SummaryPlant immunity against the majority of the microbial pathogens is conveyed by a phenomenon known as non-host resistance (NHR). This defence mechanism affords durable protection to plant species against given species of phytopathogens. We investigated the genetic basis of NHR in Arabidopsis against the wheat powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt). Both primary and appressorial germ tubes were produced from individual Bgt conidia on the surface of the Arabidopsis leaves. Attempted infection occasionally resulted in successful penetration, which led to the development of an abnormal unilateral haustorium. Inoculation of a series of Arabidopsis defence-related mutants with Bgt resulted in the attenuation of reactive oxygen intermediate (ROI) production and salicylic acid (SA)-dependent defence gene expression in eds1, pad4 and nahG plants, which are known to be defective in some aspects of host resistance. Furthermore, Bgt often developed bilateral haustoria in the mutant Arabidopsis lines that closely resembled those formed in wheat. A similar decrease in NHR was observed following treatment of the wild-type Arabidopsis plants with cytochalasin E, an inhibitor of actin micro®lament polymerisation. In eds1 mutants, inhibition of actin polymerisation severely compromised NHR in Arabidopsis against Bgt. This permitted completion of the Bgt infection cycle on these plants. Therefore, actin cytoskeletal function and EDS1 activity, in combination, are major contributors to NHR in Arabidopsis against wheat powdery mildew.

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A full-length cDNA (CaSUI1) was isolated from a Coffea arabica cDNA library from beans during maturation. Its putative translational product is highly homologous to the SUI1 protein of Saccharomyces cerevisiae which functions in concert with eIF2 and the initiator tRNA-Methionin in directing the ribosome to the proper start site of translation. The corresponding gene from coffee was also cloned and sequenced. Its organization is very similar to what observed for the same gene from rice (Oryza sativa) particularly regarding the position and length of its introns. The expression of CaSUI1 gene was also checked and showed that it was highly expressed in all coffee tissues analyzed, therefore confirming the essential role of the SUI1 protein in cell housekeeping functions.Key words: Coffea arabica, constitutive gene expression, GOS2, SUI1, translational initiation.Clonagem de uma seqüência completa de cDNA e gene de Coffea arabica codificando uma proteína homóloga ao fator de tradução SUI1 de levedura: análise da expressão em órgãos da planta: Uma seqüência completa de cDNA (CaSUI1) foi isolada de uma biblioteca de cDNA de frutos de Coffea arabica coletados no estádio de maturação. O produto putativo de transcrição é altamente homólogo à proteína SUI1 de Saccharomyces cerevisiae, que funciona em combinação com eIF2 e o iniciador tRNA-Metionina no direcionamento do ribossomo para o sítio correto de iniciação da tradução. O gene correspondente de café também foi clonado e sequenciado. Sua organização é similar ao que foi descrito para o mesmo gene em arroz (Oryza sativa), particularmente em relação à posição e ao comprimento de seus introns. A expressão do gene CaSUI1 também foi estudada, observando-se alta expressão em todos os tecidos de café analisados, confirmando, portanto, o papel essencial da proteína de SUI1 em funções básicas de manutenção celular. Palavras-chave:Coffea arabica, expressão gênica constitutiva, GOS2, iniciação de tradução, SUI1.

The aim of this project was to undertake large scale transcript profiling of endophyte and plant genes during symbiosis, and to determine the impact of targeted endophyte gene deletions on expression of plant and endophyte genes. We have designed and developed an Affymetrix NimbleExpress™ GeneChip® representing expressed sequence tags (ESTs) of the fungal endophyte Neotyphodium lolii Lp19 and its ryegrass host, Lolium perenne. In total, 8511 genes were represented on the microarrays with approximately eleven 25 base pair oligonucleotides per gene. Experiments were conducted to analyse differential expression of genes from endophyte-infected and endophyte-free plant material, and from endophytes grown in culture. In some symbioses, endophytes had targeted mutations in genes involved in signalling, synthesis of secondary metabolites or in genes of unknown function. Here we describe the processes which guided design of the GeneChip®, the results of quality control assessments of hybridised arrays and considerations concerning statistical analyses of gene expression. Keywords: Affymetrix, GeneChip®, NimbleExpress, Neotyphodium lolii, Epichloë festucae, ryegrass, Lolium perenne, endophyte, symbiosis.

As a first step towards a functional genomics approach to gain a greater understanding of this important symbiosis, we have generated, sequenced and analysed two EST libraries from cultures of N. lolii and six in planta subtracted EST libraries enriched for differentially expressed genes. A total of 12871 ESTs were sequenced which, after filtering for quality, clustered into 1066 contigs and 3230 singletons to give a set of 4296 unique sequences or unigenes. BLASTX analysis revealed that 60% of fungal sequences derived from cultures were of unknown function with a sub-set of these corresponding to orphans. For the in planta-derived ESTs, most of the sequences with homologs in the public databases (98%) were of ryegrass origin. Comparisons made against fully sequenced genomes revealed that most fungal ESTs were homologous to genes present in both pathogenic and non-pathogenic ascomycete filamentous fungi, whereas the subtracted libraries comprised mostly plant genes. A range of sequences having significant homology to demonstrated pathogenicity/virulence genes in other fungal pathosystems were also identified, as well as some ESTs with proven roles in endophyte secondary metabolism. Keywords: ESTs, cDNA, Neotyphodium lolii, Lolium perenne, symbiosis, mutualism, suppression subtractive hybridisation

Neotyphodium lolii is a fungal endophyte that lives entirely within the intercellular spaces of its grass host, perennial ryegrass (Lolium perenne, L.). Infection is symptomless and the endophyte relies on the host plant for dissemination via the seed. The association is mutually beneficial since the endophyte confers a number of biotic and abiotic advantages to the host. This paper presents an overview of the functional genomics approaches we are using at AgResearch to dissect the molecular basis of this symbiosis and will broadly describe the fields of genomics, transcriptomics, proteomics and metabolomics, as applied to this system. We have used isogenic ryegrass lines infected or uninfected with endophyte in combination with a suite of molecular biology tools, including Expressed Sequence Tags (ESTs), cDNA and Affymetrix GeneChip® microarray analysis, 2D-gel electrophoresis (to identify novel proteins associated with symbiosis), and metabolic profiling. By using a multidisciplinary approach we aim to identify genes which are important in both the establishment and maintenance of symbiosis, as well as elucidate how endophyte confers enhancements to its host. Keywords: Neotyphodium, Epichloë, symbiosis, functional genomics

In fungal pathogenesis the cAMP signalling cascade is usually essential for virulence. Deletion of the adenylate cyclase gene, the enzyme that synthesises cAMP, often results in an attenuated or avirulent phenotype. Our aim was to identify the signalling mechanisms regulating colonisation of perennial ryegrass (Lolium perenne) by the fungal symbiont Epichloë festucae Fl1. We have identified genes from several signalling networks, and here report on the outcomes of targeted disruption of the E. festucae Fl1 adenylate cyclase gene (acyA). A dual genome (endophyte/ ryegrass) Affymetrix GeneChip® has been synthesised and we are undertaking large scale transcript profiling of the L. perenne/ E. festucae ΔacyA symbiotum to identify target genes regulated by the endophyte cAMP signalling network. Keywords: cAMP, adenylate cyclase, acyA, Neotyphodium lolii, Epichloë festucae, symbiosis, Affymetrix GeneChip