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

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THE UNIVERISTY OF MANITOBA FACULTY OF GRADUATE STUDIES ************ Molecular mechanisms of potato defense suppression by Phytophthora infestans This thesis submitted to the Faculty of Graduate Studies of the University of Manitoba in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Science. Maria Antonia Henriquez © 2010 Permission has been granted to the library of the University of Manitoba to lend or sell copies of this thesis. I agree that the Libraries of this University may make it freely available for inspection. This reproduction or copy of this thesis has been made available by authority of the copyright owner for the purpose of private study and research, and may only be reproduced and copied as permitted by copyright laws or with express written authorization from the copyright owner. II
ABSTRACT Henriquez Maria Antonia. Ph.D., The University of Manitoba, September, 2010. Molecular mechanisms of potato defense suppression by Phytophthora infestans Supervisor: Dr. Fouad Daayf. Manitoba is the second largest potato producer in Canada after Prince Edward Island. This crop faces many disease problems including the most famous and devastating one, late blight, caused by the oomycete pathogen Phytophthora infestans. More than 150 years have elapsed since this disease caused the Irish potato famine, but strategies for managing it often remain unsustainable and costly. With the advent of molecular biology, genetic engineering, and now, genomics-based approaches, it may be possible to initiate studies which will address important questions regarding very complex host-pathogen interactions. Dr. Daayf’s lab has shown that the most aggressive strains of P. infestans suppress potato defense mechanisms, through transcriptional inhibition of PAL and HMGR pathways. Therefore, the objective of the current study was to carry out a molecular characterization of potato defense suppression by Phytophthora infestans. Gene expression profiling was accomplished by developing a new subtractive hybridization (SH)/cDNA-AFLP combinational approach. This approach is a gel-based subtractive hybridization profiling technique that uses the advantages of cDNA-AFLP and subtractive hybridization in order to amplify cDNA products in a polyacrylamide gel and remove the constitutively/commonly expressed sequences. Using this 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 III
Page 1: Molecular characterization of potat
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 and 42: Chapter 4: Phytophthora infestans e
Page 43 and 44: slightly up-regulated in response t
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
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CAGCTACTTGGGAGGCTGAG-3’ and DL81-
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2.4. RESULTS 2.4.1. Inoculation res
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Figure 2.1. Schematic representatio
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Figure 2.2. Example of TDFs in a po
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espectively. Transcripts DL32 and D
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Table 2.1. Similarities between seq
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US8 and US11, respectively. qRT-PCR
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and Schmittgen 2001) was used to ca
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Only in RB+US8, the DL21 precursor
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Constitutive gene expression has be
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iosynthesis via the diamino-pimelat
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Further validation of the subtracti
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CHAPTER 3: CLONING AND CHARACTERIZA
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Until recently, it was considered t
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3.3.2. Cloning of DXS cDNA Using th
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3.3.4. Bioinformatic analyses Deduc
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Table 3.1. Primers used for the syn
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StDXS1 has been submitted to the Ge
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Figure 3.2. Alignment of deduced am
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Figure 3.3. Phylogenetic tree of DX
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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,
Page 177 and 178:
Haas BJ, Kamoun S, Zody MC, Jiang R
Page 179 and 180:
Kamoun S (2005) A catalogue of the
Page 181 and 182:
McGarvey DJ, Croteau R (1995) Terpe
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Pieterse C, de Wit P, Govers F (199
Page 185 and 186:
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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