Multiple levels of gene regulation mediate differentiation of the intracellular pathogen Leishmania.

For many years, mRNA abundance has been used as the surrogate measure of gene expression in biological systems. However, recent genome-scale analyses in both bacteria and eukaryotes have revealed that mRNA levels correlate with steady-state protein abundance for only 50-70% of genes, indicating that translation and post-translation processes also play important roles in determining gene expression. What is not yet clear is whether dynamic processes such as cell cycle progression, differentiation, or response to environmental changes change the relationship between mRNA and protein abundance. Here, we describe a systems approach to interrogate promastigote-to-amastigote differentiation in the obligatory intracellular parasitic protozoan Leishmania donovani. Our results indicate that regulation of mRNA levels plays a major role early in the differentiation process, while translation and post-translational regulation are more important in the latter part. In addition, it appears that the differentiation signal causes a transient global increase in the rate of protein synthesis, which is subsequently down-regulated by phosphorylation of α-subunit of translation initiation factor 2. Thus, Leishmania dynamically changes the relationship between mRNA and protein abundance as it adapts to new environmental circumstances. It is likely that similar mechanisms play a more important role than previously recognized in regulation of gene expression in other organisms.

A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes.

A genetic map of melon enriched for fruit traits was constructed, using a recombinant inbred (RI) population developed from a cross between representatives of the two subspecies of Cucumis melo L.: PI 414723 (subspecies agrestis) and 'Dulce' (subspecies melo). Phenotyping of 99 RI lines was conducted over three seasons in two locations in Israel and the US. The map includes 668 DNA markers (386 SSRs, 76 SNPs, six INDELs and 200 AFLPs), of which 160 were newly developed from fruit ESTs. These ESTs include candidate genes encoding for enzymes of sugar and carotenoid metabolic pathways that were cloned from melon cDNA or identified through mining of the International Cucurbit Genomics Initiative database (http://www.icugi.org/). The map covers 1,222 cM with an average of 2.672 cM between markers. In addition, a skeleton physical map was initiated and 29 melon BACs harboring fruit ESTs were localized to the 12 linkage groups of the map. Altogether, 44 fruit QTLs were identified: 25 confirming QTLs described using other populations and 19 newly described QTLs. The map includes QTLs for fruit sugar content, particularly sucrose, the major sugar affecting sweetness in melon fruit. Six QTLs interacting in an additive manner account for nearly all the difference in sugar content between the two genotypes. Three QTLs for fruit flesh color and carotenoid content were identified. Interestingly, no clear colocalization of QTLs for either sugar or carotenoid content was observed with over 40 genes encoding for enzymes involved in their metabolism. The RI population described here provides a useful resource for further genomics and metabolomics studies in melon, as well as useful markers for breeding for fruit quality.

Analysis of the Leishmania donovani transcriptome reveals an ordered progression of transient and permanent changes in gene expression during differentiation.

Leishmania donovani is an intracellular protozoan parasite that causes kala-azar in humans. During infection the extracellular insect forms (promastigotes) undergo rapid differentiation to intracellular amastigotes that proliferates in phagolysosomes of mammalian macrophages. We used microarray-based expression profiling to investigate the time-course of changes in RNA abundance during promastigote-to-amastigote differentiation in a host-free system that mimics this process. These studies revealed that several hundred genes underwent an ordered progression of transient or permanent up- and down-regulation during differentiation. Genes that were permanently up-regulated in amastigotes were enriched for transporters and surface proteins, but under-represented in genes involved in protein and other metabolism. Most of these changes occurred late in the differentiation process, when morphological differentiation was essentially complete. Down-regulated genes were over-represented in those involved in cell motility, growth and/or maintenance, and these changes generally occurred earlier in the process. Genes that were transiently up- or down-regulated during differentiation included those encoding heat shock proteins, ubiquitin hydrolases, RNA binding proteins, protein kinases, a protein phosphatase, and a histone deacetylase. These results suggest that changes in mRNA abundance may be important in signal transduction, as well as protein and mRNA turnover, during differentiation. In addition to these mRNA changes, other transcripts including one or more rRNAs and snoRNAs, and non-coding RNAs from several telomeres, also showed substantial changes in abundance during the differentiation process. This paper provides the first genome-scale quantitative analysis of gene expression during the transition from promastigotes to amastigotes and demonstrates the utility of the host-free differentiation system.

Gene expression during chemically induced liver fibrosis: effect of halofuginone on TGF-beta signaling.

Hepatic fibrosis is associated with the activation of stellate cells (HSCs), the major source of extracellular matrix (ECM) proteins. Transforming growth factor-beta (TGF-beta), signaling via Smad3, is the most profibrogenic cytokine and the major promoter of ECM synthesis. Halofuginone, an inhibitor of liver fibrosis, inhibits TGF-beta-dependent Smad3 phosphorylation in human HSCs in culture. We have used transcriptional profiling to evaluate the effect of halofuginone on gene expression during the progression of thioacetamide (TAA)-induced liver fibrosis in the rat and have focused on genes that are associated with TGF-beta. TAA treatment causes alterations in the expression of 7% of liver genes. Halofuginone treatment prevents the changes in the expression of 41% of these genes and results in the inhibition of HSC activation and collagen synthesis. During the early stages of the disease, halofuginone affects genes involved in alcohol, lipid, protein, and phosphate metabolism and cell adhesion and, at later stages, in the cell cycle (cell development, differentiation, cell proliferation, and apoptosis). The activation of TGF-beta-dependent genes, such as tartrate-resistant acid phosphatase, its putative substrate osteopontin, stellate cell activation-association protein, and fibrillin-1, during chemically induced fibrosis is prevented by halofuginone. This study thus highlights the role of TGF-beta signaling in liver fibrosis and especially its potential for pharmacological intervention. Halofuginone, which has demonstrated efficacy and tolerance in animals and humans, could become an effective and novel therapy for liver fibrosis.

The defense response elicited by the pathogen Rhizoctonia solani is suppressed by colonization of the AM-fungus Glomus intraradices.

Defense responses of alfalfa roots to the pathogenic fungus Rhizoctonia solani were reduced significantly in roots simultaneously infected with the vesicular arbuscular mycorrhizal (AM) fungus Glomus intraradices. R. solani induced five- to tenfold increases in the steady-state levels of chalcone isomerase and isoflavone reductase mRNAs a doubling of root peroxidase activity and a marked autofluorescence in the infected tissue. These changes were inhibited by the presence of G. intraradices. Interestingly, germination of G. intraradices spores and hyphal elongation were sensitive to low concentrations (2 µM) of medicarpin-3-O-glucoside, an isoflavonoid phytoalexin that accumulated both in roots colonized by the pathogenic fungus as well as in AM-treated roots receiving high P, where no colonization by the beneficial fungus occurred. These data support the hypothesis that during early stages of colonization by G. intraradices, suppression of defense-related properties is associated with the successful establishment of AM symbiosis.

Mycorrhiza-induced changes in disease severity and PR protein expression in tobacco leaves

The development of leaf disease symptoms and the accumulation of pathogenesis-related (PR) proteins were monitored in leaves of tobacco (Nicotiana tabacum cv. Xanthinc) plants colonized by the arbuscular mycorrhizal fungus Glomus intraradices. Leaves of mycorrhizal plants infected with the leaf pathogens Botrytis cinerea or tobacco mosaic virus showed a higher incidence and severity of necrotic lesions than those of nonmycorrhizal controls. Similar plant responses were obtained at both low (0.1 mM) and high (1.0 mM) nutritional P levels and with mutant plants (NahG) that are unable to accumulate salicylic acid. Application of PR-protein activators induced PR-1 and PR-3 expression in leaves of both nonmycorrhizal and mycorrhizal plants; however, accumulation and mRNA steady-site levels of these proteins were lower, and their appearance delayed, in leaves of the mycorrhizal plants. Application of 0.3 mM phosphate to the plants did not mimic the delay in PR expression observed in the mycorrhizal tobacco. Together, these data strongly support the existence of regulatory processes, initiated in the roots of mycorrhizal plants, that modify disease-symptom development and gene expression in their leaves.

A Nod factor-binding lectin is a member of a distinct class of apyrases that may be unique to the legumes.

Recent studies from our laboratory have found that a root lectin from the legume Dolichos hifloris is present on the root surface, binds rhizobial Nod factor and has apyrase activity. To assess the broader significance of this lectin/nucleotide phosphohydrolase (Db-LNP), we have cloned a second related cDNA (Db-apyrase-2) from D. hiflorus, as well as related cDNAs from the legumes Lotus japonicus and Medicago sativa, and from Arabidopsis thaliana, a non-legume. The deduced amino acid sequences of these apyrases were aligned with one another and with the sequences of other apyrases from plants, animals, yeast and protozoa. Phylogenetic analysis shows that Db-LNP has closely related orthologs only in other legumes, while Db-apyrase-2 is more closely related to apyrase sequences from non-leguminous plants. We also show that the orthologs of Db-LNP from M. sativa and Pisum sativum have carbohydrate binding activity. The results suggest that legume LNPs may represent a special class of apyrases that arose by gene duplication and subsequent specialization.

Identification of lumichrome as a sinorhizobium enhancer of alfalfa root respiration and shoot growth.

Sinorhizobium meliloti bacteria produce a signal molecule that enhances root respiration in alfalfa (Medicago sativa L.) and also triggers a compensatory increase in whole-plant net carbon assimilation. Nuclear magnetic resonance, mass spectrometry, and ultraviolet-visible absorption identify the enhancer as lumichrome, a common breakdown product of riboflavin. Treating alfalfa roots with 3 nM lumichrome increased root respiration 21% (P < 0.05) within 48 h. A closely linked increase in net carbon assimilation by the shoot compensated for the enhanced root respiration. For example, applying 5 nM lumichrome to young alfalfa roots increased plant growth by 8% (P < 0.05) after 12 days. Soaking alfalfa seeds in 5 nM lumichrome before germination increased growth by 18% (P < 0.01) over the same period. In both cases, significant growth enhancement (P < 0.05) was evident only in the shoot. S. meliloti requires exogenous CO2 for growth and may benefit directly from the enhanced root respiration that is triggered by lumichrome. Thus Sinorhizobium-alfalfa associations, which ultimately form symbiotic N2-reducing root nodules, may be favored at an early developmental stage by lumichrome, a previously unrecognized mutualistic signal. The rapid degradation of riboflavin to lumichrome under many physiological conditions and the prevalence of riboflavin release by rhizosphere bacteria suggest that events demonstrated here in the S. meliloti-alfalfa association may be widely important across many plant-microbe interactions.

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO2 evolution or as O2 uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10(7) bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO2 production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO2, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.

Promotion of nod Gene Inducers and Nodulation in Common Bean (Phaseolus vulgaris) Roots Inoculated with Azospirillum brasilense Cd.

Inoculation of Phaseolus vulgaris with Azospirillum brasilense Cd promoted root hair formation in seedling roots and significantly increased total and upper nodule numbers at different concentrations of Rhizobium inoculum. In experiments carried out in a hydroponic system, A. brasilense caused an increase in the secretion of nod gene-inducing flavonoids, as was observed by nod gene induction assays of root exudates fractionated by high-performance liquid chromatography. Possible mechanisms involved in the influence of A. brasilense on this symbiotic system are discussed.

Suppression of an Isoflavonoid Phytoalexin Defense Response in Mycorrhizal Alfalfa Roots.

Isoflavonoids and steady-state mRNA levels of phenylalanine ammonia-lyase, chalcone isomerase, and isoflavone reductase were followed during a rapid, nearly synchronous infection of alfalfa (Medicago sativa L.) roots by the vesicular arbuscular fungus Glomus intraradices (Schenck & Smith) to test whether previously indicated suppression of the host defense response is regulated by changes in the steady-state mRNA level. Relative amounts of steady-state phenylalanine ammonia-lyase mRNA in the mycorrhizal roots doubled between d 14 and 18 and then immediately declined by 75% to reach and maintain a value lower than the control roots through d 21. Relative levels of chalcone isomerase mRNA in the inoculated roots increased 6-fold between d 14 and 17 and then decreased rapidly to the control level. Isoflavone reductase mRNA was not induced by mycorrhizal colonization. High-performance liquid chromatography, proton-nuclear magnetic resonance, and fast atom bombardment-mass spectrometry analyses showed consistent increases in formononetin levels and transient increases in medicarpin-3-O-glycoside and formononetin conjugates in the inoculated roots when colonization began. As colonization increased, levels of formononetin conjugates declined in mycorrhizal roots below those in uncolonized controls. Medicarpin aglycone, an alfalfa phytoalexin normally associated with pathogenic infections, was not detected at any stage. These findings supply detailed evidence that, during early colonization of plant roots by symbiotic Glomus, defense transcripts are induced and then subsequently suppressed.

A Vesicular Arbuscular Mycorrhizal Fungus (Glomus intraradix) Induces a Defense Response in Alfalfa Roots.

Flavonoid accumulation and activities of phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and chitinase were followed during early colonization of alfalfa roots (Medicago sativa L. cv Gilboa) by vesicular arbuscular (VA) fungi (Glomus intraradix). Formononetin was the only flavonoid detected that showed a consistent increase in the inoculated roots. This increase depended only on the presence of the fungus in the plant rhizosphere; no colonization of the root tissue was required. CHI and chitinase activities increased in inoculated roots prior to colonization, whereas the increase in PAL activity coincided with colonization. After reaching a maximum, activities of all enzymes declined to below those of uninoculated roots. PAL inactivation was not caused by a soluble inhibitor. Our results indicate that VA fungi initiate a host defense response in alfalfa roots, which is subsequently suppressed.