Hans-Joachim Reisener

Lignin biosynthetic enzymes in stem rust infected, resistant and susceptible near-isogenic wheat lines☆

Abstract Near-isogenic lines of the wheat cultivar Prelude, carrying either the Sr5 gene for resistance or the sr5 allele for susceptibility were inoculated with the stem rust fungus Puccinia graminis f. sp. tritici race 32, which possesses the P5 gene for avirulence. The time-course of the enzyme activities of the general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL), as well as the enzyme activities of the specific pathway leading to the biosynthesis of lignin, cinnamyl-alcohol dehydrogenase (CAD) and peroxidase (PO), were determined. The resistant and the susceptible isoline both exhibit a first maximum in the coordinately regulated enzyme activities of PAL and 4CL at a time when the fungus is still growing on the surface of the leaves. This maximum is followed by a decrease to the levels of uninoculated controls. In the resistant isoline, a second increase is observed at the time of the hypersensitive resistant reaction. In contrast, enzyme activities in susceptible plants continue to decline, even falling below control levels until the onset of sporulation when a second, late increase can be detected. The observed changes in CAD and PO activities are consistent with a feed-forward regulation by products of the general phenylpropanoid pathway. However, no late increase in PO activity can be observed in the compatible interaction. An interpretation of these results is proposed in terms of non-host resistance, basic compatibility and race-cultivar specific resistance.

A single glycoprotein from Puccinia graminis f. sp. tritici cell walls elicits the hypersensitive lignification response in wheat

Abstract Con A-binding glycoproteins were detected in germ tube walls of Puccinia graminis f. sp. tritici and appeared to be potent inducers of the hypersensitive lignification response. This response, which is typical of the large resistance reaction in wheat leaves, was preceded by a increase in extractable phenylalanine ammonia-lyase (PAL) activity, a key enzyme of the phenylpropanoid pathway. Germ tube glycoproteins were isolated from homogenized fungal cell walls by differential centrifugation and affinity chromatography on a Con A-Sepharose column. The most active glycoprotein was purified on a Mono Q anion-exchange column. The relative molecular mass of the molecule was determined in SDS-polyacrylamide gels to be 67 kD. The carbohydrate portion consists mainly of mannose (50%) and galactose (47%) and contains the active part of the glycoprotein as demonstrated by the inability of pronase and trypsin digestion to influence activity. An elicitor-active glycoprotein with identical molecular mass and Con A-binding properties was isolated from intercellular fluids of rust-infected plants of the susceptible wheat cultivar Little Club indicating that the elicitor is released from fungal cell walls during infection. We discuss the role of Con A-binding proteins as inducers of non-host resistance in cereals.

An elicitor of the hypersensitive lignification response in wheat leaves isolated from the rust fungus Puccinia graminis f. sp. tritici. II: Induction of enzymes correlated with the biosynthesis of lignin

The genuine biotic elicitor from germ tube walls of Puccinia graminis f. sp. tritici (Pgt-elicitor) induces lignification, preceded by an increase in phenylalanine ammonia-lyase (PAL) activity, when injected into the intercellular space of primary wheat leaves. This increase in PAL activity is accompanied by an increase in other enzyme activities of the general phenylpropanoid pathway and the specific pathway of lignin biosynthesis: 4-coumarate :CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (PO). Total extractable activities of 4CL, CAD and PO do not differ significantly in resistant and susceptible near isogenic wheat lines, whereas the dose response curve of induced PAL activity shows significantly higher values in the resistant isoline. This difference is not observed after injection of other biotic and abiotic elicitors of the lignification response. Although total induced PO activities 24 h after elicitor treatment are equal in both isolines, the PO isoenzyme pattern of resistant plants differs markedly from that of susceptible plants. The patterns of PO isoenzymes in the compatible and incompatible interaction 48 h after inoculation with Puccinia graminis f. sp. tritici also show differences, which closely resemble those seen after injection of the Pgt-elicitor. The patterns observed after injection of an artificial biotic elicitor clearly differ from those of the natural interaction.

An Elicitor of the Hypersensitive Lignification Response in Wheat Leaves Isolated from the Rust Fungus Puccinia graminis f. sp. tritici I. Partial Purification and Characterization

Several biotic and abiotic clicitors of the hypersensitive reaction in wheat leaves are described. The elicitors induce enhanced activity of phenylalanine ammonia-lyase with subsequent lignification, visible as a yellow autofluorescence. The deposited material stains positively with phloroglucinol. DEAE-dextran, epoxystearic acid and chitosan are strong elicitors, while the glucans tested have no activity. A biotic elicitor (genuine Pgt-elicitor) was isolated from the germ tube walls of uredospores of Puccinia graminis f. sp. tritici. The high molecular weight, water soluble elicitor is heat stable and unaffected by mild acid and mild alkaline treatments. It seems to be a glycoprotein with the carbohydrate moiety bearing the active residues, as indicated by periodate sensitivity and protease stability. The carbohydrate moiety consists mainly of glucose with some galactose and mannose. Upon ultrafiltration and gel chromatography elicitor activity was associated with fractions of molecular weight of more than 100 kD. When injected into the intercellular space of primary wheat leaves, the elicitor induces lignification, preceded by an increase in phenylalanine ammonia-lyase activity.

Suppression of the hypersensitive response in wheat stem rust interaction by reagents with affinity for wheat plasma membrane galactoconjugates.

The growth of the wheat stem rust (Puccinia graminis f.sp. tritici, race 32) on the resistant wheat cultivar Feldkrone is restricted by cell necrosis. Those epidermal cells first invaded react hypersensitively as is shown by the appearance of autofluorescence and the formation of lignin in the invaded cells. Cell necrosis was inhibited by soy-bean agglutinin (SBA), a lectin, which reacts specifically with galactose and N-acetyl-galactosamine, by Erythrina corallodendron lectin (ECO), specific for galactose, as well as by galactose oxidase, an enzyme which acts on the non-reducing terminal D-galactosyl and N-acetyl-D-galactosaminyl residues of surface galactoconjugates. As a result of this inhibition of cell necrosis, the size of the rust colony on treated leaves in-creased, i.e. the resistance of the wheat was lowered. Since previous results had shown that SBA binds to galactolipids on the surface of wheat protoplasts, we now postulate that SBA, ECO, and galactose oxidase interfere with the interaction of host plasmalemma with the fungal haustorium wall during the initial stages of infection.

Elicitor-active glycoproteins in apoplastic fluids of stemrust-infected wheat leaves

Abstract Cell walls of germ tubes from wheat stem rust ( Puccinia graminis f. sp. tritici ) contain a glycoprotein with a molecular mass of about 67000 referred to as the Pgt elicitor. This glycoprotein induces a hypersensitive-like response in wheat leaves. In elicitor-active intercellular washing fluid (IWF) from compatible wheat-stem rust interactions we detected several elicitor-active glycoproteins. One of these glycoproteins had an electrophoretic mobility identical to the Pgt elicitor. This IWF-glycoprotein exhibited elicitor activity upon elution from SDS gels. It was recognized by anti-Pgt elicitor antiserum suggesting partial structural identity between the Pgt and IWF elicitors. As with the Pgt elicitor, the elicitor-activity of the IWF glycoprotein resides in the carbohydrate moiety because periodate, but not trypsin or pronase, destroyed activity. These results suggest that the Pgt elicitor is released from hyphal cell walls into the wheat apoplast during stem rust infection.

Changes in the level of enzyme activities involved in lignin biosynthesis during the temperature sensitive resistant response of wheat (Sr6) to stem rust (P6)

Abstract Near-isogenic lines of the wheat cultivar Marquis, carrying either the Sr6-gene for resistance or the corresponding sr6-allele for susceptibility were grown either at 18°C or at 27°C. Seven-day-old plants were inoculated with uredospores of race 32 of the stem rust fungus Puccinia graminis f. sp. tritici (P6). At different times after inoculation, activities of the enzymes phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase (4CL), and peroxidases (PO) were determined. In the incompatible Sr6/P6 interaction at 18°C, increases in these enzyme activities were detected at the time of the hypersensitive resitant response. In contrast, in the compatible interactions, Sr6/P6 at 27°C and sr6/P6 at both temperatures, activities of PAL and 4CL rapidly increased at the time of beginning sporulation, whereas PO activities remained low. When plants were treated with an elicitor derived from stem rust germlings both isolines exhibited typical symptoms of the resistant reaction, i.e., chloroses, necroses, yellow autofluorescence under UV-light, and increase in PAL activity, at both temperatures. At 18°C, the Sr6/P6 interaction, thus, shows changes in lignin biosynthetic enzyme activities which are typical for highly incompatible interactions; while at 27°C, these changes reflect the fully compatible nature of this same interaction.

A wheat cell wall fragment suppresses elicitor-induced resistance responses and disturbs fungal development.

Summary A fraction prepared by Pectolyase-digestion of wheat cell walls suppressed plant responses when co-injected with a fungal elicitor of hypersensitive-like lignification.This fraction also reduced haustorial mother cell formation and prevented the development of haustoria when injected into wheat leaves prior to inoculation with the stem rust fungus.

Wheat cultivar and chromosomal selectivity of two types of eliciting preparations from rust pathogens

Abstract The activity of elicitors found in intercellular washing fluids (IWF) from infected wheat leaves and in extracts from germinating spores of both leaf rust ( Puccinia recondita f.sp. tritici ) and stem rust ( Puccinia graminis f.sp. tritici ) fungi was examined in a range of wheat cultivars drawn from Australian and German seed collections. Not all cultivars responded to these preparations. A comparison of the activity of each of the elicitor fractions from both rusts revealed that, regardless of source, the elicitors were active in the same group of cultivars. The ability of germ tube extracts to elicit both visible leaf responses and the induction of the enzyme phenylalanine ammonia-lyase was tested in two chromosome substitution lines. These experiments suggested that responsiveness to the elicitors in each case was controlled by a gene(s) in chromosome 5A of the host.

Polyamine levels in stem rust infected wheat leaves and effects of alpha-difluoromethylornithine on fungal infection.

Abstract The concentrations of the free polyamines putrescine/agmatine (Put/Agm), spermidine (Spd), and spermine (Spm)were determined in three near-isogenic lines of the wheat cultivar Prelude differing in their resistance to the wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt) race 32, over entire infection cycles. In highly resistant wheat plants, no changes in polyamine contents were detected. In moderately resistant and fully susceptible plants, Spm concentrations remained unchanged, while Put/Agm contents were increased over control plants between 3 and 7 days after inoculation. Spd concentrations in both interactions started to rise 3 days after inoculation, reaching a maximum at day 6 then declining in susceptible plants. Injection of alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, into the leaves of susceptible infected plants reduced the production of uredospores at concentrations as low as 0·0625 mM. DFMO had no effect on the germination of Pgt uredospores or growth and polyamine content of healthy wheat plants.