Antifungal metabolites (monorden, monocillins I, II, III) from Colletotrichum graminicola, a systemic vascular pathogen of maize

Colletotrichum graminicola is a systemic vascular pathogen that causes anthracnose stalk rot and leaf blight of maize. In the course of an effort to explore the potential presence and roles of C. graminicola metabolites in maize, ethyl acetate extracts of solid substrate fermentations of several C. graminicola isolates from Michigan and Illinois were found to be active against Aspergillus flavus and Fusarium verticillioides, both mycotoxin-producing seed-infecting fungal pathogens. Chemical investigations of the extract of one such isolate (NRRL 47511) led to the isolation of known metabolites monorden (also known as radicicol) and monocillins I–III as major components. Monorden and monocillin I displayed in vitro activity against the stalk- and ear-rot pathogen Stenocarpella maydis while only the most abundant metabolite (monorden) showed activity against foliar pathogens Alternaria alternata, Bipolaris zeicola, and Curvularia lunata. Using LC–HRESITOFMS, monorden was detected in steam-sterilized maize stalks and stalk residues inoculated with C. graminicola but not in the necrotic stalk tissues of wound-inoculated plants grown in an environmental chamber. Monorden and monocillin I can bind and inhibit plant Hsp90, a chaperone of R-proteins. It is hypothesized that monorden and monocillins could support the C. graminicola disease cycle by disrupting maize plant defenses and by excluding other fungi from necrotic tissues and crop residues. This is the first report of natural products from C. graminicola, as well as the production of monorden and monocillins by a pathogen of cereals.     

Evaluation of fungal contamination and mycotoxin production in maize silage

Agricultural activities involve daily use of maize silage as feed for livestock, which can be contaminated by mycotoxigenic molds. To evaluate fungal contamination, and the production of mycotoxins in maize silage we propose a multi-disciplinary approach utilizing PCR methods with genes of the aflatoxin (ver-1, omt-1 and apa-2), fumonisin (FUM1) and trichothecene (TRI6) biosynthesis pathways. To detect Aspergillus fumigatus, a 26S/intergenic spacer region of the rDNA complex was amplified. These specific PCR assays allowed three major groups of toxigenic fungi-like aflatoxin-producing Aspergilli, fumonisin and trichothecene-producing Fusaria, and the ubiquitous mold A. fumigatus, to be targeted. A multimycotoxin method is also proposed to simultaneously quantify seven mycotoxins (i.e., aflatoxin B₁, citrinin, deoxynivalenol, fumonisin B₁, gliotoxin, ochratoxin A, zearalenone) in maize silage by high-performance liquid chromatography coupled to mass spectrometry (HPLC–MS). These microbiological and analytical tools revealed three potentially toxigenic groups of fungi and A. fumigatus grown from mature maize silage (11 month old) that was collected in Normandy (France) and the mycotoxins aflatoxin B₁ (7.0–51.3 μg/kg), citrinin (10.1–14.2 μg/kg), deoxynivalenol (128.0–181.0 μg/kg) and gliotoxin (6.6–11.9 μg/kg). Results indicate that the combination of PCR and HPLC–MS can be used to assess fungal quality of maize silages.     

Precise mapping of quantitative trait loci for resistance to southern leaf blight, caused by Cochliobolus heterostrophus race O, and flowering time using advanced intercross maize lines

The intermated B73 × Mo17 (IBM) population, an advanced intercross recombinant inbred line population derived from a cross between the maize lines B73 (susceptible) and Mo17 (resistant), was evaluated in four environments for resistance to southern leaf blight (SLB) disease caused by Cochliobolus heterostrophus race O. Two environments were artificially inoculated, while two were not inoculated and consequently had substantially lower disease pressure. Four common SLB resistance quantitative trait loci (QTL) were identified in all environments, two in bin 3.04 and one each in bins 1.10 and 8.02/3. There was no significant correlation between disease resistance and days to anthesis. A direct comparison was made between SLB QTL detected in two populations, independently derived from the same parental cross: the IBM advanced intercross population and a conventional recombinant inbred line population. Several QTL for SLB resistance were detected in both populations, with the IBM providing between 5 and, in one case, 50 times greater mapping resolution.     

RNA-Seq Analysis Reveals MAPKKK Family Members Related to Drought Tolerance in Maize

The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction pathway that is involved in plant development and stress responses. As the first component of this phosphorelay cascade, mitogen-activated protein kinase kinase kinases (MAPKKKs) act as adaptors linking upstream signaling steps to the core MAPK cascade to promote the appropriate cellular responses; however, the functions of MAPKKKs in maize are unclear. Here, we identified 71 MAPKKK genes, of which 14 were novel, based on a computational analysis of the maize (Zea mays L.) genome. Using an RNA-seq analysis in the leaf, stem and root of maize under well-watered and drought-stress conditions, we identified 5,866 differentially expressed genes (DEGs), including 8 MAPKKK genes responsive to drought stress. Many of the DEGs were enriched in processes such as drought stress, abiotic stimulus, oxidation-reduction, and metabolic processes. The other way round, DEGs involved in processes such as oxidation, photosynthesis, and starch, proline, ethylene, and salicylic acid metabolism were clearly co-expressed with the MAPKKK genes. Furthermore, a quantitative real-time PCR (qRT-PCR) analysis was performed to assess the relative expression levels of MAPKKKs. Correlation analysis revealed that there was a significant correlation between expression levels of two MAPKKKs and relative biomass responsive to drought in 8 inbred lines. Our results indicate that MAPKKKs may have important regulatory functions in drought tolerance in maize.     

Early Events in the Fusarium verticillioides-Maize Interaction Characterized by Using a Green Fluorescent Protein-Expressing Transgenic Isolate

The infection of maize by Fusarium verticillioides can result in highly variable disease symptoms ranging from asymptomatic plants to severe rotting and wilting. We produced F. verticillioides green fluorescent protein-expressing transgenic isolates and used them to characterize early events in the F. verticillioides-maize interaction that may affect later symptom appearance. Plants grown in F. verticillioides-infested soil were smaller and chlorotic. The fungus colonized all of the underground parts of a plant but was found primarily in lateral roots and mesocotyl tissue. In some mesocotyl cells, conidia were produced within 14 to 21 days after infection. Intercellular mycelium was detected, but additional cells were not infected until 21 days after planting. At 25 to 30 days after planting, the mesocotyl and main roots were heavily infected, and rotting developed in these tissues. Other tissues, including the adventitious roots and the stem, appeared to be healthy and contained only a small number of hyphae. These results imply that asymptomatic systemic infection is characterized by a mode of fungal development that includes infection of certain tissues, intercellular growth of a limited number of fungal hyphae, and reproduction of the fungus in a few cells without invasion of other cells. Development of visibly rotted tissue is associated with massive production of fungal mycelium and much less organized growth.     

Expression of a rice chitinase gene in transgenic banana (��Gros Michel��, AAA genome group) confers resistance to black leaf streak disease

Transgenic banana (Musa acuminata ??Gros Michel??) integrating either of two rice chitinase genes was generated and its resistance to Black Leaf Streak disease caused by the fungus Mycosphaerella fijiensis was tested using a leaf disk bioassay. PCR screening indicated the presence of the hpt selectable marker gene in more than 90 % of the lines tested, whereas more than three quarters of the lines contained the linked rice chitinase gene resulting in a co-transformation frequency of at least 71.4 %. Further, a unique stable integration of the transgenes in each line revealed some false negative PCR results and the expected co-transformation frequency of 100 %. The transgene insert number per line ranged from 1 to 5 and single transgene insert lines (25 % of all) were identified. Considerable delay in disease development (up to 63 days post-incoculation) over a monitoring period of 108 days occurred in nine lines with extracellularly targeted chitinase out of 17 transgenic lines tested and their necrotic leaf area decreased by 73?C94 % compared to the untransformed susceptible control line. Finally, correlation between symptom development and rice chitinase expression was confirmed in two lines by Western analysis. The potential of rice chitinase genes to enhance resistance against M. fijiensis in banana was demonstrated as well as the usefulness of the leaf disk bioassay for early disease screening in transgenic banana lines.     

A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize.     

Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize

An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types), z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control), normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25–30%) appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences.