Seisuke Kimura

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.

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.

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.

Mechanical Regulation of Auxin-Mediated Growth

BACKGROUND The phytohormone auxin is a primary regulator of growth and developmental pattern formation in plants. Auxin accumulates at specific sites (e.g., organ primordia) and induces localized growth within a tissue. Auxin also mediates developmental responses to intrinsic and external physical stimuli; however, exactly how mechanics influences auxin distribution is unknown. RESULTS Here we show that mechanical strain can regulate auxin transport and accumulation in the tomato shoot apex, where new leaves emerge and rapidly grow. Modification of turgor pressure, application of external force, and artificial growth induction collectively show that the amount and intracellular localization of the auxin efflux carrier PIN1 are sensitive to mechanical alterations. In general, the more strained the tissue was, the more PIN1 was present per cell and the higher the proportion localized to the plasma membrane. Modulation of the membrane properties alone was sufficient to explain most of the mechanical effects. CONCLUSIONS Our experiments support the hypothesis that the plasma membrane acts as a sensor of tissue mechanics that translates the cell wall strain into cellular responses, such as the intracellular localization of membrane-embedded proteins. One implication of this fundamental mechanism is the mechanical enhancement of auxin-mediated growth in young organ primordia. We propose that growth-induced mechanical strain upregulates PIN1 function and auxin accumulation, thereby promoting further growth, in a robust positive feedback loop.

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.

A High-Throughput Method for Illumina RNA-Seq Library Preparation

With the introduction of cost effective, rapid, and superior quality next generation sequencing techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. We provide a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. We have also designed and report a set of 96 unique barcodes for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. Our developed protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. We also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation.

Two types of replication protein A 70 kDa subunit in rice, Oryza sativa: molecular cloning, characterization, and cellular & tissue distribution.

Replication protein A (RPA), which is comprised of three subunits, is an important factor involved in DNA replication, repair, and transcription. We isolated and characterized 70 and 32 kDa subunits of RPA from rice (Oryza sativa cv. Nipponbare) termed OsRPA70a and OsRPA32. OsRPA70a shows a low level of homology with OsRPA1 which was isolated from deepwater rice (Oryza sativa cv. Pin Gaew 56), previously. We also succeeded to isolate OsRPA70b which is homologue to OsRPA1 from Oryza sativa cv. Nipponbare. OsRPA70a shows only 33.8% sequence identity with OsRPA70b, indicating that two different types of 70 kDa RPA subunits are present in Oryza sativa cv. Nipponbare. These subunits showed differences in their expression patterns among tissues. The transcripts of OsRPA70a and OsRPA32 were expressed strongly in proliferating tissues such as root tips and young leaves that contain root apical meristem and marginal meristem, respectively, and weakly in the mature leaves which have no proliferating tissues. On the other hand, OsRPA70b was expressed mostly in the proliferating tissues. The roles of these molecules in plant DNA replication and DNA repair are discussed.

Tomato (Solanum lycopersicum): A Model Fruit-Bearing Crop

INTRODUCTIONTomato (Solanum lycopersicum) is one of the most important vegetable plants in the world. It originated in western South America, and domestication is thought to have occurred in Central America. Because of its importance as food, tomato has been bred to improve productivity, fruit quality, and resistance to biotic and abiotic stresses. Tomato has been widely used not only as food, but also as research material. The tomato plant has many interesting features such as fleshy fruit, a sympodial shoot, and compound leaves, which other model plants (e.g., rice and Arabidopsis) do not have. Most of these traits are agronomically important and cannot be studied using other model plant systems. There are 13 recognized wild tomato species that display a great variety of phenotypes and can be crossed with the cultivated tomato. These wild tomatoes are important for breeding, as sources of desirable traits, and for evolutionary studies. Current progress on the tomato genome sequencing project has generated useful information to help in the study of tomato. In addition, the tomato belongs to the extremely large family Solanaceae and is closely related to many commercially important plants such as potato, eggplant, peppers, tobacco, and petunias. Knowledge obtained from studies conducted on tomato can be easily applied to these plants, which makes tomato important research material. Because of these facts, tomato serves as a model organism for the family Solanaceae and, specifically, for fleshy-fruited plants.

ATM‐mediated phosphorylation of SOG1 is essential for the DNA damage response in Arabidopsis

Arabidopsis SOG1 (suppressor of gamma response 1) is a plant‐specific transcription factor that governs the DNA damage response. Here we report that SOG1 is phosphorylated in response to DNA damage, and that this phosphorylation is mediated by the sensor kinase ataxia telangiectasia mutated (ATM). We show that SOG1 phosphorylation is crucial for the response to DNA damage, including transcriptional induction of downstream genes, transient arrest of cell division and programmed cell death. Although the amino‐acid sequences of SOG1 and the mammalian tumour suppressor p53 show no similarity, this study demonstrates that ATM‐mediated phosphorylation of a transcription factor has a pivotal role in the DNA damage response in both plants and mammals.

DNA Damage Response in Plants: Conserved and Variable Response Compared to Animals

The genome of an organism is under constant attack from endogenous and exogenous DNA damaging factors, such as reactive radicals, radiation, and genotoxins. Therefore, DNA damage response systems to sense DNA damage, arrest cell cycle, repair DNA lesions, and/or induce programmed cell death are crucial for maintenance of genomic integrity and survival of the organism. Genome sequences revealed that, although plants possess many of the DNA damage response factors that are present in the animal systems, they are missing some of the important regulators, such as the p53 tumor suppressor. These observations suggest differences in the DNA damage response mechanisms between plants and animals. In this review the DNA damage responses in plants and animals are compared and contrasted. In addition, the function of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a plant-specific transcription factor that governs the robust response to DNA damage, is discussed.