Identification and functional analysis of Botrytis cinerea genes induced during infection of tomato

Research output: Thesisinternal PhD, WU

Abstract

<font size="3"><STRONG><p>Keyword(s): <em>Botryotinia fuckeliana</em> , grey mould, pathogenesis, tomato, differential gene expression, glutathione S-transferase, aspartic protease, ubiquitin</p></STRONG><p> </p><p> </p><p>In the past decade many new methods have been developed that improved the unbiased search for differentially expressed genes. The fine-tuning towards high throughput analysis with increased sensitivity for genes that are expressed at a low level makes these new methods very powerful.</p><p>This thesis focuses on the isolation and characterisation of novel pathogenicity genes of <em>Botrytis cinerea.</em> They were isolated based on the assumption that such genes are preferentially expressed during interaction of the pathogen with its host plant. Tomato was chosen as a host plant. Differential Display RT-PCR (DDRT-PCR: chapter 2), differential hybridisation screening of a genomic library of <em>B. cinerea</em> (chapter 3) and hybridisation with subtractive RT-PCR products (chapter 4) have been performed to isolate novel genes that are potentially involved in pathogenesis. Some of these methods are, however, error-prone and relatively insensitive. We faced the difficulty of searching for <em>B. cinerea</em> genes that are induced upon infection of a host, at a timepoint where the majority of the isolated RNAs (&gt;95 %) is of plant origin. This implies that expression of fungal genes <em>per se</em> is difficult to detect. Small differences in expression levels of fungal genes often remain undetected and expression of tomato genes may blur interpretation of the screening. Although many successful studies have been reported showing differences in gene expression with a single organism grown in two different environments, the methods that we applied on two interacting organisms were not always successful.</p><p>At the start of this project, we developed a synchronised infection method and observed that the infection of tomato leaves by <em>B. cinerea</em> occurs in three phases (chapter 2; Benito <em>et al</em> ., 1998), namely: 1) In the first 18 h after <em>B. cinerea</em> conidia are sprayed on plant tissue, no clear visual response of the plant can be observed. 2) In the period between 18 h and 72 h, lesions are visible that do not expand. 3) After 72 h, lesions are spreading and at a later stage <em>B. cinerea</em> is infesting the whole tissue. With the use of the DDRT-PCR method (chapter 2) we performed an unbiased screening for induced genes involved in the infection process. It was necessary to adjust the method to our needs. Several gene fragments were cloned and analysed, but none of them displays any sequence homology to sequences present in the public databases. DDRT-PCR, however, is a very sensitive technique that yields fragments of novel, unidentified genes. The lack of sequence homology of these fragments requires isolation of the complete genes to reveal the gene products and subsequent mutational analysis to evaluate their role in pathogenesis. The emerging sequence information of whole genomes may circumvent the first step in the future.</p><p>In parallel we chose for a straightforward differential screening of a genomic library of <em>B. cinerea</em> (chapter 3) as was successfully performed by Pieterse <em>et al</em> . (1991) for <em>Phytophthora infestans</em> . The screening resulted in the identification of two genes encoding polyubiquitin and monoubiquitin, respectively. Ubiquitin is a protein predominantly involved in labelling misfolded or damaged proteins for degradation. Induction of ubiquitin gene expression in <em>B. cinerea</em> during infection of a host plant might suggest that the fungus experiences a stress situation, presumably caused by oxidative processes occurring in the lesion. This would result in malfunctioning of proteins and protein degradation would be imperative. This might explain the need for increased levels of ubiquitin. However, the monoubiquitin gene also encodes a carboxyl extension protein that is required for ribosome synthesis. The enhanced expression of the monoubiquitin gene may also reflect the need for increased ribosome synthesis.</p><p>A glutathione-S-transferase gene (GST) was identified by a hybridisation with subtractive RT-PCR products (chapter 4). Disruption of the <em>Bcgst</em> 1 gene did not result in decreased virulence. Expression of <em>Bcgst</em> 1 was induced by adding hydrogen peroxide to liquid cultures, suggesting that the (presumably cytosolic) enzyme may be involved in protection of <em>B. cinerea</em> against oxidative damage. Under the same conditions an extracellular catalase gene is also induced (Schouten, unpublished). This catalase probably serves as a first extracellular line of defence against hydrogen peroxide, while GST serves as intracellular backup for removing residual hydrogen peroxide that has not efficiently been removed by the catalase. Therefore, catalase could mask the action of GST as no phenotype is observed for a GST-deficient mutant.</p><p>As Movahedi and Heale (1990) claimed a role for aspartic protease in the infection process, we set out for a targeted approach to isolate an aspartic protease of <em>B. cinerea</em> ( <em>Bcap</em> 1: Chapter 5). <em>Bcap</em> 1 was isolated using degenerate primers for PCR. BcAP1 is not secreted and disruption of <em>Bcap</em> 1 as described in Chapter 5 does not have any detectable phenotype on the infection process. Other aspartic proteases must be responsible for the action described by Movahedi and Heale (1990). Recently, other aspartic protease gene sequences have been discovered (ten Have and van Kan, pers. comm.; Bitton <em>et al</em> ., 1999), suggesting a small gene family of aspartic proteases. These aspartic protease genes should be investigated (either apart or as double mutants) to determine the significance of their role during pathogenesis.</p><p>The results described in this thesis and recently published data extend our knowledge of the infection strategy of <em>B. cinerea</em> . Since <em>B. cinerea</em> is a necrotrophic fungus, its infection strategy must be substantially different from that of biotrophic fungi. The infection of host plants by <em>B. cinerea</em> and the related necrotroph <em>Sclerotinia sclerotiorum</em> is not necessarily subtle. <em>B. cinerea</em> is often referred to as an opportunistic pathogen that can grow on weakened or senescent tissue. Although <em>B. cinerea</em> has a broad host range (Jarvis, 1977), a vast number of mainly monocotyledonous species cannot be invaded. The reason for this is yet unclear. Possible explanations could be that some species do not react to <em>B. cinerea</em> with a necrotic response, thereby depriving the fungus of dead tissue serving as substrate. It is also conceivable that non-hosts produce toxic compounds, which cannot be degraded by <em>B. cinerea</em> .</p><p>Essentially, three basic features can contribute to the infection strategy of a necrotroph:</p><UL><p><LI>Initiation of infection by stimulating host cell death at the site of infection. This can be facilitated by an active process caused by the necrotroph (Movahedi and Heale, 1990; von Tiedemann, 1997; Liu <em>et al</em> ., 1998), a defence-related cell-death (hypersensitive response) of the host plant (Malolepsza and Urbanek, 2000) incited by the necrotroph, or a combination.</LI></p></UL><UL><p><LI>Coinciding with a hypersensitive response, the host produces active oxygen species (AOS) and other defence compounds. Therefore, the necrotroph needs sufficient protection against, and/or neutralisation of these AOS (Cessna <em>et al</em> ., 2000) and defence compounds (Quidde <em>et al</em> ., 1998). GST and ubiquitin may play a role in this period of consolidation; GST is presumably able to diminish the oxidative stress while ubiquitin can selectively remove defective proteins.</LI></p></UL><UL><p><LI>After this period of consolidation at the primary site of infection, <em>B. cinerea</em> can exploit its hydrolytic enzymes to degrade the host tissue in order to infest and sporulate (reviewed in Prins <em>et al</em> ., Chapter 1; ten Have, 2000 and references therein). These three steps are repeated to extend the area of necrotic tissue.</LI></p></UL></font>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • de Wit, P.J.G.M., Promotor
  • van Kan, Jan, Promotor
Award date17 Oct 2001
Place of PublicationS.l.
Print ISBNs9789058084682
Publication statusPublished - 2001

Keywords

  • tomatoes
  • solanum lycopersicum
  • plant pathogenic fungi
  • botrytis cinerea
  • pathogenesis
  • gene expression
  • glutathione transferase
  • aspartic proteinases
  • ubiquitin

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