David McK. Bird

Acyl-Lipid Metabolism

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.

Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism

We have established Meloidogyne hapla as a tractable model plant-parasitic nematode amenable to forward and reverse genetics, and we present a complete genome sequence. At 54 Mbp, M. hapla represents not only the smallest nematode genome yet completed, but also the smallest metazoan, and defines a platform to elucidate mechanisms of parasitism by what is the largest uncontrolled group of plant pathogens worldwide. The M. hapla genome encodes significantly fewer genes than does the free-living nematode Caenorhabditis elegans (most notably through a reduction of odorant receptors and other gene families), yet it has acquired horizontally from other kingdoms numerous genes suspected to be involved in adaptations to parasitism. In some cases, amplification and tandem duplication have occurred with genes suspected of being acquired horizontally and involved in parasitism of plants. Although M. hapla and C. elegans diverged >500 million years ago, many developmental and biochemical pathways, including those for dauer formation and RNAi, are conserved. Although overall genome organization is not conserved, there are areas of microsynteny that may suggest a primary biological function in nematodes for those genes in these areas. This sequence and map represent a wealth of biological information on both the nature of nematode parasitism of plants and its evolution.

The Cytochrome P450 Enzyme CYP96A15 Is the Midchain Alkane Hydroxylase Responsible for Formation of Secondary Alcohols and Ketones in Stem Cuticular Wax of Arabidopsis

Most aerial surfaces of plants are covered by cuticular wax that is synthesized in epidermal cells. The wax mixture on the inflorescence stems of Arabidopsis (Arabidopsis thaliana) is dominated by alkanes, secondary alcohols, and ketones, all thought to be formed sequentially in the decarbonylation pathway of wax biosynthesis. Here, we used a reverse-genetic approach to identify a cytochrome P450 enzyme (CYP96A15) involved in wax biosynthesis and characterized it as a midchain alkane hydroxylase (MAH1). Stem wax of T-DNA insertional mutant alleles was found to be devoid of secondary alcohols and ketones (mah1-1) or to contain much lower levels of these components (mah1-2 and mah1-3) than wild type. All mutant lines also had increased alkane amounts, partially or fully compensating for the loss of other compound classes. In spite of the chemical variation between mutant and wild-type waxes, there were no discernible differences in the epicuticular wax crystals on the stem surfaces. Mutant stem wax phenotypes could be partially rescued by expression of wild-type MAH1 under the control of the native promoter as well as the cauliflower mosaic virus 35S promoter. Cauliflower mosaic virus 35S-driven overexpression of MAH1 led to ectopic accumulation of secondary alcohols and ketones in Arabidopsis leaf wax, where only traces of these compounds are found in the wild type. The newly formed leaf alcohols and ketones had midchain functional groups on or next to the central carbon, thus matching those compounds in wild-type stem wax. Taken together, mutant analyses and ectopic expression of MAH1 in leaves suggest that this enzyme can catalyze the hydroxylation reaction leading from alkanes to secondary alcohols and possibly also a second hydroxylation leading to the corresponding ketones. MAH1 expression was largely restricted to the expanding regions of the inflorescence stems, specifically to the epidermal pavement cells, but not in trichomes and guard cells. MAH1-green fluorescent protein fusion proteins localized to the endoplasmic reticulum, providing evidence that both intermediate and final products of the decarbonylation pathway are generated in this subcellular compartment and must subsequently be delivered to the plasma membrane for export toward the cuticle.

Horizontally transferred genes in plant-parasitic nematodes: a high-throughput genomic approach

BackgroundPublished accounts of horizontally acquired genes in plant-parasitic nematodes have not been the result of a specific search for gene transfer per se, but rather have emerged from characterization of individual genes. We present a method for a high-throughput genome screen for horizontally acquired genes, illustrated using expressed sequence tag (EST) data from three species of root-knot nematode, Meloidogyne species.ResultsOur approach identified the previously postulated horizontally transferred genes and revealed six new candidates. Screening was partially dependent on sequence quality, with more candidates identified from clustered sequences than from raw EST data. Computational and experimental methods verified the horizontal gene transfer candidates as bona fide nematode genes. Phylogenetic analysis implicated rhizobial ancestors as donors of horizontally acquired genes in Meloidogyne.ConclusionsHigh-throughput genomic screening is an effective way to identify horizontal gene transfer candidates. Transferred genes that have undergone amelioration of nucleotide composition and codon bias have been identified using this approach. Analysis of these horizontally transferred gene candidates suggests a link between horizontally transferred genes in Meloidogyne and parasitism.

MOLECULAR SIGNALS AND RECEPTORS: CONTROLLING RHIZOSPHERE INTERACTIONS BETWEEN PLANTS AND OTHER ORGANISMS

Rhizosphere interactions are affected by many different regulatory signals. As yet, however, only a few have been identified. Signals, by definition, contain information, react with a receptor, and elicit a response. Signals may thus represent the highest level of evolved response in rhizosphere communities and, in that sense, occupy a supreme control point. At the same time, some signals may function as modulators of downstream responses, rather than on/off switches. To assess these possibilities, several interactions between plants and soil organisms are described, starting with the molecular interactions between legu- minous plants and symbiotic bacteria of the family Rhizobiaceae, one of the best-charac- terized plant-microbe associations in the rhizosphere. We then examine other interactions between plants and soil organisms for overlap and/or connections with the rhizosphere signals utilized in the legume-Rhizobium symbiosis. Whether information currently avail- able reflects the interaction of the organisms in nature or only in the laboratory has not always been determined. Thus, the key ecological issue of how important some of the signals are under field conditions remains to be addressed. Molecular tools now available make this task less daunting than in the past, and thus a new age of experimental field ecology may soon burst forth in rhizosphere studies. By identifying the signals, receptors, and the critical control points, we can better understand the organismal dynamics in this key belowground ecosystem.

Analysis and functional classification of transcripts from the nematode Meloidogyne incognita

BackgroundPlant parasitic nematodes are major pathogens of most crops. Molecular characterization of these species as well as the development of new techniques for control can benefit from genomic approaches. As an entrée to characterizing plant parasitic nematode genomes, we analyzed 5,700 expressed sequence tags (ESTs) from second-stage larvae (L2) of the root-knot nematode Meloidogyne incognita.ResultsFrom these, 1,625 EST clusters were formed and classified by function using the Gene Ontology (GO) hierarchy and the Kyoto KEGG database. L2 larvae, which represent the infective stage of the life cycle before plant invasion, express a diverse array of ligand-binding proteins and abundant cytoskeletal proteins. L2 are structurally similar to Caenorhabditis elegans dauer larva and the presence of transcripts encoding glyoxylate pathway enzymes in the M. incognita clusters suggests that root-knot nematode larvae metabolize lipid stores while in search of a host. Homology to other species was observed in 79% of translated cluster sequences, with the C. elegans genome providing more information than any other source. In addition to identifying putative nematode-specific and Tylenchida-specific genes, sequencing revealed previously uncharacterized horizontal gene transfer candidates in Meloidogyne with high identity to rhizobacterial genes including homologs of nodL acetyltransferase and novel cellulases.ConclusionsWith sequencing from plant parasitic nematodes accelerating, the approaches to transcript characterization described here can be applied to more extensive datasets and also provide a foundation for more complex genome analyses.

Overlapping Plant Signal Transduction Pathways Induced by a Parasitic Nematode and a Rhizobial Endosymbiont

Root-knot nematodes and rhizobia establish interactions with roots characterized by the de novo induction of host structures, termed giant cells and nodules, respectively. Two transcription regulators, PHAN and KNOX, required for the establishment of meristems were previously shown to be expressed in tomato giant cells. We isolated the orthologues of PHAN and KNOX (Mt-phan and Mt-knox-1) from the model legume Medicago truncatula, and established the spatial distribution of their expression in situ. We confirmed that Mt-phan and Mt-knox-1 are expressed in lateral root initials and in nematode-induced giant cells and showed that they are expressed in nodules induced by Sinorhizobium meliloti. Expression of both genes becomes spatially restricted as the nodules develop. We further examined nematode feeding sites for the expression of two genes involved in nodule formation, ccs52 (encodes a mitotic inhibitor) and ENOD40 (encodes an early, nodulation mitogen), and found transcripts of both genes to be present in and around giant cells induced in Medicago. Collectively, these results reveal common elements of host responses to mutualistic and parasitic plant endosymbionts and imply that overlapping regulatory pathways lead to giant cells and nodules. We discuss these pathways in the context of phytohormones and parallels between beneficial symbiosis and disease.

Are roots special? Nematodes have their say

Nematodes are ubiquitous and cosmopolitan parasites of vascular plants, causing substantial crop damage. Although various species exploit all parts of the plant, roots are the major target. Nematodes deploy a broad spectrum of feeding strategies, ranging from simple grazing to the establishment of complex cellular structures (including galls) in host tissues. Various models of feeding site formation have been proposed, and a role for phytohormones has long been speculated. Based on recent molecular evidence we present several scenarios involving phytohormones in the induction of giant cells by root-knot nematodes. The origin of parasitism by nematodes, and the role of horizontal gene transfer from microbes is discussed. Throughout, parallels with aphid-plant interactions are emphasized.

The Genomes of Root-Knot Nematodes

Plant-parasitic nematodes are the most destructive group of plant pathogens worldwide and are extremely challenging to control. The recent completion of two root-knot nematode genomes opens the way for a comparative genomics approach to elucidate the success of these parasites. Sequencing revealed that Meloidogyne hapla, a diploid that reproduces by facultative, meiotic parthenogenesis, encodes approximately 14,200 genes in a compact, 54 Mpb genome. Indeed, this is the smallest metazoan genome completed to date. By contrast, the 86 Mbp Meloidogyne incognita genome encodes approximately 19,200 genes. This species reproduces by obligate mitotic parthenogenesis and exhibits a complex pattern of aneuploidy. The genome includes triplicated regions and contains allelic pairs with exceptionally high degrees of sequence divergence, presumably reflecting adaptations to the strictly asexual reproductive mode. Both root-knot nematode genomes have compacted gene families compared with the free-living nematode Caenorhabditis elegans, and both encode large suites of enzymes that uniquely target the host plant. Acquisition of these genes, apparently via horizontal gene transfer, and their subsequent expansion and diversification point to the evolutionary history of these parasites. It also suggests new routes to their control.

RNAi effector diversity in nematodes

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes.