Daniel Koenig

Spontaneous epigenetic variation in the Arabidopsis thaliana methylome

Heritable epigenetic polymorphisms, such as differential cytosine methylation, can underlie phenotypic variation. Moreover, wild strains of the plant Arabidopsis thaliana differ in many epialleles, and these can influence the expression of nearby genes. However, to understand their role in evolution, it is imperative to ascertain the emergence rate and stability of epialleles, including those that are not due to structural variation. We have compared genome-wide DNA methylation among 10 A. thaliana lines, derived 30 generations ago from a common ancestor. Epimutations at individual positions were easily detected, and close to 30,000 cytosines in each strain were differentially methylated. In contrast, larger regions of contiguous methylation were much more stable, and the frequency of changes was in the same low range as that of DNA mutations. Like individual positions, the same regions were often affected by differential methylation in independent lines, with evidence for recurrent cycles of forward and reverse mutations. Transposable elements and short interfering RNAs have been causally linked to DNA methylation. In agreement, differentially methylated sites were farther from transposable elements and showed less association with short interfering RNA expression than invariant positions. The biased distribution and frequent reversion of epimutations have important implications for the potential contribution of sequence-independent epialleles to plant evolution.

The Capsella rubella genome and the genomic consequences of rapid mating system evolution

The shift from outcrossing to selfing is common in flowering plants, but the genomic consequences and the speed at which they emerge remain poorly understood. An excellent model for understanding the evolution of self fertilization is provided by Capsella rubella, which became self compatible <200,000 years ago. We report a C. rubella reference genome sequence and compare RNA expression and polymorphism patterns between C. rubella and its outcrossing progenitor Capsella grandiflora. We found a clear shift in the expression of genes associated with flowering phenotypes, similar to that seen in Arabidopsis, in which self fertilization evolved about 1 million years ago. Comparisons of the two Capsella species showed evidence of rapid genome-wide relaxation of purifying selection in C. rubella without a concomitant change in transposable element abundance. Overall we document that the transition to selfing may be typified by parallel shifts in gene expression, along with a measurable reduction of purifying selection.

Comparative transcriptomics reveals patterns of selection in domesticated and wild tomato

Significance One of the most important technological advances by humans is the domestication of plant species for the production of food. We have used high-throughput sequencing to identify changes in DNA sequence and gene expression that differentiate cultivated tomato and its wild relatives. We also identify hundreds of candidate genes that have evolved new protein sequences or have changed expression levels in response to natural selection in wild tomato relatives. Taken together, our analyses provide a snapshot of genome evolution under artificial and natural conditions. Although applied over extremely short timescales, artificial selection has dramatically altered the form, physiology, and life history of cultivated plants. We have used RNAseq to define both gene sequence and expression divergence between cultivated tomato and five related wild species. Based on sequence differences, we detect footprints of positive selection in over 50 genes. We also document thousands of shifts in gene-expression level, many of which resulted from changes in selection pressure. These rapidly evolving genes are commonly associated with environmental response and stress tolerance. The importance of environmental inputs during evolution of gene expression is further highlighted by large-scale alteration of the light response coexpression network between wild and cultivated accessions. Human manipulation of the genome has heavily impacted the tomato transcriptome through directed admixture and by indirectly favoring nonsynonymous over synonymous substitutions. Taken together, our results shed light on the pervasive effects artificial and natural selection have had on the transcriptomes of tomato and its wild relatives.

Plant secondary siRNA production determined by microRNA-duplex structure

Processing of microRNA (miRNA) precursors results in the release of a double-stranded miRNA/miRNA* duplex. The miRNA or guide strand, is loaded onto the Argonaute (AGO) effector, and the miRNA* or passenger strand is typically degraded. The loaded AGO-containing RNA-induced silencing complex specifically recognizes a target mRNA, leading to its degradation or translational inhibition. In plants, miRNA-mediated cleavage of a target triggers in some cases the production of secondary small interfering RNAs (siRNAs), which in turn can silence other genes in trans. This alternative pathway depends on the length of the miRNA and the specific AGO in the effector complex. However, 22-nt miRNAs are sufficient, but not essential for this pathway. Using a combination of computational and experimental approaches, we show that transitivity can be triggered when the small RNA that is not retained in AGO is 22-nt long. Moreover, we demonstrate that asymmetrically positioned bulged bases in the miRNA:miRNA* duplex, regardless of miRNA or miRNA* length, are sufficient for the initiation of transitivity. We propose that the RNA-induced silencing complex reprogramming occurs during the early steps of miRNA loading, before the miRNA duplex is disassembled and the guide strand is selected.

The genome of the stress-tolerant wild tomato species Solanum pennellii

Solanum pennellii is a wild tomato species endemic to Andean regions in South America, where it has evolved to thrive in arid habitats. Because of its extreme stress tolerance and unusual morphology, it is an important donor of germplasm for the cultivated tomato Solanum lycopersicum. Introgression lines (ILs) in which large genomic regions of S. lycopersicum are replaced with the corresponding segments from S. pennellii can show remarkably superior agronomic performance. Here we describe a high-quality genome assembly of the parents of the IL population. By anchoring the S. pennellii genome to the genetic map, we define candidate genes for stress tolerance and provide evidence that transposable elements had a role in the evolution of these traits. Our work paves a path toward further tomato improvement and for deciphering the mechanisms underlying the myriad other agronomic traits that can be improved with S. pennellii germplasm.

Auxin patterns Solanum lycopersicum leaf morphogenesis.

One of the most striking aspects of plant diversity is variation in leaf shape. Much of this diversity is achieved by the modulation of leaf blade dissection to form lobes or leaflets. Here, we show that the phytohormone auxin is a crucial signal regulating the partitioned outgrowth necessary to develop a dissected leaf. In developing leaves, the asymmetric distribution of auxin, driven by active transport, delineates the initiation of lobes and leaflets and specifies differential laminar outgrowth. Furthermore, homologous members of the AUX/indole-3-acetic acid (IAA) gene family mediate the action of auxin in determining leaf shape by repressing outgrowth in areas of low auxin concentration during both simple and compound leaf development. These results provide molecular evidence that leaflets initiate in a process reminiscent of organogenesis at the shoot apical meristem, but that compound and simple leaves regulate marginal growth through an evolutionarily conserved mechanism, thus shedding light on the homology of compound and simple leaves.

WOX4 Promotes Procambial Development

The WOX (WUSCHEL-related homeobox) gene family of Arabidopsis comprises fifteen plant-specific transcriptional factors that play important development roles. Genetic, phylogenetic, and genomic analyses suggest that WOX genes generally act non-autonomously to organize stem-cell and initial-cell populations within plant meristems and organ anlagen. Previous cross-complementation analyses indicate that the functional diversification of distinct WOX paralogs may be explained largely by promoter evolution, although paralog-specific protein::protein interactions are also implicated. A recent report described WOX4 function during development of the procambium, which comprises the meristematic tissues of the plant vasculature. Here we show that WOX4 fails to complement PRS1/WOX3 function, when driven from the PRS1/WOX3 native promoter. These data suggest that WOX4 identifies different DNA targets and/or interacting proteins during development of the vasculature procambium than does PRS1/WOX3 during the specification of lateral organ initial cells. The identification of super-compound leaf phenotypes induced by overexpression of the SlWOX4 ortholog in tomato suggests a functional link between vascular patterning and leaf complexity.Plant shoot organs arise from initial cells that are recruited from meristematic tissues. Previous studies have shown that members of the WUSCHEL-related HOMEOBOX (WOX) gene family function to organize various initial cell populations during plant development. The function of the WOX4 gene is previously undescribed in any plant species. Comparative analyses of WOX4 transcription and function are presented in Arabidopsis (Arabidopsis thaliana), a simple-leafed plant with collateral vasculature, and in tomato (Solanum lycopersicum), a dissected-leafed species with bicollateral venation. WOX4 is transcribed in the developing vascular bundles of root and shoot lateral organs in both Arabidopsis and tomato. RNA interference-induced down-regulation of WOX4 in Arabidopsis generated small plants whose vascular bundles accumulated undifferentiated ground tissue and exhibited severe reductions in differentiated xylem and phloem. In situ hybridization analyses of Atwox4-RNA interference plants revealed delayed and reduced expression of both the phloem developmental marker ALTERED PHLOEM1 and HOMEOBOX GENE8, a marker of the vascular procambium. Overexpression of SlWOX4 correlated with overproliferation of xylem and phloem in transgenic tomato seedlings. The cumulative data suggest that the conserved WOX4 function is to promote differentiation and/or maintenance of the vascular procambium, the initial cells of the developing vasculature.

Natural Variation in Leaf Morphology Results from Mutation of a Novel KNOX Gene

Striking diversity in size, arrangement, and complexity of leaves can sometimes be seen in closely related species. One such variation is found between wild tomato species collected by Charles Darwin from the Galapagos Islands [1-5]. Here, we show that a single-nucleotide deletion in the promoter of the PETROSELINUM (PTS) [3] gene upregulates the gene product in leaves and is responsible for the natural variation in leaf shape in the Galapagean tomatoes. PTS encodes a novel KNOTTED1-LIKE HOMEOBOX (KNOX) gene that lacks a homeodomain. We also showed that the tomato classical mutant bipinnata (bip) [6], which recapitulates the Pts phenotype, results from the loss of function of a BEL-LIKE HOMEODOMAIN (BELL) gene, BIP. We used bimolecular fluorescence complementation and two-hybrid competition assays to show that PTS represses KNOX1 protein interactions with BIP, as well as subsequent nuclear localization of this transcriptional complex. We suggest that natural variation in leaf shape can be created with a rheostat-like mechanism that alters the KNOX1 protein interaction network specifically during leaf development. This subtle change in interaction between transcription factors leaves essential KNOX1 function in the shoot apical meristem intact and appears to be a facile way to alter leaf morphology during evolution.

Evolution of DNA Methylation Patterns in the Brassicaceae is Driven by Differences in Genome Organization

DNA methylation is an ancient molecular modification found in most eukaryotes. In plants, DNA methylation is not only critical for transcriptionally silencing transposons, but can also affect phenotype by altering expression of protein coding genes. The extent of its contribution to phenotypic diversity over evolutionary time is, however, unclear, because of limited stability of epialleles that are not linked to DNA mutations. To dissect the relative contribution of DNA methylation to transposon surveillance and host gene regulation, we leveraged information from three species in the Brassicaceae that vary in genome architecture, Capsella rubella, Arabidopsis lyrata, and Arabidopsis thaliana. We found that the lineage-specific expansion and contraction of transposon and repeat sequences is the main driver of interspecific differences in DNA methylation. The most heavily methylated portions of the genome are thus not conserved at the sequence level. Outside of repeat-associated methylation, there is a surprising degree of conservation in methylation at single nucleotides located in gene bodies. Finally, dynamic DNA methylation is affected more by tissue type than by environmental differences in all species, but these responses are not conserved. The majority of DNA methylation variation between species resides in hypervariable genomic regions, and thus, in the context of macroevolution, is of limited phenotypic consequence.

Interspecific RNA Interference of SHOOT MERISTEMLESS-Like Disrupts Cuscuta pentagona Plant Parasitism

The authors demonstrate that parasite gene-specific silencing signals originating from a transgenic host are transferred into the invading parasite, leading to reduced parasite yield, stature, and infectivity. This article also refreshes the debate on the origin of haustoria as the authors use morphological and molecular evidence to show that haustoria have both stem and root characteristics. Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA–mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection.