A method that stimulates cholera toxin (CT) production by Vibrio cholerae culture fluid was established.
SummaryA glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs : orf1 , orf2 and orf3 . Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, D orf1 and D orf2 , exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from D orf1 mutant using aspartic N -peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the D orf1 , D orf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule.
Summary The bacterial flagellin (FliC) epitopes flg22 and flgII-28 are microbe-associated molecular patterns (MAMPs). While flg22 is recognized by many plant species via the pattern recognition receptor FLS2, neither the flgII-28 receptor nor the extent of flgII-28 recognition by different plant families is known.Here we tested the significance of flgII-28 as a MAMP and the importance of allelic diversity in flg22 and flgII-28 in plant–pathogen interactions using purified peptides and a Pseudomonas syringae ΔfliC mutant complemented with different fliC alleles.Plant genotype and allelic diversity in flg22 and flgII-28 were found to significantly affect the plant immune response but not bacterial motility. Recognition of flgII-28 is restricted to a number of Solanaceous species. While the flgII-28 peptide does not trigger any immune response in Arabidopsis, mutations in both flg22 and flgII-28 have FLS2-dependent effects on virulence. However, expression of a tomato allele of FLS2 does not confer to Nicotiana benthamiana the ability to detect flgII-28 and tomato plants silenced for FLS2 are not altered in flgII-28 recognition.Therefore, MAMP diversification is an effective pathogen virulence strategy and flgII-28 appears to be perceived by a yet unidentified receptor in the Solanaceae although it has an FLS2-dependent virulence effect in Arabidopsis.
We characterized pharmacologically the hypersensitive cell death of tobacco BY-2 cells that followed treatments with Escherichia coli preparations of INF1, the major secreted elicitin of the late blight pathogen Phytophthora infestans. INF1 elicitin treatments resulted in fragmentation and 180 bp laddering of tobacco DNA as early as 3 h post-treatment. INF1 elicitin also induced rapid accumulation of H 2 O 2 typical of oxidative burst, and the expression of defense genes such as phenylalanine ammonia-lyase (PAL) gene at 1 h and 3 h after elicitin treatment, respectively. To investigate the involvement of the oxidative burst and/or the expression of defense genes in the signal transduction pathways leading to hypersensitive cell death, we analyzed the effect of several chemical inhibitors of signal transduction pathways on the various responses. The results indicated that (a) the cell death required serine proteases, Ca 21 and protein kinases, (b) the oxidative burst was involved in Ca 21 and protein kinase mediated pathways, but elicitin-induced AOS was neither necessary nor sufficient for cell death and PAL gene expression, and (c) the signaling pathway of PAL gene expression required protein kinases. These results suggest that the three signal transduction pathways leading to cell death, oxidative burst and expression of defense genes branch in the early stages that follow elicitin recognition by tobacco cells.
BackgroundThe Solanaceae family includes several economically important vegetable crops. The tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance.ResultsTo accelerate the progress in tomato genomics, we developed a collection of fully-sequenced 13,227 Micro-Tom full-length cDNAs. By checking redundant sequences, coding sequences, and chimeric sequences, a set of 11,502 non-redundant full-length cDNAs (nrFLcDNAs) was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.061%.ConclusionThe collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. Full-length cDNA sequences and their annotations are provided in the database KaFTom http://www.pgb.kazusa.or.jp/kaftom/ via the website of the National Bioresource Project Tomato http://tomato.nbrp.jp.
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