Regulation of the avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum

S.S. Snoeijers

Research output: Thesisinternal PhD, WU

Abstract

<p>During growth of a pathogen in host tissue, pathogenicity genes are usually highly expressed. A detailed understanding of how these pathogenicity genes are regulated is required to gain a better insight in the molecular communication between pathogen and host. <strong>Chapter one</strong> describes several bacterial and fungal genes, which are envisaged to be involved in pathogenicity and are induced <em>in vitro</em> during growth under nutrient-limiting conditions. Based on the data described in this chapter, <strong></strong> we speculate that in plants, pathogens encounter an environment in which nutrients are limiting. Lack of nitrogen might be one of the key factors that induce these pathogenicity genes.</p><p>The interaction between the fungus <em>Cladosporium fulvum</em> and its only host, tomato, is used as a model system to study plant-pathogen interactions. This interaction is a typical gene-for-gene relationship, that states that for each avirulence ( <em>Avr</em> ) gene in the pathogen there is a corresponding resistance ( <em>R</em> ) gene in the plant. Direct or indirect interaction between the products of <em>Avr</em> and <em>R</em> genes leads to incompatibility.</p><p>The object of the research performed in this thesis was to obtain a better understanding of the factor(s) involved in regulation of the <em>C. fulvum</em> avirulence gene <em>Avr</em> 9, which is highly expressed <em>in planta</em> during colonisation of the intercellular spaces of tomato leaves. The product of this gene is specifically recognised by tomato plants carrying matching resistance gene <em>Cf-9</em> . After recognition, the plant mounts a hypersensitive response (HR) that eventually leads to resistance against the fungus.</p><p>Before the study was initiated it was known that the <em>Avr</em> 9 gene is induced under conditions of nitrogen starvation <em>in vitro</em> . Furthermore, several (TA)GATA sequences were found to be present in the <em>Avr</em> 9 promoter. These sequences had earlier been identified as the binding sites for a wide-domain GATA-type regulator (AREA in <em>Aspergillus nidulans</em> and NIT2 in <em>Neurospora crassa</em> ), involved in nitrogen utilisation. Both observations made it likely to hypothesise that a similar regulator would be involved in induction of <em>Avr</em> 9 expression in <em>C. fulvum</em> and that nitrogen-limitation in the apoplast is the environmental factor that induces <em>Avr</em> 9 expression <em>in planta</em> .</p><p><strong>Chapter two</strong> describes the <em>Avr</em> 9 promoter activity in <em>A. nidulans</em> transformants, containing a single copy of an <em>Avr</em> 9 promoter- <em>uid</em> A (GUS) reporter gene fusion in different <em>are</em> A backgrounds ( <em>are</em> A wild-type <em>, are</em> A minus, <em>are</em> A constitutive), targeted at the <em>arg</em> B locus, following nitrogen starvation. Induction of the <em>Avr</em> 9 promoter was found to be similarly regulated in <em>A. nidulans</em> and <em>C. fulvum</em> , indicating that the AREA protein of <em>A. nidulans</em> is able to induce the <em>Avr</em> 9 promoter and that <em>C. fulvum</em> contains an AREA-like regulator that can bind to (TA)GATA sequences.</p><p><strong>Chapter three</strong> describes a mutational analysis of these (TA)GATA sequences which reveals that two TAGATA-boxes, located most proximal to the start codon, both containing two invertedly orientated TAGATA sequences, are crucial for inducibility of <em>Avr</em> 9 promoter activity in <em>A. nidulans</em> .</p><p>Mutated <em>Avr</em> 9 promoter fragments which did not show any inducibility in <em>A. nidulans</em> were fused to the <em>Avr</em> 9 coding region and introduced (not targeted) into strains of <em>C. fulvum</em> lacking <em>Avr</em> 9. However, in <em>C. fulvum</em> transformants the <em>Avr</em> 9 gene was induced when they were grown in rich, liquid media, a condition which normally suppresses <em>Avr</em> 9 gene expression. We have no Southern data on the transformants but it could be that multiple integrations have caused the loss of nitrogen-dependent <em>Avr</em> 9 regulation both <em>in vitro</em> and <em>in planta</em> . This result emphasises that for reliable promoter studies in <em>C. fulvum</em> a gene-targeting system is required.</p><p>The development of such a system for <em>C</em> . <em>fulvum</em> is described in <strong>Chapter four</strong> . For this purpose, the <em>C. fulvum</em><em>pyr</em> 1 gene was isolated. The <em>pyr</em> 1 gene codes for the enzyme orotidine-5'-monophosphate decarboxylase, which is involved in the pyrimidine biosynthetic pathway and is considered to be a versatile selection marker for filamentous fungi. The isolation of the <em>C. fulvum</em><em>pyr</em> 1 gene was based on complementation of an <em>A. nidulans</em><em>pyr</em> G-minus mutant strain which was simultaneously transformed with digested genomic DNA of <em>C. fulvum</em> containing the wild-type <em>pyr</em> 1 gene and an autonomously-replicating plasmid.</p><p><em>C. fulvum</em><em>pyr</em> 1 <sup>+</sup> transformants were obtained by introducing a vector, containing the <em>C. fulvum</em><em>pyr</em> 1 gene with a defined mutation, into a <em>C. fulvum pyr</em> 1-mutant strain. Southern blot analysis of these transformants showed that site-directed integration of this vector at the <em>pyr</em> 1 locus had occurred. Thus, targeting of constructs of interest to the <em>pyr</em> 1 locus of <em>C. fulvum</em> is feasible.</p><p>Isolation of the <em>are</em> A/ <em>nit</em> -2 homologue of <em>C. fulvum</em> , designated <em>Nrf</em> 1 (for <u>n</u> itrogen <u>r</u> esponse <u>f</u> actor 1), is described in <strong>Chapter five</strong> . The gene encodes a protein which contains a putative zinc finger DNA-binding domain that is 98% identical to the zinc finger domain present in the AREA and NIT2 proteins. Function equivalence of <em>Nrf</em> 1 to <em>are</em> A was demonstrated by complementation of an <em>A. nidulans</em><em>are</em> A-minus mutant with <em>Nrf</em> 1. Expression analysis in liquid media revealed that, in contrast to what occurs in wild-type <em>C. fulvum</em> strains, in <em>Nrf</em> 1-deficient strains the <em>Avr</em> 9 gene is not induced under conditions of nitrogen starvation. However, <em>Nrf</em> 1-deficient strains were still avirulent on tomato plants containing the <em>Cf-9</em> resistance gene, indicating that <em>in planta</em> still sufficient quantities of the AVR9 elicitor are produced. It appears that, although NRF1 is a major regulator of the <em>Avr</em> 9 gene expression, <em>in planta</em> at least one additional positive regulator of <em>Avr</em> 9 gene expression is active.</p><p>In <strong>Chapter six</strong> we studied the effect of elevated nitrogen levels on expression of <em>Avr</em> 9 in <em>C. fulvum</em> grown <em>in planta</em> . We observed <strong></strong> that tomato plants containing both the <em>Cf-9</em> gene and elevated levels of nitrate in the apoplast show partial resistance against strains of <em>C. fulvum</em> containing the <em>Avr</em> 9 gene. This implies that the elevated level of nitrate in the apoplast represses <em>Avr</em> 9 expression.</p><p>In <strong>Chapter seven</strong> the data obtained in this research project are discussed in relation to other known avirulence genes. It is still unknown why, in their host, pathogens would produce proteins that betray them. A possible role for the AVR9 elicitor as a kind of "survival protein" for the fungus during infection is discussed. Although, it appears that regulation of the <em>Avr</em> 9 gene is associated with nitrogen circuits in <em>C. fulvum</em> , regulation of <em>Avr</em> 9 by NRF1 <em>in vitro</em> and <em>in planta</em> is not similar. The isolation of additional plant factor(s) which are able to induce <em>Avr</em> 9 is a challenge for future research.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • de Wit, P.J.G.M., Promotor
  • Goosen, T., Promotor, External person
  • Joosten, Matthieu, Promotor
Award date10 Oct 2000
Place of PublicationS.l.
Print ISBNs9789058082879
Publication statusPublished - 2000

Keywords

  • tomatoes
  • solanum lycopersicum
  • passalora fulva
  • plant pathogenic fungi
  • gene expression
  • virulence
  • disease resistance

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