Rapid Changes in Polyphosphoinositide Metabolism in Pea in Response to Fungal Signals

Effects of the elicitor and/or suppressor from Mycosphaerellapinodes on polyphosphoinositide metabolism (PI metabolism) inpea were examined both in vivo and in vitro. The elicitor induceda rapid and biphasic increase in levels of phosphatidylinositol-4,5-bisphosphate(PtdInsP₂) and inositol 1,4,5-trisphosphate (IP₃) in epicotyltissues that was apparent within 15 min. A transient increasein levels of PtdInsP₂ and IP₃ was detected immediately in elicitor-treatedplasma membranes. However, the concomitant presence of suppressorwith elicitor resulted in inhibition of these increases bothin vivo and in vitro. These findings suggest that the elicitorrapidly activates phosphatidylinositol kinase, phosphatidylinositol-4-monophosphatekinase and phospholipase C, which are involved in PI metabolism,whereas the suppressor markedly inhibits these enzymes. Neomycin,a known inhibitor of phospholipase C, blocked the elicitor-inducedaccumulation both of IP₃ and pisatin and it also induced localsusceptibility in pea tissues that resembled that of the fungalsuppressor. From these results, it appears that rapid changesin PI metabolism are indispensable in the signal transductionrelated to defense responses of pea plants. (Received January 18, 1993; Accepted May 13, 1993)     

Suppression of Pisatin Production and ATPase Activity in Pea Plasma Membranes by Orthovanadate, Verapamil and a Suppressor from Mycosphaerella pinodes

A pea pathogen, Mycosphaerella pinodes, secretes both an elicitorand a suppressor for the accumulation of pisatin, a major phytoalexinof pea, into the spore germination fluid. The effects of theelicitor and the suppressor on the ATPase activity in pea plasmamembranes was examined. The ATPase was sensitive to orthovanadateand dicyclohexylcarbodiimide but insensitive to nitrate andazide; it was unaffected by the elicitor but was markedly inhibitedby the suppressor (50µg.ml^–1, bovine serum albuminequivalents) or verapamil (1OOµM). The accumulation ofpisatin induced by the elicitor was delayed for 3 to 6 h inthe presence of orthovanadate or verapamil to an extent similarto that in the presence of the suppressor. The relationshipbetween the inhibition of plasma membrane ATPase activity andthe suppression of the active defense reaction that involvesthe production of pisatin in the pea plant is discussed. (Received April 16, 1990; Accepted September 6, 1990)     

An Endogenous Suppressor of the Defense Response in Pisum sativum

Healthy pea plants contain a substance, tentatively called "endogenoussuppressor", which specifically suppresses the accumulationof pisatin in pea plants that is induced by treatment with CuCl₂or an elicitor from Mycosphaerella pinodes. This suppressorelicits the accumulation of phytoalexins in other legumes, suchas kidney bean, soybean and cowpea. The endogenous suppressorfunctions to delay the accumulation of pisatin, the activationof phenylalanine ammonialyase (PAL) and the accumulation ofmRNAs for PAL and chalcone synthase induced by the elicitorfrom M. pinodes. The substance specifically induces susceptibilityto nonpathogens, such as Mycosphaerella ligulicola and M. melonis,in pea out of four species of legume tested, but the effectis not cultivar-specific. Thus, the endogenous suppressor inhealthy pea plants suppresses a series of self-defense reactionsand induces susceptibility in pea plants in a species-specificmanner, being similar to the exogenous fungal suppressor fromthe pea pathogen, M.pinodes. (Received February 19, 1992; Accepted May 11, 1992)     

Plant Lectins Induce the Production of a Phytoalexin in Pisum sativum

The effects of several plant lectins on the production of apea phytoalexin, pisatin, were examined. Con A, PHA, PNA andPSA each induced the production of pisatin in pea epicotyl tissues,demonstrating that plant lectins can act as elicitors. The productionof pisatin in response to PHA, PNA or PSA was not affected bythe simultaneous presence of the respective hapten sugars, whereashaptens specific for Con A, such as -D-mannose and methyl--D-mannoside,abolished the induction of pisatin by Con A. These results indicatethat the elicitor effect of Con A is attributable to its abilityto bind to specific carbohydrates in pea cells. Induction ofthe production of pisatin by Con A was markedly inhibited bythe suppressor derived from a pea pathogen, Mycosphaerella pinodes,and by several inhibitors related to signal-transduction pathways.It is suggested, therefore, that the Con A-induced productionof pisatin in pea tissues might be associated with activationof a signal-transduction pathway. An additive effect on theaccumulation of pisatin was observed when Con A was presentwith a polysaccharide elicitor from M. pinodes, suggesting thatexogenous Con A does not compete with the recognition site(s)for the fungal elicitor in pea cells. The present data alsoindicate that Con A may be useful for characterization of thesignal-transduction system that leads to the synthesis of phytoalexinin pea epicotyl tissues. (Received November 16, 1994; Accepted April 20, 1995)     

Two Suppressors, Supprescins A and B, Secreted by a Pea Pathogen, Mycosphaerella pinodes

Two mucin-type glycopeptides that suppressed the productionof pisatin, a phytoalexin of pea, were purified from a pea pathogen,Mycosphaerella pinodes. The structures of Supprescin A (Mr,452) and Supprescin B (Mr, 959) were determined by an analysisof amino acid sequences and ¹³C- and ¹H-NMR. (Received April 22, 1992; Accepted June 16, 1992)     

Inhibition of ATPase Activity in Pea Plasma Membranes In Situ by a Suppressor from a Pea Pathogen, Mycosphaerella pinodes

A pathogenic fungus of pea, Mycosphaerella pinodes, secretesa so-called "suppressor" in its pycnospore germination fluid.The suppressor blocks the defense responses and induces localsusceptibility (accessibility) in pea plants to agents thatare not pathogenic in pea. The suppressor nonspecifically inhibitsthe ATPase activity in plasma membranes prepared from pea, soybean,kidney bean, cowpea and barley plants. However, cytochemicalstudies by electron microscopy indicate that the suppressorspecifically inhibits the ATPase in pea cell membranes, butnot in those of four other plant species tested. That is, thespecificity of the suppressor appears at the cell and/or tissuelevel, but is not evident in vitro. Furthermore, the inhibitoryeffect of the suppressor is temporary because the ATPase activityrecovers 9 h after the treatment. A similar effect was observedafter inoculation with M. pinodes but not with a nonpathogenof pea, M. ligulicola. The role of the suppressor in host-parasitespecificity is discussed. (Received April 9, 1991; Accepted August 6, 1991)     

Dynamics of Free Pisatin Elicitor Accumulation in Infection-droplets of Monilinia fructicola on Pea Endocarp*

Rapid elicitor accumulation in the infection-droplet of the pea-M. fructicola interaction began between 2 and 3 h after inoculation. Rapid accumulation of pisatin began between 2 and 3 h, however, low levels (0.06–0.1 μg/ml) were detected in the infection-droplet as early as 1–2 h following inoculation with the fungus. The pisatin concentration reached levels inhibitory to the fungus between 6 and 12 h (ca. 1–5 μg/ml) and the ED₅₀ value of 10 μg pisatin/ml for mycelial growth of M. fructicola was attained after 14 h. Elicitor activity in infection-droplets after 4 and 18 h was a function of inoculum concentration and pisatin accumulation in diffusate after 40 h was a function of elicitor concentration (linear doseresponse curve). However, when elicitor was applied at zero time, the rate of pisatin accumulation over the first 12 h was indistinguishable from that which occurred when M. fructicola conidia were applied to the endocarp and elicitor accumulated with time. The initial rate of pisatin accumulation therefore appears to be dependent on the pea pod and independent of any time delays associated with conidial germination and elicitor accumulation. However, the final pisatin concentration which accumulated in the infection-droplet was dependent on the, dose' of elicitor irrespective of the nature and timing of the elicitor treatment. The presence of elicitor activity was demonstrated in the interaction in space and time where and when these molecules could function as elicitors of pisatin in vivo.     

Inhibition of ATPase Activity in Pea Plasma Membranes by Fungal Suppressors from Mycosphaerella pinodes and Their Peptide Moieties

The effects of two suppressors of the defense reactions of hostplants, which had been purified from the pea pathogen Mycosphaerellapinodes, as well as the effects of peptide moieties, on theATPase activity in pea plasma membranes were examined in vitro.One of the suppressors, Supprescin B, inhibited the ATPase activityin a non-competitive manner, but the other suppressor, SupprescinA, did not. Supprescin A was observed to reduce the inhibitoryeffect of Supprescin B. A tripeptide, Ser-Ser-Gly, and a hexapeptide,Ser-Ser-Gly-Asp-Glu-Thr, which were the respective peptide moietiesof Supprescin A and B, inhibited the ATPase activity in a competitivemanner. Supprescin B and fragments of the hexapeptide, suchas Asp-Glu-Thr and Gly- Asp-Glu, inhibited not only the ATPaseactivity but also the acid phosphatase activity of plasma membranesin vitro. These results indicate that the acidic amino-acidresidues of the "Asp-Glu" moiety seem to act as inhibitors ofthe phosphatase activity. Thus, the peptide moiety of SupprescinB consists of at least two functional elements. (Received October 23, 1992; Accepted January 18, 1993)     

Elution Profiles of Pisatin Elicitor Activity from Extracts of Monilinia fructicola,Pisum sativum and Diffusates from their Interaction*

Pisatin elicitor activity was examined in diffusate of the M. fructicola–pea endocarp interaction, leachate of pea endocarp, extracts of ungerminated conidia and filtrates of conidia germinated in vitro in a simple nutrient broth, or in filter–sterilized diffusate or pea leachate. Extracts were made after 18 h incubations which represents the half–time of the primary phase of pisatin accumulation in the M. fructicola–pea model system. These were chromatographed on a Bio–Gel, P–2 column and elicitor activity in eluate fractions was located by bioassay for the ability to induce pisatin accumulation. A characteristic elution profile of pisatin elicitor activity was obtained from diffusate of the pea–M. fructicola interaction. Other preparations obtained from pea leachate, ungerminated conidial extracts or culture filtrates of the fungus germinated in a simple nutrient broth or pea leachate (6 h) did not contain the same profile of elicitor components. No further production of elicitor was detected in early diffusate (6 h) filtrates when they were incubated in vitro alone or withM. fructicola. Elicitor activity was significant in some conidial germination filtrates in vitro. The elution profiles of elicitor activity obtained from these filtrates were shown to be dependent on the physical conditions of culture used (still or shaken). Incubation of the pea leachates with M. fructicola in vitro resulted in the formation of a high molecular weight elicitor which did not correspond with that of the diffusate preparations. Its reapplication to pod tissue did not suggest that it was significantly metabolized by pea tissue to produce the same elicitors found in diffusate preparations. The results emphasize the importance of in vivo studies in the search for elicitors of phytoalexins where two living systems, the plant and the fungus are intimately involved.     

Specific Inhibition of Cell Wall-Bound ATPases by Fungal Suppressor from Mycosphaerella pinodes

Activities of phosphatases were found in the fractions whichwere solubilized from cell walls of both pea and cowpea seedlingswith 0.5 M NaCl. These phosphatases hydrolyzed triphosphonucleotidesin the order: UTP=CTP>GTP>ATP; and UTP=GTP>CTP=ATP,respectively. The activities of a pyrophosphatase and a p-nitrophenylphosphatasewere also detected in these fractions. The suppressor in thespore germination fluid of a pea pathogen,Mycosphaerella pinodes,inhibited all of these phosphatase activities in the fractionsolubilized from pea cell walls, but it rather enhanced onlythe activity of the ATPase among those phosphatases from thecowpea cell wall. Hydrolysis of ATP by a cell wall fractionof pea was also markedly inhibited by the suppressor, whilehydrolysis of ATP by similar fractions from cowpea, kidney beanand soybean were rather enhanced by the suppressor, as wellas by the elicitor. Thus, the cell wall-bound ATPases respondedto the suppressor species-specifically. These cell wall-boundATPases seemed to be different from the plasma membrane ATPasesin several respects. The results suggest that plants recognizethe fungal signals not only on their plasma membranes but alsoon their cell walls and, moreover that putative receptors forthe fungal signals might be located close to cell wall-boundATPases or might even be these ATPases themselves. (Received November 16, 1994; Accepted April 20, 1995)     

12-O-tetradecanoylphorbol-13-acetate stimulates release of arachidonic acid, prostaglandin E2 and prostaglandin F2 alpha from TPA nonproliferative variants of 3T3 cells

The Proteinase Inhibitor Inducing Factor, PIIF, a pectic polysaccharide that induces synthesis and accumulation of proteinase inhibitor proteins in tomato and potato leaves, is an effective elicitor of the phytoalexin pisatin in pea pod tissues. The levels of pisatin induced by PIIF, and the time course of elicitation, are similar to those induced by chitosans, β-1,4 glucosamine polymers, which are potent elicitors of pisatin in pea pods. Similarly, the chitosans, found in both insect and fungal cell walls, are the most potent inducers yet found of proteinase inhibitor accumulation in excised tomato cotyledons. The similarity in the induction of synthesis of proteinase inhibitors in tomato cotyledons and of pisatin in pea pods by pectic polysaccharides and chitosans suggests that the two polysaccharide types may be triggering a similar fundamental system present in pea and tomato plants that regulates the expression of genes for natural protection systems.     

Suppressors: Determinants of Specificity Produced by Plant Pathogens

Plant pathogens secrete suppressors that delay or prevent thehost defense responses, with resultant conditioning of hostcells such that they become susceptible even to avirulent ornon-pathogenic microorganisms. Suppressors have been characterizedas glycoproteins, glycopeptides, peptides and anionic and nonanionicglucans. A suppressor itself is non-toxic to plant cells and,thus, it can be distinguished from host-specific toxins producedby certain pathogens. Suppressors disturb fundamental functionsof host plasma membranes. For example, the suppressor from apea pathogen, Mycosphaerella pinodes, inhibits both the ATPaseactivity and polyphosphoinositide metabolism in pea plasma membranes,causing the temporary suppression of the signal-transductionpathway that leads to the expression of defense genes, whichencode key enzymes in the biosynthetic pathway to phytoalexin.In this review, evidence for the role of suppressors in thedetermination of plant host-parasite specificity is summarized. ³Present address: Plant Pathology Laboratory, School of Agriculture,Nagoya University, Chikusa, Nagoya, 464-01 Japan     

Non-Specific Induction of Pisatin and Local Resistance in Pea Leaves by Elicitors from Mycosphaerella pinodes, M. melonis and M. ligulicola and the Effect of Suppressor from M. pinodes

The accumulation of pisatin was induced non-specifically by elicitors prepared from the high molecular weight fraction (molecular weight: more than 10,000 daltons) of the spore germination fluid of three species of Mycosphaerella-plant pathogens in pea leaves with epidermis removed, regardless of the pathogenicity of the fungi to pea. Before the elicitation of pisatin synthesis, local resistance to infection by Mycosphaerella pinodes was induced by elicitors again non-specifically inpea leaves in which wax had been removed from the leaf surface. The substance responsible for local resistance could be extracted with ethylacetate from the elicitor-containing drop diffusate which was placed on pea leaves. The substance prevented the penetration of M. pinodes through heat-killed pea epidermis, but did not affect spore germination. The suppressor prepared from the low molecular weight fraction (molecular weight: less than 10,000 daltons) of the spore germination fluid of M.pinodes counteracted the ability of elicitors to induce both phases of resistance mechanisms.     

Functional Analysis of the Putative cis-Elements Involved in Activation by Elicitor or UV and Deactivation by Suppressor in the Promoter of a Pea Gene for Phenylalanine Ammonia-lyase (PSPAL1)

The promoter of a pea gene for phenylalanine am-monia-lyase(PSPAL 1) contains Boxes II and IV that have synergistic effectson activation by fungal elicitor or UV light. Fungal suppressorsuppressed the activation that was mediated not only by elicitorbut also by UV in pea but activated the promoter in cowpea. (Received October 25, 1996; Accepted September 27, 1997)     

Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - IFR isoflavone reductase - 2iP 6-(dimethylallylamino)-purine - MS Murashige & Skoog basal salt medium - PAL phenylalanine ammonia-lyase - PMSF phenylmethylsulfonyl fluoride - POPOP 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazole     

Orthovanadate Suppresses Accumulation of Phenylalanine Ammonia-Lyase mRNA and Chalcone Synthase mRNA in Pea Epicotyls Induced by Elicitor from Mycosphaerella pinodes

Orthovanadate delayed accumulation of mRNAs encoding phenylalanineammonia-lyase and chalcone synthase in pea epicotyls inducedby an elicitor from Mycosphaerella pinodes. However, accumulationof mRNA for a putative P-type ATPase was not affected. The relationshipbetweenthe ATPase and defense responses is discussed. ³Present address: Plant Pathology Laboratory, School of Agriculture,Nagoya University, Chikusa, Nagoya, 464-01 Japan.     

Role of Inhibitors of Fungal Growth in the Limitation of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

The phytoalexin, pisatin, was detected in host tissues 24 hafter inoculation of pea leaflets with spores of the leaf-spottingpathogens Ascochyta pisi and Mycosphaerella pinodes. Pisatincontinued to accumulate in infected tissue as A. pisi lesionsdeveloped and was present in inhibitory concentrations in thebrown tissue beyond the region colonized by the pathogen. During the formation of limited M. pinodes lesions, concentrationsof pisatin were highest 2 days after inoculation. Levels weremore variable and lower in older lesions which appeared to containno other inhibitors of germ-tube growth. Spreading lesions causedby M. pinodes on leaflets floating on water contained littleor no pisatin although little was released to the water below.These lesions did, however, contain other highly active inhibitorsof germ-tube growth. The significance of these results in terms of limitation oflesions are discussed. The ease with which M. pinodes lesionscan become progressive may reflect the greater ability of thispathogen to grow in high concentrations of pisatin under certainconditions.     

Effect of Vanadate on Microsomal ATPase Activity, Acidification of the Medium and Auxin-stimulated Growth in Pea and Cucumber

The relationship between ATPase activity, medium acidificationand auxin-stimulated growth in segments of pea stem (Pisum sativumL., cv. Alaska) and cucumber hypocotyl (Cucumis sativus L.,cv. Long Green Ridge) was investigated using sodium orthovanadate,widely used as a selective inhibitor of plasma membrane-associatedATPase activity. ATPase activity of cucumber microsomal preparationswas about seven times lower than similar preparations from pea(on a mg microsomal protein basis) and was much more effectivelyinhibited by vanadate. Similarly, acidification of the mediumby abraded cucumber segments occurred to a lesser extent thanwith pea and showed a greater inhibition by vanadate. Both growthin controls and auxin-stimulated growth of cucumber segmentswere strongly inhibited by vanadate, whereas in pea auxin-stimulatedgrowth was reduced by only half and controls showed little inhibition.Acidification of the medium by segments of both species wasfound to occur readily even in controls and showed little promotionin the presence of IAA, although growth in both species wasrapidly and significantly promoted by IAA. These results indicatethat acidification is brought about by a plasma membrane-associatedATPase, and suggest that while acidification is an essentialfactor for auxin-stimulated growth it may not be the mechanismby which the growth rate is controlled. ATPase, Cucumis sativus, indole-3-acetic acid, Pisum sativum, vanadate