Ralph L. Nicholson

Synthesis of Phytoalexins in Sorghum as a Site-Specific Response to Fungal Ingress

Sorghum produces phytoalexins that are 3-deoxyanthocyanidin flavonoids. The compounds inhibit the growth of phytopathogenic fungi in vitro. The phytoalexins appear to be synthesized in subcellular inclusions within a host epidermal cell that is about to be penetrated by a fungus. This site-restricted synthesis suggests that the phytoalexin response occurs initially in the first cells that come under fungal attack and is not simply a response of cells that surround the original infection site.

Mycosporine-alanine: A self-inhibitor of germination from the conidial mucilage ofColletotrichum graminicola

Abstract On soft substrata conidia of Colletotrichum graminicola germinated by the formation of germ tubes, whereas on hard, hydrophobic substrata they germinated by the formation of appressoria. Regardless of the substratum, conidia did not germinate at high conidium concentrations, suggesting the presence of an endogenous self-inhibitor. Bioassays to test for the inhibition of germination demonstrated the presence of a low-molecular-weight self-inhibitor in the mucilage which surrounds conidia as they are produced in acervuli. The inhibitory material exhibited an absorbance maximum of 310 nm, indicating that it is a member of the mycosporine family of compounds. The inhibitor was purified to homogeneity and shown by mass spectrometric analysis to have a molecular mass consistent with the structure corresponding to mycosporine-alanine.

cDNA Cloning of a Sorghum Pathogenesis-Related Protein (PR-10) and Differential Expression of Defense-Related Genes Following Inoculation with Cochliobolus heterostrophus or Colletotrichum sublineolum

A sorghum cDNA clone was isolated by differential screening of a cDNA library prepared from mesocotyls (cultivar DK18) inoculated with fungal pathogenes. The deduced translation product shows sequence similarity to a family of intracellular pathogenesis-related proteins (PR-10) with a potential ribonuclease function. We studied the accumulation of PR-10 and chalcone synthase (CHS) transcripts in mesocotyls following inoculation with Cochliobolus heterostrophus or Colletotrichum sublineolum. CHS is involved in phytoalexin synthesis in sorghum. Coordinate expression of PR-10 and CHS genes was localized in the area of inoculation along with the accumulation of phytoalexins. C. heterostrophus is a nonpathogen of sorghum and cytological studies indicated that cultivar DK18 is resistant to C. sublineolum, a sorghum pathogen. We demonstrated that the two fungi triggered different time courses of plant defense reactions. Inoculation with C. heterostrophus resulted in rapid accumulation of PR-10 and CHS transcripts after appressoria had become mature. Accumulation of these transcripts was delayed in plants inoculated with C. sublineolum until penetration of host tissue had been completed and infection vesicles had formed. Results suggest that different recognition events are involved in the expression of resistance to the two fungi used or that C. sublineolum suppresses the nonspecific induction of defense responses.

Preparation of the infection court by Erysiphe graminis f. sp. hordei: cutinase is a component of the conidial exudate

Abstract Conidia of Erysiphe graminis f. sp. hordei release a liquid upon contact with a substratum, either the surface of a barley leaf or cellophane. The liquid had been shown to contain esterase activity which partially erodes the surface of the cuticle of barley leaves. The present investigation demonstrates that the liquid exudate contains cutinase activity. The presence of cutinase in the conidial exudate was confirmed by enzyme assays using 3 H-cutin as the substrate. Like other fungal cutinases, the enzyme from E. graminis was inhibited by the serine esterase inhibitor diisopropyl fluorophosphate. The results are significant since they demonstrate for the first time the production of cutinase by a powdery mildew parasite. It is possible that the cutinase is involved in the erosion of the leaf cuticle which begins prior to the development of appressoria. Such erosion may be necessary either for recognition of the host surface by the appressorium or for adhesion and more efficient penetration of the leaf.

Preparation of the infection court by Erysiphe graminis: Degradation of the host cuticle☆

Abstract Contact with a surface stimulates the release of a liquid from conidia of Erysiphe graminis and the liquid has been shown to contain esterase activity. The present investigation demonstrates that the liquid exudate partially erodes the cuticle of barley leaves. The release of esterase into the liquid occurs in two stages. The second stage of release occurs between 10 and 15 min after contact and was shown to be temperature dependent and to be inhibited by cycloheximide. Cuticular erosion may provide a stimulus for subsequent morphological differentiation, such as appressorium formation, or it may enhance appressorial adhesion.

Accumulation of sorghum phytoalexins induced by Colletotrichum graminicola at the infection site

Abstract Microspectrophotometry was performed on intact, pigmented vesicle-like inclusions within living sorghum cells that were accumulating phytoalexins as a response to attempted fungal infection. The results indicate that the deoxyanthocyanidin phytoalexins are present in inclusions. Moreover, the phytoalexin concentration within a single inclusion, based on luteolinidin, was calculated to be 0·15 m . The amounts of luteolinidin and apigeninidin in cells involved in the phytoalexin response at individual infection sites were also determined. The data showed that luteolinidin accumulated to levels of 0·48–1·20 ng/cell whereas apigeninidin accumulated to levels of 0·24–0·91 ng/cell. The results of both analyses confirmed that at the infection site the deoxyanthocyanidins accumulate to levels in substantial excess of those required for inhibition of the fungus Colletotrichum graminicola .

A Stilbene Synthase Gene (SbSTS1) Is Involved in Host and Nonhost Defense Responses in Sorghum

A chalcone synthase (CHS)-like gene, SbCHS8, with high expressed sequence tag abundance in a pathogen-induced cDNA library, was identified previously in sorghum (Sorghum bicolor). Genomic Southern analysis revealed that SbCHS8 represents a single-copy gene. SbCHS8 expression was induced in sorghum mesocotyls following inoculation with Cochliobolus heterotrophus and Colletotrichum sublineolum, corresponding to nonhost and host defense responses, respectively. However, the induction was delayed by approximately 24 h when compared to the expression of at least one of the other SbCHS genes. In addition, SbCHS8 expression was not induced by light and did not occur in a tissue-specific manner. SbCHS8, together with SbCHS2, was overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) tt4 (transparent testa) mutants defective in CHS activities. SbCHS2 rescued the ability of these mutants to accumulate flavonoids in seed coats and seedlings. In contrast, SbCHS8 failed to complement the mutation, suggesting that the encoded enzyme does not function as a CHS. To elucidate their biochemical functions, recombinant proteins were assayed with different phenylpropanoid-Coenzyme A esters. Flavanones and stilbenes were detected in the reaction products of SbCHS2 and SbCHS8, respectively. Taken together, our data demonstrated that SbCHS2 encodes a typical CHS that synthesizes naringenin chalcone, which is necessary for the formation of different flavonoid metabolites. On the other hand, SbCHS8, now retermed SbSTS1, encodes an enzyme with stilbene synthase activity, suggesting that sorghum accumulates stilbene-derived defense metabolites in addition to the well-characterized 3-deoxyanthocyanidin phytoalexins.

Preparation of the infection court by Erysiphe graminis.II: Release of esterase enzyme from conidia in response to a contact stimulus

Abstract The release of esterase enzyme activity by conidia of Erysiphe graminis is described as a tropic response to the stimulus of contact with leaf or cellophane surfaces. The release of esterase was completed within 30 minutes of the contact stimulus, and activity was released in two stages. The first began within 2 minutes of conidium contact and the second began between 10 and 15 minutes after contact. A portion of the esterase activity was solubilized in aqueous buffer and a portion remained bound to the conidial wall. Preparations of soluble proteins containing esterase activity were separated by sodium dodecyl sulfate-polyacrylamide and native gel electrophoresis which revealed the presence of several proteins and three zones that possessed esterase activity. The release of enzyme activity was correlated with changes in surface morphology described to occur within the first 30 minutes of initiation by the contact stimulus. The results are discussed in the context of cuticular degradation and preparation of the infection court by the fungus prior to the time of conidium germination.

Adhesion of Fungi to the Plant Surface

The initial process of attachment of fungal propagules to a host plant is essential to the successful establishment of pathogenesis. Attachment may be involved in recognition of the host surface, serve as a base around which the infection court can be altered, and may include adhesion of the propagule. It was considered initially that attachment was purely a chance event resulting from physical entrapment of the propagule or germling. We know now that attachment involves an active process of secretion of adhesive materials by the fungus that in some cases are highly specific for the recognition of, and binding to, a particular host species. Adhesive production may occur at a specific stage of conidium or germling development, but may best be considered a general phenomenon for the establishment of the fungus prior to penetration.

Phytoalexin accumulation in sorghum: identification of an apigeninidin acyl ester

Abstract The accumulation of a new 3-deoxyanthocyanidin phytoalexin in sorghum leaves and mesocotyls was demonstrated to occur following inoculation with an isolate of Colletotrichum graminicola pathogenic to sorghum, an isolate of C. graminicola pathogenic only to corn, and an isolate of Helminthosporum maydis (a nonpathogen of sorghum). The compound was found to be a caffeic acid ester of arabinosyl-5-O-apigeninidin and was never present in uninoculated tissues. Comparison of the fungitoxicity of the compound with that of two phytoalexins identified previously (apigeninidin and luteolinidin) showed that all three of the compounds exhibited toxicity to C. graminicola at concentrations of less than 10 μ m .