Adult plant resistance in maize to northern leaf spot is a feature of partial loss-of-function alleles of Hm1.

Adult plant resistance (APR) is an enigmatic phenomenon in which resistance genes are ineffective in protecting seedlings from disease but confer robust resistance at maturity. Maize has multiple cases in which genes confer APR to northern leaf spot, a lethal disease caused by Cochliobolus carbonum race 1 (CCR1). The first identified case of APR in maize is encoded by a hypomorphic allele, Hm1A, at the hm1 locus. In contrast, wild-type alleles of hm1 provide complete protection at all developmental stages and in every part of the maize plant. Hm1 encodes an NADPH-dependent reductase, which inactivates HC-toxin, a key virulence effector of CCR1. Cloning and characterization of Hm1A ruled out differential transcription or translation for its APR phenotype and identified an amino acid substitution that reduced HC-toxin reductase (HCTR) activity. The possibility of a causal relationship between the weak nature of Hm1A and its APR phenotype was confirmed by the generation of two new APR alleles of Hm1 by mutagenesis. The HCTRs encoded by these new APR alleles had undergone relatively conservative missense changes that partially reduced their enzymatic activity similar to HM1A. No difference in accumulation of HCTR was observed between adult and juvenile plants, suggesting that the susceptibility of seedlings derives from a greater need for HCTR activity, not reduced accumulation of the gene product. Conditions and treatments that altered the photosynthetic output of the host had a dramatic effect on resistance imparted by the APR alleles, demonstrating a link between the energetic or metabolic status of the host and disease resistance affected by HC-toxin catabolism by the APR alleles of HCTR.

Regulation of stomatal tropism and infection by light in Cercospora zeae-maydis: evidence for coordinated host/pathogen responses to photoperiod?

Cercospora zeae-maydis causes gray leaf spot of maize, which has become one of the most widespread and destructive diseases of maize in the world. C. zeae-maydis infects leaves through stomata, which is predicated on the ability of the pathogen to perceive stomata and reorient growth accordingly. In this study, the discovery that light was required for C. zeae-maydis to perceive stomata and infect leaves led to the identification of CRP1, a gene encoding a putative blue-light photoreceptor homologous to White Collar-1 (WC-1) of Neurospora crassa. Disrupting CRP1 via homologous recombination revealed roles in multiple aspects of pathogenesis, including tropism of hyphae to stomata, the formation of appressoria, conidiation, and the biosynthesis of cercosporin. CRP1 was also required for photoreactivation after lethal doses of UV exposure. Intriguingly, putative orthologs of CRP1 are central regulators of circadian clocks in other filamentous fungi, raising the possibility that C. zeae-maydis uses light as a key environmental input to coordinate pathogenesis with maize photoperiodic responses. This study identified a novel molecular mechanism underlying stomatal tropism in a foliar fungal pathogen, provides specific insight into how light regulates pathogenesis in C. zeae-maydis, and establishes a genetic framework for the molecular dissection of infection via stomata and the integration of host and pathogen responses to photoperiod.

A circadian rhythm regulating hyphal melanization in Cercospora kikuchii.

Many metabolic and developmental processes in fungi are controlled by biological rhythms. Circadian rhythms approximate a daily (24 h) cycle and have been thoroughly studied in the model fungus, Neurospora crassa. However relatively few examples of true circadian rhythms have been documented among other filamentous fungi. In this study we describe a circadian rhythm underlying hyphal melanization in Cercospora kikuchii, an important pathogen of soybean. After growth in light or light : dark cycles, colonies transferred to darkness produced zonate bands of melanized hyphae interspersed with bands of hyaline hyphae. Rhythmic production of bands was remarkably persistent in the absence of external cues, lasting at least 7 d after transfer to darkness, and was compensated over a range of temperatures. As in N. crassa, blue light but not red light was sufficient to entrain the circadian rhythm in C. kikuchii, and a putative ortholog of white collar-1, one of the genes required for light responses in N. crassa, was identified in C. kikuchii. Circadian regulation of melanization is conserved in other members of the genus: Similar rhythms were identified in another field isolate of C. kikuchii as well as field isolates of C. beticola and C. sorghi, but not in wild-type strains of C. zeae-maydis or C. zeina. This report represents the first documented circadian rhythm among Dothideomycete fungi and provides a new opportunity to dissect the molecular basis of circadian rhythms among filamentous fungi.

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.

Analyses of expressed sequence tags from the maize foliar pathogen Cercospora zeae-maydis identify novel genes expressed during vegetative, infectious, and reproductive growth.

BACKGROUND: The ascomycete fungus Cercospora zeae-maydis is an aggressive foliar pathogen of maize that causes substantial losses annually throughout the Western Hemisphere. Despite its impact on maize production, little is known about the regulation of pathogenesis in C. zeae-maydis at the molecular level. The objectives of this study were to generate a collection of expressed sequence tags (ESTs) from C. zeae-maydis and evaluate their expression during vegetative, infectious, and reproductive growth. RESULTS: A total of 27,551 ESTs was obtained from five cDNA libraries constructed from vegetative and sporulating cultures of C. zeae-maydis. The ESTs, grouped into 4088 clusters and 531 singlets, represented 4619 putative unique genes. Of these, 36% encoded proteins similar (E value < or = 10(-05)) to characterized or annotated proteins from the NCBI non-redundant database representing diverse molecular functions and biological processes based on Gene Ontology (GO) classification. We identified numerous, previously undescribed genes with potential roles in photoreception, pathogenesis, and the regulation of development as well as Zephyr, a novel, actively transcribed transposable element. Differential expression of selected genes was demonstrated by real-time PCR, supporting their proposed roles in vegetative, infectious, and reproductive growth. CONCLUSION: Novel genes that are potentially involved in regulating growth, development, and pathogenesis were identified in C. zeae-maydis, providing specific targets for characterization by molecular genetics and functional genomics. The EST data establish a foundation for future studies in evolutionary and comparative genomics among species of Cercospora and other groups of plant pathogenic fungi.

Fine mapping of the Pc locus of Sorghum bicolor, a gene controlling the reaction to a fungal pathogen and its host-selective toxin.

Milo disease in sorghum is caused by isolates of the soil-borne fungus Periconia circinata that produce PC-toxin. Susceptibility to milo disease is conditioned by a single, semi-dominant gene, termed Pc. The susceptible allele (Pc) converts to a resistant form (pc) spontaneously at a gametic frequency of 10(-3) to 10(-4). A high-density genetic map was constructed around the Pc locus using DNA markers, allowing the Pc gene to be delimited to a 0.9 cM region on the short arm of sorghum chromosome 9. Physically, the Pc-region was covered by a single BAC clone. Sequence analysis of this BAC revealed twelve gene candidates. Several of the predicted genes in the region are homologous to disease resistance loci, including one NBS-LRR resistance gene analogue that is present in multiple tandem copies. Analysis of pc isolines derived from Pc/Pc sorghum suggests that one or more members of this NBS-LRR gene family are the Pc genes that condition susceptibility.

Identification and expression analysis of regulatory genes induced during conidiation in Exserohilum turcicum.

Light influences numerous developmental and biochemical processes in fungi. The objectives of this research were to characterize the influence of light on growth and conidiation and associated gene expression in the plant pathogenic ascomycete, Exserohilum turcicum. We found that vegetative growth was more extensive in light/dark cycles than in constant light or darkness as measured by analysis of ergosterol content and genomic DNA. Cultures grown under continuous white light or blue light (approximately 465-480 nm) were developmentally arrested after the formation of conidiophores, whereas those grown in continuous darkness or a light/dark cycle produced mature conidia. Incubation of conidiophore-producing cultures in darkness for a minimum of 2 h was necessary and sufficient to initiate synchronous conidiation. To identify genes that are expressed during dark-induced conidiation, we constructed subtractive cDNA libraries from cultures grown under conidiation-permissive and -repressive conditions. From 816 sequenced EST clones in the conidiation-permissive and 310 in the repressive libraries, 12 putative regulatory genes were chosen for expression analysis by quantitative real-time PCR. The majority of those genes reached maximum expression by 2 h after initiation of the dark period and then declined to initial levels by 4-24 h in darkness. Expression of two dark-induced genes remained elevated after 24 h in darkness but was reset to initial levels if cultures were returned to light. This study revealed several genes whose expression increased rapidly after dark induction of conidiation, suggesting that they encode regulators of asexual development in E. turcicum.

Malazy, a degenerate, species-specific transposable element in Cercospora zeae-maydis.

Two fungal pathogens, Cercospora zeae-maydis Groups I and II, cause gray leaf spot of maize. During the sequencing of a cosmid library from C. zeae-maydis Group I, we discovered a sequence with high similarity to Maggy, a transposable element from Magnaporthe grisea. The element from C. zeae-maydis, named Malazy, contained 194-base-pair terminal repeats and sequences with high similarity to reverse transcriptase and integrase, components of the POL gene in the gypsy-like retrotransposons in fungi. Sequences with similarity to other POL gene components, protease and ribonuclease, were not detected in Malazy. A single copy of the element was detected by PCR and Southern analyses in all six North American isolates of C. zeae-maydis Group I but was not detected in the four isolates of C. zeae-maydis Group II from three continents or in phylogenetically related species. Fragments of the core domains of reverse transcriptase and integrase contained a high frequency of stop codons that were conserved in all six isolates of Group I. Additional C:G to T:A transitions in occasional isolates usually were silent mutations, while two resulted in isolate-specific stop codons. The absence of Malazy from related species suggests that it was acquired after the divergence of C. zeae-maydis Groups I and II. The high frequency of stop codons and the presence of a single copy of the element suggest that it was inactivated soon after it was acquired. Because the element is inactive and because reading frames for other genes were not found in sequences flanking the element, Malazy does not appear to be the cause of differences leading to speciation or genetic diversity between C. zeae-maydis Groups I and II.

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.

Microcycle Conidiation in Cercospora zeae-maydis.

ABSTRACT Conidia of Cercospora zeae-maydis are the primary inoculum causing gray leaf spot of maize. On nutrient-deficient substrates, but not on water on the leaf surface, conidia germinate and develop secondary conidia on conidiophores produced from germ tubes or conidial cells. A population of conidia increases its numbers more than twofold by 2 days on the surface of a water droplet and by fourfold on trichomes. This microcycle conidiation is suppressed by hydrogen peroxide and ammonium compounds but not by nitrate compounds, amino acids, or simple sugars. Microcycle conidiation is sensitive to alpha-amanitin and cycloheximide, suggesting that new RNA and proteins must be synthesized. Upon transfer from a humid to a dry atmosphere, secondary conidia and conidiophores dehydrate and collapse. Mature, dehydrated, secondary conidia are liberated by wind speeds approximately one-third those required to liberate hydrated conidia. The dispersed secondary conidia can rehydrate and germinate normally. Because this microcycle conidiation occurs at the expense of endogenous reserves, the ability to produce secondary conidia is lost after four successive cycles without a period of growth on nutrient media. This alternative method of maintaining inoculum potential during periods of fluctuating relative humidity may have epidemiological consequences when primary conidia fail to infect.

A Rapid PCR-Based Method for the Detection of Magnaporthe oryzae from Infected Perennial Ryegrass.

Gray leaf spot caused by Magnaporthe oryzae is a serious disease of perennial ryegrass in the midwestern United States. Symptoms of gray leaf spot can be confused with those caused by other fungal diseases that also are common during periods of high temperatures and ample moisture. Because turf managers must select appropriate fungicides for remedial treatment, accurate and timely identification of the pathogen is essential for efficient and effective disease management. We developed and evaluated a polymerase chain reaction (PCR)-based method to detect M. oryzae in infected perennial ryegrass tissue. The method utilizes a commercially available kit that is used for isolation and amplification of plant DNA from leaf tissue. The kit protocol was modified and found to be reliable for the extraction of M. oryzae DNA from infected perennial ryegrass. Primers were designed to amplify a 687-bp fragment of the Pot2 transposon that is found in multiple copies in the genome of the pathogen. The protocol amplified amounts of purified DNA as low as 5 pg and consistently and specifically detected M. oryzae in single diseased leaf blades as well as in field samples of infected perennial ryegrass. The total time required for detection was approximately 4 to 8 h.

Host-selective toxins and avirulence determinants: what's in a name?

Host-selective toxins, a group of structurally complex and chemically diverse metabolites produced by plant pathogenic strains of certain fungal species, function as essential determinants of pathogenicity or virulence. Investigations into the molecular and biochemical responses to these disease determinants reveal responses typically associated with host defense and incompatibility induced by avirulence determinants. The characteristic responses that unify these disparate disease phenotypes are numerous, yet the evidence implicating a causal relationship of these responses, whether induced by host-selective toxins or avirulence factors, in determining the consequences of the host-pathogen interaction is equivocal. This review summarizes some examples of the action of host-selective toxins to illustrate the similarity in responses with those to avirulence determinants.