Nasser Yalpani

Ultraviolet light and ozone stimulate accumulation of salicylic acid, pathogenesis-related proteins and virus resistance in tobacco

In tobacco (Nicotiana tabacum L. cv. Xanthinc), salicylic acid (SA) levels increase in leaves inoculated by necrotizing pathogens and in healthy leaves located above the inoculated site. Systemic SA increase may trigger disease resistance and synthesis of pathogenesis-related proteins (PR proteins). Here we report that ultraviolet (UV)-C light or ozone induced biochemical responses similar to those induced by necrotizing pathogens. Exposure of leaves to UV-C light or ozone resulted in a transient ninefold increase in SA compared to controls. In addition, in UV-light-irradiated plants, SA increased nearly fourfold to 0.77 μg·g−1 fresh weight in leaves that were shielded from UV light. Increased SA levels were accompanied by accumulation of an SA conjugate and by an increase in the activity of benzoic acid 2-hydroxylase which catalyzes SA biosynthesis. In irradiated and in unirradiated leaves of plants treated with UV light, as well as in plants fumigated with ozone, PR proteins 1a and 1b accumulated. This was paralleled by the appearance of induced resistance to a subsequent challenge with tobacco mosaic virus. The results suggest that UV light, ozone fumigation and tobacco mosaic virus can activate a common signal-transduction pathway that leads to SA and PR-protein accumulation and increased disease resistance.

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.

Signal molecules in systemic plant resistance to pathogens and pests

Alexander J. Enyedi, Nasser Yaipani, Paul Silverman, and iiya Raskin AgBiotech Center Cook College Rutgers University New Brunswick, New Jersey 08903-0231 introduction Vertebrate animals possess an inducible antigen-anti- body immune system that acts as a defense against dis- ease. it is a lesser known fact that plants can also be immunized against disease-causing pathogens or feeding pests (Kuc, 1982). This phenomenon is the result of the development of systemic acquired resistance (SAR), a term coined by Ross (1981 b) to describe an increase in resistance to subsequent pathogen attack in inoculated and uninoculated parts of the plant. SAR in plants was documented almost 60 years ago (Chester, 1933) and fur- ther characterized in 1952 for Sweet William (Dianthus barbatus L.) infected by a carnation mosaic virus (Gilpa- trick and Weintraub, 1952). in this review, we extend the definition of SAR to include any form of resistance that develops after localized attack by viral, bacterial, or fungai pathogens or by invertebrate pests. SAR to pathogens usually develops after the appear- ance of a necrotic lesion around the inoculation site. This localized ceil suicide is called the hypersensitive response (HR). While the HR effectively traps pathogens in and around lesions, it makes the whole plant more resistant to a wide range of disease-causing microorganisms (Mad- amanchi and Kuc, 1991; White and Antoniw, 1991). For example, SAR induced by necrotizing viruses develops in uninfected tissues two to three days after inoculation, lasts for several weeks, and may provide protection against pathogenic bacteria, fungi, and other viruses. increased resistance to insect feeding was also observed in plants previously damaged by chewing pests or mechanical wounding. Although not as extensively documented as SAR to pathogens, SAR to pests was shown in some cases to be an effective deterrent to continued herbivory (re- viewed in Tailamy and Raupp, 1991). SAR provides the third and final line of plant defense against pathogens and pests. The first line consists of genetically inherited resistance mechanisms that make plants constitutiveiy resistant to the majority of pathogenic organisms and pests present in the environment. The sec- ond line of defense is activated in the immediate vicinity of the infected or wounded sites in an attempt to prevent the spread of the pathogen throughout the plant or to deter insect feeding. These local resistance responses develop more rapidly than SAR and involve ceil wall and cuticle strengthening, synthesis of toxins (phytoaiexins), antifeed- ants, and the production of defense-related proteins, which include the pathogenesis-related (PR) proteins de- scribed below. In addition to long-distance signal moie-

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.

Elucidation of the Final Reactions of DIMBOA-Glucoside Biosynthesis in Maize: Characterization of Bx6 and Bx7

Benzoxazinoids were identified in the early 1960s as secondary metabolites of the grasses that function as natural pesticides and exhibit allelopathic properties. Benzoxazinoids are synthesized in seedlings and stored as glucosides (glcs); the main aglucone moieties are 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) and 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA). The genes of DIBOA-glc biosynthesis have previously been isolated and the enzymatic functions characterized. Here, the enzymes for conversion of DIBOA-glc to DIMBOA-glc are identified. DIBOA-glc is the substrate of the dioxygenase BENZOXAZINLESS6 (BX6) and the produced 2,4,7-trihydroxy-2H-1,4-benzoxazin-3-(4H)-one-glc is metabolized by the methyltransferase BX7 to yield DIMBOA-glc. Both enzymes exhibit moderate Km values (below 0.4 mm) and kcat values of 2.10 s−1 and 0.25 s−1, respectively. Although BX6 uses a glucosylated substrate, our localization studies indicate a cytoplasmic localization of the dioxygenase. Bx6 and Bx7 are highest expressed in seedling tissue, a feature shared with the other Bx genes. At present, Bx6 and Bx7 have no close relatives among the members of their respective gene families. Bx6 and Bx7 map to the cluster of Bx genes on the short arm of chromosome 4.

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 hosts salicylic acid levels act as a signal for the activation of at least some of these induced defenses.

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.