor 2 (RYR2), calmodulin (CALM), argininosuccinate synthase and delta-1-pyrroline-5-carboxylate synthetase ALDH18A1 (P5CS). Downregulated transcripts included the testis-specific serine/threonine-protein kinase 3, aquaporin-7 (AQP7) and glycerol kinase GlpK (GK). The 10 related transcripts involved in the developmental biological process were identified by GO (Gene Ontology) annotation. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways indicated that peroxisome proliferator-activated receptors (PPARs) and calcium signaling pathways were significantly (P<0.001) associated with normal testis physiology. The differential expression of select genes implicated in reproductive processes was verified by qRT-PCR, which was consistent with the expression trend of transcriptome sequencing (RNA-seq). Differentially expressed candidate genes RYR2, CALM, P5CS, AQP7 and GK were identified by transcriptional analysis in mulard and Pekin duck testes. These were important for the normal development of the male duck reproductive system. These data provide a framework for the further exploration of the molecular and genetic mechanisms of sterility in mulard ducks. Highlights. The mulard duck is an intergeneric sterile hybrid offspring resulting from mating between Muscovy and Pekin ducks. The transcriptomes of testis tissue from mulard and Pekin ducks were systematically characterized, and differentially expressed genes were screened, in order to gain insights into potential gonad gene expression mechanisms contributing to genetic sterility in mulard ducks.

Time course of myogenic and metabolic gene expression in response to acute exercise in human skeletal muscle

2005 ◽  
Vol 98 (5) ◽  
pp. 1745-1752 ◽  
Author(s):  
Yifan Yang ◽  
Andrew Creer ◽  
Bozena Jemiolo ◽  
Scott Trappe
Keyword(s):  
Gene Expression ◽  
Time Course ◽  
Acute Exercise ◽  
Vastus Lateralis ◽  
Gene Induction ◽  
Mrna Levels ◽  
Metabolic Gene ◽  
Expression Studies ◽  
Metabolic Gene Expression ◽  
Gene Expression Studies

The aim of this study was to examine the time course activation of select myogenic (MRF4, Myf5, MyoD, myogenin) and metabolic (CD36, CPT1, HKII, and PDK4) genes after an acute bout of resistance (RE) or run (Run) exercise. Six RE subjects [25 ± 4 yr (mean ± SD), 74 ± 14 kg, 1.71 ± 0.11 m] and six Run subjects (25 ± 4 yr, 72 ± 5 kg, 1.81 ± 0.07 m, 63 ± 8 ml·kg−1·min−1) were studied. Eight muscle biopsies were taken from the vastus lateralis (RE) and gastrocnemius (Run) before, immediately after, and 1, 2, 4, 8, 12 and 24 h after exercise. RE increased mRNA of MRF4 (3.7- to 4.5-fold 2–4 h post), MyoD (5.8-fold 8 h post), myogenin (2.6- and 3.5-fold 8–12 h post), HKII (3.6- to 10.5-fold 2–12 h post), and PDK4 (14- to 26-fold 2–8 h post). There were no differences in Myf5, CD36, and CPT1 mRNA levels 0–24 h post-RE. Run increased mRNA of MyoD (5.0- to 8.0-fold), HKII (12- to 16-fold), and PDK4 (32- to 52-fold) at 8–12 h postexercise. There were no differences in MRF4, Myf5, myogenin, CD36 and CPT1 mRNA levels 0–24 h post-Run. These data indicate a myogenic and metabolic gene induction with RE and Run exercise. The timing of the gene induction is variable and generally peaks 4–8 h postexercise with all gene expression not significantly different from the preexercise levels by 24 h postexercise. These data provide basic information for the timing of human muscle biopsy samples for gene-expression studies involving exercise.

scholarly journals Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 331-340 ◽  
Author(s):  
WE Kaminski ◽  
E Jendraschak ◽  
K Baumann ◽  
R Kiefl ◽  
S Fischer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Time Course ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Constitutive Expression ◽  
Ionophore A23187 ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Human Mononuclear Cells

Abstract Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

Gene expression after resumption of development of Artemia franciscana cryptobiotic embryos

1994 ◽  
Vol 72 (3-4) ◽  
pp. 78-83 ◽  
Author(s):  
Ricardo Escalante ◽  
Alberto García-Sáez ◽  
Maria-Asunción Ortega ◽  
Leandro Sastre
Keyword(s):  
Gene Expression ◽  
Time Course ◽  
Developmental Stages ◽  
Artemia Franciscana ◽  
Mrna Levels ◽  
Muscle Actin ◽  
Mrna Accumulation ◽  
Α Subunit ◽  
Steady State Levels ◽  
Cyst Development

The steady-state levels of six different mRNAs have been studied during Artemia franciscana development. Some of these mRNAs are present in the cryptobiotic cyst, like those coding for cytoplasmic actins, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, and the Na+,K+-ATPase α-subunit isoform coded by the clone pArATNa136. The expression of these mRNAs is markedly induced during cyst development. A small increase in mRNA levels can be observed for some genes at very early stages of development (2 h). The main increase is observed between 4 and 16 h of development for all these genes, although the time course of mRNA accumulation is different for each one of the genes studied. Some other genes, like those coding for muscle actin (actin 3) or the Na+,K+-ATPase α-subunit isoform coded by the cDNA clone α2850, are not expressed in the cyst before resumption of development and their expression is induced after 10 or 6 h of development, respectively. These data on the kinetic of mRNA accumulation provide the information required to determine transcriptionally active developmental stages, necessary to study in more detail the mechanisms of transcriptional regulation during activation of cryptobiotic cysts and resumption of embryonic development.Key words: Artemia, gene expression, actin, Na,K-ATPase, Ca2+-ATPase.

scholarly journals Gene Expression of Indoleamine 2,3 Dioxygenase 1, Insulin-Growth Factor 1 and Red/IK Cytokine in Alopecia Areata

2014 ◽  
Vol 6 (3) ◽  
pp. 263-266
Author(s):  
Simona Corina ȘENILĂ ◽  
Ovidiu BĂLĂCESCU ◽  
Loredana BĂLĂCESCU ◽  
Elisabeta CANDREA ◽  
Loredana UNGUREANU ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hair Follicle ◽  
Mhc Class I ◽  
Alopecia Areata ◽  
Fas Ligand ◽  
Immune Privilege ◽  
Mrna Levels ◽  
Insulin Growth Factor ◽  
Qrt Pcr ◽  
Anagen Hair

Alopecia areata (AA) is a chronic, T-cell mediated autoimmune disease directed against the hair follicle, which partially evolves due to a loss of the immune privilege of the anagen hair follicle. The immune privilege is maintained by several factors, including a downregulation of MHC class I and II, local immunosupressants and expression of Fas ligand. The purpose of the study was to evaluate several factors involved in the collapse and restoration of the immune privilege. We investigated IDO1, IGF1 and red/IK gene expression in lesional and perilesionalscalp biopsies from alopecia areata patients. Seven paired punch-biopsies were taken from the active edge of alopecic plaque and from the perilesional scalp. Expression of IDO1, IGF1 and red/IK genes was performed by qRT-PCR. In lesional tissue, IGF1, IDO1 and red/IK genes showed an increase in the mRNA levels as compared with the perilesional scalp. By comparing the pairs of data for the investigated genes, IDO1was statistically upregulated in the lesional area. No significant differences were observed between the gene expression in mild or severe AA, from the lesional or perilesional areas. IDO1 mRNA expression was higher in patients with a relapse duration of less than 6 months as compared to patients with a relapse duration of more than 6 months; levels of IGF1 and red/IK mRNA are increased in lesionals compared to perilesional scalp area.

Expression of IGFBP-1 in normal and cirrhotic human livers

1994 ◽  
Vol 141 (2) ◽  
pp. 377-382 ◽  
Author(s):  
R J M Ross ◽  
J Rodriguez-Arnao ◽  
A Donaghy ◽  
J Bentham ◽  
A Clark ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Liver ◽  
Chronic Liver ◽  
Hepatic Regeneration ◽  
Mrna Levels ◽  
Insulin Levels ◽  
Time Of Surgery ◽  
Group 3 ◽  
Group 2 ◽  
Circulating Levels

Abstract Cirrhosis of the liver, a condition characterised by hepatocyte regeneration, is also associated with elevated insulin levels and insulin resistance. In animal models hepatic regeneration is associated with increased IGFBP-1 gene expression. Insulin is known to be an inhibitor of IGFBP-1 gene expression and circulating insulin levels in man demonstrate a negative correlation with IGFBP-1 levels. To further our understanding of the regulation of IGFBP-1 in cirrhosis we have studied steady state levels of IGFBP-1 mRNA in human liver from three groups of patients: Group 1, tissue obtained at the time of harvesting donor liver for orthotopic liver transplantation (n=4); group 2, patients undergoing major liver resection with no histological evidence of chronic liver disease (n=4); and group 3, patients undergoing orthotopic transplantation for chronic liver failure (n=9). Simultaneous samples of serum were taken at the time of surgery in some patients and in these patients IGFBP-1 mRNA levels were related to circulating levels of IGFBP-1 and insulin. IGFBP-1 mRNA was detectable in all the human liver samples with the greatest levels seen from the normal livers of group 2 patients. Insulin levels were elevated in the cirrhotic group 3 patients compared to a normal range as were IGFBP-1 levels. There was no relationship between circulating levels of IGFBP-1 and IGFBP-1 gene expression. In conclusion, IGFBP-1 mRNA is present in human adult liver at the time of surgery and also in cirrhotic liver despite high levels of insulin suggesting that there are factors other than insulin regulating IGFBP-1 gene expression. Journal of Endocrinology (1994) 141, 377–382

Secretoneurin is a potential paracrine factor from lactotrophs stimulating gonadotropin release in the goldfish pituitary

2010 ◽  
Vol 299 (5) ◽  
pp. R1290-R1297 ◽  
Author(s):  
E. Zhao ◽  
Caleb L. Grey ◽  
Dapeng Zhang ◽  
Jan A. Mennigen ◽  
Ajoy Basak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Time Course ◽  
Pcr Analysis ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Paracrine Factor ◽  
Lh Release

Secretoneurin (SN) is a functional neuropeptide derived from the evolutionarily conserved part of precursor protein secretogranin II (SgII). In the time course study, SN (10 nM) stimulates luteinizing hormone (LH) production and secretion after 6 h of static incubation of goldfish pituitary cells. Due to the existence of SN-immunoreactivity (SN-IR) in goldfish lactotrophs, endogenous SN might exert a paracrine effect on LH in the pituitary. In an in vitro immunoneutralization experiment, coincubation with anti-SN antiserum reduces the stimulatory effect of salmon gonadotropin-releasing hormone (sGnRH) on LH release by 64%. Using Western blot analysis, we demonstrate that sGnRH significantly increases the expression of the major SgII-derived peptide (∼57 kDa, with SN-IR) and prolactin (PRL) after 12 h in the static culture of goldfish pituitary cells. Furthermore, there exists a significant correlation between the levels of these two proteins ( R = 0.76, P = 0.004). Another ∼30 kDa SgII-derived peptide containing SN is only observed in sGnRH-treated pituitary cells. Consistent with the Western blot analysis results, real-time RT-PCR analysis shows that a 12-h treatment with sGnRH induced 1.6- and 1.7-fold increments in SgII and PRL mRNA levels, respectively. SgII gene expression was also associated with PRL gene expression ( R = 0.66; P = 0.02). PRL cells loaded with the calcium-sensitive dye, fura 2/AM, respond to sGnRH treatment with increases in intracellular Ca2+ concentration level, suggesting a potential mechanism of GnRH on PRL cells and thus SgII processing and SN secretion. Taken together, endogenous lactotroph-generated SN, under the control of hypothalamic GnRH, exerts a paracrine action on neighboring gonadotrophs to stimulate LH release.

scholarly journals TcpH Influences Virulence Gene Expression in Vibrio cholerae by Inhibiting Degradation of the Transcription Activator TcpP

2004 ◽  
Vol 186 (24) ◽  
pp. 8309-8316 ◽  
Author(s):  
Nancy A. Beck ◽  
Eric S. Krukonis ◽  
Victor J. DiRita
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Time Course ◽  
Virulence Gene ◽  
Degradation Pathway ◽  
Mrna Levels ◽  
Transcription Activator ◽  
Virulence Gene Expression ◽  
Protein Levels ◽  
Periplasmic Domain

ABSTRACT Expression of toxT, the transcription activator of cholera toxin and pilus production in Vibrio cholerae, is the consequence of a complex cascade of regulatory events that culminates in activation of the toxT promoter by TcpP and ToxR, two membrane-localized transcription factors. Both are encoded in operons with genes whose products, TcpH and ToxS, which are also membrane localized, are hypothesized to control their activity. In this study we analyzed the role of TcpH in controlling TcpP function. We show that a mutant of V. cholerae lacking TcpH expressed virtually undetectable levels of TcpP, although tcpP mRNA levels remain unaffected. A time course experiment showed that levels of TcpP, expressed from a plasmid, are dramatically reduced over time without co-overexpression of TcpH. By contrast, deletion of toxS did not affect ToxR protein levels. A fusion protein in which the TcpP periplasmic domain is replaced with that of ToxR remains stable, suggesting that the periplasmic domain of TcpP is the target for degradation of the protein. Placement of the periplasmic domain of TcpP on ToxR, an otherwise stable protein, results in instability, providing further evidence for the hypothesis that the periplasmic domain of TcpP is a target for degradation. Consistent with this interpretation is our finding that derivatives of TcpP lacking a periplasmic domain are more stable in V. cholerae than are derivatives in which the periplasmic domain has been truncated. This work identifies at least one role for the periplasmic domain of TcpP, i.e., to act as a target for a protein degradation pathway that regulates TcpP levels. It also provides a rationale for why the V. cholerae tcpH mutant strain is avirulent. We hypothesize that regulator degradation may be an important mechanism for regulating virulence gene expression in V. cholerae.

scholarly journals Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

2020 ◽  
Author(s):  
Liz M. Florez ◽  
Reiny W. A. Scheper ◽  
Brent M. Fisher ◽  
Paul W. Sutherland ◽  
Matthew D. Templeton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reference Genes ◽  
Time Course ◽  
Gene Expression Analysis ◽  
Virulence Genes ◽  
Functional Characterization ◽  
Post Inoculation ◽  
In Planta ◽  
Qrt Pcr

AbstractEuropean canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Real-time quantitative reverse transcription PCR (qRT-PCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate qRT-PCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

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