Molecular and cellular biology of resistance to Phytophthora infestans in Solanum species

Research output: Thesisinternal PhD, WU

Abstract

<p>Ever since the late blight epidemics of the mid-nineteenth century, man has endeavored to protect his potato crop. Every year, extensive chemical protection is applied, which is expensive and may be harmful to the natural environment. The use of resistant potato cultivars would provide an elegant alternative, however breeding for late blight resistance has not yet resulted in adequately resistant cultivars. In addition to the demand for a high level of resistance, the resistance should be durable. The causal agent of the late blight disease is <em>Phytophthora infestans</em> , a biotrophic oomycete pathogen. Oomycetes are often incorrectly referred to as fungi, but they evolved the ability to infect plants independently from fungi, and may therefore have distinct mechanisms for interacting with plants. Insight in the mechanisms of resistance to <em>P. infestans</em> may assist the breeders in their operation to achieve durable resistance.</p><p>Genetic resistance can be determined at the subspecies or variety level (race-specific resistance), or at the species or genus level (nonhost resistance). Nonhost resistance is full resistance, and is present in most plant species. In addition, resistance may be a quantitative trait (partial resistance). A rich pool of diverse resistances to <em>P. infestans</em> has been found in wild <em>Solanum</em> species, with levels ranging from full resistance to various levels of partial resistance. Some old potato cultivars also exhibit partial resistance, which proved to be durable. To achieve a durable late blight resistance, a better understanding of the molecular basis of the various types of resistances is essential. To this end, we compiled a set of <em>Solanum</em> species with various types and levels of resistance to <em>P. infestans</em> , and studied the cellular and molecular aspects of the resistance mechanisms present in these plants.</p><p>Laboratory studies at the cellular and molecular level require an experimental assay, which is both comparable to the natural situation, and assures a high percentage of successful infections. To this end, we developed a resistance assay with detached leaves under controlled conditions in the laboratory and compared this assay with a field trial. The tested growing conditions of the plants did not affect the resistance to <em>P. infestans.</em> Leaves on intact plants however, were more resistant than detached leaves. The incubation conditions of the detached leaves in the laboratory assay rather than the detachment itself appeared to affect the resistance expression. However, on intact plants the infection frequency was too low for molecular studies. Since the ranking of resistance levels within a set of twenty plant clones was similar under laboratory and field conditions, the laboratory assay proved adequate to study the <em>Solanum</em> - <em>P. infestans</em> interaction.</p><p>A cytological survey of the interaction between twenty <em>Solanum</em> clones and three <em>P. infestans</em> isolates provided the first impression of the nature of the resistance responses. Potato cultivars with race-specific resistance ( <em>R</em> ) genes displayed the hypersensitive response (HR), a programmed cell death of plant cells, upon inoculation with <em>P. infestans</em> . Through this rapid cell death, the biotrophic pathogen became localized between dead cells, and was prevented from further growth. Also durably resistant potato cultivars without known <em>R</em> genes, wild <em>Solanum</em> species, and nonhosts displayed the HR. Interestingly, in highly resistant <em>Solanum</em> species such as <em>S. berthaultii</em> and <em>S circaeifolium,</em> and nonhosts such as <em>Arabidopsis thaliana</em> and <em>S. nigrum</em> (black nightshade), the HR was extremely fast and effective, resulting in very localized cell death. In partially resistant plants, the HR was delayed, and resulted in larger HR lesions. Occasionally, hyphae were able to escape from these lesions and established a biotrophic interaction with the host. The effectiveness of the HR in restricting growth of the pathogen differed considerably between clones, and correlated with resistance levels. In addition to the HR, local depositions of callose and phenolic compounds occurred, which may function as physical barriers. Although these responses did not correlate with resistance levels, they may influence the balance between growth of the pathogen and induction of the HR. Ultimately, this fine balance may determine resistance at the cellular level, and illustrates the quantitative nature of the resistance to <em>P. infestans</em> at the plant or field level <em>.</em></p><p>The HR is initiated upon recognition of pathogen elicitors by plant cell receptors or <em>R</em> gene products as suggested by the gene-for-gene hypothesis. Several types of <em>R</em> genes are recognized in plants, including the nucleotide binding site leucine-rich repeat (NBS-LRR) type, and the Pto-like serine/threonine protein kinase type. The <em>Pto</em> gene was originally identified from wild relatives of tomato. We exploited our <em>Solanum</em> collection to identify <em>Pto</em> -like sequences, and studied evolutionary scenarios for <em>Pto</em> -like genes. Polymerase chain reaction (PCR) amplifications using primers based on conserved and variable regions of <em>Pto</em> yielded 32 intact <em>Pto</em> -like sequences from six <em>Solanum</em> species, and revealed an extensive <em>Pto</em> family. <em>Pto</em> -like transcripts were also detected in leaf tissue of all tested plants. The kinase consensus and autophosphorylation residues were highly conserved, in contrast to the kinase activation domain which is involved in ligand recognition in Pto. Phylogenetic analyses distinguished nine classes of <em>Pto</em> -like genes, and revealed that orthologues (homologues separated by a speciation process) were more similar than paralogues (homologues generated by a gene duplication event). This suggests that the <em>Pto</em> gene family evolved through a series of ancient gene duplication events prior to speciation in <em>Solanum</em> . The phylogenetic data are in line with recent results on the NBS-LRR class of <em>R</em> genes, and suggest that <em>Pto-</em> like genes are ancient, and highly diverse.</p><p>Various levels of nonspecific resistance were revealed in <em>Solanum</em> species after inoculation with five <em>P. infestans</em> isolates. In partially resistant plants where hyphal escape occurred, the lesions expanded often slower than in susceptible plants. Here, defense mechanisms other than HR are thought to operate; this might for example involve systemic acquired resistance (SAR). SAR can be induced by various signals, but also basal levels of SAR may vary between plants. When we monitored basal expression levels of SAR marker genes in healthy leaves <em>,</em> we found variation between the <em>Solanum</em> clones in constitutive mRNAs levels of the pathogenesis-related ( <em>PR</em> ) genes <em>PR-1</em> , <em>PR-2</em> , and <em>PR-5</em> . At the genus level, there was no correlation between basal <em>PR</em> mRNA levels and nonspecific resistance to <em>P. infestans</em> . In contrast, a positive correlation was found at the species level in <em>S. arnezii x hondelmannii</em> , <em>S. microdontum</em> , <em>S. sucrense</em> and <em>S. tuberosum</em> . In <em>S. tuberosum</em> cultivars, the levels of <em>PR</em> gene expression were the highest in resistant 'Robijn', intermediate in partially resistant 'Première', 'Estima' and 'Ehud', and the lowest in susceptible 'Bintje'. These results suggest that constitutive expression of <em>PR</em> genes may contribute to nonspecific resistance to <em>P. infestans</em> in <em>Solanum</em> . Therefore, <em>PR</em> mRNA levels could serve as molecular markers in potato breeding programs.</p><p>In conclusion, diverse resistance reactions to <em>P. infestans</em> operate at various levels in <em>Solanum</em> species, including specific and nonspecific mechanisms. The ubiquitous association of the HR in all types of resistance suggests that numerous <em>R</em> genes are present in <em>Solanum</em> against the oomycete <em>P. infestans</em> . A remaining challenge is the identification and transfer of these <em>R</em> genes into commercially grown potato cultivars.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • de Wit, P.J.G.M., Promotor
  • Govers, F., Promotor, External person
  • Colon, L.T., Promotor
Award date5 Jan 2001
Place of PublicationS.l.
Print ISBNs9789058083500
Publication statusPublished - 2001

Keywords

  • potatoes
  • solanum tuberosum
  • solanum
  • wild relatives
  • phytophthora infestans
  • plant pathogenic fungi
  • disease resistance
  • molecular biology

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