Larry D. Dunkle

RAS2 regulates growth and pathogenesis in Fusarium graminearum.

Fusarium graminearum is a ubiquitous pathogen of cereal crops, including wheat, barley, and maize. Diseases caused by F. graminearum are of particular concern because harvested grains frequently are contaminated with harmful mycotoxins such as deoxynivalenol (DON). In this study, we explored the role of Ras GTPases in pathogenesis. The genome of F. graminearum contains two putative Ras GTPase-encoding genes. The two genes (RAS1 and RAS2) showed different patterns of expression under different conditions of nutrient availability and in various mutant backgrounds. RAS2 was dispensable for survival but, when disrupted, caused a variety of morphological defects, including slower growth on solid media, delayed spore germination, and significant reductions in virulence on wheat heads and maize silks. Intracellular cAMP levels were not affected by deletion of RAS2 and exogenous treatment of the ras2 mutant with cAMP did not affect phenotypic abnormalities, thus indicating that RAS2 plays a minor or no role in cAMP signaling. However, phosphorylation of the mitogen-activated protein (MAP) kinase Gpmk1 and expression of a secreted lipase (FGL1) required for infection were reduced significantly in the ras2 mutant. Based on these observations, we hypothesize that RAS2 regulates growth and virulence in F. graminearum by regulating the Gpmk1 MAP kinase pathway.

CZK3, a MAP Kinase Kinase Kinase Homolog in Cercospora zeae-maydis, Regulates Cercosporin Biosynthesis, Fungal Development, and Pathogenesis

The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

Essentiality of the ketone function for toxicity of the host-selective toxin produced by Helminthosporium carbonum☆

Abstract The cyclic tetrapeptide, cyclo-(-Pro-Ala-Ala-Aoe), which is a host-selective toxin (HC-toxin) produced by the maize pathogen, Helminthosporium carbonum race 1, was reduced with sodium borohydride. Reduction converted the 2-amino-8-oxo-9,10-epoxydecanoic acid (Aoe) residue to a 2-amino-8-hydroxy-9,10-epoxydecanoic acid (Ahe) residue. Two isomers were isolated and shown by NMR to be diastereomers of cyclo-(-Pro-Ala-Ala-Ahe) that differed by their configurations of carbon atom number eight of the Ahe residues. Neither isomer, alone nor as a 1:1 mixture, was toxic to lines of maize sensitive to HC-toxin. Consequently the ketone group of the Aoe residue in HC-toxin appears to be necessary for the toxicity of this host-selective toxin.

Accumulation of host-specific toxin produced by Cochliobolus carbonum during pathogenesis of maize†

During penetration and colonization of susceptible maize leaves, the foliar pathogen Cochliobolus carbonum race 1 released the cyclic tetrapeptide toxin required for genotype-specific pathogenicity. Inoculation fluids on the leaf surface and extracts of leaf tissue contained the host-specific toxin by 12 and 18 h, respectively, after inoculation. Quantities of the toxin in inoculated leaf tissue increased during the development of necrotic lesions. The following preparations did not contain detectable quantities of the host-specific toxin: extracts of leaf tissue from susceptible or resistant isolines inoculated with the weakly virulent race 2; leaf extracts of the resistant genotype inoculated with race 1; and extracts of non-germinated conidia of either race. The results indicate that the pathogen produces and releases the toxin by the time of leaf penetration and that, in the susceptible interaction, the toxin accumulates by the time defence responses in the host must be suppressed.

Reversible effects of inhibitory diffusates from maize inoculated with Cochliobolus carbonum.

Abstract The localized resistance of maize to Cochliobolus carbonum race 1 induced by a prior inoculation with the weakly virulent race 2 is associated with the release of inhibitory compounds from affected leaf cells into the infection court after penetration. In this study, diffusates containing this inhibitory material harvested from the inoculated leaf surface were found to inhibit conidial germination and accompanying metabolic and biosynthetic activities—oxygen uptake and uridine and leucine incorporation. Addition of carboxyl-containing compounds (amino acids or salts of organic acids) to the germination medium prevented and reversed these inhibitory effects. Similarly, in the host-pathogen interaction, these compounds prevented the action of the diffusates, abolished the induced resistance, and promoted disease development in both resistant and susceptible isolines of maize by race 2 of C. carbonum . In this respect, sodium acetate mimicked the phenotypic effect of the host-specific toxin produced by race 1, which is essential for pathogenicity of C. carbonum . In contrast to the toxin, however, the effect of sodium acetate was not genotype-specific, suggesting that the inhibitory compounds produced by maize leaf cells are components of a general resistance response whose activity is suppressed by acidic compounds and whose synthesis by susceptible genotypes is suppressed by HC-toxin.

Purification and distribution of pathotoxin-enhanced proteins in sorghum*

The synthesis of a group of four 16 kDa proteins in sorghum [ Sorghum bicolor (L.) Moench] roots is enhanced by treatment with peritoxin, the host-selective toxin produced by the root rot fungus Periconia circinata (Mangin) Sacc. The proteins were purified by two-dimensional gel electrophoresis and partially characterized, and their distribution within the sorghum plant and in other plant species was determined. The four proteins were found to be charge isomers indistinguishable by electrophoretic analysis of the products of protease digestion and CNBr cleavage. In Western blots with polyclonal antibodies, the proteins were detected in all organs of mature sorghum plants in quantities that decreased from the roots upward toward the inflorescence. Multiple cross-reacting proteins from 15–21 kDa were also detected in all grass species tested and in some other monocotyledons. None of the dicotyledons analysed contained cross-reacting proteins. The results suggest that the 16 kDa proteins are conserved proteins whose constitutive synthesis is regulated and whose function is important in several plant species, especially grasses. The presence of these proteins in other plant species suggests that they are not the direct cause of disease symptoms and plant cell death in peritoxin-treated sorghum plants.

Molecular variability in the maize grey leaf spot pathogens in Brazil

Isolates of Cercospora species from leaves displaying symptoms of grey leaf spot were collected in maize-producing areas of south-central Brazil in 2001 and 2002. Restriction digests of the internal transcribed spacer region of rDNA detected the presence of the same two Cercospora species described on maize in the United States, namely C. zeae-maydis and the recently described species, C. zeina. Genetic variability among isolates was assessed by analysing 104 amplified fragment length polymorphism loci. Cluster analysis confirmed the genetic separation of isolates into two species with a mean similarity of 35%. Similarity levels within species were high, averaging 93% and 92% among isolates of C. zeae-maydis and C. zeina, respectively. The mean genetic similarity between C. zeae-maydis and C. zeina and two isolates of C. sorghi f. sp. maydis was 45% and 35%, respectively. Results of this study showed that populations of the grey leaf spot pathogens in Brazil are similar to those in the United States regarding species composition and that C. zeina is also present in Brazil.

Phenylpropanoid accumulation and symptom expression in the lethal leaf spot mutant of maize

Symptom expression in the lethal leaf spot (LLS) mutant of maize ( Zea mays L.), a disease lesion mimic conditioned by the homozygous recessive lls gene, was investigated to assess the rate of lesion development and to compare the phenolic compounds that accumulate in affected LLS leaf tissue with those that accumulate in wild-type sib-plants inoculated with the fungus Cochliobolus heterostrophus . Several esters of phenylpropanoids, including caffeic acid and ferulic acid, accumulated during lesion development in both the LLS mutant and in lesions incited by C. heterostrophus . A p -coumaric acid ester accumulated in the LLS lesions during their development but was not detected in uninoculated LLS controls or in leaves inoculated with the fungus. Unlike typical lesions incited by fungal pathogens and unlike the lesions in other disease mimics of maize, the lesions in LLS plants did not become delimited and attain a finite size. Lesions in LLS plants expanded at increasing rates until no living tissue remained on the leaf blade. Thus, LLS lesions expanded regardless of the accumulation of phenylpropanoid derivatives that are associated with lesion delimitation in fungal-induced lesions. The data suggest that lesion termination signals or factors proposed to restrict lesion expansion in disease mimics are absent from LLS plants.

Factors regulating the synthesis of a cyclic peptide pathotoxin produced by Cochliobolus carbonum

The cyclic tetrapeptide, HC-toxin, is a host-selective virulence determinant produced by the foliar pathogen of maize, Cochliobolus carbonum race 1. HC-toxin is synthesized by a multifunctional peptide synthetase (HTS) encoded by the HTS1 gene. Analyses of culture filtrates identified low toxin-producing and high toxin-producing strains among field isolates of the pathogen. These strains were analyzed by reverse transcription PCR to determine whether synthesis of the toxin is directly influenced by HTS1 transcript levels. The results indicated that expression of HTS1 was up-regulated and reached maximal levels several days before the toxin was detected in the culture medium and that HTS1 transcript levels were not correlated with toxigenicity. These observations suggest that genes in addition to HTS1 are involved in regulation of HC-toxin biosynthesis. The toxin-producing ability of low toxin-producing isolates was enhanced following inoculation and reisolation from leaves of susceptible maize genotypes, suggesting that additional factors within the host environment influence toxin production during pathogenesis.