Biomedical subjects
Publications and source records attributed to J Ryals.
Sequencing of the Arabidopsis thaliana genome is complete. The analytical tools for determining gene function by altering and monitoring gene expression are relatively well developed, and are generating large volumes of valuable data. Recent advances in techniques for the analysis of small molecules allow researchers to apply biochemical profiling as another powerful approach to functional genomics and metabolic research.
The NIM1 (for noninducible immunity, also known as NPR1) gene is required for the biological and chemical activation of systemic acquired resistance (SAR) in Arabidopsis. Overexpression of NIM1 in wild-type plants (hereafter referred to as NIM1 plants or lines) results in varying degrees of resistance to different pathogens. Experiments were performed to address the basis of the enhanced disease resistance responses seen in the NIM1 plants. The increased resistance observed in the NIM1 lines correlated with increased NIM1 protein levels and rapid induction of PR1 gene expression, a marker for SAR induction in Arabidopsis, following pathogen inoculation. Levels of salicylic acid (SA), an endogenous signaling molecule required for SAR induction, were not significantly increased compared with wild-type plants. SA was required for the enhanced resistance in NIM1 plants, however, suggesting that the effect of NIM1 overexpression is that plants are more responsive to SA or a SA-dependent signal. This hypothesis is supported by the heightened responsiveness that NIM1 lines exhibited to the SAR-inducing compound benzo(1,2,3)-thiadiazole-7-car-bothioic acid S-methyl ester. Furthermore, the increased efficacy of three fungicides was observed in the NIM1 plants, suggesting that a combination of transgenic and chemical approaches may lead to effective and durable disease-control strategies.
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We have used an antisense expression technology in Arabidopsis based on the yeast GAL4/UAS transactivation system (Guyer et al., Genetics, 1998; 149:633-639) to reduce levels of protoporphyrinogen IX oxidase (PPO), the last common enzyme of the biosynthesis of the haem group and chlorophyll. Plants expressing the antisense PPO gene presented growth alterations and their leaves showed necrotic lesions that appeared similar to lesions characteristic of the pathogen-induced hypersensitive reaction, and seen in the so-called lesion-mimic mutants. Plants expressing the antisense gene also had high endogenous salicylic acid levels, constitutive expression of the PR-1 gene, and were resistant to Peronospora parasitica, consistent with the activation of systemic acquired resistance (SAR). Treatment of wild-type plants with sublethal concentrations of herbicides that inhibit PPO also induced defence responses that conferred enhanced tolerance to P. parasitica. This effect was not observed in NahG and nim1 plants, which are compromised in their ability to activate SAR. These results demonstrate that genetic or chemical disruption of a metabolic pathway can lead to the induction of a set of defence responses including activation of SAR.
Wheat cDNAs that encode proteins PR-1.1 and PR-1.2 were cloned. Deduced amino acid sequences were homologous to those of pathogen-induced, basic PR-1 proteins from plants. Although expression of PR1.1 and PR1.2 genes was induced upon infection with either compatible or incompatible isolates of the fungal pathogen Erysiphe graminis, these genes did not respond to activators of systemic acquired resistance (SAR), such as salicylic acid (SA), benzothiadiazole (BTH), or isonicotinic acid (INA).
The Arabidopsis PR-1 gene is one of a suite of genes induced co-ordinately during the onset of systemic acquired resistance (SAR), a plant defense pathway triggered by pathogen infection or exogenous application of chemicals such as salicylic acid (SA) and 2,6-dichloroisonicotinic acid (INA). We have characterized cis-acting regulatory elements in the PR-1 promoter involved in INA induction using deletion analysis, linker-scanning mutagenesis, and in vivo footprinting. Compared to promoter fragments of 815 bp or longer (which show greater than 10-fold inducibility after INA treatment), induction of a 698 bp long promoter fragment is reduced by half and promoter fragments of 621 bp or shorter have lost all inducibility. Additionally, two 10-bp linker-scanning mutations centered at 640 bp and 610 bp upstream from the transcription initiation site are each sufficient to abolish chemical inducibility of a GUS reporter fusion. The -640 linker-scanning mutation encompasses a region highly homologous to recognition sites for transcription factors of the basic leucine zipper class, while the -610 linker-scanning mutation contains a sequence similar to a consensus recognition site for the transcription factor NF-kappa B. Furthermore, several inducible in vivo footprints located at or nearby these motifs demonstrate significant and highly reproducible changes in DNA accessibility following SAR induction. This in vivo signature of protein-DNA interactions after INA induction is tightly correlated with the functionally important regions of the promoter identified by mutation analysis.
Touch stimulation of wheat (Triticum aestivum L.) seedlings led to a strong and dose-dependent increase in the level of lipoxygenase mRNA transcripts. The touch-induced response occurred within 1 h and was transient. A similar response was observed after wind treatment and wounding. The mechanical strain-regulated lipoxygenase might translate mechanical strain into lipoxygenase pathway-dependent growth responses.
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.
Benzothiadiazole (BTH) is a novel chemical activator of disease resistance in tobacco, wheat and other important agricultural plants. In this report, it is shown that BTH works by activating SAR in Arabidopsis thaliana. BTH-treated plants were resistant to infection by turnip crinkle virus, Pseudomonas syringae pv 'tomato' DC3000 and Peronospora parasitica. Chemical treatment induced accumulation of mRNAs from the SAR-associated genes, PR-1, PR-2 and PR-5. BTH treatment induced both PR-1 mRNA accumulation and resistance against P. parasitica in the ethylene response mutants, etr1 and ein2, and in the methyl jasmonate-insensitive mutant, jar1, suggesting that BTH action is independent of these plant hormones. BTH treatment also induced both PR-1 mRNA accumulation and P. parasitica resistance in transgenic Arabidopsis plants expressing the nahG gene, suggesting that BTH action does not require salicylic acid accumulation. However, because BTH-treatment failed to induce either PR-1 mRNA accumulation or P. parasitica resistance in the non-inducible immunity mutant, nim1, it appears that BTH activates the SAR signal transduction pathway.
Systemic acquired resistance is an important component of the disease resistance repertoire of plants. In this study, a novel synthetic chemical, benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), was shown to induce acquired resistance in wheat. BTH protected wheat systemically against powdery mildew infection by affecting multiple steps in the life cycle of the pathogen. The onset of resistance was accompanied by the induction of a number of newly described wheat chemically induced (WCI) genes, including genes encoding a lipoxygenase and a sulfur-rich protein. With respect to both timing and effectiveness, a tight correlation existed between the onset of resistance and the induction of the WCI genes. Compared with other plant activators, such as 2,6-dichloroisonicotinic acid and salicylic acid, BTH was the most potent inducer of both resistance and gene induction. BTH is being developed commercially as a novel type of plant protection compound that works by inducing the plant's inherent disease resistance mechanisms.
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.
Transgenic tobacco plants that express the bacterial nahG gene encoding salicylate hydroxylase have been shown to accumulate very little salicylic acid and to be defective in their ability to induce systemic acquired resistance (SAR). In recent experiments using transgenic NahG tobacco and Arabidopsis plants, we have also demonstrated that salicylic acid plays a central role in both disease susceptibility and genetic resistance. In this paper, we further characterize tobacco plants that express the salicylate hydroxylase enzyme. We show that tobacco mosaic virus (TMV) inoculation of NahG tobacco leaves induces the accumulation of the nahG mRNA in the pathogen infected leaves, presumably due to enhanced stabilization of the bacterial mRNA. SAR-associated genes are expressed in the TMV-infected leaves, but this is localized to the area surrounding necrotic lesions. Localized acquired resistance (LAR) is not induced in the TMV-inoculated NahG plants suggesting that LAR, like SAR, is dependent on SA accumulation. When SA is applied to nahG-expressing leave's SAR gene expression does not result. We have confirmed earlier reports that the salicylate hydroxylase enzyme has a narrow substrate specificity and we find that catechol, the breakdown product of salicylic acid, neither induces acquired resistance nor prevents the SA-dependent induction of the SAR genes.
Significant insight has been gained in the past year into the roles of salicylic acid (SA) in plant-pathogen interactions. The ability to accumulate SA has been shown to be essential for systemic acquired resistance in tobacco plants. Further experiments have shown that SA is apparently not a systemic, vascular-mobile signal, but rather is required for signal transduction at the local level. Its mode of action may include inhibition of catalase activity, leading to increased levels of hydrogen peroxide.
cDNA clones encoding imidazoleglycerolphosphate dehydratase (IGPD; EC 4.2.1.19) from Arabidopsis thaliana were isolated by complementation of a bacterial auxotroph. The predicted primary translation product shared significant identity with the corresponding sequences from bacteria and fungi. As in yeast, the plant enzyme is monofunctional, lacking the histidinol phosphatase activity present in the Escherichia coli protein. IGPD mRNA was present in major organs at all developmental stages assayed. The Arabidopsis genome appears to contain two genes encoding this enzyme, based on DNA gel blot and polymerase chain reaction analysis.
With the aid of partial amino acid sequences determined for cinnamate 4-hydroxylase (P450C4H) purified from mung bean seedlings, two cDNA clones were isolated and their inserts were completely sequenced. The nucleotide sequences of the two clones were nearly identical and contained an open reading frame predicted to encode a polypeptide consisting of 505 amino acid residues. The partial sequences determined from the purified P450C4H closely corresponded to the primary structures deduced from the cDNA sequences. This is the first isolation of cDNA clones encoding a higher plant P450 possessing clear physiological activity. Comparison to known cytochromes P450 indicated that P450C4H belongs to a novel P450 gene family.
Pathogenesis-related protein-1a (PR-1a) is a protein of unknown function that is strongly induced during the onset of systemic acquired resistance (SAR) in tobacco. The expression of PR-1a is under complex regulation that is controlled at least partially by the rate of transcription. In this study, we demonstrated that 661 bp of 5' flanking DNA was sufficient to impart tobacco mosaic virus and salicylic acid inducibility to a reporter gene. The PR-1a promoter did not respond significantly to treatments with either auxin or cytokinin. Experiments with the protein synthesis inhibitor cycloheximide indicated that protein synthesis is required for salicylate-dependent mRNA accumulation. At flowering, the PR-1a gene was expressed primarily in the mesophyll and epidermal tissues of the leaf blade and the sepals of the flower. Several artifacts, most importantly ectopic expression in pollen, were associated with the use of the beta-glucuronidase reporter gene.
Complementary DNA clones encoding acidic and basic isoforms of the class III chitinase were isolated from Nicotiana tabacum. The clones share ca. 65% identity, are equally homologous to the class III chitinases from cucumber and Arabidopsis, and are members of small gene families in tobacco. An acidic class III chitinase was purified from the intercellular fluid of tobacco leaves infected with tobacco mosaic virus (TMV). Partial amino acid sequencing of the protein confirmed that it was encoded by one of the cDNA clones. The mRNAs of the class III chitinases are coordinately expressed in response to TMV infection, both in infected and uninfected tissue. The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.
The fungicidal class I chitinases are believed to be important in the induced defense response of plants. We isolated and partially characterized genomic clones representing two members, CHN14 and CHN50, of the gene subfamily encoding these enzymes in Nicotiana tabacum L. cv. Havana 425. The coding sequences of genes CHN14, CHN50, and CHN48, which was cloned earlier, are identical at 79-95% of the positions. Tobacco is an amphidiploid species derived from ancestors most closely related to the present-day species N. sylvestris and N. tomentosiformis. Southern analysis of genomic DNA, comparison of deduced amino acid sequences, and partial sequencing of the purified enzymes suggest that the gene pairs CHN48/CHN50 and CHN14/CHN14' are homeologues. Gene CHN48, which encodes chitinase A (Mr ca. 34 kDa), and gene CHN14 are derived from N. tomentosiformis; whereas gene CHN50, which encodes chitinase B (Mr ca. 32 kDa), and gene CHN14' are derived from N. sylvestris. Class I chitinases are induced in leaves of plants treated with ethylene or infected with the fungal pathogen Cercospora nicotianae and in cultured cells transferred to medium without added auxin and cytokinin. RNase protection assays show that under these conditions transcripts encoded by the homeologues CHN48 and CHN50 account for greater than 90% of the total chitinase mRNA. The less abundant transcript, CHN48, consistently showed a greater degree of induction than CHN50. Expression of the homeologues CHN14 and CHN14' represented less than 10% of the total chitinase mRNA. They showed a pattern of hormonal regulation similar to CHN48 and CHN50, but transcripts of these genes were not detected in leaves infected with C. nicotianae. Therefore the two sets of homeologues are regulated in the same way by hormones and respond differently to infection by a pathogen.