RESUMEN

Helminthosporium solani causes silver scurf, which affects the quality of potato. The biocontrol agent Clonostachys rosea greatly limited the severity of silver scurf symptoms and amount of H. solani genomic DNA in laboratory experiments. Transcriptomic analysis during interaction showed that H. solani gene expression was highly reduced when coinoculated with the biocontrol agent C. rosea, whereas gene expression of C. rosea was clearly boosted as a response to the pathogen. The most notable upregulated C. rosea genes were those encoding proteins involved in cellular response to oxidative stress, proteases, G-protein signaling, and the methyltransferase LaeA. The most notable potato response to both fungi was downregulation of defense-related genes and mitogen-activated protein kinase kinase kinases. At a later stage, this shifted, and most potato defense genes were turned on, especially those involved in terpenoid biosynthesis when H. solani was present. Some biocontrol-activated defense-related genes in potato were upregulated during early interaction with C. rosea alone that were not triggered by H. solani alone. Our results indicate that the reductions of silver scurf using C. rosea are probably due to a combination of mechanisms, including mycoparasitism, biocontrol-activated stimulation of plant defense mechanisms, microbial competition for nutrients, space, and antibiosis.

Asunto(s)

Helminthosporium/genética , Hypocreales/genética , Control Biológico de Vectores , Solanum tuberosum/genética , Solanum tuberosum/microbiología , Transcriptoma/genética , ADN de Plantas/metabolismo , Regulación hacia Abajo/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Helminthosporium/crecimiento & desarrollo , Tubérculos de la Planta/genética , ARN de Planta/metabolismo , Terpenos/metabolismo , Regulación hacia Arriba/genética

RESUMEN

Defensins form an antimicrobial peptides (AMP) family, and have been widely studied in various plants because of their considerable inhibitory functions. However, their roles in rice (Oryza sativa L.) have not been characterized, even though rice is one of the most important staple crops that is susceptible to damaging infections. Additionally, a previous study identified 598 rice genes encoding cysteine-rich peptides, suggesting there are several uncharacterized AMPs in rice. We performed in silico gene expression and coexpression network analyses of all genes encoding defensin and defensin-like peptides, and determined that OsDEF7 and OsDEF8 are coexpressed with pathogen-responsive genes. Recombinant OsDEF7 and OsDEF8 could form homodimers. They inhibited the growth of the bacteria Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, and Erwinia carotovora subsp. atroseptica with minimum inhibitory concentration (MIC) ranging from 0.6 to 63µg/mL. However, these OsDEFs are weakly active against the phytopathogenic fungi Helminthosporium oryzae and Fusarium oxysporum f.sp. cubense. This study describes a useful method for identifying potential plant AMPs with biological activities.

Asunto(s)

Defensinas/genética , Oryza/genética , Péptidos/genética , Enfermedades de las Plantas/genética , Antiinfecciosos/aislamiento & purificación , Simulación por Computador , Cisteína/genética , Defensinas/aislamiento & purificación , Fusarium/genética , Fusarium/patogenicidad , Regulación de la Expresión Génica de las Plantas , Helminthosporium/genética , Helminthosporium/patogenicidad , Oryza/microbiología , Péptidos/aislamiento & purificación , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Xanthomonas/genética , Xanthomonas/patogenicidad

RESUMEN

Spliceosomal twin introns, "stwintrons", have been defined as complex intervening sequences that carry a second intron ("internal intron") interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an "internal intron" interrupted the donor sequence of an "external intron". Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing.

Asunto(s)

Empalme Alternativo , Helminthosporium/genética , Empalmosomas/genética , Secuencia Conservada , Intrones/genética , ARN Mensajero/genética

RESUMEN

Morphological, cultural and physiological characteristics of 19 Helminthosporium-like hyphomycetes isolated from the White Sea were studied. Taxonomic status of the isolates was verified using molecular genetics techniques. One of the isolates was identified as Alternatia sp., while the rest of marine isolates belonged to the species Paradendriphiella salina (G.K. Sutherl.) Woudenb. & Crous. The specific features of the isolates studied were characterized as adaptive. Optimum salinity for their growth was 1-2% NaCl, which is lower than the value for the known open ocean isolates. This is probably due to relatively low salinity of White Sea (22-24 per thousand) as compared with the ocean water (35 per thousand). While the temperature optimum for growth was 22 per thousand, growth and sporulation occurred at 6 degrees C, which has not been reported for marine fungi isolated from warmer seawater. All isolates studied grew and sporulated efficiently on the medium supplied with the Fucus algae extract and in the sea water layer. Conidia of the isolates submerged in the sea water were propagated efficiently, unlike the soil-born fungi. Holoblastic conidiogenesis was demonstrated by light and scanning electron microscopy, confirming the separation of P. salina from the genus Scolecobasidium.

Asunto(s)

Helminthosporium/clasificación , Helminthosporium/crecimiento & desarrollo , Filogenia , ARN Ribosómico 16S/genética , Microbiología del Agua , Organismos Acuáticos , Helminthosporium/efectos de los fármacos , Helminthosporium/genética , Salinidad , Tolerancia a la Sal/fisiología , Cloruro de Sodio/farmacología , Temperatura

RESUMEN

A new fungal isolate that grows endophytically in sweet sorghum was identified as Helminthosporium velutinum Link ex Ficinus & Schubert. Light-microscopy of cross-sections of colonized sweet sorghum roots showed that the intercellular, pigmented hyphae of the fungus was mostly limited to the epidermal layer and formed outer mantle-like structures. This endophyte has the ability to significantly increase sweet sorghum biomass. This is the first report of Helminthosporium as an endophyte and could help realize sustainable the biomass production for biofuel purposes.

Asunto(s)

Helminthosporium/clasificación , Helminthosporium/aislamiento & purificación , Sorghum/microbiología , Biomasa , ADN de Hongos/química , ADN de Hongos/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Helminthosporium/citología , Helminthosporium/genética , Histocitoquímica , Hifa/citología , Microscopía , Datos de Secuencia Molecular , Filogenia , Raíces de Plantas/microbiología , Análisis de Secuencia de ADN , Sorghum/crecimiento & desarrollo

RESUMEN

A xylanase gene (htxyl2) was cloned from Helminthosporium turcicum, the cause of northern leaf blight of maize by screening the genomic library from the fungus using a approximately 500 bp PCR fragment of the gene as a probe. The gene is a second xylanase gene cloned from the fungus and it is different from the previously cloned xylanase gene (htxyl1). The two genes are grouped into separate clades in a phylogenetic tree based on DNA sequences. The patterns of htxyl1 and htxyl2 transcript accumulation were studied in vitro with different carbon and nitrogen sources and during infection of maize by the pathogen. In culture high htxyl1 transcript was detected in mycelium obtained from medium containing birchwood xylan and birchwood xylan plus xylose as sole carbon sources. On the other hand htxyl2 transcript was detected in a medium containing birchwood xylan plus xylose but not in the medium where only birchwood xylan was used as the sole carbon source. Addition of glucose to the basal inducing media had inhibitory effect to the expression of both genes. The htxyl1 transcript accumulation declined sharply with an increase in concentration of ammonium sulphate and glutamic acid, while increased htxyl2 transcript was observed with an increase in concentrations of the two nitrogen sources. Abundant htxyl2 transcript was detected at early and late stages of infection of maize by Helminthosporium turcicum. On the other hand no detectable htxyl1 transcript was found in northern blot under the test conditions. This study demonstrated that in H. turcicum xylanolytic system htxyl1 and htxyl2 are differentially expressed and might play important roles in the saprophytic and pathogenic phases of the fungus, respectively.

Asunto(s)

Endo-1,4-beta Xilanasas/biosíntesis , Endo-1,4-beta Xilanasas/genética , Helminthosporium/genética , Zea mays/microbiología , Secuencia de Aminoácidos , Animales , Northern Blotting , Clonación Molecular , Endo-1,4-beta Xilanasas/aislamiento & purificación , Regulación Fúngica de la Expresión Génica/genética , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Semillas/microbiología , Alineación de Secuencia , Transcripción Genética

RESUMEN

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium- tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase ( hph) and the enhanced green fluorescent protein ( EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium- tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium- tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.

Asunto(s)

Agrobacterium tumefaciens/genética , Helminthosporium/genética , Transformación Genética , Zea mays/microbiología , Agrobacterium tumefaciens/metabolismo , Southern Blotting , ADN Bacteriano , Higromicina B/farmacología , Plásmidos/genética

RESUMEN

Silver scurf caused by Helminthosporium solani causes significant economic losses in table stock, seed and processing potatoes. Specific polymerase chain reaction (PCR) primers, Hs1F1/Hs2R1, from H. solani were used for the amplification of a 447-bp product from 20 tissue samples and 54 single spore H. solani isolates, from eastern Canada (27 isolates), western Canada (13 isolates) and North Dakota in USA (14 isolates), but not from other potato fungal pathogens. In addition to PCR analysis, all 54 isolates were studied using conventional detection methods, visual disease symptoms and/or colony morphology and microscopic examination of the morphology of conidiophores and conidia. The PCR assay successfully detected H. solani and the PCR results correlated well with assessments based on conventional techniques. The detection of H. solani by PCR (1 day) is rapid and offers an alternative to the time consuming conventional diagnostic techniques (4-5 weeks). Nested PCR assay was necessary for the detection of H. solani in soils and thus can provide a sensitive technique to study the epidemiology of silver scurf in soils.

Asunto(s)

ADN de Hongos/análisis , Helminthosporium/aislamiento & purificación , Enfermedades de las Plantas/microbiología , Microbiología del Suelo , Solanum tuberosum/microbiología , Cartilla de ADN , ADN de Hongos/aislamiento & purificación , Helminthosporium/genética , Tallos de la Planta/microbiología , Reacción en Cadena de la Polimerasa/métodos , Esporas Fúngicas/genética

RESUMEN

We have cloned and sequenced a novel alcohol oxidase (Hv-p68) from the filamentous fungus Helminthosporium (Cochliobolus) victoriae that copurifies with mycoviral double-stranded RNAs. Sequence analysis revealed that Hv-p68 belongs to the large family of FAD-dependent glucose methanol choline oxidoreductases and that it shares significant sequence identity (>67%) with the alcohol oxidases of the methylotrophic yeasts. Unlike the intronless alcohol oxidases from methylotrophic yeasts, a genomic fragment of the Hv-p68 gene was found to contain four introns. Hv-p68, purified from fungal extracts, showed only limited methanol oxidizing activity, and its expression was not induced in cultures supplemented with methanol as the sole carbon source. Northern hybridization analysis indicated that overexpression of Hv-p68 is associated with virus infection, because significantly higher Hv-p68 mRNA levels (10- to 20-fold) were detected in virus-infected isolates compared with virus-free ones. We confirmed by Northwestern blot analysis that Hv-p68 exhibits RNA binding activity and demonstrated that the RNA-binding domain is localized within the N-terminal region that contains a typical ADP-binding beta-alpha-beta fold motif. The Hv-p68 gene, or closely similar genes, was present in all species of the genus Cochliobolus but absent in the filamentous fungus, Penicillium chrysogenum, as well as in two nonmethylotrophic yeasts examined. This study represents the first reported case that a member of the FAD-dependent glucose methanol choline oxidoreductase family, Hv-p68, may function as an RNA-binding protein.

Asunto(s)

Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/fisiología , Proteínas Fúngicas , Genes Fúngicos , Helminthosporium/enzimología , Helminthosporium/virología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Totivirus/genética , Oxidorreductasas de Alcohol/biosíntesis , Secuencia de Aminoácidos , Ascomicetos/enzimología , Ascomicetos/genética , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Helminthosporium/genética , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , ARN Bicatenario/metabolismo , ARN Mensajero/biosíntesis , ARN Viral/metabolismo , Proteínas de Unión al ARN/biosíntesis , Homología de Secuencia de Aminoácido , Transfección

RESUMEN

A cellular protein that co-purifies with mycoviral dsRNA was isolated from the plant pathogenic fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae) infected with two viruses, the totivirus Helminthosporium victoriae 190S virus and the chrysovirus-like Helminthosporium victoriae 145S virus (Hv145SV). The cellular protein, which was, designated Hv-p68, accumulated to higher levels in virus-infected isolates compared to virus-free ones. The majority of the Hv145S dsRNAs were found in association with Hv-p68 and not packaged in virions. Hv-p68 could also be detected as a minor component of the virus capsid. Evidence is presented that Hv-p68 occurs in vivo as an octamer and that it possesses RNA-binding activities. Based on partial amino acid sequence analysis, Hv-p68 was shown to share significant sequence identity with alcohol oxidases from methylotrophic yeasts. Hv-p68 is proposed to play a role in viral RNA packaging/replication and in regulating viral pathogenesis.

Asunto(s)

Helminthosporium/química , Helminthosporium/virología , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , Oxidorreductasas de Alcohol/química , Secuencia de Aminoácidos , Cápside/metabolismo , Cápside/ultraestructura , Centrifugación por Gradiente de Densidad , Helminthosporium/enzimología , Helminthosporium/genética , Microscopía Electrónica , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Sondas ARN/genética , Sondas ARN/metabolismo , Virus ARN/genética , Virus ARN/aislamiento & purificación , Virus ARN/metabolismo , Virus ARN/ultraestructura , ARN Bicatenario/genética , ARN Bicatenario/aislamiento & purificación , ARN Viral/genética , ARN Viral/aislamiento & purificación , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/ultraestructura , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ensamble de Virus

RESUMEN

The fungal maize pathogen Cochliobolus carbonum produces a phytotoxic and cytostatic cyclic peptide, HC-toxin, of structure cyclo(D-prolyl-L-alanyl-D-alanyl-L-Aeo), in which Aeo stands for 2-amino-9,10-epoxi-8-oxodecanoic acid. Here we report the isolation of a gene, TOXC, that is present only in HC-toxin-producing (Tox2+) fungal strains. TOXC is present in most Tox2+ strains in three functional copies, all of which are on the same chromosome as the gene encoding HC-toxin synthetase. When all copies of TOXC are mutated by targeted gene disruption, the fungus grows and sporulates normally in vitro but no longer makes HC-toxin and is not pathogenic, indicating that TOXC has a specific role in HC-toxin production and hence virulence. The TOXC mRNA is 6.5 kb and the predicted product has 2,080 amino acids and a molecular weight of 233,000. The primary amino acid sequence is highly similar (45 to 47% identity) to the beta subunit of fatty acid synthase from several lower eukaryotes, and contains, in the same order as in other beta subunits, domains predicted to encode acetyl transferase, enoyl reductase, dehydratase, and malonyl-palmityl transferase. The most plausible function of TOXC is to contribute to the synthesis of the decanoic acid backbone of Aeo.

Asunto(s)

Ácido Graso Sintasas/genética , Genes Fúngicos , Helminthosporium/genética , Helminthosporium/metabolismo , Micotoxinas/biosíntesis , Péptidos Cíclicos/biosíntesis , Zea mays/microbiología , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos/genética , Eliminación de Gen , Helminthosporium/patogenicidad , Datos de Secuencia Molecular , Fenotipo , Mapeo Restrictivo , Virulencia/genética

RESUMEN

The complete nucleotide sequence, 5178 bp, of the totivirus Helminthosporium vicotoriae 190S virus (Hv190SV) double-stranded RNA, was determined. Computer-assisted sequence analysis revealed the presence of two large overlapping ORFs; the 5'-proximal large ORF (ORF1) codes for the coat protein (CP) with a predicted molecular mass of 81 kDa, and the 3'-proximal ORF (ORF2), which is in the -1 frame relative to ORF1, codes for an RNA-dependent RNA polymerase (RDRP). Unlike many other totiviruses, the overlap region between ORF1 and ORF2 lacks known structural information required for translational frameshifting. Using an antiserum to a C-terminal fragment of the RDRP, the product of ORF2 was identified as a minor virion-associated polypeptide of estimated molecular mass of 92 kDa. No CP-RDRP fusion protein with calculated molecular mass of 165 kDa was detected. The predicted start codon of the RDRP ORF (2605-AUG-2607) overlaps with the stop codon (2606-UGA-2608) of the CP ORF, suggesting RDRP is expressed by an internal initiation mechanism. Hv190SV is associated with a debilitating disease of its phytopathogenic fungal host. Knowledge of its genome organization and expression will be valuable for understanding its role in pathogenesis and for potential exploitation in the development of biocontrol measures.

Asunto(s)

Proteínas de la Cápside , Helminthosporium/genética , ARN Bicatenario/química , Secuencia de Aminoácidos , Secuencia de Bases , Cápside/química , Cápside/genética , Clonación Molecular , Codón , Análisis Mutacional de ADN , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , ARN Viral/química , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética

Asunto(s)

Hongos/patogenicidad , Micotoxinas/toxicidad , Enfermedades de las Plantas/etiología , Alternaria/genética , Alternaria/metabolismo , Alternaria/patogenicidad , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/toxicidad , Hongos/genética , Hongos/metabolismo , Helminthosporium/genética , Helminthosporium/metabolismo , Helminthosporium/patogenicidad , Interacciones Huésped-Parásitos , Micotoxinas/biosíntesis , Micotoxinas/química , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Péptidos Cíclicos/toxicidad

RESUMEN

Race 1 isolates of Cochliobolus carbonum are pathogenic on certain maize lines due to production of a host-selective cyclic tetrapeptide, HC-toxin. Flanking HTS1, which encodes the central enzyme in HC-toxin biosynthesis, a gene was identified and named TOXA. Like HTS1, TOXA occurred only in isolates of the fungus that make HC-toxin and was present as two linked copies in most toxin-producing isolates. HTS1 and TOXA were transcribed in the opposite orientation and their transcriptional start sites were 386 bp apart. The predicted product of TOXA was a 58 kDa hydrophobic protein with 10-13 membrane-spanning regions. The sequence was highly similar to several members of the major facilitator superfamily that confer resistance to tetracycline, methylenomycin, and other antibiotics. Although it was possible to mutate one copy or the other of TOXA by targeted gene disruption, numerous attempts to disrupt both copies in a single strain were unsuccessful, suggesting that TOXA is an essential gene in strains that synthesize HC-toxin. On the basis of its presence only in HC-toxin-producing strains, its proximity to HTS1 and its predicted amino acid sequence, we propose that TOXA encodes an HC-toxin efflux pump which contributes to self-protection against HC-toxin and/or the secretion of HC-toxin into the extracellular milieu.

Asunto(s)

Proteínas Portadoras/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Helminthosporium/genética , Proteínas de Transporte de Membrana , Péptidos Cíclicos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/metabolismo , Cruzamientos Genéticos , ADN de Hongos/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Marcación de Gen , Helminthosporium/metabolismo , Datos de Secuencia Molecular , Péptidos Cíclicos/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transcripción Genética , Transformación Genética , Zea mays/microbiología