John D. Hipskind

Constitutive Accumulation of a Resveratrol-Glucoside in Transgenic Alfalfa Increases Resistance to Phoma medicaginis

Alfalfa (Medicago sativa) was transformed with a peanut (Arachis hypogaea) cDNA encoding resveratrol synthase (RS) transcriptionally regulated by an enhanced Cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants accumulated a new compound, not present in wild-type or vector-transformed alfalfa, that was identified as trans-resveratrol-3-O-beta-D-glucopyranoside (RGluc) by high-pressure liquid chromatography (HPLC), UV, 1H- and 13C-nuclear magnetic resonance (NMR) analyses. RGluc concentration was highest in the youngest leaves (>15 microg per g fresh weight) and oldest stem internode segments (>10 microg per g fresh weight) while roots contained only trace amounts (<0.2 microg per g fresh weight). RS transcript levels were highest in leaves and stems, with comparatively little transcript accumulation in the roots, while an inverse pattern was observed for chalcone synthase (CHS) transcript levels. CHS directly competes with RS for the metabolic precursors p-coumaroyl CoA and malonyl CoA, and may also contribute to the developmental variations in RGluc levels by limiting the availability of substrates. Agar-plate bioassays indicated that both RGluc and resveratrol greatly inhibit hyphal growth of the alfalfa fungal pathogen Phoma medicaginis. Subsequently, RGluc-containing leaves were wound inoculated and showed a significant reduction (relative to control leaves) in the size of necrotic lesions, intensity of adjacent chlorosis, and number of fungal reproductive structures (pycnidia). Decreasing sporulation of this pathogen may greatly reduce disease spread and severity throughout the field.

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.

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 .

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 .

Phytoalexin synthesis in the juvenile sorghum leaf

Abstract Infection of the juvenile sorghum leaf by Colletotrichum graminicola (a pathogen) and Helminthosporium maydis (a non-pathogen) resulted in the rapid accumulation of a phytoalexin pigment complex previously identified from the sorghum mesocotyl. The principal pigments have been identified as the 3-deoxyanthocyanidins, apigeninidin and luteolinidin, but the complex includes several as yet unidentified pigment components. After the onset of penetration, components of the complex rapidly accumulated to nanogram levels within a highly restricted area at the infection site. Because of the speed of synthesis, occurrence in response to attempted infection, and previously reported fungitoxicity of the components, we propose that synthesis of the pigment complex constitutes a defense response in the juvenile sorghum leaf. The results show that the preformed cyanogenic glycoside, dhurrin, is not the only source of resistance of sorghum seedlings to fungal infection.

Protection against phenol toxicity by the spore mucilage of Colletotrichum graminicola, an aid to secondary spread

Abstract Phenolic compounds inhibitory to the germination of spores of Colletotrichum graminicola were shown to leach from necrotic lesions on corn leaves caused by the fungus. Primary components of the phenolic mixture were identified as esters and glycosides of p -coumaric and ferulic acids as well as the free compounds themselves. Spores of C. graminicola produced in acervuli on infected leaves were shown to be surrounded by a mucilaginous matrix as is the case when the fungus is cultured in vitro . It is suggested that the mucilage protects spores from the inhibitory effects of the phenols by the presence of proline-rich proteins that have been shown to have a high binding affinity for a variety of phenols.

Isolation of a cDNA encoding a novel leucine-rich repeat motif from Sorghum bicolor inoculated with fungi.

A sorghum cDNA clone has been isolated that encodes a protein containing six imperfect leucine-rich repeats (LRRs) of approximately 22 amino acids in length. The putative protein, designated SLRR, also contains a signal peptide, and six potential N-glycosylation sites. Comparisons of SLRR and its LRR consensus sequences found significant homology to the extracellular binding domains of receptor-protein kinases RLK5 and TMK1 of Arabidopsis, and some plant disease resistance genes. Results from RNA gel blot analyses showed that SLRR mRNA accumulates rapidly in mesocotyls and juvenile leaves by 6 h postinoculation with the fungus Colletotricum graminicola. Further experiments suggest that the gene encoding SLRR is neither systemically induced by fungal inoculation, nor transcriptionally activated in a host-fungal-pathogen-specific manner. The presence of LRRs strongly suggests that the SLRR protein is involved in protein-ligand binding and therefore may be a component of a signal transduction pathway.

Elicitor of sorghum phytoalexin synthesis from Colletotrichum graminicola

Abstract Extracts of conidia and the conidial mucilage of Colletotrichum graminicola contained materials that elicited the accumulation of deoxyanthocyanidin phytoalexins in sorghum mesocotyls and juvenile sorghum leaves. Elicitor activity was associated with peptide and carbohydrate components extracted from conidia and with peptides removed from conidia by repeated washing. The accumulation in mesocotyl tissue of deoxyanthocyanidin phytoalexins occurred linearly over time in response to a single elicitor concentration and the amount of deoxyanthocyanidins that accumulated also increased linearly in response to increasing concentrations of crude elicitor preparations. Elicitor activity was highly specific for sorghum in that crude preparations did not elicit a visible response from nine other plant species. Neither wounding by cutting nor gentle abrasion of the sorghum tissue was sufficient to elicit the accumulation of the deoxyanthocyanidin phytoalexins.

Analysis of anthocyanins and 3-deoxyanthocyanidins by plasma desorption mass spectrometry

Abstract A technique for the mass spectrometric analysis of anthocyanins, 3-hydroxyanthocyanidins and 3-deoxyanthoeyanidins in Sorghum bicolor by plasma desorption was developed. Sensitivities to ng levels with minimum sample preparation and analysis were achieved. The technique provides a sensitive means for the measurement of these flavonoid pigments as well as a means to screen for very small quantities of the compounds within plant tissues.

Effects of elicitor from Colletotrichum graminicola on the response of sorghum to Periconia circinata and its pathotoxin

Abstract The effects of an elicitor (CG-elicitor) from Colletotrichum graminicola and the host-specific pathotoxin (PC-toxin) produced by Periconia circinata were studied to determine the interactions of responses associated with resistance and susceptibility, respectively. Roots of sorghum ( Sorghum bicolor ) accumulated 3-deoxyanthocyanidin phytoalexins in response to CG-elicitor but not in response to PC-toxin over a range of concentrations. Elicitation of the phytoalexins prior to treatment with PC-toxin had no effect on the genotype-specific induction of electrolyte leakage or on the toxin-enhanced synthesis of a specific group of 16 kDa proteins. Similarly, prior treatment with the elicitor did not prevent infection and development of milo disease symptoms in susceptible seedlings inoculated with conidia of P. circinata . However, treatment of roots with the CG-elicitor enhanced the synthesis of the 16 kDa proteins in both resistant and susceptible genotypes without expression of disease symptoms. Thus, the activities of PC-toxin and CG-elicitor are separable and independent. PC-toxin apparently does not produce disease symptoms by inducing phytoalexins, and induction of phytoalexins does not prevent pathogenesis by P. circinata or the detrimental effects of PC-toxin.