David Greenshields¹, Guosheng Liu², Gopalan Selvaraj³, Yangdou Wei²

1Sainsbury Laboratory, Norwich, United Kingdom, ²University of Saskatchewan, Saskatoon, Canada, ³NRC Plant Biotechnology Institute, Saskatoon, Canada

Iron is an essential element for both plants and fungal pathogens. In plants, iron is generally bound to proteins and small molecules during transport and storage, which prevents the generation of toxic free radicals via iron-mediated redox reactions. During fungal attack, however, monocot plants target substantial amounts of cellular iron to the apoplast, where it mediates an oxidative burst at sites of attempted pathogen penetration. In order to scavenge plant host iron, fungal pathogens have evolved at least two distinct iron acquisition strategies. One iron uptake strategy involves the secretion and subsequent uptake of small iron chelators called siderophores and the other system uses cell surface reductases which reduce bound plant ferric iron to ferrous iron for uptake. Several recent reports show that fungal plant pathogens require either reductive or siderophore-mediated iron uptake during infection. Interestingly, a pathogen’s preferred mode of iron uptake appears to be related to its pathogenic lifestyle: biotrophs seem to favour reductive iron uptake and necrotrophs seem to favour the use of siderophores.

Hans van Etten¹, Jeff Coleman¹, Catherine Wassman¹, Evans Kaimoyo¹, Tomoyoshi Akashi², Shin-ichi Ayabe²

1University of Arizona, Arizona, United States, ²Nihon University, Kanagawa, Japan

Shortly after the first elucidation of the structure of a phytoalexin (pisatin) was reported in 1960, I. A. M. Cruickshank surveyed the sensitivity of 45 fungal species to this phytoalexin. He found that only five species were tolerant of pisatin and all were pea pathogens, thereby establishing the concept that tolerance to a phytoalexin could be a host-specific virulence trait. Past research has demonstrated that necrotrophic pea pathogens have a specific cytochromone P450, pisatin demethylase (PDA) that detoxifies pisatin. Orthologs for the PDA of Nectria haematococca have been found only in other pea pathogens. In N. haematococca, PDA is in a cluster of other genes for pea pathogenicity and current results suggest this cluster, and the chromosome carrying the cluster, arose by horizontal gene transfer. In addition to PDA, a specific pisatin-induced ABC transporter (NhABC1) that contributes to pisatin tolerance has been characterized in N. haematococca. Mutation in either PDA or NhABC1 reduces virulence on pea; double mutants are greatly reduced in pisatin tolerance and virulence on pea. NhABC1 is similar to other ABC transporters that have been shown to be involved in pathogenicity suggesting these are common genes for pathogenicity.

The synthesis of pisatin, an isoflavonoid derivative, is restricted mainly to the relatives of Pisum. The last step in pisatin biosynthesis is a methylation and the gene responsible for this methylation is a homolog of the methyltransferase that catalyzes an early common step in isoflavonoid biosynthesis. Two homologs of this gene are found in all pisatin-producing legumes examined and in P. sativum, the two genes are clustered. Sequence comparisons suggest a change in one or two amino acids account for the different catalytic properties of the enzymes. We propose that in fungi, the evolution of pathogenicity on pea involved the acquisition of multiple mechanisms to tolerate pisatin and that these mechanisms involve specific classes of cytochromone P450s and ABC transporters. We also suggest that in pea, the evolution of a chemical-based disease-resistance mechanism involved a duplication of a common isoflavonoid biosynthetic gene and that the duplicated copy catalyzes a new reaction in pea.

The fungus Ustilago maydis is a biotrophic maize pathogen that does not use aggressive virulence strategies and needs the living plant tissue for completion of its life cycle. We now provide evidence how such a biotrophic relationship is established. The U. maydis genome codes for 178 novel proteins which are predicted to be secreted. Many of the respective genes are clustered in the genome and are upregulated during pathogenic development. These gene clusters have crucial roles during discrete stages of biotrophic growth. We now show that U. maydis is eliciting distinct defense responses when individual clusters/genes are deleted, suggesting that the respective gene products suppress this reaction. Maize gene expression profiling has allowed us to classify these defense responses and provides leads to where suppression may operate. We describe where the crucial secreted effector molecules localize, their interaction partners and how this may suppress the respective plant defense responses.

Prasanna Kankanala¹, Gloria Mosquera¹, Chang Hyun Khang¹, Romain Berruyer⁴, Martha Giraldo¹, Kirk Czymmek³, Sook-Young Park², Seogchan Kang², Barbara Valent¹

1Kansas State University, Manhattan, KS, United States, ²Pennsylvania State University, University Park, PA, United States, ³University of Delaware, Newark, DE, United States, ⁴Université d'Angers, 49045 Angers, France

The filamentous ascomycete fungus Magnaporthe oryzae is the hemibiotropic pathogen that causes rice blast disease leading to severe crop losses annually. M. oryzae penetrates the host surface barrier though an appressorium and sequentially invades living plant cells using intracellular invasive hyphae (IH) that grow from cell to cell. Using live-cell imaging of a highly compatible interaction, we reported that IH are tightly wrapped in plant-derived extra-invasive hyphal membrane (EIHM). IH appear to seek out plasmodesmata for moving from one cell to another. The EIHM encloses distinct membrane caps at the tips of IH as they begin to grow in new cells. To identify and characterize effector proteins secreted by IH into live plant cells to control host cellular processes, we developed a reproducible procedure to obtain infected sheath tissue with 20% IH RNA at 36 hpi, when most IH were still growing in the first-invaded rice cell. Hybridizations with the whole genome M. oryzae microarray identified many IH-specific putative secreted proteins of unknown function. Hybridization with a rice microarray showed that highly induced rice genes encoded membrane-associated proteins, signal transduction components and transcription factors. These results were consistent with the hypothesis that IH secrete novel effector proteins into rice cells to reprogram host gene expression. Gene replacement experiments have shown no major phenotypes associated with putative effectors. To study effector secretion in planta, we fused known and putative blast effectors to green fluorescent protein (GFP). Predicted effector signal peptides directed secretion of GFP from the fungus into the membrane caps. As the fungus grew into the cells the membrane caps moved from the tips to the sides of IH. We named these regions in the EIHM where fluorescence concentrated as Blast Interfacial Complexes (BICs). Fusion proteins accumulated in BICs as long as IH grew in the cell. Correlative light and electron microscopy suggest that BICs are complex membrane-rich and vesicle-rich structures between the fungal wall and EIHM. We will discuss a potential role for BICs in blast effector secretion.

Linda Johnson, Albert Koulman, Michael Christensen, Richard Johnson, Geoffrey Lane, Christine Voisey, Anar Khan, Zaneta Park, Gregory Bryan, Susanne Rasmussen

We have identified an extracellular siderophore of novel structure and an intracellular siderophore (ferricrocin) from symbiotic fungal endophytes (genera Epichloë and Neotyphodium) of cool-season grasses. In nature, these fungi are never free-living, but form mutually beneficial associations where they are confined to intercellular spaces of leaf sheaths and blades. Targeted gene replacement of a non-ribosomal peptide synthetase, sidN, from E. festucae eliminated biosynthesis of an extracellular siderophore that was induced under iron-depleted conditions. Structural characterisation indicated a novel structure with similarities to fusarinine-type siderophores. Associations of ∆sidN mutants with Lolium perenne (perennial ryegrass) were no longer mutualistic; detrimental gross plant phenotypic changes were observed. Siderophore absence caused malfunctioning of the characteristic synchronized growth of the fungus with its plant partner, leading to a loss of mutualism and an apparent switch to antagonistic growth. Endophyte metabolism is also affected since the biosynthesis of host controlled endophyte alkaloids, ergovaline and lolitrem, are significantly increased in sidN mutant plants. Further investigation of the molecular mechanisms controlling global regulation of iron in the symbiotum, and how this influences endophyte secondary metabolism were studied by using Affymetrix Gene Chips®. Endophyte genes falling under the GO term classifications of catalytic activity, chromatin, subtilase activity and others were significantly affected. On the host side, some of the perennial ryegrass genes affected were associated with response to osmotic stress, cell morphogenesis and alcohol dehydrogenase activity. We hypothesise that competition for iron is a critical factor in controlling fungal growth and hence for mutualism with grasses. SidN siderophore absence most likely disrupts iron homeostasis in the whole symbiotum.

Valerie Jaulneau, Marc Cazaux, Joanne Wong Sak Hoi, Christophe Jacquet, Bernard Dumas

In phytopathogenic fungi, STE12-like genes encode transcription factors essential for appressorium-mediated host penetration. Recently, we showed that a STE12-like factor, named CLSTE12, regulates production of extracellular proteins such as pectinases and cell-wall associated proteins in the bean pathogen Colletotrichum lindemuthianum (Wong et al., 2007). In the present study, we have investigated the role of CLSTE12 in the production of fungal effectors which induce plant defence responses in the case of host and non-host interactions. This study was done by using the model legume Medicago truncatula, a non-host plant for C. lindemuthianum, but a host for the related Colletotrichum specie, C. trifolii. Two M. truncatula lines showing contrasted phenotype towards C. trifolii infection were used: Jemalong is resistant C. trifolii strains, whereas F83005.5 is susceptible (Ameline-Torregrosa et al., 2008). A STE12-like gene was isolated from C. trifolii, named CTSTE12 (Colletotrichum trifolii STE12) and a disrupted mutant was obtained by homologous recombination. C. lindemuthianum and C. trifolii strains (wild-type strains and STE12-like mutants) were used to inoculate the two Medicago truncatula lines. Inoculation of M. truncatula lines with a wild-type C. lindemuthianum strain induced the development of a hypersensitive response (HR) characterized by a necrotic spot at the site of inoculation and the induction defence gene coding PR proteins and enzymes involved in the synthesis of phytoalexins. Inoculation with CLSTE12 mutants or with the wild type strain induced similar responses in both lines. C. trifolii strains disrupted for CTSTE12 were non-pathogenic on the susceptible lines confirming the role of STE12-like proteins in pathogenesis. Interestingly, these strains were still able to produce a HR on the resistant lines, and also on the susceptible line. Together, these results demonstrate that perception of adapted and non-adapted Colletotrichum species by the plant occurs before the appressorium-mediated penetration through a STE12-like independent mechanism, and that suppression of this response on the susceptible plant required a functional STE12-like gene.

Ameline-Torregrosa, C. et al. (2008) Mol Plant-Microbe Interact 21 (1), 61-69; Wong Sak Hoi, J.W. et al. (2007) Mol Microbiol 64 (1), 68-82

Francis Parlange¹, Guillaume Daverdin¹, Isabelle Fudal¹, Marie-Line Kuhn¹, Françoise Blaise¹, Bruno Grezes-Besset², Marie-Hélène Balesdent¹, Thierry Rouxel¹

1INRA-Bioger, Versailles, France, ²BIOGEMMA, Mondonville, France

Leptosphaeria maculans is the ascomycete fungus responsible for one of the most damaging disease of oilseed rape (Brassica napus), stem canker of crucifers, and develops gene-for-gene interactions with its host. Both avirulence (AvrLm) genes in the fungus and resistance (Rlm) genes in the plant are genetically clustered. In the course of the map-based cloning of AvrLm7, part of the AvrLm3-AvrLm4-AvrLm7-AvrLm9 genetic cluster, we identified three BAC clones containing the AvrLm7 locus, and delineating a region of 238 kb. Structural features of the region were reminiscent of those previously found on another chromosome for the genomic regions encompassing AvrLm1 and AvrLm6, i.e., alternate of G+C-equilibrated, gene-rich isochores, and A+T-rich, recombinant-deficient, gene-poor isochores corresponding to mosaics of RIP (Repeat Induced Point mutation)-degenerated and truncated transposons and retrotransposons. In the AvrLm7 region, three novel transposable elements, including a DNA transposon were identified. Following complementation of a virulent isolate, AvrLm7 was found to be the only ORF isolated within a 60 kb A+T-rich isochore. Surprisingly, the AvrLm7 sequence was shown to induce resistance responses in plants harbouring either Rlm7 or Rlm4. AvrLm7, thus renamed AvrLm4-7, encodes a 143 amino-acid cystein-rich protein, predicted to be secreted outside of the fungus cells and strongly induced during the early stages of plant infection. Sequencing of 30 AvrLm4-AvrLm7 or avrLm4-AvrLm7 alleles in L. maculans field isolates, coupled with restriction analyses on 223 field isolates and targeted point mutagenesis strongly suggested that one single base mutation, leading to the change of a glycine residue to an arginine residue, was responsible for the loss of the AvrLm4 specificity whereas the AvrLm7 recognition by the plant was unaltered.

Natalia Requena, Hannah Kuhn, Silke Kloppholz, Nina Rieger, Torsten Klug

Several key plant components of the signaling cascade leading to arbuscular mycorrhiza initiation have been identified in the last years, including the receptor kinase NORK, thanks to genetic and molecular approaches. At the fungal side of the symbiosis, advances have mainly taking place using molecular cloning approaches that led to the identification of some genes regulated in response to plant signals. In this project we are trying to identify fungal effector molecules able to trigger the initiation of the recognition of the fungal partner in the plant using complementary approaches. An in vitro bioassay based on the expression of a reporter gene under the control of an early mycorrhiza-induced M. truncatula gene has being established using hairy root explants. With this system, both genetic and biochemical techniques are being used to identify arbuscular mycorrhizal secreted proteins triggering induction of reporter genes. Different developmental stages of the fungal life cycle have been used, including pre-symbiotic mycelium, appressoria, arbuscules and plant-induced extraradical mycelium. Fungal cDNAs are then screened for secretion peptides, which drive expression and secretion of a truncated invertase gene and promote yeast growth on sucrose (YSST= yeast signal sequence trap method). Subsequently, candidate effectors are functionally validated in planta for their ability to turn on the promoter-reporter construct in the bioassay. In parallel yeast two hybrid libraries of fungal material have been established and screened for interacting partners of the symbiosis receptor kinase NORK. Two possibly secreted proteins have been identified that interact with the LRR domain of NORK and will be validated in planta as effector molecules.