Katherine Petsch

Genic and nongenic contributions to natural variation of quantitative traits in maize

The complex genomes of many economically important crops present tremendous challenges to understand the genetic control of many quantitative traits with great importance in crop production, adaptation, and evolution. Advances in genomic technology need to be integrated with strategic genetic design and novel perspectives to break new ground. Complementary to individual-gene-targeted research, which remains challenging, a global assessment of the genomic distribution of trait-associated SNPs (TASs) discovered from genome scans of quantitative traits can provide insights into the genetic architecture and contribute to the design of future studies. Here we report the first systematic tabulation of the relative contribution of different genomic regions to quantitative trait variation in maize. We found that TASs were enriched in the nongenic regions, particularly within a 5-kb window upstream of genes, which highlights the importance of polymorphisms regulating gene expression in shaping the natural variation. Consistent with these findings, TASs collectively explained 44%-59% of the total phenotypic variation across maize quantitative traits, and on average, 79% of the explained variation could be attributed to TASs located in genes or within 5 kb upstream of genes, which together comprise only 13% of the genome. Our findings suggest that efficient, cost-effective genome-wide association studies (GWAS) in species with complex genomes can focus on genic and promoter regions.

Plant MicroRNAs Display Differential 3′ Truncation and Tailing Modifications That Are ARGONAUTE1 Dependent and Conserved Across Species

Analysis of hen1 methyltransferase mutants of Arabidopsis, rice, and maize shows widespread 3′ truncation and tailing of microRNAs in patterns that are different among microRNA families but highly conserved across plant species, suggesting an important endogenous function in wild-type plants. Plant small RNAs are 3′ methylated by the methyltransferase HUA1 ENHANCER1 (HEN1). In plant hen1 mutants, 3′ modifications of small RNAs, including oligo-uridylation (tailing), are associated with accelerated degradation of microRNAs (miRNAs). By sequencing small RNAs of the wild type and hen1 mutants from Arabidopsis thaliana, rice (Oryza sativa), and maize (Zea mays), we found 3′ truncation prior to tailing is widespread in these mutants. Moreover, the patterns of miRNA truncation and tailing differ substantially among miRNA families but are conserved across species. The same patterns are also observable in wild-type libraries from a broad range of species, only at lower abundances. ARGONAUTE (AGO1), even with defective slicer activity, can bind these truncated and tailed variants of miRNAs. An ago1 mutation in hen1 suppressed such 3′ modifications, indicating that they occur while miRNAs are in association with AGO1, either during or after RNA-induced silencing complex assembly. Our results showed AGO1-bound miRNAs are actively 3′ truncated and tailed, possibly reflecting the activity of cofactors acting in conserved patterns in miRNA degradation.

Mendelian and Non-Mendelian Regulation of Gene Expression in Maize

Transcriptome variation plays an important role in affecting the phenotype of an organism. However, an understanding of the underlying mechanisms regulating transcriptome variation in segregating populations is still largely unknown. We sought to assess and map variation in transcript abundance in maize shoot apices in the intermated B73×Mo17 recombinant inbred line population. RNA–based sequencing (RNA–seq) allowed for the detection and quantification of the transcript abundance derived from 28,603 genes. For a majority of these genes, the population mean, coefficient of variation, and segregation patterns could be predicted by the parental expression levels. Expression quantitative trait loci (eQTL) mapping identified 30,774 eQTL including 96 trans-eQTL “hotspots,” each of which regulates the expression of a large number of genes. Interestingly, genes regulated by a trans-eQTL hotspot tend to be enriched for a specific function or act in the same genetic pathway. Also, genomic structural variation appeared to contribute to cis-regulation of gene expression. Besides genes showing Mendelian inheritance in the RIL population, we also found genes whose expression level and variation in the progeny could not be predicted based on parental difference, indicating that non-Mendelian factors also contribute to expression variation. Specifically, we found 145 genes that show patterns of expression reminiscent of paramutation such that all the progeny had expression levels similar to one of the two parents. Furthermore, we identified another 210 genes that exhibited unexpected patterns of transcript presence/absence. Many of these genes are likely to be gene fragments resulting from transposition, and the presence/absence of their transcripts could influence expression levels of their ancestral syntenic genes. Overall, our results contribute to the identification of novel expression patterns and broaden the understanding of transcriptional variation in plants.

Genome-Wide Analysis of leafbladeless1-Regulated and Phased Small RNAs Underscores the Importance of the TAS3 ta-siRNA Pathway to Maize Development

Maize leafbladeless1 (lbl1) encodes a key component in the trans-acting short-interfering RNA (ta-siRNA) biogenesis pathway. Correlated with a great diversity in ta-siRNAs and the targets they regulate, the phenotypes conditioned by mutants perturbing this small RNA pathway vary extensively across species. Mutations in lbl1 result in severe developmental defects, giving rise to plants with radial, abaxialized leaves. To investigate the basis for this phenotype, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects the accumulation of small RNAs in all major classes, and reveal unexpected crosstalk between ta-siRNA biogenesis and other small RNA pathways regulating transposons. Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model. Our analysis identified nine TAS loci, all belonging to the conserved TAS3 family. Information from RNA deep sequencing and PARE analyses identified the tasiR-ARFs as the major functional ta-siRNAs in the maize vegetative apex where they regulate expression of AUXIN RESPONSE FACTOR3 (ARF3) homologs. Plants expressing a tasiR-ARF insensitive arf3a transgene recapitulate the phenotype of lbl1, providing direct evidence that deregulation of ARF3 transcription factors underlies the developmental defects of maize ta-siRNA biogenesis mutants. The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3. Our data indicate that diversity in TAS pathways and their targets cannot fully account for the phenotypic differences conditioned by ta-siRNA biogenesis mutants across plant species. Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well.

Targeted forward mutagenesis by transitive RNAi

A novel technique is described that targets specific populations of transcripts for homology-based gene silencing using transitive RNAi. This approach is designed to target a subset of the transcriptome in order to identify genes involved in a particular localized process, such as photosynthesis. As a proof-of-concept approach, mesophyll cells from Arabidopsis thaliana were laser-microdissected from whole leaves to generate a focused cDNA library that was bi-directionally cloned into a transitive RNAi vector that had been designed to induce silencing of homologous, endogenous genes. Approximately 15% of the transformant plants identified from both sense and antisense libraries exhibited visible phenotypes indicative of photosynthetic defects. Amplification from the genome and sequencing of cDNA inserts identified candidate genes underlying the phenotypes. For 10 of 11 such mutants, re-transformation with an RNAi construct corresponding to the candidate gene recapitulated the original mutant phenotype, and reduction of corresponding endogene transcripts was confirmed. In addition, one of the re-transformed transgenes also silenced transcripts of closely related family members, thereby demonstrating the utility of this approach for mutagenesis of redundant gene functions. Preliminary results using tissue-specific transitive RNAi forward mutagenesis of the Arabidopsis vegetative shoot apical meristem demonstrate the broad applicability of this forward mutagenesis technique for a variety of plant cell types.

Novel DICER-LIKE1 siRNAs Bypass the Requirement for DICER-LIKE4 in Maize Development

A DCL1-dependent siRNA pathway bypasses the requirement for DCL4 in development, which can reduce selective constraints on DCL4, allowing it to diversify in response to viral suppressors. Dicer enzymes function at the core of RNA silencing to defend against exogenous RNA or to regulate endogenous genes. Plant DICER-LIKE4 (DCL4) performs dual functions, acting in antiviral defense and in development via the biogenesis of trans-acting short-interfering RNAs (siRNAs) termed tasiR-ARFs. These small RNAs play an essential role in the grasses, spatially defining the expression domain of AUXIN RESPONSE FACTOR3 (ARF3) transcription factors. However, contrary to tasiR-ARFs’ essential function in development, DCL4 proteins exhibit strong evidence of recurrent adaptation typical of host factors involved in antiviral immunity. Here, we address how DCL4 balances its role in development with pressures to diversify in response to viral attack. We show that, in contrast to other tasiR-ARF biogenesis mutants, dcl4 null alleles have an uncharacteristically mild phenotype, correlated with normal expression of select arf3 targets. Loss of DCL4 activity yields a class of 22-nucleotide tasiR-ARF variants associated with the processing of arf3 transcripts into 22-nucleotide secondary siRNAs by DCL1. Our findings reveal a DCL1-dependent siRNA pathway that bypasses the otherwise adverse developmental effects of mutations in DCL4. This pathway is predicted to have important implications for DCL4’s role in antiviral defense by reducing the selective constraints on DCL4 and allowing it to diversify in response to viral suppressors.

Correction: Mendelian and Non-Mendelian Regulation of Gene Expression in Maize

[This corrects the article DOI: 10.1371/journal.pgen.1003202.].

Correction to: Genome-wide discovery and characterization of maize long non-coding RNAs

The original version [1] of this article unfortunately contained a mistake. The additive effects of the eQTLs of lncRNAs were flipped, meaning that the base allele in the contrast to derive the additive effects should have been B73, rather than Mo17, due to the original coding of biallele SNPs as “0s” and “1s”. Going through the entire analysis procedure, it was determined that the mistake was made while tabulating the eQTL results from QTL Cartographer.

Mutagenesis by Transitive RNAi

Transitive RNAi is a posttranscriptional mechanism of gene silencing that is based on the phenomenon of “transitivity.” This term refers to the spreading of silencing outside of the initial target sequence and is associated with transgene-induced posttranscriptional gene silencing (PTGS). Transitive RNAi is triggered by placing an inverted repeat sequence immediately 3′ of the sense transgene that is to be targeted. Placement of the inverted repeat in this region is thought to increase the efficiency by which RDR6 initiates copying of the transgene to generate double-stranded RNA (dsRNA). In a proof-of-concept approach, we showed that select subsets of genes can be manipulated with transitive RNAi in a high-throughput forward mutagenesis approach (Plant J 61:873–882, 2010). Laser microdissection of Arabidopsis mesophyll cells and en masse cloning of the resulting cDNA libraries into transitive RNAi vectors demonstrated that approximately 15% of genes in the pilot study could generate visible phenotypes, resulting in photosynthetic defects. The capacity for transitive RNAi to silence multiple members of gene family members demonstrated the utility of this approach for forward mutagenesis of redundant gene functions. Targeted silencing of a focused population of gene transcripts by transitive RNAi provides an efficient and complementary approach to procedures that target the entire genome. The ability of RNAi to target closely related genes holds promise for its use in forward mutagenesis of polyploid plants, which exhibit high levels of genetic redundancy. Advantages of transitive RNAi as a forward genetic approach, as well as potential drawbacks to this method, are discussed.