Structure and function of the (a)virulence protein NIP1 of Rhynchosporium secalis

K.A.E. van 't Slot

Research output: Thesisexternal PhD, WU


<font size="3"><em><p>Rhynchosporium secalis</em> is the causal agent of leaf scald on barley, rye and severa l other grasses. The fungus belongs to the class of <em>Deuteromycetes</em> , also referred to as <em>Fungi imperfecti</em> , indicating that a sexual stage has not been observed in nature. Germinating fungal spores are capable of penetrating the cuticle. Then the fungal hypha ramifies as mycelium beneath the cuticle causing the collapse of epidermal cells. In a later stage of the infection, mesophyll cells start to collapse and disease symptoms become visible. Only at this stage, a few fungal hyphae can be detected between dead mesophyll cells. In resistant barley cultivars the infection is halted after the collapse of a few epidermal cells. This is caused by the onset of a defense mechanism by the plant, which involves the massive production of pathogenesis-related (PR) proteins. The response of the plant is triggered upon recognition of the fungus. It was established that <em>R. secalis</em> strains carrying the avirulence gene <em>AvrRrs1</em> , which codes for the NIP1 protein, are unable to infect barley plants carrying the resistance gene <em>Rrs1</em> . In this thesis, we describe the biochemical and biological properties of the NIP1 protein, its interaction with factors present on the plant plasma membrane and its tertiary structure.</p><p>NIP1 consists of 60 amino acid (a.a.) residues, including 10 cysteines, all of which are involved in intramolecular disulfide bonds. The a.a. sequence of NIP1 shows no similarity to sequences in the databases. By sequencing a limited set of NIP1 alleles from field isolates, four NIP1 isoforms were deduced displaying only a limited number of a.a. alterations. Two of these, types I and II, although differing in three amino acid positions, are able to induce PR protein biosynthesis in <em>Rrs1</em> barley as well as leaf necrosis independent of the resistance genotype. Types III and IV both differ from type II in the single a.a. alterations G45R and S23P, respectively. We engineered type I NIP1 protein into type III and IV-like proteins (types III* and IV*) by exchanging the activity-blocking type III and IV amino acids.</p><p>Application of NIP1 to <em>Rrs1</em> -plants is sufficient to elicit the production of PR proteins in a manner comparable to amounts induced after infection by the fungus. In addition to a function as elicitor, NIP1 is able to cause necrosis on barley plants independent of their resistance genotypes, and a role for NIP1 in virulence was therefore proposed. For several virulence factors involved in other plant-pathogen interactions an additional role as elicitor of defense responses has been shown. In viruses, all proteins encoded by the few genes in the genome are expected to be important for viral replication and multiplication. Several plants have developed defense responses that are initiated upon recognition of specific viral gene products. Bacteria, fungi, oomycetes and nematodes have specialized genes for pathogenicity and virulence, some of which can induce resistance in plants as they recognize inflicted activities of the intruding pathogen. The number of pathogen factors for which dual functions in virulence and avirulence is described is accumulating rapidly. The emerging picture is that plant recognition systems targeted against important pathogen virulence factors may possess common characteristics. This opens up multiple possibilities to utilize natural mechanisms in engineering more durable resistance against a larger number of pathogens.</p><p>A heterologous expression system was set up in <em>E. coli</em> , allowing the production of milligram quantities of NIP1. In this expression system, NIP1 is expressed as a fusion protein, where a histidine tag is fused to the N-terminus of NIP1. In order to obtain NIP1 containing the correct disulfide bond pattern, an unfolding / refolding procedure, involving the use of a cysteine / cystine redox couple was applied. The protease Xa cleavage site between the histidine tag and NIP1 allowed removal of the His-tag. The NIP1 protein produced by this heterologous expression procedure has elicitor activity similar to the native NIP1 protein purified from <em>R. secalis</em> . In addition, the expression system allowed the expression and purification of mutated NIP1 proteins.</p><p>To identify putative NIP1 binding sites, NIP1 was labeled with radioactive iodine. Binding studies using <sup>125</SUP>I-NIP1 and barley membrane fractions revealed the presence of a single class of high-affinity NIP1 binding sites on barley plasma membranes. Binding of NIP1 to membrane fractions was specific, reversible, and saturable. The equilibrium dissociation constant, <em>K</em><sub>d</sub> , was determined at 5.6 nM and the concentration of binding sites was calculated to be 255 fmol per mg of microsomal membrane protein. The binding site was detected in both resistant ( <em>Rrs1</em> ) and susceptible ( <em>rrs1</em> ) barley plants, suggesting that the <em>Rrs1</em> gene does not encode the NIP1 binding site. In addition, a binding site with very similar affinity for NIP1 was detected in microsomes from the related plant species wheat, rye, oats and maize, but not from <em>Arabidopsis thaliana</em> . These results correlate with the toxic effect of NIP1 on leaves of the cereal plant species , while no toxic effect was observed in leaves of <em>Arabidopsis thaliana</em> . Two NIP1 isoforms type III* and type IV* both competed successfully for the binding site, although they are not active as elicitors of PR5 biosynthesis. We conclude from these data that these mutant proteins bind efficiently to the binding site, but are unable to activate downstream signaling. In contrast, NIP1 type II is characterized by a drastically increased <em>K</em><sub>d</sub> value. An only slight decrease in elicitor activity and H <sup>+</SUP>-ATPase-stimulating activity of this isoform indicates that binding of NIP1 is not the limiting step in the signal transduction pathway.</p></font><p>The three-dimensional structure of NIP1 was determined by NMR spectroscopy. The disulfide bond pattern of NIP1 was determined with biochemical techniques, based on partial reduction of individual disulfide bridges and subsequent analysis of the released fragments after cleavage by mass spectroscopy. The structure of NIP1 can be regarded as a protein consisting of two motifs. The orientation of these motifs with respect to each other is well defined due to the stabilizing effect of the disulfide bond connecting the two motifs. The N-terminal motif contains an anti-parallel<FONT FACE="Symbol">b</font>-sheet comprised of 2<FONT FACE="Symbol">b</font>-strands, whereas the C-terminal domain consists of three antiparallel<FONT FACE="Symbol">b</font>strands and a relatively large flexible region. NIP1 has a novel fold; no proteins with homologous structures were described so far. The activity-blocking mutations S23P and G45R both have a high impact on the structure.</p><font size="3"><p>The perception of NIP1 by barley plants may comply with the guard hypothesis, which proposes a third component to be required for perception of an elicitor, and that this additional component is the virulence target of the AVR protein. In the presence of the R <em></em> protein, binding of the corresponding elicitor to the virulence target, will result in the onset of defense responses, whereas in susceptible plants binding will result in transmission of its virulence function
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • de Wit, P.J.G.M., Promotor
  • Knogge, W., Promotor, External person
Award date15 Apr 2002
Place of PublicationS.l.
Print ISBNs9789058086259
Publication statusPublished - 2002


  • barley
  • hordeum vulgare
  • rhynchosporium secalis
  • plant pathogenic fungi
  • host parasite relationships
  • disease resistance
  • pathogenesis-related proteins
  • virulence

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