BSPP Presidential Meeting 2000

Plant-pathogen Interactions:
Understanding Mechanisms of Resistance and Pathogenicity for Disease Control

Session VI: Targets for intervention and disease control

Strategies to control Fusarium ear blight on cereals
Kim Hammond-Kosack, Martin Urban, Wendy Phillips, Steve Daniels, Richard Kemp, Pierre Lecocq, Pat Ouimet1 and John Pitkin1
Cereal Technology Group, Monsanto UK Ltd, The Maris Centre, Trumpington, Cambridge CB2 2LQ, UK Tel 01223 - 849243 Fax 01223 844425 and 1`Monsanto SA , St. Louis, Missouri, USA.

Fusarium fungal diseases of wheat, maize and other cereals are on the increase in both Europe and North America and they pose a serious threat to both cereal yield and grain quality (1,2). There are at least 5 underlying causes:

  • Current fungicides are ineffective.
  • Only a few resistance breeding sources are recognised and in almost all lines the resistance trait is under oligogenic control.
  • Fusarium infected cereal grains become contaminated with a range of mycotoxins. In both the EU and USA mycotoxin levels are continuously monitored in both animal feed grains and foods. If mycotoxin levels in grain samples exceed those permitted grain cannot be sold or exported.
  • At least 4 different Fusarium species (F. graminearum, F. culmorum, F. poae and M. nivalis) attack wheat in Europe and this makes visual diagnosis difficult. The predominant problem species in the diverse geographical regions across Europe are still not known.
  • Fusarium graminearum (Fg) attacks both maize (corn) and wheat. Changing crop rotation practices in Europe, in combination with the reduction in straw stubble burning, now means that wheat invariably follows the maize crop and maize stubble can become very heavily colonised by Fg. The primary source of inoculum each year is ascospores released from infected stubble (3).

Targets for intervention and disease control : Both plant and pathogen targets are sought in an attempt to control Fusarium ear blight.

On the plant side two key questions will be addressed. Firstly, what is the mechanistic difference between so called " type 1 resistance" which prohibits Fusarium infection of ears and so called "type 2 resistance " which minimises hyphal spread within ear tissue once infection occurs. No race-specific resistance has been detected in the numerous wheat germplasms / Fusarium isolates interactions investigated. A second key question, is what is the relationship between resistance to Fusarium and the eventual mycotoxin levels found in the harvested grain.

On the pathogen side of the interaction we wish to determine which Fusarium genes are required to cause disease on cereal ears. In particular, are the same sorts of pathogenicity genes required by foliar attacking fungi also required by fungi that primarily cause disease on floral organs. A homologue of the Magnaporthe grisea PMK1 gene has been isolated from F. graminearum. The M. grisea PMK1 gene encoding for a MAP kinase is required to cause blast disease on rice leaves (4). A number of other research groups have shown that when a homologue of the PMK1 gene is knocked out in other foliar attacking fungal species, their is either a loss or severe reduction in disease causing ability.

References :

1. US Wheat and Barley Scab Initiative Web-site
2. McMullen, Jones, R. and Gallenberg, D (1997) Plant Disease 81: 1340-1348
Fernando, WGD, Paulitz, TC, Seaman, WL, Dutilleul, P and Miller, JD (1997) Phytopathology 87: 414-421
4. Xu, JR and Hamer JE ( 1996) Genes Dev 10: 2696-2706

Powdery mildew in perspective: development and differentiation, sensing and signalling
Sarah Jane Gurr
Department of Plant Sciences, University of Oxford, OX1 3RB, UK.

Erysiphe graminis f.sp. hordei (Egh) (syn. Blumeria graminis), the causal agent of barley powdery mildew, is an obligate biotroph. Arrival of the conidium on the host leaf triggers a precise developmental sequence culminating in appressorium maturation and attempted penetration. Firstly, a primary germ tube (PGT) emerges from the spore body, followed by second-formed germ tube, which elongates to become the appressorial germ tube (AGT). At maturity, the tip of the AGT distal to the conidium hooks and an appressorial peg penetrates the host. Such developmental precision may be due to multiple, plant-derived cues and to endogenous tactile and chemical signals (see also Carver, this meeting).

Signal transduction in Egh seems more complex than in other phytopathogenic fungi. For example, Egh lacks the simple cAMP-mediated trigger for differentiation seen in Magnaporthe grisea1. Instead, we have evidence that cAMP levels cycle during differentiation3 and that high levels of PKA activity are required to initiate AGT elongation but low PKA levels for hooking of the AGT tip1,2,3. Futhermore, the work indicates that cAMP generation alone is not sufficient to trigger the complete programme of germling differentiation in this obligate biotroph and that additional signal transduction pathways, such as via protein kinase C, PKC, or via a mitogen-activated protein kinase cascade (MAPK) may be required.

Recently, we cloned, characterised and transcript-profiled two Egh MAPK genes, mpk1 and mpk2 (Zhang & Gurr, unpubl.), alongside two pkc genes, pkc1 and pkc25,6, cpka12,5 and the cell wall genes chs1 and chs26,7. Various agonist / antagonist studies are underway but our challenge will be to assign a function to these genes by transformation4 and also to use them to study the interplay between different signal transduction pathways and their potential roles in the maintenance of cell integrity7. Such proposed interplay between pathways is not without precedent in phytopathogenic fungi.

1. Hall, A.A., Gurr, S.J. (2000). Initiation of appressorial germ tube differentiation and appressorial hooking: distinct morphological events regulated by cAMP signalling in Blumeria graminis f.sp hordei. Physiol. Mol. Plant Pathol.. 56 : (1) 39-46.
2. Hall, A.A., Bindslev, L., Rouster, J., Rasmussen, S.W., Oliver, R., Gurr, S.J. (1999). Involvement of cAMP and protein kinase A in conidial differentiation by Erysiphe graminis f. sp hordei. Mol. Plant Microbe Interact. 12 (11) 960-968.
3. Kinane, J., Dalvin, S., Bindslev, L., Hall, A.A., Gurr, S.J., Oliver, R. (2000). Evidence that the cAMP pathway controls emergence of both primary and appressorial germ tubes of barley powdery mildew. Mol. Plant Microbe Interact. 13 : (5) 494-502.
4. Chaure, P., Gurr, S.J. and Spanu, P. (2000) Stable transformation of the obligate biotrophic powdery mildew fungus Erysiphe graminis. Nature Biotechnology. 18 , 2 205-207
5. Zhang, Z., Gurr, S. J., (2000). The Erysiphe graminis protein kinase C genes pkc1, pkc2 and catalytic subunit of protein kinase A, cpka gene are differentially regulated during germling morphogenesis (submitted)
6. Zhang, Z., Gurr, S. J. (2000). Walking into the unknown: a 'step down' PCR-based technique leading to the direct sequence analysis of flanking genomic DNA. Gene 253 (2), 145-150.
7. Zhang, Z., Hall, A., Perfect, E., Gurr, S. J. (2000). Differential expression of two Blumeria graminis chitin synthase genes. Molecular Plant Pathology 1 (2), 125-138.

NIM1/NPR1 over-expression as a component of an integrated disease control strategy
Robert Dietrich, Leslie Friedrich, Laura Weislo, Mike Willits, and John Salmeron
Syngenta, 3054 Cornwallis Road, Research Triangle Park, NC 27709 USA

Salicylic acid (SA) and the product of the NIM1 gene (also known as NPR1) are essential components of the systemic acquired resistance (SAR) signal transduction pathway in Arabidopsis. SA levels increase in inoculated plants just prior to the induction of SAR. Endogenous SA is required for the biological induction of SAR, and exogenous treatment with SA results in enhanced resistance that has all the characteristics of SAR. Plants with mutations in the NIM1 gene are insensitive to SA, indicating that NIM1 acts downstream of SA in the pathway. Experiments were done to test the effect of increasing NIM1 protein levels in a plant. Over-expression of the NIM1 gene in Arabidopsis results in constitutive enhanced resistance in some, though not all, transgenic lines. This enhanced resistance appears to be due to increased sensitivity of the plants to SA or an SA dependent signal. Similarly, NIM1over-expressing plants are hyper-responsive to BTH, a synthetic chemical that induces SAR. SAR was induced in all NIM1 over-expressing lines following BTH treatment at concentrations ten-fold lower than required for induction of SAR in untransformed parental lines. In addition, increased fungicide efficacy is seen on plants with elevated NIM1 protein levels. These results suggest that an integrated approach combining NIM1 over-expression with low-level BTH and fungicide treatment could result in an effective and durable disease control strategy.

Signals and receptors in plant-pathogen interactions
Thorsten Nuernberger, Beatrix Blume, Frederic Brunner, Guido Fellbrich, Justin Lee, Annette Romanski, Jason Rudd, Dierk Scheel, Anne Varet, Birgit Kluesener1 & John Mansfield2
Institute of Plant Biochemistry Halle, Germany. Guy Cornelis, University of Louvain, Belgium. Sakari Kauppinen, NOVO Nordisk, Bagsvaerd, Denmark
University of Bochum, Germany
Imperial College at Wye, UK

Our research aims at analyzing the molecular basis for activation of resistance responses in non-host plants. A primary challenge to the innate immune response of plants is the discrimination of a large number of potential pathogens from self, with a restricted number of receptors. This challenge has been met by the evolution of a variety of receptors that recognize conserved motifs on pathogens. Such motifs are expected to have essential roles in the biology of the invading pathogens, and may therefore be evolutionarily conserved. We have identified a calcium-dependent cell wall transglutaminase (TGase, EC from Phytophthora sojae, which serves as a recognition determinant for the activation of non-host resistance in parsley. Transcripts encoding TGase were expressed and enzyme activity was detectable in 10 Phytophthora species analyzed. A surface-exposed fragment (Pep-13) of this protein, which was previously found to be sufficient for receptor-mediated activation of pathogen defense is fully conserved in all Phytophthora TGases. Mutational analysis within the Pep-13 sequence revealed amino acid residues indispensible for both transglutaminase activity and activation of plant defense. Thus, receptors recognizing conserved "epitope-like" motifs essential for the biological function of pathogen-derived molecules, appear to be present in plants.

A 100-kDa monomeric plasma membrane protein constitutes the Pep-13 receptor, which mediates transcriptional activation of pathogenesis-related genes and phytoalexin production in parsley. Ligand-induced receptor activation results in rise of cytoplasmic [Ca2+] via influx through plasma membrane [Ca2+] channels. [Ca2+]-dependent rapid production of superoxide anions and activation of at least two MAP kinase cascades are implicated in activation of pathogen defense by Pep-13. A 24-kD cell wall protein (NPP1) present in various phytopathogenic Phytophthora species was identified, which triggers phytoalexin production and cell death in parsley cells as well as hypersensitive cell death-like lesion formation in parsley leaves. NPP1 did not compete for binding of [125I]Pep-13 to its binding site. Rapidly induced elevation of cytoplasmic [Ca2+] in elicitor-treated parsley cells, formation of an oxidative burst, and activation of MAP kinase cascades by either elicitor suggests initiation of the same signaling cascade via distinct receptors.

NPP1 simulated pathogen infection in Arabidopsis thaliana Col-0. Plants infiltrated with 1 M NPP1 developed lesions reminiscent of the hypersensitive response, apposition of callose and transcriptional activation of PR1. NPP1 also induced ethylene production in Arabidopsis cells. Intriguingly, NPP1-induced PR gene expression was suppressed in the ethylene-insensitive mutant, ein2. Currently, a conditional lethal screen for Arabidopsis mutants resistant to NPP1 is performed.

The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and the ability to elicit the hypersensitive reaction in resistant plants. HrpZ from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (harpinPsph), is secreted in a hrp-dependent manner in Psph and exported by the type III secretion system in the mammalian pathogen, Yersinia enterocolitica. Structural properties of harpinPsph resemble those of proteins assumed to interact with membranes, such as Y. enterocolitica YopB. Although hrpZPsph failed to restore pathogenicity of a yopB-deficient Y.enterocolitica strain, harpinPsph was found to stably associate with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2nM purified recombinant harpinPsph to planar lipid bilayers provoked an ion current of large unitary conductivity (207pS). HarpinPsph-related proteins from P. s. pv. tomato or syringae triggered ion currents similar to those stimulated by harpinPsph. The harpinPsph-mediated ion-conducting pore was permeable for cations but did not mediate Cl- fluxes. We propose, that harpins may either mediate nutrient release from host cells or assist delivery of virulence factors into the plant cell.

Tobacco plasma membranes harbor a binding site for harpinPsph, which mediates activation of plant defense responses in a receptor-like manner. Binding of [125I]harpinPsph to tobacco microsomal membranes (KD=425 nM) and protoplasts (KD=380 nM) was specific, reversible, and saturable. A close correlation between the abilities of harpinPsph fragments to elicit expression of the defense-related tobacco gene, HIN1, and to compete for binding of [125I]harpinPsph to its binding site was found. The harpinPsph binding site was insensitive to protease treatment, and could not be visualized by chemical crosslinking/autoradiography. Thus, binding of harpinPsph to specific lipid or other non-proteinaceous constituents of plant membranes is likely. HarpinPsph-induced salicylic acid-responsive MAP kinase activity and HIN1 transcript accumulation was independent of extracellular [Ca2+]. However, use of the MAP kinase kinase inhibitor, U0126, revealed that MAPK activity was essential for HIN1 expression in harpinPsph-treated tobacco cells. Thus, a receptor-mediated MAPK-dependent signaling pathway may mediate activation of plant defense responses induced by harpinPsph.