Maize rhm1 resistance to Bipolaris maydis is associated with few differences in pathogenesis-related proteins and global mRNA profiles.

The maize rhm1 mutant resists Bipolaris maydis, the causal agent of Southern corn leaf blight, by producing small necrotic lesions surrounded by chlorotic haloes. The rhm1 and wild-type lesions contain viable fungus in equal frequency, but fungal sporulation was markedly inhibited on rhm1. The levels of the pathogenesis-related (PR) proteins chitinase, PR1, and peroxidase differ little between rhm1 and wild type, with or without B. maydis inoculation. The global mRNA profiles surveyed revealed hundreds of cDNA fragments that were twofold or more induced or suppressed in rhm1 and wild-type plants following B. maydis inoculation. Nonetheless, between rhm1 and wild type, only 0.4 to 0.7% of the cDNA fragments were expressed differentially by twofold or more. Among the up-regulated genes in rhm1 was beta-glucosidase glu1, which prompted a test of whether rhm1 resistance depends upon the antimicrobial compound 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one or other hydroxamic acids whose glucosyl conjugates are preferred substrates for the Glu1 enzyme. Double mutants of rhm1 and bx1, a hydroxamic acid-deficient mutant, indicate that rhm1 resistance is hydroxamic acid independent. The rhm1 resistance presently appears to operate via a mechanism unlike those of previously described resistance genes.

Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco.

Salicylic acid (SA) has been shown to act as a signal molecule that is produced by many plants subsequent to the recognition of potentially pathogenic microbes. Increases in levels of SA often trigger the activation of plant defenses and can result in increased resistance to subsequent challenge by pathogens. We observed that the polyketide 6-methylsalicylic acid (6-MeSA), a compound that apparently is not endogenous to tobacco, can mimic SA. Tobacco leaves treated with 6-MeSA show enhanced accumulation of the pathogenesis-related (PR) proteins PR1, beta-1,3-glucanase, and chitinase and also develop increased resistance to tobacco mosaic virus. We transformed tobacco with 6msas, the 6-methylsalicylic acid synthase (6MSAS) gene from Penicillium patulum, to generate plants that constitutively accumulate 6-MeSA. Analysis of primary transformants and the first generation progeny of 6MSAS tobacco revealed that plants can be engineered to accumulate significant amounts of 6-MeSA as a conjugate. Levels of total 6-MeSA increased with plant age. Increased 6-MeSA accumulation correlated with increased levels of PR1 and chitinase proteins and resulted in enhanced resistance of NN genotype 6MSAS tobacco to tobacco mosaic virus. Our results demonstrate that a multistep biosynthetic pathway can be engineered into plants using a single fungal polyketide synthase gene. The functional expression of 6msas can be used to activate disease resistance pathways that normally are induced by SA.

Prohibitins, stomatins, and plant disease response genes compose a protein superfamily that controls cell proliferation, ion channel regulation, and death.

Prohibitins, stomatins, and a group of plant defense response genes are demonstrated to belong to a novel protein superfamily. This superfamily is bound by similar primary and secondary predicted protein structures and hydropathy profiles. A PROSITE-formatted regular expression was generated that is highly predictive for identifying members of this superfamily using PHI-BLAST. The superfamily is named PID (proliferation, ion, and death) because prohibitins are involved in proliferation and cell cycle control, stomatins are involved in ion channel regulation, and the plant defense-related genes are involved in cell death. The plant defense gene family is named HIR (hypersensitive induced reaction) because its members are associated with hypersensitive reactions involving cell death and pathogen resistance. For this study, eight novel maize genes were introduced: four closely related to prohibitins (Zm-phb1, Zm-phb2, Zm-phb3, and Zm-phb4), one to stomatins (Zm-stm1), and three to a gene implicated in plant disease responses (Zm-hir1, Zm-hir2, and Zm-hir3). The maize Zm-hir3 gene transcript is up-regulated in a disease lesion mimic mutation (Les9), supporting a role in maize defense responses. Members of this gene superfamily are involved in diverse functions, but their structural similarity suggests a conserved molecular mechanism, which we postulate to be ion channel regulation.

A porphyrin pathway impairment is responsible for the phenotype of a dominant disease lesion mimic mutant of maize.

The maize lesion mimic gene Les22 is defined by dominant mutations and characterized by the production of minute necrotic spots on leaves in a developmentally specified and light-dependent manner. Phenotypically, Les22 lesions resemble those that are triggered during a hypersensitive disease resistance response of plants to pathogens. We have cloned Les22 by using a Mutator-tagging technique. It encodes uroporphyrinogen decarboxylase (UROD), a key enzyme in the biosynthetic pathway of chlorophyll and heme in plants. Urod mutations in humans are also dominant and cause the metabolic disorder porphyria, which manifests itself as light-induced skin morbidity resulting from an excessive accumulation of photoexcitable uroporphyrin. The phenotypic and genetic similarities between porphyria and Les22 along with our observation that Les22 is also associated with an accumulation of uroporphyrin revealed what appears to be a case of natural porphyria in plants.

Benzoic acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid biosynthesis.

Benzoic acid 2-hydroxylase (BA2H) catalyzes the biosynthesis of salicylic acid from benzoic acid. The enzyme has been partially purified and characterized as a soluble protein of 160 kDa. High-efficiency in vivo labeling of salicylic acid with 18O2 suggested that BA2H is an oxygenase that specifically hydroxylates the ortho position of benzoic acid. The enzyme was strongly induced by either tobacco mosaic virus inoculation or benzoic acid infiltration of tobacco leaves and it was inhibited by CO and other inhibitors of cytochrome P450 hydroxylases. The BA2H activity was immunodepleted by antibodies raised against SU2, a soluble cytochrome P450 from Streptomyces griseolus. The anti-SU2 antibodies immunoprecipitated a radiolabeled polypeptide of around 160 kDa from the soluble protein extracts of L-[35S]-methionine-fed tobacco leaves. Purified BA2H showed CO-difference spectra with a maximum at 457 nm. These data suggest that BA2H belongs to a novel class of soluble, high molecular weight cytochrome P450 enzymes.

Pathway of Salicylic Acid Biosynthesis in Healthy and Virus-Inoculated Tobacco.

Salicylic acid (SA) is a likely endogenous regulator of localized and systemic disease resistance in plants. During the hypersensitive response of Nicotiana tabacum L. cv Xanthi-nc to tobacco mosaic virus (TMV), SA levels rise dramatically. We studied SA biosynthesis in healthy and TMV-inoculated tobacco by monitoring the levels of SA and its likely precursors in extracts of leaves and cell suspensions. In TMV-inoculated leaves, stimulation of SA accumulation is accompanied by a corresponding increase in the levels of benzoic acid. 14C-Tracer studies with cell suspensions and mock-or TMV-inoculated leaves indicate that the label moves from trans-cinnamic acid to SA via benzoic acid. In healthy and TMV-inoculated tobacco leaves, benzoic acid induced SA accumulation. o-Coumaric acid, which was previously reported as a possible precursor of SA in other species, did not increase SA levels in tobacco. In healthy tobacco tissue, the specific activity of newly formed SA was equal to that of the supplied [14C]benzoic acid, whereas in TMV-inoculated leaves some isotope dilution was observed, presumably because of the increase in the pool of endogenous benzoic acid. We observed accumulation of pathogen-esis-related-1 proteins and increased resistance to TMV in benzoic acid- but not in o-coumaric acid-treated tobacco leaves. This is consistent with benzoic acid being the immediate precursor of SA. We conclude that in healthy and virus-inoculated tobacco, SA is formed from cinnamic acid via benzoic acid.

Induction of Benzoic Acid 2-Hydroxylase in Virus-Inoculated Tobacco.

Salicylic acid (SA) plays an important role in the induction of plant resistance to pathogens. An accompanying article (N. Yalpani, J. Leon, M.A. Lawton, I. Raskin [1993] Plant Physiol 103: 315-321) shows that SA is synthesized via the decarboxylation of cinnamic acid to benzoic acid (BA), which is, in turn, hydroxylated to SA. Leaf extracts of tobacco (Nicotiana tabacum L. cv Xanthi-nc) catalyze the 2-hydroxylation of BA to SA. The monooxygenase catalyzing this reaction, benzoic acid 2-hydroxylase (BA2H), required NAD(P)H or reduced methyl viologen as an electron donor. BA2H activity was detected in healthy tobacco leaf extracts (1-2 nmol h-1 g-1 fresh weight) and was significantly increased upon inoculation with tobacco mosaic virus (TMV). This increase paralleled the levels of free SA in the leaves. Induction of BA2H activity was restricted to tissue expressing a hypersensitive response at 24[deg]C. TMV induction of BA2H activity and SA accumulation were inhibited when inoculated tobacco plants were incubated at 32[deg]C. However, when inoculated plants were incubated for 4 d at 32[deg]C and then transferred to 24[deg]C, they showed a 15-fold increase in BA2H activity and a 65-fold increase in free SA content compared with healthy plants incubated at 24[deg]C. Treatment of leaf tissue with the protein synthesis inhibitor cycloheximide blocked the induction of BA2H activity by TMV. The effect of TMV inoculation on BA2H could be duplicated by infiltrating leaf discs of healthy plants with BA. This response was observed even when applied levels of BA were much lower than the levels observed in vivo after virus inoculation. Feeding tobacco leaves with phenylalanine, cinnamic acid, or o-coumaric acid (putative precursors of SA) failed to trigger the induction of BA2H activity. BA2H appears to be a pathogen-inducible protein with an important regulatory role in SA accumulation during the development of induced resistance to TMV in tobacco.

Salicylic acid: a systemic signal in induced plant disease resistance.

Some plants respond to infection by pathogens with both localized and systemic resistance responses. These prevent the spread of the disease-causing organism and reduce the severity of a subsequent infection. Recent evidence suggests that systemic increases in the host's salicylic acid levels act as a signal for the activation of at least some of these induced defenses.

Salicylic Acid induces cyanide-resistant respiration in tobacco cell-suspension cultures.

Cyanide-resistant, alternative respiration in Nicotiana tabacum L. cv Xanthi-nc was analyzed in liquid suspension cultures using O(2) uptake and calorimetric measurements. In young cultures (4-8 d after transfer), cyanide inhibited O(2) uptake by up to 40% as compared to controls. Application of 20 mum salicylic acid (SA) to young cells increased cyanide-resistant O(2) uptake within 2 h. Development of KCN resistance did not affect total O(2) uptake, but was accompanied by a 60% increase in the rate of heat evolution from cells as measured by calorimetry. This stimulation of heat evolution by SA was not significantly affected by 1 mm cyanide, but was reduced by 10 mm salicylhydroxamic acid (SHAM), an inhibitor of cyanide-resistant respiration. Treatment of SA-induced or uninduced cells with a combination of cyanide and SHAM blocked most of the O(2) consumption and heat evolution. Fifty percent of the applied SA was taken up within 10 min, with most of the intracellular SA metabolized in 2 h. 2,6-Dihydroxybenzoic and 4-hydroxybenzoic acids also induced cyanide-resistant respiration. These data indicate that in tobacco cell-suspension culture, SA induces the activity and the capacity of cyanide-resistant respiration without affecting the capacity of the cytochrome c respiration pathway.

Induction of UDP-Glucose:Salicylic Acid Glucosyltransferase in Oat Roots.

A UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) (GTase) from oat (Avena sativa L. cv Dal) root extracts was assayed in vitro using [(14)C]salicylic acid (SA) and an ion exchange column to separate SA from beta-glucosylsalicylic acid. The GTase, present at a very low constitutive level, was inducible to 23 times the constitutive level. When excised roots were exposed to SA at pH 6.5, the specific activity of the enzyme increased within 1.5 h, peaked after 8 to 10 h, and then declined. The increase in specific activity depended on the concentration of SA in the induction medium. Among 16 phenolics and phenolic derivatives tested, GTase induction showed high specificity toward SA and acetylsalicylic acid. Specific activity of the enzyme was induced to higher levels in roots from 7-d-old seedlings than roots from younger plants. GTase activity was less inducible in basal compared with median or apical root sections. Induction of GTase activity was a result of de novo RNA and protein synthesis. Candidate peptides for the GTase were identified by comparison of two-dimensional electrophoresis gels of proteins labeled with [(35)S]methionine during incubation of roots in the presence or the absence of SA and a gel of a partially purified GTase preparation.

Partial purification and properties of an inducible uridine 5'-diphosphate-glucose-salicylic Acid glucosyltransferase from oat roots.

A salicylic acid (SA)-inducible uridine 5'-diphosphate (UDP)-glucose:SA 3-O-glucosyltransferase was extracted from oat (Avena sativa L. cv Dal) roots. Reverse phase high-performance liquid chromatography or anion exchange chromatography was used to separate SA from the product, beta-O-d-glucosylsalicylic acid. The soluble enzyme was purified 176-fold with 5% recovery using a combination of pH fractionation, anion exchange, gel filtration, and chromatofocusing chromatography. The partially purified protein had a native molecular weight of about 50,000, an apparent isoelectric point at pH 5.0, and maximum activity at pH 5.5. The enzyme had a K(m) of 0.28 mm for UDP-glucose and was highly specific for this sugar donor. More than 20 hydroxybenzoic and hydroxycinnamic acid derivatives were assayed as potential glucose acceptors. UDP-glucose:SA 3-O-glucosyltransferase activity was highly specific toward SA (K(m) = 0.16 mm). The enzyme was inhibited by UDP and uridine 5'-triphosphate but not by up to 7.5 mm uridine 5'-monophosphate.

Localization, conjugation, and function of salicylic acid in tobacco during the hypersensitive reaction to tobacco mosaic virus.

Salicylic acid (SA) is hypothesized to be a natural signal that triggers the systemic induction of pathogenesis-related proteins and disease resistance in tobacco. When Xanthi-nc (NN genotype) tobacco was inoculated with tobacco mosaic virus (TMV) there was an increase in endogenous SA in both inoculated and virus-free leaves. The highest levels of SA were detected in and around necrotic lesions that formed in response to TMV. Chemical and enzymatic hydrolysis of extracts from TMV-inoculated leaves demonstrated the presence of a SA conjugate tentatively identified as O-beta-D-glucosyl-SA. The SA conjugate was detected only in leaves that contained necrotic lesions and was not detected in phloem exudates or uninoculated leaves of TMV-inoculated Xanthi-nc tobacco. When exogenous SA was fed to excised tobacco leaves, it was metabolized within 10 hr. However, this reduction in free SA did not prevent the subsequent accumulation of the PR-1 family of pathogenesis-related proteins. The absence of SA accumulation in TMV-inoculated tobacco plants incubated at 32 degrees C was not a result of the glucosylation of SA. The addition of SA to the medium elevated levels of SA in the leaves of virus-free tobacco grown hydroponically. Increasing the endogenous level of SA in leaves to those naturally observed during systemic acquired resistance resulted in increased resistance to TMV, expressed as a reduction in lesion area. These data further support the hypothesis that SA is a likely natural inducer of pathogenesis-related proteins and systemic acquired resistance in TMV-inoculated Xanthi-nc tobacco.

Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco.

Systemic induction of pathogenesis-related (PR) proteins in tobacco, which occurs during the hypersensitive response to tobacco mosaic virus (TMV), may be caused by a minimum 10-fold systemic increase in endogenous levels of salicylic acid (SA). This rise in SA parallels PR-1 protein induction and occurs in TMV-resistant Xanthi-nc tobacco carrying the N gene, but not in TMV-susceptible (nn) tobacco. By feeding SA to excised leaves of Xanthi-nc (NN) tobacco, we have shown that the observed increase in endogenous SA levels is sufficient for the systemic induction of PR-1 proteins. TMV infection became systemic and Xanthi-nc plants failed to accumulate PR-1 proteins at 32 degrees C. This loss of hypersensitive response at high temperature was associated with an inability to accumulate SA. However, spraying leaves with SA induced PR-1 proteins at both 24 and 32 degrees C. SA is most likely exported from the primary site of infection to the uninfected tissues. A computer model predicts that SA should move rapidly in phloem. When leaves of Xanthi-nc tobacco were excised 24 hr after TMV inoculation and exudates from the cut petioles were collected, the increase in endogenous SA in TMV-inoculated leaves paralleled SA levels in exudates. Exudation and leaf accumulation of SA were proportional to TMV concentration and were higher in light than in darkness. Different components of TMV were compared for their ability to induce SA accumulation and exudation: three different aggregation states of coat protein failed to induce SA, but unencapsidated viral RNA elicited SA accumulation in leaves and phloem. These results further support the hypothesis that SA acts as an endogenous signal that triggers local and systemic induction of PR-1 proteins and, possibly, some components of systemic acquired resistance in NN tobacco.

Competition for in vitro [h]gibberellin a(4) binding in cucumber by substituted phthalimides: comparison with in vivo gibberellin-like activity.

Certain N-substituted phthalimides (NSPs) have gibberellin (GA)-like activity in a number of GA bioassays. The interaction between representative NSPs and a protein fraction from cucumber (Cucumis sativus L.) hypocotyls that has GA-binding characteristics consistent with those expected of GA receptors was studied. Analysis of in vitro equilibrium saturation data indicated the presence of only one class of high affinity [(3)H]GA(4) binding sites (K(d) approximately 30 nanomolar, n = 0.25 picomole per milligram of protein). In the presence of 6 or 60 micromolar 1-[3-chlorophthalimido]-cyclohexanecarboximide (AC-94,377), the K(d) for [(3)H]GA(4) increased, whereas the maximum number of saturable [(3)H]GA(4) binding sites did not change significantly. The dissociation of [(3)H]GA(4) from its binding sites was complex and was best described by a bi-exponential equation. AC-94,377 did not affect the rates of [(3)H]GA(4) dissociation from its binding sites. These results implied that AC-94,377 and [(3)H]GA(4) compete for binding to the same sites. A correlation was observed between the activity of over 20 NSPs in the cucumber hypocotyl bioassay and their in vitro affinity for the GA binding sites. Our observations lend further support to the notion that certain GA binding proteins in cucumber cytosol are GA receptors and also provide a molecular explanation for the GA-like in vivo activity of some NSPs.

Competition for in vitro [h]gibberellin a(4) binding in cucumber by gibberellins and their derivatives.

The gibberellin (GA) binding properties of a cytosol fraction from hypocotyls of cucumber (Cucumis sativus L. cv National Pickling) were examined using a DEAE filter paper assay, [(3)H]GA(4), and over 20 GAs, GA derivatives and other growth regulators. The results demonstrate structural specificity of the binding protein for gamma-lactonic C-19 GAs with a 3 beta-hydroxyl and a C-6 carboxyl group. Additional hydroxylations of the A, C, or D ring of the ent-gibberellane skeleton and methylation of the C-6 carboxyl impede or abolish binding affinity. Bioassay data are generally supported by the in vitro results but significantly GA(9) and GA(36), both considered to be precursors of GA(4) in cucumber, show no affinity for the binding protein. The results are discussed in relation to the active site of the putative GA(4) receptor in cucumber.