Targeted suppression of gibberellin biosynthetic genes ZmGA20ox3 and ZmGA20ox5 produces a short stature maize ideotype.

Maize is one of the world's most widely cultivated crops. As future demands for maize will continue to rise, fields will face ever more frequent and extreme weather patterns that directly affect crop productivity. Development of environmentally resilient crops with improved standability in the field, like wheat and rice, was enabled by shifting the architecture of plants to a short stature ideotype. However, such architectural change has not been implemented in maize due to the unique interactions between gibberellin (GA) and floral morphology which limited the use of the same type of mutations as in rice and wheat. Here, we report the development of a short stature maize ideotype in commercial hybrid germplasm, which was generated by targeted suppression of the biosynthetic pathway for GA. To accomplish this, we utilized a dominant, miRNA-based construct expressed in a hemizygous state to selectively reduce expression of the ZmGA20ox3 and ZmGA20ox5 genes that control GA biosynthesis primarily in vegetative tissues. Suppression of both genes resulted in the reduction of GA levels leading to inhibition of cell elongation in internodal tissues, which reduced plant height. Expression of the miRNA did not alter GA levels in reproductive tissues, and thus, the reproductive potential of the plants remained unchanged. As a result, we developed a dominant, short-stature maize ideotype that is conducive for the commercial production of hybrid maize. We expect that the new maize ideotype would enable more efficient and more sustainable maize farming for a growing world population.

Transcriptome analysis of the barley-deoxynivalenol interaction: evidence for a role of glutathione in deoxynivalenol detoxification.

Trichothecenes are a major group of toxins produced by phytopathogenic fungi, including Fusarium graminearum. Trichothecenes inhibit protein synthesis in eukaryotic cells and are toxicologically relevant mycotoxins for humans and animals. Because they promote plant disease, the role of host responses to trichothecene accumulation is considered to be an important aspect of plant defense and resistance to fungal infection. Our overall objective was to examine the barley response to application of the type B trichothecene deoxynivalenol (DON). We found that DON is diluted by movement from the application site to acropetal and basipetal florets. A susceptible barley genotype converted DON to DON-3-O-glucoside, indicating that UDP-glucosyltransferases capable of detoxifying DON must exist in barley. RNA profiling of DON-treated barley spikes revealed strong upregulation of gene transcripts encoding ABC transporters, UDP-glucosyltransferases, cytochrome P450s, and glutathione-S-transferases. We noted that transcripts encoding cysteine synthases were dramatically induced by DON, and that toxin-sensitive yeast on glutathione- or cysteine-supplemented media or carrying a gene that encodes a cysteine biosynthetic enzyme exhibit DON resistance, suggesting that preventing glutathione depletion by increasing cysteine supply could play a role in ameliorating the impact of DON. Evidence for nonenzymatic formation of DON-glutathione adducts in vitro was found using both liquid chromatography-mass spectrometry and nuclear magnetic resonance analysis, indicating that the formation of DON-glutathione conjugates in vivo may reduce the impact of trichothecenes. Our results indicate that barley exhibits multiple defense mechanisms against trichothecenes.

Validation of a candidate deoxynivalenol-inactivating UDP-glucosyltransferase from barley by heterologous expression in yeast.

Resistance to the virulence factor deoxynivalenol (DON) due to formation of DON-3-O-glucoside (D3G) is considered to be an important component of resistance against Fusarium spp. which produce this toxin. Multiple candidate UDP-glycosyltransferase (UGT) genes from different crop plants that are either induced by Fusarium spp. or differentially expressed in cultivars varying in Fusarium disease resistance have been described. However, UGT are encoded by a very large gene family in plants. The study of candidate plant UGT is highly warranted because of the potential relevance for developing Fusarium-spp.-resistant crops. We tested Arabidopsis thaliana genes closely related to a previously identified DON-glucosyltransferase gene by heterologous expression in yeast and showed that gene products with very high sequence similarity can have pronounced differences in detoxification capabilities. We also tested four candidate barley glucosyltransferases, which are highly DON inducible. Upon heterologous expression of full-length cDNAs, only one gene, HvUGT13248, conferred DON resistance. The conjugate D3G accumulated in the supernatant of DON-treated yeast transformants. We also present evidence that the product of the TaUGT3 gene recently proposed to encode a DON-detoxification enzyme of wheat does not protect yeast against DON.

The genetics of barley low-tillering mutants: low number of tillers-1 (lnt1).

Barley (Hordeum vulgare L.) carrying recessive mutations at the Low number of tillers1 (Lnt1) gene does not develop secondary tillers and only develops one to four tillers by maturity. Double mutant analysis determined that the lnt1 mutant was epistatic to five of the six low and high tillering mutants tested. Double mutants of lnt1 and the low tillering mutant intermedium-b (int-b) resulted in a uniculm plant, indicating a synergistic interaction and that Lnt and Int-b function in separate tillering pathways. RNA profiling identified 70 transcripts with either increased or decreased abundance in the lnt1 mutant compared to wild-type. One gene with reduced transcript levels in the lnt1 mutant was the BELL-like homeodomain transcription factor JuBel2. The JuBel2 allele in the lnt1.a mutant contained a frameshift mutation that eliminated most of the predicted polypeptide, indicating that the Lnt1 gene encodes JuBel2. Previous studies with the low-tillering mutant absent lower laterals (als) showed that the tillering phenotypes and genetic interactions of als and lnt1 with other tillering mutants were very similar. However, the transcriptomes were very different; many transcripts annotated as stress and defense response exhibited increased abundance in the als mutant. This difference suggests a functional separation between Als and Lnt1 in the genetic control of tillering.

The genetics of barley low-tillering mutants: absent lower laterals (als).

Barley (Hordeum vulgare L.) carrying the recessive mutation absent lower laterals (als) exhibits few tillers and irregular inflorescence development. To gain an increased understanding of the genetic control of tillering in barley, we conducted morphological, genetic, and transcriptome analysis of the als mutant. Axillary buds for primary tillers, but not for secondary tillers, developed in als plants. Double mutant combinations of als with one low-tillering and four high-tillering mutants resulted in a tillering phenotype similar to als, indicating that als was epistatic to these tillering genes. However, double mutant combinations of als with another low-tillering mutant, intermedium-b, reduced tiller numbers, indicating there were at least two genetic pathways regulating tillering in barley. Next, we used simple sequence repeat markers to map the Als gene on the long arm of barley chromosome 3H, Bin 11. Finally, the Affymetrix Barley1 GeneChip was used to identify differentially accumulated transcripts in als compared to wild-type. Forty percent of the transcripts with twofold or greater accumulation in als tissues corresponded to stress and defense response genes. This finding suggested that a tillering pathway may modulate the stress response.

Transcriptome analysis of trichothecene-induced gene expression in barley.

Fusarium head blight, caused primarily by Fusarium graminearum, is a major disease problem on barley (Hordeum vulgare L.). Trichothecene mycotoxins produced by the fungus during infection increase the aggressiveness of the fungus and promote infection in wheat (Triticum aestivum L.). Loss-of-function mutations in the TRI5 gene in F. graminearum result in the inability to synthesize trichothecenes and in reduced virulence on wheat. We examined the impact of pathogen-derived trichothecenes on virulence and the transcriptional differences in barley spikes infected with a trichothecene-producing wild-type strain and a loss-of-function tri5 trichothecene nonproducing mutant. Disease severity, fungal biomass, and floret necrosis and bleaching were reduced in spikes inoculated with the tri5 mutant strain compared with the wild-type strain, indicating that the inability to synthesize trichothecenes results in reduced virulence in barley. We detected 63 transcripts that were induced during trichothecene accumulation, including genes encoding putative trichothecene detoxification and transport proteins, ubiquitination-related proteins, programmed cell death-related proteins, transcription factors, and cytochrome P450s. We also detected 414 gene transcripts that were designated as basal defense response genes largely independent of trichothecene accumulation. Our results show that barley exhibits a specific response to trichothecene accumulation that can be separated from the basal defense response. We propose that barley responds to trichothecene accumulation by inducing at least two general responses. One response is the induction of genes encoding trichothecene detoxification and transport activities that may reduce the impact of trichothecenes. The other response is to induce genes encoding proteins associated with ubiquitination and cell death which may promote successful establishment of the disease.

Transcriptome analysis of the barley-Fusarium graminearum interaction.

Fusarium head blight (FHB) of barley (Hordeum vulgare L.) is caused by Fusarium graminearum. FHB causes yield losses and reduction in grain quality primarily due to the accumulation of trichothecene mycotoxins such as deoxynivalenol (DON). To develop an understanding of the barley-F. graminearum interaction, we examined the relationship among the infection process, DON concentration, and host transcript accumulation for 22,439 genes in spikes from the susceptible cv. Morex from 0 to 144 h after F. graminearum and water control inoculation. We detected 467 differentially accumulating barley gene transcripts in the F. graminearum-treated plants compared with the water control-treated plants. Functional annotation of the transcripts revealed a variety of infection-induced host genes encoding defense response proteins, oxidative burst-associated enzymes, and phenylpropanoid pathway enzymes. Of particular interest was the induction of transcripts encoding potential trichothecene catabolic enzymes and transporters, and the induction of the tryptophan biosynthetic and catabolic pathway enzymes. Our results define three stages of E graminearum infection. An early stage, between 0 and 48 h after inoculation (hai), exhibited limited fungal development, low DON accumulation, and little change in the transcript accumulation status. An intermediate stage, between 48 and 96 hai, showed increased fungal development and active infection, higher DON accumulation, and increased transcript accumulation. A majority of the host gene transcripts were detected by 72 hai, suggesting that this is an important timepoint for the barley-F. graminearum interaction. A late stage also identified between 96 and 144 hai, exhibiting development of hyphal mats, high DON accumulation, and a reduction in the number of transcripts observed. Our study provides a baseline and hypothesis-generating dataset in barley during F. graminearum infection and in other grasses during pathogen infection.

Comparative structural and functional characterization of sorghum and maize duplications containing orthologous myb transcription regulators of 3-deoxyflavonoid biosynthesis.

Sequence characterization of the genomic region of sorghum yellow seed 1 shows the presence of two genes that are arranged in a head to tail orientation. The two duplicated gene copies, y1 and y2 are separated by a 9.084 kbp intergenic region, which is largely composed of highly repetitive sequences. The y1 is the functional copy, while the y2 may represent a pseudogene; there are several sequence indels and rearrangements within the putative coding region of y2. The y1 gene encodes a R2R3 type of Myb domain protein that regulates the expression of chalcone synthase, chalcone isomerase and dihydroflavonol reductase genes required for the biosynthesis of 3-deoxyflavonoids. Expression of y1 can be observed throughout the plant and it represents a combination of expression patterns produced by different alleles of the maize p1. Comparative sequence analysis within the coding regions and flanking sequences of y1, y2 and their maize and teosinte orthologs show local rearrangements and insertions that may have created modified regulatory regions. These micro-colinearity modifications possibly are responsible for differential patterns of expression in maize and sorghum floral and vegetative tissues. Phylogenetic analysis indicates that sorghum y1 and y2 sequences may have arisen by gene duplication mechanisms and represent an evolutionarily parallel event to the duplication of maize p2 and p1 genes.

Development of a Fusarium graminearum Affymetrix GeneChip for profiling fungal gene expression in vitro and in planta.

Recently the genome sequences of several filamentous fungi have become available, providing the opportunity for large-scale functional analysis including genome-wide expression analysis. We report the design and validation of the first Affymetrix GeneChip microarray based on the entire genome of a filamentous fungus, the ascomycetous plant pathogen Fusarium graminearum. To maximize the likelihood of representing all putative genes (approximately 14,000) on the array, two distinct sets of automatically predicted gene calls were used and integrated into the online F. graminearum Genome DataBase. From these gene sets, a subset of calls was manually annotated and a non-redundant extract of all calls together with additional EST sequences and controls were submitted for GeneChip design. Experiments were conducted to test the performance of the F. graminearum GeneChip. Hybridization experiments using genomic DNA demonstrated the usefulness of the array for experimentation with F. graminearum and at least four additional pathogenic Fusarium species. Differential transcript accumulation was detected using the F. graminearum GeneChip with treatments derived from the fungus grown in culture under three nutritional regimes and in comparison with fungal growth in infected barley. The ability to detect fungal genes in planta is surprisingly sensitive even without efforts to enrich for fungal transcripts. The Plant Expression Database (PLEXdb, http://www.plexdb.org) will be used as a public repository for raw and normalized expression data from the F. graminearum GeneChip. The F. graminearum GeneChip will help to accelerate exploration of the pathogen-host pathways that may involve interactions between pathogenicity genes in the fungus and disease response in the plant.

Genietic and molecular characterization of Candystripel transposition events in sorghum.

In sorghum, the Candystripe1 (Cs1) transposable element causes a variegated pericarp phenotype due to its excision activity from the yl (yellow seed1) locus. The Y1 is a transcription regulator which is required for the biosynthesis of red 3-deoxyflavonoid pigments. Somatic variability in the transposition behavior of Cs1 was observed via biochemical analysis of 3-deoxyflavonoids in the leaf tissues of the Y1-cs alleles. Using somatic excisions of Cs1 as a tool, we establish that the Cs1 is active in young seedlings and the y1 locus is also functional in these tissues. Several somatic and germinal excision events were characterized and sequence analysis of independent events predominantly showed 2-bp footprints. Further, with the goal of ur.derstanding the properties of Cs1 that would facilitate the development of a transposon tagging system in sorghum, germinal excisions of Cs1 from y1 were used as a marker. Transposition of Cs1 was followed by characterization of putative insertion events. Genetic linkage between mutant phenotypes and the cosegregating restriction fragments of Cs1 provided additional evidence that Cs1 is an active transposable element in sorghum.