Disease cycle of potato late blight - MSpace at the University of ...

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CHAPTER 2: IDENTIFICATION AND CLONING OF DIFFERENTIALLY EXPRESSED GENES INVOLVED IN THE INTERACTION BETWEEN POTATO AND PHYTOPHTHORA INFESTANS USING A SUBTRACTIVE HYBRIDIZATION AND CDNA-AFLP COMBINATIONAL APPROACH (Henriquez, M.A. and Daayf F. 2010. Journal of Integrative Plant Biology 52: 453–467) 2.1. ABSTRACT Using a subtractive hybridization (SH)/cDNA-AFLP combinational approach, differentially expressed genes involved in the potato-Phytophthora infestans interaction were identified. These included genes potentially controlling pathogenesis or avr genes in P. infestans as well as those potentially involved in potato resistance or susceptibility to this pathogen. Forty-one differentially expressed transcript-derived fragments (TDFs), resulting from the interaction, were cloned and sequenced. Two TDFs, suggested as potential pathogenicity factors, have sequence similarity to N-succinyl diaminopimelate aminotransferase and a transcriptional regulator, TetR family gene. Two other TDFs, suggested as potential avr genes, have sequence similarity to an EST sequence from Avr4/Cf-4/Avr9/Cf-9 and a P. infestans avirulence-associated gene. The expression and origin of the genes were confirmed using Southern blots, Northern blots and qRT-PCR. Potential resistance gene DL81 was induced at 12 hpi in the moderately resistant cultivar, whereas it was down-regulated as early as 6 hpi in the susceptible cultivar. On the other hand, DL21 was induced at 6 hpi (3.38-fold) in response to the highly aggressive isolate (US8) and strongly up-regulated thereafter (25.13-fold at 120 hpi.), whereas it was only 25
slightly up-regulated in response to the weakly aggressive isolate US11 (3.82-fold at 96 hpi), suggesting its potential involvement as a susceptibility gene. 2.2. INTRODUCTION Potato late blight caused by the oomycete Phytophthora infestans (Mont.) de Bary (Fry and Goodwin 1997) results in economic losses of $3.25 billion per year in potato- growing areas worldwide (Pel et al. 2009). This disease can be initiated either directly with germinating sporangia or through zoospores, and affects leaves, stems, and tubers (Judelson and Blanco 2005). The re-emergence of late blight as an important disease in the US and Canada occurred in the 1980s and 1990s, respectively, due to changes in the population structure of P. infestans, with a shift from the pre-existing A1-mating type, metalaxyl-susceptible to new metalaxyl-insensitive genotypes, highly aggressive A1 and A2 genotypes (Daayf and Platt 2000). In Canada, the A1 genotype US-1 has been completely displaced by the A2 genotype US8 of P. infestans (Daayf et al. 2000; Daayf et al. 2001), which is more virulent and exhibits highly increased aggressiveness (Kato et al. 1997). Extensive literature is available about incompatible interactions between potato and P. infestans leading to plant resistance, whereas the molecular mechanisms of compatibility leading to disease have been investigated less (Restrepo et al. 2005). It has been suggested that potato susceptibility to P. infestans is a result of the suppression of potato resistance reactions by glucans released from P. infestans (Garas et al. 1979; Doke et al. 1980; Currier 1981; Andreu et al. 1998; Ozeretskovskaya et al. 2001). Also, P. 26
Page 1 and 2: Molecular characterization of potat
Page 3 and 4: ABSTRACT Henriquez Maria Antonia. P
Page 5 and 6: ACKNOWLEDGEMENT I would like to tha
Page 7 and 8: CHAPTER 2: IDENTIFICATION AND CLONI
Page 9 and 10: CHAPTER 4: PHYTOPHTHORA INFESTANS E
Page 11 and 12: LIST OF TABLES Table 2.1. Similarit
Page 13 and 14: Figure 4.1. Effect of EPA elicitor
Page 15 and 16: SH: subtractive hybridization TDFs:
Page 17 and 18: FORWARD This thesis has been writte
Page 19 and 20: the use of moderately resistant var
Page 21 and 22: subtropical and temperate environme
Page 23 and 24: FALL Disease Disease cycle cycle of
Page 25 and 26: plant intercellular space (apoplast
Page 27 and 28: signaling cascades, alteration in g
Page 29 and 30: The recent research target in late
Page 31 and 32: (lignin), UV protection (quercetin,
Page 33 and 34: 1.2.5.4.2 Terpenoids pathways Terpe
Page 35 and 36: Isopentenyl diphosphate (IPP) is th
Page 37 and 38: hemiterpenes C5 (1 isoprene unit),
Page 39 and 40: (Tian et al. 2005; Tian et al. 2007
Page 41: Chapter 4: Phytophthora infestans e
Page 45 and 46: The objective of the current study
Page 47 and 48: days growth in Pea Broth Medium at
Page 49 and 50: 2.3.5. SH- Second Strand synthesis
Page 51 and 52: Second Strand for digestion. Howeve
Page 53 and 54: CAGCTACTTGGGAGGCTGAG-3’ and DL81-
Page 55 and 56: 2.4. RESULTS 2.4.1. Inoculation res
Page 57 and 58: Figure 2.1. Schematic representatio
Page 59 and 60: Figure 2.2. Example of TDFs in a po
Page 61 and 62: espectively. Transcripts DL32 and D
Page 63 and 64: Table 2.1. Similarities between seq
Page 65 and 66: US8 and US11, respectively. qRT-PCR
Page 67 and 68: and Schmittgen 2001) was used to ca
Page 69 and 70: Only in RB+US8, the DL21 precursor
Page 71 and 72: Constitutive gene expression has be
Page 73 and 74: iosynthesis via the diamino-pimelat
Page 75 and 76: Further validation of the subtracti
Page 77 and 78: CHAPTER 3: CLONING AND CHARACTERIZA
Page 79 and 80: Until recently, it was considered t
Page 81 and 82: 3.3.2. Cloning of DXS cDNA Using th
Page 83 and 84: 3.3.4. Bioinformatic analyses Deduc
Page 85 and 86: Table 3.1. Primers used for the syn
Page 87 and 88: StDXS1 has been submitted to the Ge
Page 89 and 90: Figure 3.2. Alignment of deduced am
Page 91 and 92: Figure 3.3. Phylogenetic tree of DX
Page 93 and 94:
StDXS1 transcripts in RB+US8 were i
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Fold induction 7 6 5 4 3 2 1 1 0 2
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tentative consensus-TC of Solanum t
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Hevea brasiliensis (HbDXS) two DXS
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This work opens up new perspectives
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4.2 INTRODUCTION Plant pathogens ha
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catalyze the first steps in the bra
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type A2) (kindly provided by Dr. Ad
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synthesis pool from each treatment,
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using a Stratagene Mx3005p cycler,
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with the glucan fraction and then i
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4.4.2. Analysis of variance Data co
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4.4.3 Gene expression analysis in t
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Relative Expression 1.6 1.4 1.2 1 0
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strains D-03 (lineage US11, A1 mati
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Table 4.6. RT-PCR analysis of the r
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There was a down-regulation of StDX
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Figure 4.6. Transcript profiles of
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cultivar Defender (DF+GL+US8). Expl
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(Ac-MVA) pathway, participate in es
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4.5.2.2 Russet Burbank defense resp
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Russet Burbank. With the exception
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CHAPTER 5: ALTERED METABOLIC PROFIL
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pathogen interactions, the cell wal
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at 0, 12, 72, and 120 hours post in
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5.4 RESULTS 5.4.1 Disease developme
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Finally, an early stage of the inte
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Figure 5.3. Chromatograpic profiles
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µg Catechin /g FW µg Catechin /g
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levels of flavonoid (P3) in RB+US11
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Terpenoid (T1) was detected in Russ
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control plant, would be associated
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Flavonoids possess a wide range of
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explanation of such a specific supp
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diaminopimelate aminotransferase wh
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the DOXP-MEP pathway, we detected c
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• Sesquiterpene cyclase (SC) gene
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good disease control strategy. For
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REFERENCES Abramovitch R, Kim Y, Ch
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Chappell J (1995) Biochemistry and
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Daayf F, Platt HW, Peters RD (2000)
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Enyedi AJ, Yalpani N, Silverman P,
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Haas BJ, Kamoun S, Zody MC, Jiang R
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Kamoun S (2005) A catalogue of the
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McGarvey DJ, Croteau R (1995) Terpe
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Pieterse C, de Wit P, Govers F (199
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Sels J, Mathys J, De Coninck BMA, C
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van Loon LC, Rep M, Pieterse CMJ (2
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Yao KN, Deluca V, Brisson N (1995)
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CGCGGCGCGGCGGCCGGGTCATTTGCCCAGCTTTC
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Sequence GGCATGAAATGCATCGGGTGGAGTAA
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TCCCTTGGGCATGGTCCCCTCAGTTATACTTCGTG
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Sequence CTGAGGCGAGTGGCAGAAACTAATAC
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EST#: DL127 Sequence CCTGGGTGGGGCCG
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TCAGTCGCTATTTTAGGTGGCGATGGTAGCGTACT
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MALCAYAFPGILNRTAVVSDSSKTAPLFSEWIHGT
Page 205 and 206:
Appendix 3 (Chapter 3) Defender Eco
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