The Arabidopsis NIMIN proteins affect NPR1 differentially.

NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) is the central regulator of the pathogen defense reaction systemic acquired resistance (SAR). NPR1 acts by sensing the SAR signal molecule salicylic acid (SA) to induce expression of PATHOGENESIS-RELATED (PR) genes. Mechanistically, NPR1 is the core of a transcription complex interacting with TGA transcription factors and NIM1-INTERACTING (NIMIN) proteins. Arabidopsis NIMIN1 has been shown to suppress NPR1 activity in transgenic plants. The Arabidopsis NIMIN family comprises four structurally related, yet distinct members. Here, we show that NIMIN1, NIMIN2, and NIMIN3 are expressed differentially, and that the encoded proteins affect expression of the SAR marker PR-1 differentially. NIMIN3 is expressed constitutively at a low level, but NIMIN2 and NIMIN1 are both responsive to SA. While NIMIN2 is an immediate early SA-induced and NPR1-independent gene, NIMIN1 is activated after NIMIN2, but clearly before PR-1. Notably, NIMIN1, like PR-1, depends on NPR1. In a transient assay system, NIMIN3 suppresses SA-induced PR-1 expression, albeit to a lesser extent than NIMIN1, whereas NIMIN2 does not negatively affect PR-1 gene activation. Furthermore, although binding to the same domain in the C-terminus, NIMIN1 and NIMIN2 interact differentially with NPR1, thus providing a molecular basis for their opposing effects on NPR1. Together, our data suggest that the Arabidopsis NIMIN proteins are regulators of the SAR response. We propose that NIMINs act in a strictly consecutive and SA-regulated manner on the SA sensor protein NPR1, enabling NPR1 to monitor progressing threat by pathogens and to promote appropriate defense gene activation at distinct stages of SAR. In this scenario, the defense gene PR-1 is repressed at the onset of SAR by SA-induced, yet instable NIMIN1.

NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) and some NPR1-related proteins are sensitive to salicylic acid.

NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1; also known as NIM1) is a master regulator of systemic acquired resistance (SAR). SAR is induced by salicylic acid (SA), leading to the expression of PATHOGENESIS-RELATED (PR) genes. Current evidence suggests that NPR1 is part of a transcription complex tethered to activation sequence-1 (as-1)-like cis-acting elements in PR-1 gene promoters through TGA transcription factors, and that SA-dependent PR-1 gene expression is regulated by NIM1-INTERACTING (NIMIN) proteins. In Arabidopsis, NPR1 is active only after SA induction. Regulation of Arabidopsis NPR1 activity has been proposed to comprise cysteine-156 (Cys-156), mediating SA-induced cytoplasmic oligomer-nuclear monomer exchange, and Cys-521 and Cys-529, mediating SA-dependent transcriptional activation. Tobacco NPR1 does not harbour these residues. To understand the function of tobacco NPR1, we analysed its biochemical capabilities in a heterologous system: yeast. Tobacco NPR1 differs from Arabidopsis NPR1 in its subcellular localization and its transactivation potential. Yet, both tobacco and Arabidopsis NPR1, as well as tobacco NIM1-like1, alter some of their biochemical activities in response to SA. Whereas the addition of SA to yeast growth medium induces transcriptional activity in tobacco NPR1, its interaction with NIMIN2-type proteins is suppressed. The effects of SA are specific, sensitive and occur coordinately. They are abolished completely by mutation of the arginine residue within the invariable penta-amino acid motif LENRV, as present in the nonfunctional Arabidopsis nim1-4 allele. Furthermore, NPR1 proteins with the LENRV domain coincidently harbour a broad and strongly conserved NIMIN1/NIMIN2 binding site. Our data suggest that NPR1 and some NPR1-like proteins are sensitive to the plant hormone SA, altering some of their biochemical capabilities to enable stimulus-dependent gene expression. The sensitivity of NPR1 proteins to SA, together with their differential interaction with diverse NIMIN proteins, seems a plausible molecular basis for the timely and coordinated activation of PR genes during SAR.

Epidemiologic aspects and laboratory features of enterovirus infections in Western Germany, 2000-2005.

From 2000 to 2005, a total of 1,096 enterovirus infections were diagnosed either by isolation of virus from cell culture or by RT-PCR (5'non-coding region (NCR)). Typing of viruses (n = 674) was carried out by immunofluorescence with monoclonal antibodies, neutralization test or molecular methods. Seasons with high enterovirus activity were characterized by high prevalence of echovirus 30 (62.2% in 2000, 25.5% in 2001) and echovirus 13 (34.5% in 2001). In contrast, in the 2003 season, which had very low enterovirus activity, these types were rare. During this season, cell culture sensitivity (human colonic carcinoma cells and human embryonic lung fibroblasts (HEL)) was exceptionally low. In order to determine the type of "non-cultivable" enteroviruses, purified RNA from selected stool samples was subjected to direct molecular typing. VP1/2A-specific fragments were amplified by RT-PCR, cloned and sequenced. The predominant virus identified was coxsackie A. Consequently, rhabdomyosarcom cells were introduced into the daily routine, which improved the isolation of enteroviruses. Echovirus 30 was again most commonly isolated during seasons 2004 and 2005 with increasing enterovirus activity. In conclusion, high prevalence of echovirus 30 and 13 is indicative of seasons with high enterovirus activity. The type of circulating enteroviruses may influence isolation of enterovirus from cell culture. RT-PCR (VP1/2A) combined with cloning and sequencing of amplicons is a useful tool for viral typing directly from stool samples. In cases of severe enterovirus infection, virological diagnosis should not solely rely on virus isolation from cell culture.

Sequence and annotation of the 288-kb ATCV-1 virus that infects an endosymbiotic chlorella strain of the heliozoon Acanthocystis turfacea.

Acanthocystis turfacea chlorella virus (ATCV-1), a prospective member of the family Phycodnaviridae, genus Chlorovirus, infects a unicellular, eukaryotic, chlorella-like green alga, Chlorella SAG 3.83, that is a symbiont in the heliozoon A. turfacea. The 288,047-bp ATCV-1 genome is the first virus to be sequenced that infects Chlorella SAG 3.83. ATCV-1 contains 329 putative protein-encoding and 11 tRNA-encoding genes. The protein-encoding genes are almost evenly distributed on both strands and intergenic space is minimal. Thirty-four percent of the viral gene products resemble entries in the public databases, including some that are unexpected for a virus. For example, these unique gene products include ribonucleoside-triphosphate reductase, dTDP-d-glucose 4,6 dehydratase, potassium ion transporter, aquaglyceroporin, and mucin-desulfating sulfatase. Comparison of ATCV-1 protein-encoding genes with the prototype chlorella virus PBCV-1 indicates that about 80% of the ATCV-1 genes are present in PBCV-1.

Tobacco NIMIN2 proteins control PR gene induction through transient repression early in systemic acquired resistance.

SUMMARY: NPR1 (for Nonexpressor of PR genes; also known as NIM1) is a positive regulator of systemic acquired resistance (SAR) in Arabidopsis, which controls the induction of Pathogenesis-Related (PR) genes by salicylic acid (SA). NPR1 interacts with members of two protein families, TGA transcription factors and NIMIN (for NIM1-interacting) proteins. In Arabidopsis, NIMIN1, NIMIN2 and NIMIN3 constitute a small gene family of structurally related, yet distinct members. To unravel the biological significance of NIMIN interaction with NPR1, we searched a tobacco yeast two-hybrid cDNA library for NPR1- and NIMIN2-binding proteins. One NPR1 cDNA clone and three clones encoding NIMIN proteins were isolated. Although clearly similar to At NPR1, Nt NPR1 does not interact with At NIMIN3. Furthermore, all Nt NIMIN proteins identified are structurally related to At NIMIN2, thus forming a small NIMIN2 subfamily in tobacco. cDNA clones encoding At NIMIN1 or At NIMIN3 homologues were not identified. The function of NIMIN2 proteins was studied by expression of Nt NIMIN2a chimeric genes in tobacco. While constitutive NIMIN2a over-expression delayed PR-1 protein induction, suppression of NIMIN2 transcripts enhanced the accumulation of PR-1 proteins. In both cases, the effects of altered NIMIN2 transcript levels became evident foremost early in SAR. Notably, Nt NIMIN2 gene expression is elevated prior to the induction of the PR-1a gene. Together, the data suggest that, in tobacco, NIMIN2 proteins control PR-1 gene expression, and that NIMIN2-mediated control is exerted through transient PR-1 repression before SAR has fully developed. Furthermore, although sharing conserved domains and functions, tobacco and Arabidopsis NPR1 and NIMIN proteins are clearly distinct.

Comparison of 11 herpesvirus isolates from tortoises using partial sequences from three conserved genes.

Herpesviruses are an important cause of epidemic disease in tortoises. There are at least two serologically distinct herpesviruses capable of infecting tortoises. Methods for the diagnosis of herpesvirus infections in tortoises include virus isolation and a number of different PCRs. We have compared 11 virus isolates collected from various species in different countries over several years using sequences from three different viral genes. During this study we used four different PCR protocols described for the diagnosis of herpesvirus infections in tortoises. The protocols used included two based on portions of the DNA polymerase gene, one targeting the UL5 homologue, and one targeting the UL39 homologue. Comparison of the methods showed that the tortoise herpesvirus-specific protocols were all serotype specific. Sequences of the obtained amplicons were compared with one another and with sequences of herpesviruses available in GenBank. The sequence alignments showed that the tortoise herpesviruses were most closely related to members of the subfamily Alphaherpesvirinae. They also showed that the tortoise isolates could be clearly divided into two genogroups.

Isolation and characterization of a new type of chlorovirus that infects an endosymbiotic Chlorella strain of the heliozoon Acanthocystis turfacea.

A novel virus, named Acanthocystis turfacea Chlorella virus (ATCV), that infects endosymbiotic Chlorella algae of the heliozoon Acanthocystis turfacea was isolated from freshwater samples. Electron microscopic analysis of ATCV revealed that the viral capsid has a distinct icosahedral shape with a diameter of 140-190 nm. Filamentous structures extending from some of the virus vertices, which may aid attachment of the virus to host cells, were also observed. The capsid is made up of one major coat protein of about 50 kDa and contains a large dsDNA genome. ATCV is a member of the genus Chlorovirus, which belongs to the family Phycodnaviridae, a group of large, icosahedral, dsDNA-containing viruses that infect algae and are ubiquitous in natural environments. However, ATCV is clearly distinct from the prototype Chlorovirus, Paramecium bursaria Chlorella virus (PBCV-1), in some aspects of its genome structure and gene content and therefore must be regarded as a member of a new group of Chlorella viruses.

Salicylic acid (SA)-dependent gene activation can be uncoupled from cell death-mediated gene activation: the SA-inducible NIMIN-1 and NIMIN-2 promoters, unlike the PR-1a promoter, do not respond to cell death signals in tobacco.

SUMMARY Tobacco pathogenesis-related (PR) genes of group 1 are induced during pathogen defence (hypersensitive response, HR, and systemic acquired resistance, SAR), after exogenous application of salicylic acid (SA), and by developmental cues. Likewise, SA enhances transcripts for Arabidopsis NIMIN-1 and NIMIN-2, which interact with NPR1/NIM1, a key regulator of SAR. To further illuminate gene activation during pathogen defence, reporter gene expression from the NIMIN-1 and NIMIN-2 promoters was analysed in transgenic tobacco plants in direct comparison to PR-1 gene expression. NIMIN[GUS] chimeric genes were highly sensitive to SA, whereas NIMIN[GUS], unlike PR1a[GUS], expression was only weak in necrotic tissue exhibiting HR. Furthermore, PR-1a, but not NIMIN, promoter constructs were activated systemically in response to local cell death elicited by expression of the proapoptotic Bax gene. Conversely, NIMIN-1[GUS] expression was completely suppressed during pathogen defence in plants depleted from SA, whereas PR-1 proteins still accumulated in necrotic tissue. These findings demonstrate that SA-dependent gene activation can be uncoupled from cell death-induced gene activation. Whereas PR-1a induction during the HR and SAR responses is mediated by HR-associated signals and SA, activation of the NIMIN-1 and NIMIN-2 promoters in infected tobacco relies on SA, but not on cell death signals.

Salicylic acid and the hypersensitive response initiate distinct signal transduction pathways in tobacco that converge on the as-1-like element of the PR-1a promoter.

Tobacco pathogenesis-related protein 1a (PR-1a) is induced in plants during the hypersensitive response (HR) after exposure of plants to salicylic acid (SA) and by developmental cues. Gene activation by these diverse stimuli is mediated via an as-1-like element in the PR-1a upstream region. To further analyze the significance of this cis-acting sequence, an authentic as-1 element from the cauliflower mosaic virus 35S RNA promoter was inserted into the PR-1a promoter in place of the as-1-like motif. Reporter gene analysis in transgenic tobacco plants demonstrated that as-1 can functionally replace the as-1-like element in the PR-1a promoter in response to all stimuli. However, reporter gene induction from the as-1 carrying promoter was enhanced in response to SA compared to the wild-type promoter, and the ratio of reporter gene activities in SA treated leaf tissue to tissue exhibiting the HR increased with the as-1 promoter construct. Our findings support a model where PR-1a gene expression relies on at least two distinct signal transduction pathways initiated by SA and by a yet unknown signal produced during the HR, that promote different, albeit related, transcription complexes on the PR-1a as-1-like element. Analysis of PR-1 proteins in plants expressing salicylate hydroxylase yielded additional evidence that an HR dependent pathway leads to high level PR-1 gene induction in tobacco.

Molecular characterization of an Enterovirus 71 causing neurological disease in Germany.

Enterovirus 71 (EV71) is mainly known as a cause of hand-foot-and-mouth disease (HFMD) but sometimes associated with neurological disease, even as fatal brainstem encephalitis. In Europe, EV71 infections are extremely rare, in contrast to the worldwide situation. This is the first report of molecular characterization of an EV71 strain isolated in Europe that had caused neurological disease. The german strain is closest related to sublineage B2 strains isolated in the United States, which where mainly associated with neurological disease. Phylogenetic analysis also showed that the strain must have been imported to Germany several years ago, and continues to circulate since then.