Dual culture of atoxigenic and toxigenic strains of Aspergillus flavus to gain insight into repression of aflatoxin biosynthesis and fungal interaction.

Application of atoxigenic strains to compete against toxigenic strains of Aspergillus flavus strains has emerged as one of the practical strategies for reducing aflatoxin contamination in corn, peanut, and tree nuts. The actual mechanism that results in aflatoxin reduction is not fully understood. Real-time RT-PCR and relative quantification of gene expression protocol were applied to elucidate the molecular mechanism. Transcriptional analyses of aflatoxin biosynthetic gene cluster in dual culture of toxigenic and atoxigenic A. flavus strains were carried out. Six targeted genes, aflR, aflJ, omtA, ordA, pksA, and vbs, were downregulated to variable levels depending on paired strains of toxigenic and atoxigenic A. flavus. Consistent with the decreased gene expression levels, the aflatoxin concentrations in dual cultures were reduced significantly in comparison with toxigenic cultures. Fluorescent images showed fungal hyphae in dual culture displayed green fluorescent, and contacts of live hyphae were seen. A coconut agar plate assay was used to show that toxigenic A. flavus colony produced blue fluorescence under long UV exposure, suggesting that aflatoxin is exported outside fungal hyphae. Furthermore, the assay was applied to demonstrate the potential role of thigmo-regulation in fungal interaction.

Structural and functional evolution of an auxin efflux carrier PIN1 and its functional characterization in common wheat.

Particularly PIN1, PIN protein-mediated rate-limiting auxin distribution plays a critical role in plant differentiation. Although well-characterized in Arabidopsis, little is known about the structural and functional relationship of the PIN1 gene among other plants. Here, we report that the gene structure remained conserved among bryophytes and angiosperms while the gene size varied by ~ 17%. Although the positions were conserved, highly variable intron phase suggests preference for specific regions in the gene sequence for independent events of intron insertion. Significant variation was observed across gene length for insertions and deletions that were mainly localized to the exonic regions flanking intron 1, possibly demarcating the sequences prone to deletions/duplications. The N and C-terminals showed a higher protein sequence similarity (~ 80%) compared to the central hydrophilic loop (~ 26%). In addition to the signature domains and motifs, we identified four novel uncharacterized motifs in the central divergent loop of PIN1 protein. Three different homo-loci, one each on chromosome groups 4, 6, and 7, were identified in wheat each showing dramatically different expression patterns during different plant developmental stages. Virus-induced gene silencing of the TaPIN1 gene resulted up to 26% reduction in plant height. Because of its direct role in controlling plant height along with a higher expression during stem elongation, the TaPIN1 gene can be manipulated to regulate plant height.

An Ethylmethane Sulfonate Mutant Resource in Pre-Green Revolution Hexaploid Wheat.

Mutagenesis is a powerful tool used for studying gene function as well as for crop improvement. It is regaining popularity because of the development of effective and cost efficient methods for high-throughput mutation detection. Selection for semi-dwarf phenotype during green revolution has reduced genetic diversity including that for agronomically desirable traits. Most of the available mutant populations in wheat (Triticum aestivum L.) were developed in post-green revolution cultivars. Besides the identification and isolation of agronomically important alleles in the mutant population of pre-green revolution cultivar, this population can be a vital resource for expanding the genetic diversity for wheat breeding. Here we report an Ethylmethane Sulfonate (EMS) generated mutant population consisting of 4,180 unique mutant plants in a pre-green revolution spring wheat cultivar 'Indian'. Released in early 1900s, 'Indian' is devoid of any known height-reducing mutations. Unique mutations were captured by proceeding with single M2 seed from each of the 4,180 M1 plants. Mutants for various phenotypic traits were identified by detailed phenotyping for altered morphological and agronomic traits on M2 plants in the greenhouse and M3 plants in the field. Of the 86 identified mutants, 75 (87%) were phenotypically stable at the M4 generation. Among the observed phenotypes, variation in plant height was the most frequent followed by the leaf morphology. Several mutant phenotypes including looped peduncle, crooked plant morphology, 'gritty' coleoptiles, looped lower internodes, and burnt leaf tips are not reported in other plant species. Considering the extent and diversity of the observed mutant phenotypes, this population appears to be a useful resource for the forward and reverse genetic studies. This resource is available to the scientific community.

Sequencing-based high throughput mutation detection in bread wheat.

BACKGROUND: Forward genetic approaches have limited use for agronomic traits that can't be reliably scored on a single plant basis. Thus, mutants in wheat and other crops are more useful for gene function studies by reverse genetic approach. With a long-term goal to develop a sequence-based mutation detection resource in hexaploid wheat, we conducted a feasibility study to accurately differentiate induced mutations from the homoeologs' sequence variations present among the three wheat genomes. RESULTS: A reduced representation ApeKI library consisting of 21 Ethylmethane Sulfonate (EMS) induced mutants and two wild type cv. Indian plants was developed using individual barcode adapters and sequenced. A novel bioinformatics pipeline was developed to identify sequence variants using 178,464 wheat unigenes as a reference wheat transcriptome. In total, 14,130 mutational changes [Single Nucleotide Polymorphisms (SNPs) and Insertions/Deletions (INDELs)] and 150,511 homoeologous sequence changes were detected. On an average, 662 SNPs (ranging from 46 to 1,330) and 10 small INDELs (ranging from 0 to 23) were identified for each of the mutants. A mutation frequency of one per 5 Kb was observed with 70 % being transitions and 30 % transversions. The pipeline was tested using the known sequence changes in the three wheat genes. Genes present in the distal regions of the chromosomes were found to be more prone to EMS compared to genes present in the proximal regions. Redefined parameters identified a total of 28,348 mutational changes (1,349/plant). CONCLUSIONS: We conclude that sequencing based mutation detection is a valuable method to identify induced mutations at large.

Silencing of a metaphase I-specific gene results in a phenotype similar to that of the Pairing homeologous 1 (Ph1) gene mutations.

Although studied extensively since 1958, the molecular mode of action of the Pairing homeologous 1 (Ph1) gene is still unknown. In polyploid wheat, the diploid-like chromosome pairing is principally controlled by the Ph1 gene via preventing homeologous chromosome pairing (HECP). Here, we report a candidate Ph1 gene (C-Ph1) present in the Ph1 locus, transient as well as stable silencing of which resulted in a phenotype characteristic of the Ph1 gene mutants, including HECP, multivalent formation, and disrupted chromosome alignment on the metaphase I (MI) plate. Despite a highly conserved DNA sequence, the C-Ph1 gene homeologues showed a dramatically different structure and expression pattern, with only the 5B copy showing MI-specific expression, further supporting our claim for the Ph1 gene. In agreement with the previous reports about the Ph1 gene, the predicted protein of the 5A copy of the C-Ph1 gene is truncated, and thus perhaps less effective. The 5D copy is expressed around the onset of meiosis; thus, it may function during the earlier stages of chromosome pairing. Along with alternate splicing, the predicted protein of the 5B copy is different from the protein of the other two copies because of an insertion. These structural and expression differences among the homeologues concurred with the previous observations about Ph1 gene function. Stable RNAi silencing of the wheat gene in Arabidopsis showed multivalents and centromere clustering during meiosis I.