Many oomycete and fungal plant pathogens are obligate biotrophs, which extract nutrients only from living plant tissue and cannot grow apart from their hosts. Although these pathogens cause substantial crop losses, little is known about the molecular basis or evolution of obligate biotrophy. Here, we report the genome sequence of the oomycete Hyaloperonospora arabidopsidis (Hpa), an obligate biotroph and natural pathogen of Arabidopsis thaliana. In comparison with genomes of related, hemibiotrophic Phytophthora species, the Hpa genome exhibits dramatic reductions in genes encoding (i) RXLR effectors and other secreted pathogenicity proteins, (ii) enzymes for assimilation of inorganic nitrogen and sulfur, and (iii) proteins associated with zoospore formation and motility. These attributes comprise a genomic signature of evolution toward obligate biotrophy.

Plant innate immunity against invasive biotrophic pathogens depends on the intracellular defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). We show here that Arabidopsis thaliana EDS1 interacts in vivo with another protein, SENESCENCE-ASSOCIATED GENE101 (SAG101), discovered through a proteomic approach to identify new EDS1 pathway components. Together with PHYTOALEXIN-DEFICIENT4 (PAD4), a known EDS1 interactor, SAG101 contributes intrinsic and indispensable signaling activity to EDS1-dependent resistance. The combined activities of SAG101 and PAD4 are necessary for programmed cell death triggered by the Toll-Interleukin-1 Receptor type of nucleotide binding/leucine-rich repeat immune receptor in response to avirulent pathogen isolates and in restricting the growth of normally virulent pathogens. We further demonstrate by a combination of cell fractionation, coimmunoprecipitation, and fluorescence resonance energy transfer experiments the existence of an EDS1-SAG101 complex inside the nucleus that is molecularly and spatially distinct from EDS1-PAD4 associations in the nucleus and cytoplasm. By contrast, EDS1 homomeric interactions were detected in the cytoplasm but not inside the nucleus. These data, combined with evidence for coregulation between individual EDS1 complexes, suggest that dynamic interactions of EDS1 and its signaling partners in multiple cell compartments are important for plant defense signal relay.

The protective barrier provided by stratified squamous epithelia relies on the cornified cell envelope (CE), a structure synthesized at late stages of keratinocyte differentiation. It is composed of structural proteins, including involucrin, loricrin, and the small proline-rich (SPRR) proteins, all encoded by genes localized at human chromosome 1q21. The genetic characterization of the SPRR locus reveals that the various members of this multigene family can be classified into two distinct groups with separate evolutionary histories. Whereas group 1 genes have diverged in protein structure and are composed of three different classes (SPRR1 (2؋), SPRR3, and SPRR4), an active process of gene conversion has counteracted diversification of the protein sequences of group 2 genes (SPRR2 class, seven genes). Contrasting with this homogenization process, all individual members of the SPRR gene family show specific in vivo and in vitro expression patterns and react selectively to UV irradiation. Apparently, creation of regulatory rather than structural diversity has been the driving force behind the evolution of the SPRR gene family. Differential regulation of highly homologous genes underlines the importance of SPRR protein dosage in providing optimal barrier function to different epithelia, while allowing adaptation to diverse external insults.

Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by a wide range of plant-associated microorganisms. They are best known for their cytotoxicity in dicot plants that leads to the induction of rapid tissue necrosis and plant immune responses. The biotrophic downy mildew pathogen Hyaloperonospora arabidopsidis encodes 10 different noncytotoxic NLPs (HaNLPs) that do not cause necrosis. We discovered that these noncytotoxic NLPs, however, act as potent activators of the plant immune system in Arabidopsis thaliana. Ectopic expression of HaNLP3 in Arabidopsis triggered resistance to H. arabidopsidis, activated the expression of a large set of defense-related genes, and caused a reduction of plant growth that is typically associated with strongly enhanced immunity. N-and C-terminal deletions of HaNLP3, as well as amino acid substitutions, pinpointed to a small central region of the protein that is required to trigger immunity, indicating the protein acts as a microbe-associated molecular pattern (MAMP). This was confirmed in experiments with a synthetic peptide of 24 aa, derived from the central part of HaNLP3 and corresponding to a conserved region in type 1 NLPs that induces ethylene production, a well-known MAMP response. Strikingly, corresponding 24-aa peptides of fungal and bacterial type 1 NLPs were also able to trigger immunity in Arabidopsis. The widespread phylogenetic distribution of type 1 NLPs makes this protein family (to our knowledge) the first proteinaceous MAMP identified in three different kingdoms of life.plant immunity | microbe-associated molecular pattern | Nep1-like protein | plant-associated microbe | biotrophic pathogen

The genome of the downy mildew pathogen Hyaloperonospora arabidopsidis encodes necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP). Although NLP are widely distributed in eukaryotic and prokaryotic plant pathogens, it was surprising to find these proteins in the obligate biotrophic oomycete H. arabidopsidis. Therefore, we analyzed the H. arabidopsidis NLP (HaNLP) family and identified 12 HaNLP genes and 15 pseudogenes. Most of the 27 genes form an H. arabidopsidis-specific cluster when compared with other oomycete NLP genes, suggesting this class of effectors has recently expanded in H. arabidopsidis. HaNLP transcripts were mainly detected during early infection stages. Agrobacterium tumefaciens-mediated transient expression and infiltration of recombinant NLP into tobacco and Arabidopsis leaves revealed that all HaNLP tested are noncytotoxic proteins. Even HaNLP3, which is most similar to necrosis-inducing NLP proteins of other oomycetes and which contains all amino acids that are critical for necrosis-inducing activity, did not induce necrosis. Chimeras constructed between HaNLP3 and the necrosis-inducing PsojNIP protein demonstrated that most of the HaNLP3 protein is functionally equivalent to PsojNIP, except for an exposed domain that prevents necrosis induction. The early expression and species-specific expansion of the HaNLP genes is suggestive of an alternative function of noncytolytic NLP proteins during biotrophic infection of plants.

Biotrophic plant pathogens secrete effector proteins that are important for infection of the host. The aim of this study was to identify effectors of the downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) that are expressed during infection of its natural host Arabidopsis thaliana. Infection-related transcripts were identified from Expressed Sequence Tags (ESTs) derived from leaves of the susceptible Arabidopsis Ws eds1-1 mutant inoculated with the highly virulent Hpa isolate Waco9. Assembly of 6364 ESTs yielded 3729 unigenes, of which 2164 were Hpa-derived. From the translated Hpa unigenes, 198 predicted secreted proteins were identified. Of these, 75 were found to be Hpa-specific and six isolate Waco9-specific. Among 42 putative effectors identified there were three Elicitin-like proteins, 16 Cysteine-rich proteins and 18 host-translocated RXLR effectors. Sequencing of alleles in different Hpa isolates revealed that five RXLR genes show signatures of diversifying selection. Thus, EST analysis of Hpa-infected Arabidopsis is proving to be a powerful method for identifying pathogen effector candidates expressed during infection. Delivery of the Waco9-specific protein RXLR29 in planta revealed that this effector can suppress PAMP-triggered immunity and enhance disease susceptibility. We propose that differences in host colonization can be conditioned by isolate-specific effectors.

Terminal differentiation of keratinocytes involves the sequential expression of several major proteins which can be identified in distinct cellular layers within the mammalian epidermis and are characteristic for the maturation state of the keratinocyte. Many of the corresponding genes are clustered in one specific human chromosomal region 1q21. It is rare in the genome to find in such close proximity the genes belonging to at least three structurally different families, yet sharing spatial and temporal expression specificity, as well as interdependent functional features. This DNA segment, termed the epidermal differentiation complex, contains 27 genes, 14 of which are specifically expressed during calcium-dependent terminal differentiation of keratinocytes (the majority being structural protein precursors of the cornified envelope) and the other 13 belong to the S100 family of calcium binding proteins with possible signal transduction roles in the differentiation of epidermis and other tissues. In order to provide a bacterial clone resource that will enable further studies of genomic structure, transcriptional regulation, function and evolution of the epidermal differentiation complex, as well as the identification of novel genes, we have constructed a single 2.45 Mbp long continuum of genomic DNA cloned as 45 p1 artificial chromosomes, three bacterial artificial chromosomes, and 34 cosmid clones. The map encompasses all of the 27 genes so far assigned to the epidermal differentiation complex, and integrates the physical localization of these genes at a high resolution on a complete NotI and SalI, and a partial EcoRI restriction map. This map will be the starting resource for the large-scale genomic sequencing of this region by The Sanger Center, Hinxton, U.K.

Among the three major POU proteins expressed in human skin, Oct-1, Tst-1/Oct-6, and Skn-1/Oct-11, only the latter induced SPRR2A, a marker of keratinocyte terminal differentiation. In this study, we have identified three Skn-1 isoforms, which encode proteins with various N termini, generated by alternative promoter usage. These isotypes showed distinct expression patterns in various skin samples, internal squamous epithelia, and cultured human keratinocytes. Skn-1a and Skn1d1 bound the SPRR2A octamer site with comparable affinity and functioned as transcriptional activators. Skn-1d2 did not affect SPRR2A expression. Skn-1a, the largest protein, functionally cooperated with Ese-1/ Elf-3, an epithelial-specific transcription factor, previously implicated in SPRR2A induction. This cooperativity, which depended on an N-terminal pointed-like domain in Skn-1a, was not found for Skn-1d1. Actually, Skn-1d1 counteracted the cooperativity between Skn-1a and Ese-1. Apparently, the human Skn-1 locus encodes multifunctional protein isotypes, subjected to biochemical cross-talk, which are likely to play a major role in the fine-tuning of keratinocyte terminal differentiation.