In vivo detection of experimental ulcerative colitis in rats using fiberoptic confocal imaging (FOCI).

Fiberoptic confocal imaging (FOCI) is a noninvasive microscopic technique that enables subsurface imaging of living tissue in vivo. The aim of the present study was to assess the suitability of FOCI for the in vivo detection of early subsurface changes in the mucosal architecture of the colon in a rat model of ulcerative colitis. Mild colitis was induced in Sprague-Dawley rats (180-250 g) by the oral ingestion of 5% (w/v) dextran sulfate sodium (DSS; Mr 40,000 Da) in drinking water. Control animals were provided with water ad libitum. After three, five or seven days of oral consumption of DSS, the mucosal surface of the colon of anesthetised rats was surgically exposed. Morphological changes in the mucosa were examined (Optiscan F900e personal confocal system with rigid endomicroscope attachment; excitation 488 nm argon ion laser, detection above 515 nm) following the topical application of a fluorescent dye (fluorescein, eosin, or acridine orange). Confocal images were correlated with conventional histology and clinical parameters including occult blood and stool consistency. Histological evaluation of colon sections demonstrated that DSS-induced colitis was characterized by focal loss of mucous crypts, loss of epithelial cells, and neutrophilic infiltration into the mucosa. The extent of mucosal damage was positively correlated with the time of ingestion of DSS. Morphological changes associated with disease activity could be detected microscopically in vivo using FOCI but were not evident by visual inspection of the colon surface. Acridine orange enabled imaging of the colonic crypts at the surface of the mucosa. Morphological changes associated with colitis, including inflammatory cell infiltrate, crypt loss, and crypt distortion, could be detected using this fluorophore. Application of fluorescein and eosin enabled subsurface imaging of the lamina propria surrounding the crypts; however, no change in structure was detected in association with colitic disease activity. This study has shown that the topical application of acridine orange enables in vivo imaging of early colitis in a rat model. FOCI may be suitable for the diagnosis and monitoring of human inflammatory bowel disease.

Salicylic acid and NIM1/NPR1-independent gene induction by incompatible Peronospora parasitica in arabidopsis.

To identify pathogen-induced genes distinct from those involved in systemic acquired resistance, we used cDNA-amplified fragment length polymorphism to examine RNA levels in Arabidopsis thaliana wild type, nim1-1, and salicylate hydroxylase-expressing plants after inoculation with an incompatible isolate of the downy mildew pathogen Peronospora parasitica. Fifteen genes are described, which define three response profiles on the basis of whether their induction requires salicylic acid (SA) accumulation and NIM1/NPR1 activity, SA alone, or neither. Sequence analysis shows that the genes include a calcium binding protein related to TCH3, a protein containing ankyrin repeats and potential transmembrane domains, three glutathione S-transferase gene family members, and a number of small, putatively secreted proteins. We further characterized this set of genes by assessing their expression patterns in each of the three plant lines after inoculation with a compatible P. parasitica isolate and after treatment with the SA analog 2,6-dichloroisonicotinic acid. Some of the genes within subclasses showed different requirements for SA accumulation and NIM1/NPR1 activity, depending upon which elicitor was used, indicating that those genes were not coordinately regulated and that the regulatory pathways are more complex than simple linear models would indicate.

Abnormal callose response phenotype and hypersusceptibility to Peronospoara parasitica in defence-compromised arabidopsis nim1-1 and salicylate hydroxylase-expressing plants.

To investigate the impact of induced host defenses on the virulence of a compatible Peronospora parasitica strain on Arabidopsis thaliana, we examined growth and development of this pathogen in nim1-1 mutants and transgenic salicylate hydroxylase plants. These plants are unable to respond to or accumulate salicylic acid (SA), respectively, are defective in expression of systemic acquired resistance (SAR), and permit partial growth of some normally avirulent pathogens. We dissected the P. parasitica life cycle into nine stages and compared its progression through these stages in the defense-compromised hosts and in wild-type plants. NahG plants supported the greatest accumulation of pathogen biomass and conidiophore production, followed by nim1-1 and then wild-type plants. Unlike the wild type, NahG and nim1-1 plants showed little induction of the SAR gene PR-1 after colonization with P parasitica, which is similar to our previous observations. We examined the frequency and morphology of callose deposits around parasite haustoria and found significant differences between the three hosts. NahG plants showed a lower fraction of haustoria surrounded by thick callose encasements and a much higher fraction of haustoria with callose limited to thin collars around haustorial necks compared to wild type, whereas nim1-1 plants were intermediate between NahG and wild type. Chemical induction of SAR in plants colonized by P. parasitica converted the extrahaustorial callose phenotype in NahG to resemble closely the wild-type pattern, but had no effect on nim1-1 plants. These results suggest that extrahaustorial callose deposition is influenced by the presence or lack of SA and that this response may be sensitive to the NIM1/NPR1 pathway. Additionally, the enhanced susceptibility displayed by nim1-1 and NahG plants shows that even wild-type susceptible hosts exert defense functions that reduce disease severity and pathogen fitness.

Harpin induces disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene.

Harpin, the product of the hrpN gene of Erwinia amylovora, elicits the hypersensitive response and disease resistance in many plants. Harpin and known inducers of systemic acquired resistance (SAR) were tested on five genotypes of Arabidopsis thaliana to assess the role of SAR in harpin-induced resistance. In wild-type plants, harpin elicited systemic resistance to Peronospora parasitica and Pseudomonas syringae pv. tomato, accompanied by induction of the SAR genes PR-1 and PR-2. However, in experiments with transgenic Arabidopsis plants containing the nahG gene which prevents accumulation of salicylic acid (SA), harpin neither elicited resistance nor activated SAR gene expression. Harpin also failed to activate SAR when applied to nim1 (non-inducible immunity) mutants, which are defective in responding to SA and regulation of SAR. In contrast, mutants compromised in responsiveness to methyl jasmonate and ethylene developed the same resistance as did wild-type plants. Thus, harpin elicits disease resistance through the NIM1-mediated SAR signal transduction pathway in an SA-dependent fashion. The site of action of harpin in the SAR regulatory pathway is upstream of SA.

Salicylate-independent lesion formation in Arabidopsis lsd mutants.

In many interactions of plants with pathogens, the primary host defense reaction is accompanied by plant cell death at the site of infection. The resulting lesions are correlated with the establishment of an inducible resistance in plants called systemic acquired resistance (SAR), for which salicylic acid (SA) accumulation is a critical signaling event in Arabidopsis and tobacco. In Arabidopsis, the lesions simulating disease (lsd) mutants spontaneously develop lesions in the absence of pathogen infection. Furthermore, lsd mutants express SAR marker genes when lesions are present and are resistant to the same spectrum of pathogens as plants activated for SAR by necrogenic pathogen infection. To assess the epistatic relationship between SA accumulation and cell death, transgenic Arabidopsis unable to accumulate SA due to the expression of the salicylate hydroxylase (nahG) gene were used in crosses with the dominant mutants lsd2 or lsd4. Progeny from the crosses were inhibited for SAR gene expression and disease resistance. However, these progeny retained the spontaneous cell death phenotype similar to siblings not expressing nahG. Because lesions form in the absence of SA accumulation for isd2 and lsd4, a model is suggested in which lesion formation in these two mutants is determined prior to SA accumulation in SAR signal transduction. By contrast, the loss of SAR gene expression and disease resistance in nahG-expressing lsd mutants indicates that these traits are dependent upon SA accumulation in the SAR signal transduction pathway.

Evidence that the Pseudomonas syringae pv. syringae hrp-linked hrmA gene encodes an Avr-like protein that acts in an hrp-dependent manner within tobacco cells.

A 25-kb DNA region, previously cloned from Pseudomonas syringae pv. syringae 61 in cosmid pHIR11, enables nonpathogenic bacteria such as Pseudomonas fluorescens and Escherichia coli to elicit the hypersensitive response (HR) in tobacco (Nicotiana tabacum). hrmA is located within this region, adjacent to a conserved cluster of hrp genes, and is essential for nonpathogens to elicit the HR. DNA sequence analysis suggested that hrmA was the second of two genes in an operon and was preceded by an open reading frame (ORF), ORF1, which is predicted to encode a 10.9-kDa protein. DNA gel blot analysis revealed that sequences hybridizing with a DNA fragment internal to hrmA were absent from P. syringae pv. syringae B728a, P. syringae pv. tabaci 11528, and P. syringae pv. glycinea race 4 U1, but present in P. syringae pv. tomato DC3000. A 2.4-kb BamHI-AvrII fragment carrying hrmA, ORF1, and native regulatory sequences was subcloned into broad-host-range vector pDSK519 and electroporated into P. syringae pv. syringae B728a and P. syringae pv. tabaci 11528. The presence of the hrmA locus had no apparent effect on the ability of P. syringae pv. syringae B728a to cause brown spot of bean, but it caused P. syringae pv. tabaci 11528 to elicit the defense-associated HR rather than disease in N. tabacum cvs. Xanthi N and Xanthi NC and N. clevelandii. Furthermore, N. debeyii, N. glutinosa, N. rustica, and N. tabacum cvs. Petit Havana and Samsun responded with the HR to P. fluorescens(pHIR11). In contrast, N. benthamiana-P. syringae pv. tabaci interactions were unaffected by the presence of HrmA, and P. fluorescens(pHIR11) did not elicit the HR in N. benthamiana. The hrmA ORF was subcloned into pFLAG-CTC, which expressed HrmA with a C-terminal FLAG synthetic epitope fusion. Escherichia coli MC4100 cells carrying the functional hrp cluster and the hrmA-FLAG derivative secreted the HrpZ harpin, but not HrmA-FLAG, to the medium, as indicated by immunoblot analysis with M2 anti-FLAG and polyclonal anti-HrpZ antibodies. The hrmA ORF was also subcloned into plant expression vector pFF19 and then biolistically delivered, along with pFF19G (expressing beta-glucuronidase), into suspension-cultured tobacco cells. Histochemical staining 24 h later revealed substantial beta-glucuronidase activity in cells receiving pFF19G and pFF19 but not in those receiving pFF19G and pFF19-HrmA. Thus, internal production of HrmA was deleterious to tobacco cells.

The Arabidopsis NIM1 protein shows homology to the mammalian transcription factor inhibitor I kappa B.

The NIM1 (for noninducible immunity) gene product is involved in the signal transduction cascade leading to both systemic acquired resistance (SAR) and gene-for-gene disease resistance in Arabidopsis. We have isolated and characterized five new alleles of nim1 that show a range of phenotypes from weakly impaired in chemically induced pathogenesis-related protein-1 gene expression and fungal resistance to very strongly blocked. We have isolated the NIM1 gene by using a map-based cloning procedure. Interestingly, the NIM1 protein shows sequence homology to the mammalian signal transduction factor I kappa B subclass alpha. NF-kappa B/I kappa B signaling pathways are implicated in disease resistance responses in a range of organisms from Drosophila to mammals, suggesting that the SAR signaling pathway in plants is representative of an ancient and ubiquitous defense mechanism in higher organisms.

Recent advances in systemic acquired resistance research--a review.

Little is known about the signal transduction events that lead to the establishment of the broad-spectrum, inducible plant immunity called systemic acquired resistance (SAR). Salicylic acid (SA) accumulation has been shown to be essential for the expression of SAR and plays a key role in SAR signaling. Hydrogen peroxide has been proposed to serve as a second messenger of SA. However, our results do not support such a role in the establishment of SAR. Further elucidation of SAR signal transduction has been facilitated by the identification and characterization of mutants. The lesions simulating disease (lsd). resistance response mutant class exhibits spontaneous lesions similar to those that occur during the hypersensitive response. Interestingly, some lsd mutants lose their lesioned phenotype when SA accumulation is prevented by expression of the nahG gene (encoding salicylate hydroxylase), thereby providing evidence for a feedback loop in SAR signal transduction. Characterization of a mutant non-responsive to SAR activator treatments has provided additional evidence for common signaling components between SAR and gene-for-gene resistance.

Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance.

Plants possess multiple resistance mechanisms that guard against pathogen attack. Among these are inducible systems such as systemic acquired resistance (SAR). SAR is activated by pathogen exposure and leads to an increase in salicylic acid (SA), high-level expression of SAR-related genes, and resistance to a spectrum of pathogens. To identify components of the signal transduction pathways regulating SAR, a mutant screen was developed that uses 2,6-dichloroisonicotinic acid as an activator of SAR gene expression and pathogen resistance, followed by assays for resistance to the fungal pathogen Peronospora parasitica. Mutants from this screen were subsequently examined to assess their defense responses. We describe here a recessive mutation that causes a phenotype of insensitivity to chemical and biological inducers of SAR genes and resistance. These data indicate the existence of a common signaling pathway that couples these diverse stimuli to induction of SAR genes and resistance. Because of its non-inducible immunity phenotype, we call this mutant nim1. Although nim1 plants fail to respond to SA, they retain the ability to accumulate wild-type levels of SA, a probable endogenous signal for SAR. Further, the ability of nim1 plants to support growth of normally incompatible races of a fungal pathogen indicates a role for this pathway in expression of genetically determined resistance, consistent with earlier findings for transgenic plants engineered to break down SA. These results suggest that the wild-type NIM1 gene product functions in a pathway regulating acquired resistance, at a position downstream of SA accumulation and upstream of SAR gene induction and expression of resistance.

Signal transduction in systemic acquired resistance.

Systemic acquired resistance (SAR) is an important component of plant defense against pathogen infection. Accumulation of salicylic acid (SA) is required for the induction of SAR. However, SA is apparently not the translocated signal but is involved in transducing the signal in target tissues. Interestingly, SA accumulation is not required for production and release of the systemic signal. In addition to playing a pivotal role in SAR signal transduction, SA is important in modulating plant susceptibility to pathogen infection and genetic resistance to disease. It has been proposed that SA inhibition of catalase results in H2O2 accumulation and that therefore H2O2 serves as a second messenger in SAR signaling. We find no accumulation of H2O2 in tissues expressing SAR; thus the role of H2O2 in SAR signaling is questionable.

A central role of salicylic Acid in plant disease resistance.

Transgenic tobacco and Arabidopsis thaliana expressing the bacterial enzyme salicylate hydroxylase cannot accumulate salicylic acid (SA). This defect not only makes the plants unable to induce systemic acquired resistance, but also leads to increased susceptibility to viral, fungal, and bacterial pathogens. The enhanced susceptibility extends even to host-pathogen combinations that would normally result in genetic resistance. Therefore, SA accumulation is essential for expression of multiple modes of plant disease resistance.

Arabidopsis mutants simulating disease resistance response.

We describe six Arabidopsis mutants, defining at least four loci, that spontaneously form necrotic lesions on leaves. Lesions resemble those resulting from disease, but occur in the absence of pathogen. In five mutants, lesion formation correlates with expression of histochemical and molecular markers of plant disease resistance responses and with expression of genes activated during development of broad disease resistance in plants (systemic acquired resistance [SAR]). We designate this novel mutant class Isd (for lesions simulating disease resistance response). Strikingly, four Isd mutants express substantial resistance to virulent fungal pathogen isolates. Isd mutants vary in cell type preferences for lesion onset and spread. Lesion formation can be conditional and can be induced specifically by biotic and chemical activators of SAR in Isd1 mutants.

Gene for the ribulose-1,5-bisphosphate carboxylase small subunit protein of the marine chromophyte Olisthodiscus luteus is similar to that of a chemoautotrophic bacterium.

The photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] small subunit protein is encoded by the gene rbcS in the chloroplast genome of the unicellular alga Olisthodiscus luteus. This observation contrasts sharply with that seen in terrestrial plants and green algae, where rbcS is nuclear-localized. In this study, the O. luteus rbcS gene has been sequenced. The predicted primary structure of the protein sequence is 139 amino acids in length and lacks an N-terminal signal sequence. Unexpectedly, the O. luteus rbcS amino acid sequence shows the greatest similarity (56% identity) to that of the chemolithotrophic bacterium Alcaligenes eutrophus. A comparison of the N-terminal amino acid rbcS sequence of A. eutrophus to those of O. luteus and brown alga Fucus species shows extensive sequence similarity (68.3% identity). This observation suggests that the rbcS genes of these organisms are evolutionary homologues and may provide useful information in the study of small-subunit function.

Chloroplast ribosomal DNA organization in the chromophytic alga Olisthodiscus luteus.

There are almost no data describing chloroplast genome organization in chromophytic (chlorophyll a/c) plants. In this study chloroplast ribosomal operon placement and gene organization has been determined for the golden-brown alga Olisthodiscus luteus. Ribosomal RNA genes are located on the chloroplast DNA inverted repeat structure. Nucleotide sequence analysis, demonstrated that in contrast to the larger spacer regions in land plants, the 16S-23S rDNA spacer of O. luteus is only 265 bp in length. This spacer contains tRNA(Ile) and tRNA(Ala) genes which lack introns and are separated by only 3 bp. The sequences of the tRNA genes and 16S and 23S rDNA termini flanking the spacer were examined to determine homology between O. luteus, chlorophytic plant chloroplast DNA, and prokaryotes.