Sorghum 3-Deoxyanthocyanidin Flavonoids Confer Resistance against Corn Leaf Aphid.

In this study we examined the role of sorghum flavonoids in providing resistance against corn leaf aphid (CLA) Rhopalosiphum maidis. In sorghum, accumulation of these flavonoids is regulated by a MYB transcription factor, yellow seed1 (y1). Functional y1 alleles accumulate 3-deoxyflavonoids (3-DFs) and 3-deoxyanthocyanidins (3-DAs) whereas null y1 alleles fail to accumulate these compounds. We found that significantly higher numbers of alate CLA adults colonized null y1 plants as compared to functional y1 plants. Controlled cage experiments and pairwise choice assays demonstrated that apterous aphids preferred to feed and reproduce on null y1 plants. These near-isogenic sorghum lines do not differ in their epicuticular wax content and were also devoid of any leaf trichomes. Significantly higher mortality of CLA was observed on artificial aphid diet supplemented with flavonoids obtained from functional y1 plants as compared to null y1 plants or the relevant controls. Our results demonstrate that the proximate mechanism underlying the deleterious effects on aphids is y1-regulated flavonoids which are important defense compounds against CLA.

The Dominant and Poorly Penetrant Phenotypes of Maize Unstable factor for orange1 Are Caused by DNA Methylation Changes at a Linked Transposon.

The maize (Zea mays) mutant Unstable factor for orange1 (Ufo1) has been implicated in the epigenetic modifications of pericarp color1 (p1), which regulates the production of the flavonoid pigments phlobaphenes. Here, we show that the ufo1 gene maps to a genetically recalcitrant region near the centromere of chromosome 10. Transcriptome analysis of Ufo1-1 mutant and wild-type plants identified a candidate gene in the mapping region using a comparative sequence-based approach. The candidate gene, GRMZM2G053177, is overexpressed by >45-fold in multiple tissues of Ufo1-1, explaining the dominance of Ufo1-1 and its phenotypes. In the mutant stock, GRMZM2G053177 has a unique transcript originating within a CACTA transposon inserted in its first intron, and it is missing the first four codons of the wild-type transcript. GRMZM2G053177 expression is regulated by the DNA methylation status of the CACTA transposon, explaining the incomplete penetrance and poor expressivity of Ufo1-1 Transgenic overexpression lines of GRMZM2G053177 (Ufo1-1) phenocopy the p1-induced pigmentation in coleoptiles, tassels, leaf sheaths, husks, pericarps, and cob glumes. Transcriptome analysis of Ufo1 versus wild-type tissues revealed changes in several pathways related to abiotic and biotic stress. Thus, this study addresses the enigma of Ufo1 identity in maize, which had gone unsolved for more than 50 years.

Turnabout Is Fair Play: Herbivory-Induced Plant Chitinases Excreted in Fall Armyworm Frass Suppress Herbivore Defenses in Maize.

The perception of herbivory by plants is known to be triggered by the deposition of insect-derived factors such as saliva and oral secretions, oviposition materials, and even feces. Such insect-derived materials harbor chemical cues that may elicit herbivore and/or pathogen-induced defenses in plants. Several insect-derived molecules that trigger herbivore-induced defenses in plants are known; however, insect-derived molecules suppressing them are largely unknown. In this study, we identified two plant chitinases from fall armyworm (Spodoptera frugiperda) larval frass that suppress herbivore defenses while simultaneously inducing pathogen defenses in maize (Zea mays). Fall armyworm larvae feed in enclosed whorls of maize plants, where frass accumulates over extended periods of time in close proximity to damaged leaf tissue. Our study shows that maize chitinases, Pr4 and Endochitinase A, are induced during herbivory and subsequently deposited on the host with the feces. These plant chitinases mediate the suppression of herbivore-induced defenses, thereby increasing the performance of the insect on the host. Pr4 and Endochitinase A also trigger the antagonistic pathogen defense pathway in maize and suppress fungal pathogen growth on maize leaves. Frass-induced suppression of herbivore defenses by deposition of the plant-derived chitinases Pr4 and Endochitinase A is a unique way an insect can co-opt the plant's defense proteins for its own benefit. It is also a phenomenon unlike the induction of herbivore defenses by insect oral secretions in most host-herbivore systems.

A sorghum MYB transcription factor induces 3-deoxyanthocyanidins and enhances resistance against leaf blights in maize.

Sorghum responds to the ingress of the fungal pathogen Colletotrichum sublineolum through the biosynthesis of 3-deoxyanthocyanidin phytoalexins at the site of primary infection. Biosynthesis of 3-deoxyanthocyanidins in sorghum requires a MYB transcription factor encoded by yellow seed1 (y1), an orthologue of the maize gene pericarp color1 (p1). Maize lines with a functional p1 and flavonoid structural genes do not produce foliar 3-deoxyanthocyanidins in response to fungal ingress. To perform a comparative metabolic analysis of sorghum and maize 3-deoxyanthocyanidin biosynthetic pathways, we developed transgenic maize lines expressing the sorghum y1 gene. In maize, the y1 transgene phenocopied p1-regulated pigment accumulation in the pericarp and cob glumes. LC-MS profiling of fungus-challenged Y1-maize leaves showed induction of 3-deoxyanthocyanidins, specifically luteolinidin. Y1-maize plants also induced constitutive and higher levels of flavonoids in leaves. In response to Colletotrichum graminicola, Y1-maize showed a resistance response.

Comparative proteomics analysis by DIGE and iTRAQ provides insight into the regulation of phenylpropanoids in maize.

The maize pericarp color1 (p1) gene encodes a Myb transcription factor that regulates the accumulation of 3-deoxyflavonoid pigments called phlobaphenes. The Unstable factor for orange1 (Ufo1) is a dominant epigenetic modifier of the p1 that results in ectopic pigmentation in pericarp. Presence of Ufo1-1 correlates with pleiotropic growth and developmental defects. To investigate the Ufo1-1-induced changes in the proteome, we conducted comparative proteomics analysis of P1-wr; Ufo1-1 pericarps using the 2-D DIGE and iTRAQ techniques. Most of the identified proteins were found to be involved in glycolysis, protein synthesis and modification, flavonoid and lignin biosynthesis and defense responses. Further, immunoblot analysis of internode protein extracts demonstrated that caffeoyl CoA O-methyltransferase (COMT) is post-transcriptionally down regulated in P1-wr; Ufo1-1 plants. Consistent with the down regulation of COMT, the concentrations of p-coumaric acid, syringaldehydes, and lignin are reduced in P1-wr; Ufo1-1 internodes. The reductions in these phenylpropanoids correlate with the bent stalk and stunted growth of P1-wr; Ufo1-1 plants. Finally, over-expression of the p1 in transgenic plants is also correlated with a lodging phenotype and reduced COMT expression. We conclude that ectopic expression of p1 can result in developmental defects that are correlated with altered regulation and synthesis of phenylpropanoid compounds including lignin. BIOLOGICAL SIGNIFICANCE: Transcription factors have specific expression patterns that ensure that the biochemical pathways under their control are active in relevant tissues. Plant breeders can select for alleles of transcription factors that produce desirable expression patterns to improve a plant's growth, development, and defense against insects and pathogens. The resulting de novo accumulation of metabolites in plant tissues in significant quantities could have beneficial and/or detrimental consequences. To understand this problem we investigated how the aberrant expression of a classically-studied transcription factor pericarp color1 (p1) which regulates phenylpropanoid metabolism, affects the maize proteome in pericarp tissue. We utilized a dominant mutant Unstable factor for orange 1-1 (Ufo1-1) which reduces the epigenetic suppression of p1 in various tissues throughout the maize plant. Our proteomic analysis shows how, in the presence of Ufo1-1, key enzymes of the glycolytic and shikimic acid pathways were modulated to produce substrates required for flavonoid synthesis. The finding that the presence of Ufo1-1 affected the expression levels of various enzymes in the lignin pathway was of particular interest. We show that lignin was reduced in Ufo1-1 plants expressing p1 and was associated with the post-transcriptional down regulation of CoA O-methyltransferase (COMT) enzyme. We further correlated the down-regulation of COMT with plant bending phenotype in Ufo1-1 plants expressing p1 and to a stalk lodging phenotype of transgenic p1 plants. This study demonstrates that although there can be adverse consequences to aberrantly overexpressing transcription factors, there might also be benefits such as being able to reduce lignin content for biofuel crops. However, more research will be required to understand the genetic and epigenetic regulation of transcription factors and how their expression can be optimized to obtain desired traits in preferred tissue types. This article is part of a Special Issue entitled: Translational Plant Proteomics.

A partial chromosomal deletion caused by random plasmid integration resulted in a reduced virulence phenotype in Fusarium graminearum.

Fusarium graminearum (teleomorph: Gibberella zeae) is an Ascomycete fungal plant pathogen which infects a range of agriculturally important crops, including wheat, barley, and maize. A random plasmid insertion mutagenesis approach was used to analyze the pathogenicity of the PH-1 strain, for which full genomic information is available. Fungal transformants were initially screened for their ability to infect wheat ears. From a total of 1,170 transformants screened, eight were confirmed to be highly reduced in pathogenicity toward wheat ears and roots. These were designated disease-attenuated F. graminearum (daf) mutants. The in vitro growth rate and appearance of each daf mutant was equivalent to the parental strain. Deoxynivalenol (DON) was not detected in threshed grain recovered from ears inoculated with the daf10 mutant. Plasmid rescue and sequencing of the mutant daf10 revealed a deletion of approximately 350 kb from one end of chromosome 1. This chromosome segment is predicted to contain 146 genes. Microarray analysis of daf10 gene expression during growth in DON-inducing conditions confirmed the large deletion. The identities of the genes deleted and their potential role in DON production, pathogenesis, and other life processes are discussed.

Flavonoid phytoalexin-dependent resistance to anthracnose leaf blight requires a functional yellow seed1 in Sorghum bicolor.

In Sorghum bicolor, a group of phytoalexins are induced at the site of infection by Colletotrichum sublineolum, the anthracnose fungus. These compounds, classified as 3-deoxyanthocyanidins, have structural similarities to the precursors of phlobaphenes. Sorghum yellow seed1 (y1) encodes a MYB transcription factor that regulates phlobaphene biosynthesis. Using the candystripe1 transposon mutagenesis system in sorghum, we have isolated functional revertants as well as loss-of-function alleles of y1. These near-isogenic lines of sorghum show that, compared to functionally revertant alleles, loss of y1 lines do not accumulate phlobaphenes. Molecular characterization of two null y1 alleles shows a partial internal deletion in the y1 sequence. These null alleles, designated as y1-ww1 and y1-ww4, do not accumulate 3-deoxyanthocyanidins when challenged with the nonpathogenic fungus Cochliobolus heterostrophus. Further, as compared to the wild-type allele, both y1-ww1 and y1-ww4 show greater susceptibility to the pathogenic fungus C. sublineolum. In fungal-inoculated wild-type seedlings, y1 and its target flavonoid structural genes are coordinately expressed. However, in y1-ww1 and y1-ww4 seedlings where y1 is not expressed, steady-state transcripts of its target genes could not be detected. Cosegregation analysis showed that the functional y1 gene is genetically linked with resistance to C. sublineolum. Overall results demonstrate that the accumulation of sorghum 3-deoxyanthocyanidin phytoalexins and resistance to C. sublineolum in sorghum require a functional y1 gene.

Characterization of two polyketide synthase genes involved in zearalenone biosynthesis in Gibberella zeae.

Zearalenone, a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. Zearalenone is a polyketide derived from the sequential condensation of multiple acetate units by a polyketide synthase (PKS), but the genetics of its biosynthesis are not understood. We cloned two genes, designated ZEA1 and ZEA2, which encode polyketide synthases that participate in the biosynthesis of zearalenone by Gibberella zeae (anamorph Fusarium graminearum). Disruption of either gene resulted in the loss of zearalenone production under inducing conditions. ZEA1 and ZEA2 are transcribed divergently from a common promoter region. Quantitative PCR analysis of both PKS genes and six flanking genes supports the view that the two polyketide synthases make up the core biosynthetic unit for zearalenone biosynthesis. An appreciation of the genetics of zearalenone biosynthesis is needed to understand how zearalenone is synthesized under field conditions that result in the contamination of grain.

Functional analysis of the polyketide synthase genes in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum).

Polyketides are a class of secondary metabolites that exhibit a vast diversity of form and function. In fungi, these compounds are produced by large, multidomain enzymes classified as type I polyketide synthases (PKSs). In this study we identified and functionally disrupted 15 PKS genes from the genome of the filamentous fungus Gibberella zeae. Five of these genes are responsible for producing the mycotoxins zearalenone, aurofusarin, and fusarin C and the black perithecial pigment. A comprehensive expression analysis of the 15 genes revealed diverse expression patterns during grain colonization, plant colonization, sexual development, and mycelial growth. Expression of one of the PKS genes was not detected under any of 18 conditions tested. This is the first study to genetically characterize a complete set of PKS genes from a single organism.

Ejection mechanics and trajectory of the ascospores of Gibberella zeae (anamorph Fuarium graminearum).

Since wind speed drops to zero at a surface, forced ejection should facilitate spore dispersal. But for tiny spores, with low mass relative to surface area, high ejection speed yields only a short range trajectory, so pernicious is their drag. Thus, achieving high speeds requires prodigious accelerations. In the ascomycete Gibberella zeae, we determined the launch speed and kinetic energy of ascospores shot from perithecia, and the source and magnitude of the pressure driving the launch. We asked whether the pressure inside the ascus suffices to account for launch speed and energy. Launch speed was 34.5 ms-1, requiring a pressure of 1.54 MPa and an acceleration of 870,000 g--the highest acceleration reported in a biological system. This analysis allows us to discount the major sugar component of the epiplasmic fluid, mannitol, as having a key role in driving discharge, and supports the role of potassium ion flux in the mechanism.