Identification of potato genes involved in Phytophthora infestans resistance by transposon mutagenesis

L.J.G. van Enckevort

Research output: Thesisinternal PhD, WU

Abstract

<p>The late blight disease, caused by the oomycete <em>Phytophthora infestans</em> (Mont.) de Bary, is a serious threat to the potato crop every growing season. This has, for example, led to the disastrous Irish famine in the middle of the 19 <sup>th</sup> century, and continued in the 20 <sup>th</sup> century to remain a serious problem for potato growers. Since the early 1980s <em>P. infestans</em> populations changed more rapidly and epidemics even increased in their severity. Resistance breeding stimulated the introduction of resistance genes ( <em>R</em> genes) from wild <em>Solanum</em> species into cultivated potato, <em>Solanum tuberosum</em> , but newly occurring virulent races of <em>P. infestans</em> circumvented these <em>R</em> gene mediated resistances and no cultivars with durable resistance were obtained. At the moment, methods using fungicides supervised by spraying control via decision support systems are the only available control measures.</p><p>Characteristic for <em>R</em> gene type mediated resistance reactions is the hypersensitive response (HR) leading to local cell death causing necrotic spots at the site of attempted infection. Genetic analysis of HR mediated resistances showed that activation of HR is highly specific and induced upon recognition between a specific <em>R</em> gene in the plant and a corresponding avirulence gene ( <em>Avr</em> gene) in the pathogen. Insights in the molecular mechanisms underlying this HR resistance reaction in <em>Solanum</em> species might facilitate the development of potato cultivars that are more durable in maintaining a resistance phenotype.</p><p>A two component <em>Ac-Ds</em> transposon tagging strategy in diploidised potato was developed to identify and isolate genes involved in the <em>R1</em> gene mediated resistance response to <em>P. infestans</em> . Transposable elements are molecular genetic tools to mutate and identify genes. The transposable elements <em>Ac</em> and <em>Ds</em> were first characterised in maize and their molecular isolation led to the identification of maize genes that were tagged by these elements. The autonomous <em>Ac</em> element is able to transpose by itself and also to induce transposition of the non-autonomous <em>Ds</em> element that is transposase defective. Introduction of these elements in heterologous species demonstrated their utility for isolating genes in self-fertilising plant species. Also in the highly heterozygous and tetraploid potato, the <em>Ac</em> and <em>Ds</em> transposable elements were shown to be functional. A cell autonomous visual marker gene for potato, the granule bound starch synthase gene ( <em>GBSS</em> gene), enabled a refined characterisation of <em>Ac</em> transposition in potato. Further molecular characterisation showed high levels of <em>Ac-Ds</em> transposition both somatically and germinally, so that suitable populations could be generated for tagging purposes.</p><p>The production of clones homozygous for the gene of interest that are normally required for efficient tagging strategies, turned out to be time consuming in potato due to self-incompatibility at the diploid level. Therefore, an alternative method based on somatic transposition was developed for the direct selection of transposition events instead of recovering germinally transmitted transpositions. Highly chimaeric <em>Ac-Ds</em> seedlings with active <em>Ds</em> transposition linked to the <em>R1</em> resistance gene on chromosome 5 of potato were selected. Protoplasts were isolated from actively transposing seedlings and using the hygromycin excision selection marker, regenerants could be selected with new independent <em>Ds</em> insertions. The resulting <em>R1</em> resistant transposon mutagenised population of almost 2000 hygromycin resistant regenerants formed an ideal start for the identification of an <em>R1</em> tagged mutant, or other <em>Ds</em> insertional mutants with an altered <em>R1</em> resistance response.</p><p>The somatically regenerated tagging population was analysed for the <em>P. infestans</em><em>R1</em> type HR resistance response, using a detached leaf assay for <em>P.</em><em>infestans</em> inoculation. In a primary screening, 33 potential <em>R1</em> resistance variants showing partial susceptibility to <em>P. infestans</em> race 0 were identified. These results were further quantified using stringent inoculation conditions on replicate samples leading to the identification of four putative mutants with a distinctly altered <em>R1</em> resistance response. In these putative mutants less than 50% of the inoculated leaves showed the <em>R1</em> type HR response and clear colonisation with sporulation of <em>P. infestans</em> was observed. The flanking sequences of the <em>Ds</em> insertion sites in these putative <em>R1</em> mutants were analysed and in two cases a potential biological correlation between the insertion sequences and the phenotype was evident. One putative mutant contained a <em>Ds</em> insertion in a region with auxin and abscisic acid response cis-elements homologous to a specific region (TAPIR) of the tomato defence related genes <em>TAPG2</em> and <em>TAP1</em> .</p><p>The second <em>P. infestans</em><em>R1</em> resistance mutant, mutant 1000 with a striking susceptible phenotype was characterised in more detail. Two <em>Ds</em> insertions were identified and the insertion site flanking sequences both showed high homology to serine/threonine protein kinases. The <em>Ds</em> insertion sites turned out to be homologous but not identical, indicating two independent <em>Ds</em> insertions in homologous but not identical genes. Both sequences showed protein identity to all the conserved regions of serine/threonine protein kinases and they contained a conserved intron position. The closest homology was to the serine/threonine protein kinase domain of the <em>Xanthomonas</em> resistance gene <em>Xa21</em> , which is involved in the induction of a HR resistance response in rice. This indicates that the isolated <em>Solanum tuberosum</em> protein kinase (StPK) homologs are candidate genes involved in resistance gene activity in potato. Further specific molecular analyses identified at least 11 homologs by sequence, which probably belong to a large family of serine/threonine protein kinases in potato. Both homologs in which the <em>Ds</em> transposons are inserted were present in susceptible parental potato clones. Therefore, it is unlikely that the isolated sequences represent the <em>R1</em> gene itself. The mutated StPKs were designated <em>rpr1</em> and <em>rpr2</em> , <u>r</u> equired for <em><u>P</u> hytophthora infestans</em><u>r</u> esistance gene 1 and 2. Studying these mutants and the StPKs involved might help in understanding the pathway leading to HR resistance in potato.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Jacobsen, Evert, Promotor
  • Pereira, A., Promotor, External person
Award date4 Dec 2000
Place of PublicationS.l.
Print ISBNs9789058083432
Publication statusPublished - 2000

Keywords

  • potatoes
  • solanum tuberosum
  • phytophthora infestans
  • plant pathogenic fungi
  • disease resistance
  • transposable elements
  • transposition
  • mutagenesis
  • gene tagging
  • selection
  • plant breeding

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