PdeH, a High-Affinity cAMP Phosphodiesterase, Is a Key Regulator of Asexual and Pathogenic Differentiation in

English version České info

Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases) and hydrolysis (by cAMP phosphodiesterases). We functionally characterized gene-deletion mutants of a high-affinity (PdeH) and a low-affinity (PdeL) cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHΔ showed enhanced conidiation (2–3 fold), precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHΔ pdeLΔ mutant showed reduced conidiation, exhibited dramatically increased (∼10 fold) cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHΔ defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling during M. oryzae development is crucial for successful establishment and spread of the blast disease in rice.

Vyšlo v časopise: PdeH, a High-Affinity cAMP Phosphodiesterase, Is a Key Regulator of Asexual and Pathogenic Differentiation in. PLoS Pathog 6(5): e32767. doi:10.1371/journal.ppat.1000897
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000897

Souhrn

Zdroje

1. PierceK

PremontR

LefkowitzR

2002 Seven-transmembrane receptors. Nature Reviews Molecular Cell Biology 639 650

2. HouslayMD

BaillieGS

MauriceDH

2007 cAMP-Specific phosphodiesterase-4 enzymes in the cardiovascular system: a molecular toolbox for generating compartmentalized cAMP signaling. Circ Res 100 950 966

3. BeavoJ

FrancisSH

HouslayM

2006 Cyclic nucleotide Phosphodiesterases in Health and Disease. CRC Press,Boca Raton,Florida

4. HouslayMD

MilliganG

1997 Tailoring cAMP-signalling responses through isoform multiplicity. Trends Biochem Sci 22 217 224

5. HouslayMD

2001 PDE4 cAMP-specific phosphodiesterases. Prog Nucleic Acid Res Mol Biol 69 249 315

6. HouslayMD

SullivanM

BolgerGB

1998 The multienzyme PDE4 cyclic adenosine monophosphate-specific phosphodiesterase family: intracellular targeting, regulation, and selective inhibition by compounds exerting anti-inflammatory and antidepressant actions. Adv Pharmacol 44 225 342

7. HuY

LiuE

BaiX

ZhangA

2009 The localization and concentration of the PDE2-encoded high-affinity cAMP phosphodiesterase is regulated by cAMP-dependent protein kinase A in the yeast Saccharomyces cerevisiae. FEMS Yeast Res

8. NamyO

Duchateau-NguyenG

RoussetJP

2002 Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae. Mol Microbiol 43 641 652

9. BenderAT

BeavoJA

2006 Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev 58 488 520

10. CooperDM

2003 Regulation and organization of adenylyl cyclases and cAMP. Biochem J 375 517 529

11. OmoriK

KoteraJ

2007 Overview of PDEs and their Regulation. Circulation research 100 309 327

12. ContiM

BeavoJ

2007 Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem 76 481 511

13. LemaireK

Van de VeldeS

Van DijckP

TheveleinJM

2004 Glucose and sucrose act as agonist and mannose as antagonist ligands of the G protein-coupled receptor Gpr1 in the yeast Saccharomyces cerevisiae. Mol Cell 16 293 299

14. JonesDL

PettyJ

HoyleDC

HayesA

OliverSG

2004 Genome-Wide Analysis of the Effects of Heat Shock on a Saccharomyces cerevisiae Mutant With a Constitutively Activated cAMP-Dependent Pathway. Comp Funct Genomics 5 419 431

15. JonesDL

PettyJ

HoyleDC

HayesA

RagniE

2003 Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway. Physiol Genomics 16 107 118

16. PanX

HeitmanJ

1999 Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Cell Biol 19 4874 4887

17. ParkJI

GrantCM

DawesIW

2005 The high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae is the major determinant of cAMP levels in stationary phase: involvement of different branches of the Ras-cyclic AMP pathway in stress responses. Biochem Biophys Res Commun 327 311 319

18. SchneperL

KraussA

MiyamotoR

FangS

BroachJR

2004 The Ras/protein kinase A pathway acts in parallel with the Mob2/Cbk1 pathway to effect cell cycle progression and proper bud site selection. Eukaryot Cell 3 108 120

19. DaveyJ

1998 Fusion of a fission Yeast. Yeast 1529 1566

20. YamamotoM

1996 The molecular control mechanisms of meiosis in fission yeast. Trends Biochemical Sciences 18 22

21. DeVotiJ, SG

BeachD

McLeodM

1991 Interaction between ran1+ protein kinase and cAMP dependent protein kinase as negative regulators of fission yeast meiosis. EMBO J 3759 3768

22. HiguchiT

WatanabeY

YamamotoM

2002 Protein kinase A regulates sexual development and gluconeogenesis through phosphorylation of the Zn finger transcriptional activator Rst2p in fission yeast. Mol Cell Biol 22 1 11

23. BahnYS

StaabJ

SundstromP

2003 Increased high-affinity phosphodiesterase PDE2 gene expression in germ tubes counteracts CAP1-dependent synthesis of cyclic AMP, limits hypha production and promotes virulence of Candida albicans. Mol Microbiol 50 391 409

24. JungWH

StatevaLI

2003 The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans. Microbiology 149 2961 2976

25. JungWH

WarnP

RagniE

PopoloL

NunnCD

2005 Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans. Yeast 22 285 294

26. HarcusD

NantelA

MarcilA

RigbyT

WhitewayM

2004 Transcription profiling of cyclic AMP signaling in Candida albicans. Mol Biol Cell 15 4490 4499

27. BahnYS

MolendaM

StaabJF

LymanCA

GordonLJ

2007 Genome-wide transcriptional profiling of the cyclic AMP-dependent signaling pathway during morphogenic transitions of Candida albicans. Eukaryot Cell 6 2376 2390

28. HuG

SteenBR

LianT

ShamAP

TamN

2007 Transcriptional regulation by protein kinase A in Cryptococcus neoformans. PLoS Pathog 3 e42 doi:10.1371/journal.ppat.0030042

29. D'SouzaCA

HeitmanJ

2001 Conserved cAMP signaling cascades regulate fungal development and virulence. FEMS Microbiol Rev 25 349 364

30. HicksJK

BahnYS

HeitmanJ

2005 Pde1 phosphodiesterase modulates cyclic AMP levels through a protein kinase A-mediated negative feedback loop in Cryptococcus neoformans. Eukaryot Cell 4 1971 1981

31. AlspaughJA

PerfectJR

HeitmanJ

1997 Cryptococcus neoformans mating and virulence are regulated by the G-protein alpha subunit GPA1 and cAMP. Genes Dev 11 3206 3217

32. KozubowskiL

LeeSC

HeitmanJ

2009 Signalling pathways in the pathogenesis of Cryptococcus. Cell Microbiol 11 370 380

33. Pukkila-WorleyR

AlspaughJA

2004 Cyclic AMP signaling in Cryptococcus neoformans. FEMS Yeast Res 4 361 367

34. LarrayaLM

BoyceKJ

SoA

SteenBR

JonesS

2005 Serial analysis of gene expression reveals conserved links between protein kinase A, ribosome biogenesis, and phosphate metabolism in Ustilago maydis. Eukaryot Cell 4 2029 2043

35. RegenfelderE

SpelligT

HartmannA

LauensteinS

BolkerM

1997 G proteins in Ustilago maydis: transmission of multiple signals? EMBO J 16 1934 1942

36. DurrenbergerF

WongK

KronstadJW

1998 Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustilago maydis. Proc Natl Acad Sci U S A 95 5684 5689

37. GoldSE

BrogdonSM

MayorgaME

KronstadJW

1997 The Ustilago maydis regulatory subunit of a cAMP-dependent protein kinase is required for gall formation in maize. Plant Cell 9 1585 1594

38. LeeN

KronstadJW

2002 ras2 Controls morphogenesis, pheromone response, and pathogenicity in the fungal pathogen Ustilago maydis. Eukaryot Cell 1 954 966

39. BannoS

OchiaiN

NoguchiR

KimuraM

YamaguchiI

2005 A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa. Genes Genet Syst 80 25 34

40. BencinaM

PannemanH

RuijterGJ

LegisaM

VisserJ

1997 Characterization and overexpression of the Aspergillus niger gene encoding the cAMP-dependent protein kinase catalytic subunit. Microbiology 143 (Pt 4) 1211 1220

41. BrakhageAA

LiebmannB

2005 Aspergillus fumigatus conidial pigment and cAMP signal transduction: significance for virulence. Med Mycol 43 Suppl 1 S75 82

42. GrosseC

HeinekampT

KniemeyerO

GehrkeA

BrakhageAA

2008 Protein kinase A regulates growth, sporulation, and pigment formation in Aspergillus fumigatus. Appl Environ Microbiol 74 4923 4933

43. IveyFD

KaysAM

BorkovichKA

2002 Shared and independent roles for a Galpha(i) protein and adenylyl cyclase in regulating development and stress responses in Neurospora crassa. Eukaryot Cell 1 634 642

44. KaysAM

RowleyPS

BaasiriRA

BorkovichKA

2000 Regulation of conidiation and adenylyl cyclase levels by the Galpha protein GNA-3 in Neurospora crassa. Mol Cell Biol 20 7693 7705

45. KaysAM

BorkovichKA

2004 Severe impairment of growth and differentiation in a Neurospora crassa mutant lacking all heterotrimeric G alpha proteins. Genetics 166 1229 1240

46. LiebmannB

MullerM

BraunA

BrakhageAA

2004 The cyclic AMP-dependent protein kinase a network regulates development and virulence in Aspergillus fumigatus. Infect Immun 72 5193 5203

47. SaudoharM

BencinaM

van de VondervoortPJ

PannemanH

LegisaM

2002 Cyclic AMP-dependent protein kinase is involved in morphogenesis of Aspergillus niger. Microbiology 148 2635 2645

48. LiebmannB

GattungS

JahnB

BrakhageAA

2003 cAMP signaling in Aspergillus fumigatus is involved in the regulation of the virulence gene pksP and in defense against killing by macrophages. Mol Genet Genomics 269 420 435

49. LafonA

HanKH

SeoJA

YuJH

d'EnfertC

2006 G-protein and cAMP-mediated signaling in Aspergilli: a genomic perspective. Fungal Genet Biol 43 490 502

50. UnoI

MatsumotoK

IshikawaT

1983 Characterization of a cyclic nucleotide phosphodiesterase-deficient mutant in yeast. J Biol Chem 258 3539 3542

51. LondesboroughJ

1978 The high-Km cyclic AMP phosphodiesterase of baker's yeast is a zinc metalloenzyme [proceedings]. Biochem Soc Trans 6 1218 1220

52. LondesboroughJC

1975 Soluble and membrane-bound cyclic AMP diesterase activity with a low Michaelis constant in baker's yeast. FEBS Lett 50 283 287

53. LondesboroughJ

SuorantaK

1983 The zinc-containing high Km cyclic nucleotide phosphodiesterase of bakers' yeast. J Biol Chem 258 2966 2972

54. MaP

WeraS

Van DijckP

TheveleinJM

1999 The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling. Mol Biol Cell 10 91 104

55. HoffmanCS

2005 Glucose sensing via the protein kinase A pathway in Schizosaccharomyces pombe. Biochem Soc Trans 33 261 264

56. WeraS

MaP

TheveleinJM

1997 Glucose exerts opposite effects on mRNA versus protein and activity levels of Pde1, the low-affinity cAMP phosphodiesterase from budding yeast, Saccharomyces cerevisiae. FEBS Lett 420 147 150

57. WilsonRB

RenaultG

JacquetM

TatchellK

1993 The pde2 gene of Saccharomyces cerevisiae is allelic to rca1 and encodes a phosphodiesterase which protects the cell from extracellular cAMP. FEBS Lett 325 191 195

58. WentzingerL

SeebeckT

2006 Protozoalphosphodiesterases.

BeavoJA

FrancisS

HouslayM

277 300 In: Phosphodiesterases in Health and Disease; CRC Press

59. SassP

FieldJ

NikawaJ

TodaT

WiglerM

1986 Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 83 9303 9307

60. MatviwH

LiJ

YoungD

1993 The Schizosaccharomyces pombe pde1/cgs2 gene encodes a cyclic AMP phosphodiesterase. Biochem Biophys Res Commun 194 79 82

61. HoyerLL

CieslinskiLB

McLaughlinMM

TorphyTJ

ShatzmanAR

1994 A Candida albicans cyclic nucleotide phosphodiesterase: cloning and expression in Saccharomyces cerevisiae and biochemical characterization of the recombinant enzyme. Microbiology 140 (Pt 7) 1533 1542

62. MasciarelliS

HornerK

LiuC

ParkSH

HinckleyM

2004 Cyclic nucleotide phosphodiesterase 3A-deficient mice as a model of female infertility. J Clin Invest 114 196 205

63. FevreEM

PicozziK

JanninJ

WelburnSC

MaudlinI

2006 Human African trypanosomiasis: Epidemiology and control. Adv Parasitol 61 167 221

64. OuSH

1985 Rice Diseases: Commonwealth Mycological Institute, Kew, Surrey, England.

65. LeeK

SinghP

ChungWC

AshJ

KimTS

2006 Light regulation of asexual development in the rice blast fungus, Magnaporthe oryzae. Fungal Genet Biol 43 694 706

66. LeungH

ShiZ

1994 Genetic regulation of sporulation in the rice blast fungus. 35 50 Zeigler, RS, Leong,SA, Teng, PS (Eds), Rice Blast Disease CAB international, Oxon

67. LauGW

HamerJE

1998 Acropetal: a genetic locus required for conidiophore architecture and pathogenicity in the rice blast fungus. Fungal Genet Biol 24 228 239

68. BeckermanJL

EbboleDJ

1996 MPG1, a gene encoding a fungal hydrophobin of Magnaporthe grisea, is involved in surface recognition. Mol Plant Microbe Interact 9 450 456

69. LeeYH

DeanRA

1993 cAMP Regulates Infection Structure Formation in the Plant Pathogenic Fungus Magnaporthe grisea. Plant Cell 5 693 700

70. LiuH

RamanujamR

NaqviN

2009 Surface sensing and signaling during initiation of rice blast disease. In Advances in Genetics, Genomics and Control of Rice Blast Disease 23 32

71. LiuH

SureshA

WillardFS

SiderovskiDP

LuS

2007 Rgs1 regulates multiple Galpha subunits in Magnaporthe pathogenesis, asexual growth and thigmotropism. Embo J 26 690 700

72. ChoiW

DeanRA

1997 The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell 9 1973 1983

73. CharbonneauH

BeierN

WalshKA

BeavoJA

1986 Identification of a conserved domain among cyclic nucleotide phosphodiesterases from diverse species. Proc Natl Acad Sci U S A 83 9308 9312

74. BalhaderePV

TalbotNJ

2001 PDE1 encodes a P-type ATPase involved in appressorium-mediated plant infection by the rice blast fungus Magnaporthe grisea. Plant Cell 13 1987 2004

75. TalbotNJ

1995 Having a blast: exploring the pathogenicity of Magnaporthe grisea. Trends Microbiol 3 9 16

76. Veneault-FourreyC

BarooahM

EganM

WakleyG

TalbotNJ

2006 Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science 312 580 583

77. McCahillA

CampbellL

McSorleyT

SoodA

LynchMJ

2008 In cardiac myocytes, cAMP elevation triggers the down-regulation of transcripts and promoter activity for cyclic AMP phosphodiesterase-4A10 (PDE4A10). Cell Signal 20 2071 2083

78. RileyBB

BarclaySL

1990 Conditions that alter intracellular cAMP levels affect expression of the cAMP phosphodiesterase gene in Dictyostelium. Proc Natl Acad Sci U S A 87 4746 4750

79. MosqueraG

GiraldoMC

KhangCH

CoughlanS

ValentB

2009 Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1-4 as Biotrophy-associated secreted proteins in rice blast disease. Plant Cell 21 1273 1290

80. SkamniotiP

GurrSJ

2007 Magnaporthe grisea cutinase2 mediates appressorium differentiation and host penetration and is required for full virulence. Plant Cell 19 2674 2689

81. MitchellTK

DeanRA

1995 The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea. Plant Cell 7 1869 1878

82. SoanesDM

KershawMJ

CooleyRN

TalbotNJ

2002 Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea. Mol Plant Microbe Interact 15 1253 1267

83. NishimuraM

HayashiN

JwaNS

LauGW

HamerJE

2000 Insertion of the LINE retrotransposon MGL causes a conidiophore pattern mutation in Magnaporthe grisea. Mol Plant Microbe Interact 13 892 894

84. OdenbachD

BrethB

ThinesE

WeberRW

AnkeH

2007 The transcription factor Con7p is a central regulator of infection-related morphogenesis in the rice blast fungus Magnaporthe grisea. Mol Microbiol 64 293 307

85. LiuS

DeanRA

1997 G protein alpha subunit genes control growth, development, and pathogenicity of Magnaporthe grisea. Mol Plant Microbe Interact 10 1075 1086

86. NishimuraM

ParkG

XuJR

2003 The G-beta subunit MGB1 is involved in regulating multiple steps of infection-related morphogenesis in Magnaporthe grisea. Mol Microbiol 50 231 243

87. HanKH

SeoJA, JHY

2004 Regulators of G-protein signalling in Aspergillus nidulans: RgsA downregulates stress response and stimulates asexual sporulation through attenuation of GanB (Gα) signalling. Molecular Microbiology 53 529 540

88. ChoiGH

ChenB

NDL

1995 Virus-mediated or transgenic suppression of a G protein α subunit and attenuation of fungal virulence. Proc Natl Acad Sci U S A 92 305 309

89. SegersGC, DLN

2003 Constitutively activated Gα negatively regulates virulence, reproduction and hydrophobin gene expression in the chestnut blight fungus Cryphonectria parasitica. Fungal Genet Biol 38 198 208

90. RosenS

YuJH

AdamsTH

1999 The Aspergillus nidulans sfaD gene encodes a G protein beta subunit that is required for normal growth and repression of sporulation. Embo J 18 5592 5600

91. HicksJK

YuJH

KellerNP

AdamsTH

1997 Aspergillus sporulation and mycotoxin production both require inactivation of the FadA G alpha protein-dependent signaling pathway. Embo J 16 4916 4923

92. AdachiK

HamerJE

1998 Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea. Plant Cell 10 1361 1374

93. XuJ-R

UrbanM

SweigardJA

HamerJE

1997 The CPKA Gene of Magnaporthe grisea Is Essential for Appressorial Penetration. Molecular Plant Microbe Interactions 10 187

94. HouslayMD

2009 Underpinning compartmentalised cAMP signalling through targeted cAMP breakdown. Trends Biochem Sci

95. BaillieGS

2009 Compartmentalized signalling: spatial regulation of cAMP by the action of compartmentalized phosphodiesterases. Febs J 276 1790 1799

96. HouslayMD

AdamsDR

2003 PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization. Biochem J 370 1 18

97. ShakurY

WilsonM

PooleyL

LobbanM

GriffithsSL

1995 Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum. Biochem J 306 (Pt 3) 801 809

98. BaillieGS

ScottJD

HouslayMD

2005 Compartmentalisation of phosphodiesterases and protein kinase A: opposites attract. FEBS Lett 579 3264 3270

99. WilsonD

Tutulan-CunitaA

JungW

HauserNC

HernandezR

2007 Deletion of the high-affinity cAMP phosphodiesterase encoded by PDE2 affects stress responses and virulence in Candida albicans. Mol Microbiol 65 841 856

100. LondesboroughJ

JonkkariL

1982 Low Km cyclic AMP phosphodiesterase of yeast may be bound to ribosomes associated with the nucleus. Mol Cell Biochem 46 65 71

101. HustonE

LynchMJ

MohamedA

CollinsDM

HillEV

2008 EPAC and PKA allow cAMP dual control over DNA-PK nuclear translocation. Proc Natl Acad Sci U S A 105 12791 12796

102. SoundararajanS

JeddG

LiX

Ramos-PamplonaM

ChuaNH

2004 Woronin body function in Magnaporthe grisea is essential for efficient pathogenesis and for survival during nitrogen starvation stress. Plant Cell 16 1564 1574

103. NaqviNI

BonmanJM

MackillDJ

NelsonRJ

ChattooBB

1995 Identification of RAPD markers linked to a major gene for blast resistance in rice. Molecular Breeding 1 341 348

104. VogelJ

SomervilleS

2000 Isolation and characterization of powdery mildew-resistant Arabidopsis mutants. Proc Natl Acad Sci U S A 97 1897 1902

105. Ramos-PamplonaM

NaqviNI

2006 Host invasion during rice-blast disease requires carnitine-dependent transport of peroxisomal acetyl-CoA. Mol Microbiol 61 61 75

106. HarrisSD

MorrellJL

HamerJE

1994 Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis. Genetics 136 517 532

107. SambrookJ

FritschEF

ManiatisT

1989 Molecular Cloning:A laboratory Manual. Cold Spring Harbor, NY Cold Spring Laboratory Press

108. AltschulSF

MaddenTL

SchafferAA

ZhangJ

ZhangZ

1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25 3389 3402

109. ThompsonJD

HigginsDG

GibsonTJ

1994 CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22 4673 4680

110. LivakKJ

SchmittgenTD

2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 402 408