Richard D. Durbin

Phytotoxins and Plant Pathogenesis

In a broad sense, “phytotoxins” can be considered as microbial metabolites, other than enzymes, that damage or are harmful to plants at very low concentrations (1–3, 23, 25). Many plant pathogenic bacteria and fungi produce phytotoxins both in culture and in their hosts during the infection process. In several cases — especially if they are produced during the early stages of plant disease development — these compounds have a function in pathogenesis and cause part or even all of the symptoms of the disease (4, 5, 20). For microbial products which are not phytotoxic per se but have a role in pathogenesis, the term aggressions has been proposed (1).

The stimulation of coupling factor 1 ATPase by tentoxin

Tentoxin at 10--1000 micrometer causes a marked species-selective stimulation of coupling factor 1 Ca2+-dependent ATPase activity (Ka 6.3 . 10(3) M-1). This effect decreases the Km for ATP to about 0.3 mM and increases V 2.75-fold. Above 1.6 micrometer tentoxin the rate of coupled electron transport was reduced to basal without uncoupling.

Tagetitoxin, a toxin produced by Pseudomonas syringae pv. tagetis: purification and partial characterization

A toxin of Pseudomonas syringae pv. tagetis which causes apical chlorosis in marigolds and zinnia has been produced in culture, isolated, purified and partially characterized. The toxin was found to contain phosphorus and sulphur and an amino function, and was homogeneous by chromatography and electrophoresis. Because it was distinctly different from other Pseudomonas toxins, in particular phaseolotoxin, the new name tagetitoxin is proposed for the new toxin. Tagetitoxin was unstable in dilute acid but was not affected at mildly basic pH or by alkaline phosphatase. Tagetitoxin gave an apical chlorosis on a zinnia seedling when 10 ng was supplied in 20 μl.

Tentoxin An uncompetitive inhibitor of lettuce chloroplast coupling factor 1

The interaction of tentoxin [cyclo-(-L-leucyl-N-methyl-(Z)-dehydrophenylalanyl-glycyl-N-methyl-L-alanyl-)] with solubilized lettuce chloroplast coupling factor 1 was characterized by direct binding studies, measurement of the time course of ATPase inhibition, and steady-state enzyme kinetics. Neither substrates, products or Ca2+ competed with the tentoxin binding site, nor did they induce any large change in tentoxin affinity. The inhibition of lettuce chloroplast coupling factor 1 ATPase was found to be the time dependent, and at equilibrium the affinities estimated by equilibrium ultrafiltration and enzyme inhibition were similar (1.8 . 10(8) M-1). The steady-state kinetics best fit an uncompetitive pattern suggesting that the inhibited steps follow an irreversible step occurring after ATP binding.

The isolation and properties of a tabtoxin-hydrolysing aminopeptidase from the periplasm of Pseudomonas syringae pv. tabaci

A tabtoxin-hydrolysing enzyme was partially purified from the periplasm of several strains of Pseudomonas syringae pv. tabaci . It is an aminopeptidase with a broad pH optimum. The enzyme was obtained by osmotic shock and then partially purified by ammonium sulphate precipitation; it became unstable with further purificiation (Bio-Gel, DEAE-Sephadex and Phenyl-Sepharose chromatography). Its activity is sensitive to EDTA but not to n -ethylmaleimide or phenylmethyl-sulphonyl fluoride. Although, tabtoxin hydrolysis absolutely requires zinc for activity in vivo , other divalent cations are partially effective in restoring activity to an EDTA-treated enzyme preparation. Bacterial strains which could not be activated by zinc to hydrolyse tabtoxin did not have a zinc-activatable peptidase in their periplasm. This enzyme appears to be responsible for the conversion of tabtoxin in the bacterium to the biologically active form, tabtoxinine- β -lactam. It may also function as part of a mechanism for self-protection against tabtoxinine- β -lactam.

Evidence for a catalytic function of the coupling factor 1 protein reconstituted with chloroplast thylakoid membranes.

The effects of tentoxin on the ATPase activities of coupling factor 1 proteins (CF1) and photophosphorylation with isolated chloroplasts and chloroplasts reconstituted with coupling factor proteins have been examined. 1. The calcium-dependent ATPase activities of coupling factors isolated from spinach, lettuce and Nicotiana otophora are completely inhibited by tentoxin. The ATPase activities of coupling factors isolated from Nicotiana tabacum and Nicotiana knightiana are not affected by tentoxin. 2. Phenazine methosulfate-catalyzed cyclic photophosphorylation with chloroplasts isolated from spinach, lettuce and N. otophora is completely inhibited by tentoxin, whereas chloroplasts isolated from N. knightiana and N. tabacum are relatively insensitive to tentoxin. 3. Spinach chloroplasts, partially depleted in CF1, can be reconstituted with coupling factors isolated from a wide variety of plants including lettuce, radish, N. tabacum, N. knightiana and N. otophora. 4. Spinach chloroplasts reconstituted with spinach, lettuce and N. otophora CF1 retain their sensitivity to tentoxin; however, when reconstituted with N. knightiana and N. tabacum coupling factor proteins, a significant fraction of the reconstituted rate remains tentoxin insensitive. These data are interpreted as evidence that coupling factors that reconstitute with spinach thylakoid membranes have both a catalytic and structural function.

Tagetitoxin affects plastid development in seedling leaves of wheat

Ultrastructural and biochemical approaches were used to investigate the mode of action of tagetitoxin, a nonhost-specific phytotoxin produced by Pseudomonas syringae pv. tagetis (Hellmers) Young, Dye and Wilkie, which causes chlorosis in developing — but not mature — leaves. Tagetitoxin has no effect on the growth rate or morphology of developing leaves of wheat (Triticum aestivum L.) seedlings. Its cytological effects are limited to plastid aberrations; in both light-and dark-grown leaves treated with toxin, internal plastid membranes fail to develop normally and plastid ribosomes are absent, whereas mitochondrial and cytoplasmic ribosomes are unaffected. The activity of a plastid stromal enzyme, ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39), which is co-coded by nuclear and chloroplast genes, is markedly lower in extracts of both light-and dark-grown toxin-treated leaves, whereas the activity of another stromal enzyme, NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-G-3P-DH, EC 1.2.1.13), which is coded only by the nuclear genome, is significantly lower in extracts of light-grown, but not of dark-grown, treated leaves. The mitochondrial enzymes fumarase (EC 4.2.1.2) and cytochrome-c oxidase (EC 1.9.3.1) are unaffected by toxin in dark-grown leaves, but fumarase activity is reduced in light-grown ones. Four peroxisomal enzyme activities are lowered by toxin treatment in both light- and dark-grown leaves. Light- and dark-grown, toxintreated leaves contain about 50% and 75%, respectively, of the total protein of untreated leaves. There are threefold and twofold increases in free amino acids in light-grown and dark-grown treated leaves, respectively. In general, the effects of tagetitoxin are more extensive and exaggerated in light-grown than in dark-grown leaves. We conclude that tagetitoxin interferes primarily with a light-independent aspect of chloroplast-specific metabolism which is important in plastid biogenesis.

Synthesis of tryptophol and O-acetyltryptophol from tryptophan by Ceratocystis fagacearum

Abstract Tryptophol (TOL) and O -acetyltryptophol ( O AcTOL) were identified as tryptophan metabolites of C. fagacearum ; O AcTOL was the only metabo

The binding of tentoxin to a tryptic digest of chloroplast coupling factor 1

Abstract The binding of tentoxin to lettuce chloroplast coupling factor 1 and its inhibition of Ca +2 -dependent ATPase involves the α and β subunits which remain after trypsin treatment. The tentoxin-binding properties of the digest are not greatly altered from those previously reported for the untreated protein.

Synergistic activation of an Mg-specific ATPase activity in chloroplast coupling factor by octylglucoside and tentoxin

Abstract (1) Octylglucoside stimulates an Mg 2+ -specific ATPase activity with CF 1 preparations from different higher plants and the alga Chlamydomonas reinhardii . (2) Tentoxin at high concentrations (10 −4 –10 −3 M) in the presence of octylglucoside further stimulates the Mg 2+ -ATPase activity of CF 1 from tentoxin-sensitive species and inhibits the activity of CF 1 from tentoxin-resistant species. The extent of tentoxin stimulation and inhibition varies among species. A maximal stimulation of over 2-fold was obtained with spinach CF 1 and a maximal inhibition of 50% was obtained with C. reinhardii CF 1 . In Nicotiana spp., tentoxin had only a marginal effect on the Mg 2+ -ATPase activity induced by octylglucoside.