Gary Stacey

Genome sequence of the palaeopolyploid soybean

Soybean (Glycine max) is one of the most important crop plants for seed protein and oil content, and for its capacity to fix atmospheric nitrogen through symbioses with soil-borne microorganisms. We sequenced the 1.1-gigabase genome by a whole-genome shotgun approach and integrated it with physical and high-density genetic maps to create a chromosome-scale draft sequence assembly. We predict 46,430 protein-coding genes, 70% more than Arabidopsis and similar to the poplar genome which, like soybean, is an ancient polyploid (palaeopolyploid). About 78% of the predicted genes occur in chromosome ends, which comprise less than one-half of the genome but account for nearly all of the genetic recombination. Genome duplications occurred at approximately 59 and 13 million years ago, resulting in a highly duplicated genome with nearly 75% of the genes present in multiple copies. The two duplication events were followed by gene diversification and loss, and numerous chromosome rearrangements. An accurate soybean genome sequence will facilitate the identification of the genetic basis of many soybean traits, and accelerate the creation of improved soybean varieties.

A LysM Receptor-Like Kinase Plays a Critical Role in Chitin Signaling and Fungal Resistance in Arabidopsis

Chitin, a polymer of N-acetyl-d-glucosamine, is found in fungal cell walls but not in plants. Plant cells can perceive chitin fragments (chitooligosaccharides) leading to gene induction and defense responses. We identified a LysM receptor-like protein (LysM RLK1) required for chitin signaling in Arabidopsis thaliana. The mutation in this gene blocked the induction of almost all chitooligosaccharide-responsive genes and led to more susceptibility to fungal pathogens but had no effect on infection by a bacterial pathogen. Additionally, exogenously applied chitooligosaccharides enhanced resistance against both fungal and bacterial pathogens in the wild-type plants but not in the mutant. Together, our data indicate that LysM RLK1 is essential for chitin signaling in plants (likely as part of the receptor complex) and is involved in chitin-mediated plant innate immunity. The LysM RLK1-mediated chitin signaling pathway is unique, but it may share a conserved downstream pathway with the FLS2/flagellin- and EFR/EF-Tu–mediated signaling pathways. Additionally, our work suggests a possible evolutionary relationship between the chitin and Nod factor perception mechanisms due to the similarities between their potential receptors and between the signal molecules perceived by them.

Plant-microbe interactions

Molecular cloning of plant disease resistance genes-- Gregory B. Martin Transgenic plants for disease control-- Luis Herrera-Hestrella, Laura Silva Rosales, and Rafael Rivera-Bustamante Systemic acquired resistance-- Urs Neuenschwander, Kay Lawton, and John Ryals Interactions of grasses with endophytic Epichloe species and hybrids-- Christopher L. Schardl Pathogenesis and sexual development of the smut fungi-- J.W. Kronstad Current concepts in the use of introduced bacteria for biological disease control: mechanisms and antifungal metabolites-- Linda S. Thomashow and David M. Weller Legume signals to rhizobial symbionts: a new approach for defining rhizosphere colonization-- Donald A. Philips and Wolfgang R. Streit Nodulation factors-- Jean Claude Prome and Nathalie Demont

Legumes symbioses : Absence of Nod genes in photosynthetic bradyrhizobia

Leguminous plants (such as peas and soybeans) and rhizobial soil bacteria are symbiotic partners that communicate through molecular signaling pathways, resulting in the formation of nodules on legume roots and occasionally stems that house nitrogen-fixing bacteria. Nodule formation has been assumed to be exclusively initiated by the binding of bacterial, host-specific lipochito-oligosaccharidic Nod factors, encoded by the nodABC genes, to kinase-like receptors of the plant. Here we show by complete genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains, BTAi1 and ORS278, that canonical nodABC genes and typical lipochito-oligosaccharidic Nod factors are not required for symbiosis in some legumes. Mutational analyses indicated that these unique rhizobia use an alternative pathway to initiate symbioses, where a purine derivative may play a key role in triggering nodule formation.

An integrated transcriptome atlas of the crop model Glycine max, and its use in comparative analyses in plants.

Soybean (Glycine max L.) is a major crop providing an important source of protein and oil, which can also be converted into biodiesel. A major milestone in soybean research was the recent sequencing of its genome. The sequence predicts 69,145 putative soybean genes, with 46,430 predicted with high confidence. In order to examine the expression of these genes, we utilized the Illumina Solexa platform to sequence cDNA derived from 14 conditions (tissues). The result is a searchable soybean gene expression atlas accessible through a browser (http://digbio.missouri.edu/soybean_atlas). The data provide experimental support for the transcription of 55,616 annotated genes and also demonstrate that 13,529 annotated soybean genes are putative pseudogenes, and 1736 currently unannotated sequences are transcribed. An analysis of this atlas reveals strong differences in gene expression patterns between different tissues, especially between root and aerial organs, but also reveals similarities between gene expression in other tissues, such as flower and leaf organs. In order to demonstrate the full utility of the atlas, we investigated the expression patterns of genes implicated in nodulation, and also transcription factors, using both the Solexa sequence data and large-scale qRT-PCR. The availability of the soybean gene expression atlas allowed a comparison with gene expression documented in the two model legume species, Medicago truncatula and Lotus japonicus, as well as data available for Arabidopsis thaliana, facilitating both basic and applied aspects of soybean research.

Identification of Four Soybean Reference Genes for Gene Expression Normalization

Gene expression analysis requires the use of reference genes constitutively expressed independently of tissues or environmental conditions. Housekeeping genes (e.g., actin, tubulin, ribosomal, polyubiquitin, and elongation factor 1‐α) are commonly used as reference genes with the assumption that they are uniformly expressed. In many cases, however, this assumption was shown to be incorrect. To provide reliable reference genes in soybean [Glycine max (L.)], we surveyed a set of genes that showed little variation in a nodulation study across a series of soybean microarray experiments. More than 200 putative reference genes were identified. We focused on 18 for further analysis using additional cDNA and Affymetrix arrays and quantitative reverse‐transcription polymerase chain reactions. Taken together, these experiments allowed us to test the expression stability of these genes in 130 different conditions, confirming four soybean genes as new reference genes (annotated as ATP‐binding cassette [ABC] transporter, F‐box protein family, metalloprotease, and CDPK‐related protein kinase). These genes should be useful for normalization of gene expression studies in soybean, an important crop plant.

Identification of a Plant Receptor for Extracellular ATP

ATP Receptor in Arabidopsis As well as its role as an intracellular energy source, extracellular adenosine triphosphate (ATP) has diverse functions as a signaling molecule. ATP receptors have been identified in animal cells, but searches based on structural homology have not identified ATP receptors in plants. Choi et al. (p. 290) have now identified an ATP receptor in the model plant Arabidopsis thaliana by tracking down the cause of mutations that leave mutant plants unresponsive to ATP signals. The receptor identified carries an intracellular kinase domain and an extracellular lectin domain. An Arabidopsis lectin receptor kinase, DORN1, is the plant receptor for extracellular adenosine triphosphate. Extracellular adenosine 5′-triphosphate (ATP) is an essential signaling molecule that is perceived in mammals by plasma membrane P2-type purinoceptors. Similar ATP receptors do not exist in plants, although extracellular ATP has been shown to play critical roles in plant growth, development, and stress responses. Here, we identify an ATP-insensitive Arabidopsis mutant, dorn1 (Does not Respond to Nucleotides 1), defective in lectin receptor kinase I.9 (Arabidopsis Information Resource accession code At5g60300). DORN1 binds ATP with high affinity (dissociation constant of 45.7 ± 3.1 nanomolar) and is required for ATP-induced calcium response, mitogen-activated protein kinase activation, and gene expression. Ectopic expression of DORN1 increased the plant response to physical wounding. We propose that DORN1 is essential for perception of extracellular ATP and likely plays a variety of roles in plant stress resistance.

Identification of 118 Arabidopsis Transcription Factor and 30 Ubiquitin-Ligase Genes Responding to Chitin, a Plant-Defense Elicitor

Chitin, found in the cell walls of true fungi and the exoskeleton of insects and nematodes, is a well-established elicitor of plant defense responses. In this study, we analyzed the expression patterns of Arabidopsis thaliana transcription factor (TF) and ubiquitin-ligase genes in response to purified chitooctaose at different treatment times (15, 30, 60, 90, and 120 min after treatment), using both quantitative polymerase chain reaction and the Affymetrix Arabidopsis whole-genome array. A total of 118 TF genes and 30 ubiquitin-ligase genes were responsive to the chitin treatment. Among these genes, members from the following four TF families were overrepresented: APETALA2/ethylene-reponsive element binding proteins (27), C2H2 zinc finger proteins (14), MYB domain-containing proteins (11), and WRKY domain transcription factors (14). Transcript variants from a few of these genes were found to respond differentially to chitin, suggesting transcript-specific regulation of these TF genes.

Quorum sensing in plant-associated bacteria

N-acyl homoserine lactone (AHL)-mediated quorum sensing by bacteria regulates traits that are involved in symbiotic, pathogenic and surface-associated relationships between microbial populations and their plant hosts. Recent advances demonstrate deviations from the classic LuxR/LuxI paradigm, which was first developed in Vibrio. For example, LuxR homologs can repress as well as activate gene expression, and non-AHL signals and signal mimics can affect the expression of genes that are controlled by quorum sensing. Many bacteria utilize multiple quorum-sensing systems, and these may be modulated via post-transcriptional and other global regulatory mechanisms. Microbes inhabiting plant surfaces also produce and respond to a diverse mixture of AHL signals. The production of AHL mimics by plants and the identification of AHL degradative pathways suggest that bacteria and plants utilize this method of bacterial communication as a key control point for influencing the outcome of their interactions.

Loss-of-Function Mutations in Chitin Responsive Genes Show Increased Susceptibility to the Powdery Mildew Pathogen Erysiphe cichoracearum

Chitin is a major component of fungal walls and insect exoskeletons. Plants produce chitinases upon pathogen attack and chito-oligomers induce defense responses in plants, though the exact mechanism behind this response is unknown. Using the ATH1 Affymetrix microarrays consisting of about 23,000 genes, we examined the response of Arabidopsis (Arabidopsis thaliana) seedlings to chito-octamers and hydrolyzed chitin after 30 min of treatment. The expression patterns elicited by the chito-octamer and hydrolyzed chitin were similar. Microarray expression profiles for several genes were verified via northern analysis or quantitative reverse transcription-PCR. We characterized T-DNA insertion mutants for nine chito-oligomer responsive genes. Three of the mutants were more susceptible to the fungal pathogen, powdery mildew, than wild type as measured by conidiophore production. These three mutants included mutants of genes for two disease resistance-like proteins and a putative E3 ligase. The isolation of loss-of-function mutants with enhanced disease susceptibility provides direct evidence that the chito-octamer is an important oligosaccharide elicitor of plant defenses. Also, this study demonstrates the value of microarray data for identifying new components of uncharacterized signaling pathways.