The chloroplast genome of the desiccation-tolerant moss Pseudocrossidium replicatum (Taylor) R.H. Zander.

Mosses in conjunction with hornworts and liverworts are collectively referred to as bryophytes. These seedless, nonvascular plants are the closest extant relatives of early terrestrial plants and their study is essential to understand the evolutionary first steps of land plants. Here we report the complete chloroplast (cp) genome sequence of Pseudocrossidium replicatum, a moss belonging to the Pottiaceae family that is common in the central highlands of Mexico, in South America, in southern USA, and in Kenia. The cp genome (plastome) of P. replicatum is 123,512 bp in size, comprising inverted repeats of 9,886 bp and single-copy regions of 85,146 bp (LSC) and 18,594 bp (SSC). The plastome encodes 82 different proteins, 31 different tRNAs, and 4 different rRNAs. Phylogenetic analysis using 16 cp protein-coding genes demonstrated that P. replicatum is closely related to Syntrichia ruralis, and the most basal mosses are Takakia lepidozioides followed by Sphagnum palustre. Our analysis indicates that during the evolution of the mosses' plastome, eight genes were lost. The complete plastome sequence reported here can be useful in evolutionary and population genetics.

Functional characterization of the three genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize.

The 1-deoxy-D-xylulose 5-phosphate synthase (DXS) enzyme catalyses the first biosynthetic step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. In plants the MEP pathway is involved in the synthesis of the common precursors to the plastidic isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate, in plastids. DXS is recognized as limiting this pathway and is a potential target for manipulation to increase various isoprenoids such as carotenoids. In Zea mays three dxs genes exist that encode plastid-targeted functional enzymes. Evidence is provided that these genes represent phylogenetically distinctive clades conserved among plants preceding monocot-dicot divergence. There is differential accumulation for each dxs gene transcript, during development and in response to external signals such as light. At the protein level, the analysis demonstrates that in Z. mays, DXS protein is feedback regulated in response to the inhibition of the pathway flow. The results support that the multilevel regulation of DXS activity is conserved in evolution.

Pore formation by Cry toxins.

Bacillus thuringiensis (Bt) bacteria produce insecticidal Cry and Cyt proteins used in the biological control of different insect pests. In this review, we will focus on the 3d-Cry toxins that represent the biggest group of Cry proteins and also on Cyt toxins. The 3d-Cry toxins are pore-forming toxins that induce cell death by forming ionic pores into the membrane of the midgut epithelial cells in their target insect. The initial steps in the mode of action include ingestion of the protoxin, activation by midgut proteases to produce the toxin fragment and the interaction with the primary cadherin receptor. The interaction of the monomeric CrylA toxin with the cadherin receptor promotes an extra proteolytic cleavage, where helix alpha-1 of domain I is eliminated and the toxin oligomerization is induced, forming a structure of 250 kDa. The oligomeric structure binds to a secondary receptor, aminopeptidase N or alkaline phosphatase. The secondary receptor drives the toxin into detergent resistant membrane microdomains formingpores that cause osmotic shock, burst of the midgut cells and insect death. Regarding to Cyt toxins, these proteins have a synergistic effect on the toxicity of some Cry toxins. Cyt proteins are also proteolytic activated in the midgut lumen of their target, they bind to some phospholipids present in the mosquito midgut cells. The proposed mechanism of synergism between Cry and Cyt toxins is that Cyt1Aa function as a receptor for Cry toxins. The Cyt1A inserts into midgut epithelium membrane and exposes protein regions that are recognized by Cry11Aa. It was demonstrated that this interaction facilitates the oligomerization of Cry11Aa and also its pore formation activity.

Defense and death responses to pore forming toxins.

Pore forming toxins (PFT) are important virulence factors produced by bacteria to kill eukaryotic cells by forming holes in the cellular membrane. They represent a diverse group of proteins with a wide range of target cells. Although the amino acid sequence is not conserved among the different PFT, many of them share some aspects of their mechanism of action. In general, the mode of action of PFT involves receptor recognition, activation by proteases, and aggregation into oligomeric-structures that insert into the membrane to form ionic pores. Beside the pore formation activity, PFT may have other effects during its interaction with their target cells such as intra-cellular signaling or transport of other enzymatic components, as in the case of anthrax or diphtheria toxins produced by Bacillus anthracis and Corynebacterium diphtheria, respectively (Parker and Feil, 2005). Although PFT have evolved as a pathogenic mechanism, some of them have great impact in society since they have different applications in biotechnology or are used as therapeutic agents, or as tools in the study of cell biology (Schiavo and van der Goot, 2001). On the other side, their target organisms have evolved different mechanisms to counter toxin action. Understanding the mechanism of action of PFT as well as the host responses to toxin action would provide ways to deal with these pathogens or with emerging pathogens and more importantly to improve the action of toxins that have biotechnological applications. In this review we will describe the intracellular effects induced by some PFT and the cellular responses evolved by eukaryotic cell to overcome PFT action.

The mitogenome of Pseudocrossidium replicatum, a desiccation-tolerant moss.

Bryophytes are the earliest plant group on Earth. They are a fundamental component of many ecosystems around the World. Some of their main roles are related to soil development, water retention, and biogeochemical cycling. Bryophytes include liverworts, hornworts, and mosses. The sequencing of chloroplast and mitochondria genomes has been useful to elucidate the taxonomy of this heterogeneous plant group. To date, despite their ecological importance only 41 mosses mitogenomes have been deposited in the GenBank. Here, the complete mitochondria genome sequence of Pseudocrossidium replicatum, a moss of the Pottiaceae family isolated in Tlaxcala, Mexico, is reported. The mitochondrial genome size of P. replicatum comprises 105,495 bp and contains the groups of genes described for other bryophytes mitogenomes. Our phylogenetic analysis shows that during the evolution of the mosses' mitogenome, nad7, rps4, rpl16, and rpl10 genes were lost independently in several lineages. The complete mitogenome sequence reported here would be a useful tool for our comprehension of the evolutionary and population genetics of this group of plants.

Autophagy mediates hydrotropic response in Arabidopsis thaliana roots.

This work shows that autophagy plays a key role in the hydrotropic curvature of Arabidopsis thaliana roots. An analysis of GFP-ATG8a transgenic plants showed that autophagosomes accumulated in the root curvature 2 h after the transfer of seedlings to Normal Medium-Water Stress Medium (NM-WSM). Autophagy flux was required for root bending. Remarkably, several atg mutants did not show hydrotropic curvature in NM-WSM or the splitting-agar system. Hyper, an H2O2 sensor showed that H2O2 preferentially accumulated in the root curvature at a similar rate as the autophagosomes did during hydrotropic response. Peroxidase and ROBH activity inhibition affected, negatively or positively root curvature. This data suggested H2O2 balance was required for root bending. Malondialdehyde, a metabolite used as an indicator of oxidative stress, accumulated at the same rate during the development of the curvature in NM-WSM. These results suggest that autophagy is required for the hydrotropic response in NM-WSM. We discuss the possible regulatory role of H2O2 on autophagy during the hydrotropic response that might relieve oxidative stress provoked by water stress. NM-WSM is water stress system suitable for studying hydrotropic responses on a short-term basis.

Genome Sequence of Enterotoxigenic Escherichia coli Strain FMU073332.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of bacterial diarrheal illness, affecting practically every population worldwide, and was estimated to cause 120,800 deaths in 2010. Here, we report the genome sequence of ETEC strain FMU073332, isolated from a 25-month-old girl from Tlaltizapán, Morelos, México.

Robust root growth in altered hydrotropic response1 (ahr1) mutant of Arabidopsis is maintained by high rate of cell production at low water potential gradient.

Hydrotropism is the directional root growth response determined by water stimulus. In a water potential gradient system (WPGS) the roots of the Arabidopsis wild type have a diminished root growth compared to normal medium (NM). In contrast, the altered hydrotropic response1 (ahr1) mutant roots maintain their robust growth in the same WPGS. The aims of this work were to ascertain how ahr1 roots could sustain growth in the WPGS, with a special focus on the integration of cellular processes involved in the signaling that determines root growth during abiotic stress and their relation to hydrotropism. Cellular analysis of the root apical meristem of ahr1 mutant contrary to the wild type showed an absence of changes in the meristem length, the elongation zone length, the length of fully elongated cells, and the cell cycle duration. The robust and steady root growth of ahr1 seedlings in the WPGS is explained by the mutant capacity to maintain cell production and cell elongation at the same level as in the NM. Analysis of auxin response at a transcriptional level showed that roots of the ahr1 mutant had a lower auxin response when grown in the WPGS, compared to wild type, indicating that auxin signaling participates in attenuation of root growth under water stress conditions. Also, wild type plants exhibited a high increase in proline content while ahr1 mutants showed minimum changes in the Normal Medium→Water Stress Medium (NM→WSM), a lower water potential gradient system than the WPGS. Accordingly, in this condition, gene expression of Δ1-6 Pyrroline-5-Carboxylate Synthetase1 (P5CS1) involved in proline synthesis strongly increased in wild type but not in ahr1 seedlings. The ahr1 phenotype shows unique features since the mutant root cells continue to proliferate and grow in the presence of a progressively negative water potential gradient at a level comparable to wild type growing in the NM. As such, it represents an exceptional resource for understanding hydrotropism.

Aedes aegypti Mos20 cells internalizes cry toxins by endocytosis, and actin has a role in the defense against Cry11Aa toxin.

Bacillus thuringiensis (Bt) Cry toxins are used to control Aedes aegypti, an important vector of dengue fever and yellow fever. Bt Cry toxin forms pores in the gut cells, provoking larvae death by osmotic shock. Little is known, however, about the endocytic and/or degradative cell processes that may counteract the toxin action at low doses. The purpose of this work is to describe the mechanisms of internalization and detoxification of Cry toxins, at low doses, into Mos20 cells from A. aegypti, following endocytotic and cytoskeletal markers or specific chemical inhibitors. Here, we show that both clathrin-dependent and clathrin-independent endocytosis are involved in the internalization into Mos20 cells of Cry11Aa, a toxin specific for Dipteran, and Cry1Ab, a toxin specific for Lepidoptera. Cry11Aa and Cry1Ab are not directed to secretory lysosomes. Instead, Mos20 cells use the Rab5 and Rab11 pathways as a common mechanism, most probably for the expulsion of Cry11Aa and Cry1Ab toxins. In conclusion, we propose that endocytosis is a mechanism induced by Cry toxins independently of specificity, probably as part of a basal immune response. We found, however, that actin is necessary for defense-specific response to Cry11Aa, because actin-silenced Mos20 cells become more sensitive to the toxic action of Cry11A toxin. Cry toxin internalization analysis in insect cell lines may contribute to a better understanding to Cry resistance in mosquitoes.

Evolution of Bacillus thuringiensis Cry toxins insecticidal activity.

Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate-limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.

Role of MAPK p38 in the cellular responses to pore-forming toxins.

Understanding the mechanism of action of pore-forming toxins (PFTs) produced by different bacteria, as well as the host responses to toxin action, would provide ways to deal with these pathogenic bacteria. PFTs affect the permeability of target cells by forming pores in their plasma membrane. Target organisms may overcome these effects by triggering intracellular responses that have evolved as defense mechanisms to PFT. Among them it is well documented that stress-activated protein kinases, and specially MAPK p38 pathway, play a crucial role triggering defense responses to several PFTs in different eukaryotic cells. In this review we describe different intracellular effects induced by PFTs in eukaryotic cells and highlight diverse responses activated by p38 pathway.

Tobacco plants expressing the Cry1AbMod toxin suppress tolerance to Cry1Ab toxin of Manduca sexta cadherin-silenced larvae.

Cry toxins produced by Bacillus thuringiensis bacteria are insecticidal proteins used worldwide in the control of different insect pests. Alterations in toxin-receptor interaction represent the most common mechanism to induce resistance to Cry toxins in lepidopteran insects. Cry toxins bind with high affinity to the cadherin protein present in the midgut cells and this interaction facilitates the proteolytic removal of helix α-1 and pre-pore oligomer formation. Resistance to Cry toxins has been linked with mutations in the cadherin gene. One strategy effective to overcome larval resistance to Cry1A toxins is the production of Cry1AMod toxins that lack helix α-1. Cry1AMod are able to form oligomeric structures without binding to cadherin receptor and were shown to be toxic to cadherin-silenced Manduca sexta larvae and Pectinophora gossypiella strain with resistance linked to mutations in a cadherin gene. We developed Cry1AbMod tobacco transgenic plants to analyze if Cry1AMod toxins can be expressed in transgenic crops, do not affect plant development and are able to control insect pests. Our results show that production of the Cry1AbMod toxin in transgenic plants does not affect plant development, since these plants exhibited healthy growth, produced abundant seeds, and were virtually undistinguishable from control plants. Most importantly, Cry1AbMod protein produced in tobacco plants retains its functional toxic activity against susceptible and tolerant M. sexta larvae due to the silencing of cadherin receptor by RNAi. These results suggest that CryMod toxins could potentially be expressed in other transgenic crops to protect them against both toxin-susceptible and resistant lepidopteran larvae affected in cadherin gene.

The mitogen-activated protein kinase p38 is involved in insect defense against Cry toxins from Bacillus thuringiensis.

The insecticidal Cry toxins are pore-forming toxins produced by the bacteria Bacillus thuringiensis that disrupt insect-midgut cells. In this work we analyzed the response of two different insect orders, the Lepidopteran Manduca sexta and Dipteran Aedes aegypti to highly specific Cry toxins, Cry1Ab and Cry11Aa, respectively. One pathway activated in different organisms in response to a variety of pore-forming toxins is the mitogen-activated protein kinase p38 pathway (MAPK p38) that activates a complex defense response. We analyzed the MAPK p38 activation by immunodetection of its phosphorylated isoform, and the induction of p38 by RT-PCR, real-time PCR quantitative assays and immunodetection. We show that MAPK p38 is activated at postraductional level after Cry toxin intoxication in both insect orders. We detected the p38 induction at the transcriptional and traductional level, and observed a different response. In these three levels, we found that both insects respond to Cry toxin action but M. sexta responses more strongly than A. aegypti. Gene silencing of MAPK p38 in vivo, resulted in both insect species becoming hypersensitive to Cry toxin action, suggesting that the MAPK p38 pathway is involved in insect defense against Bt Cry toxins. This finding may have biotechnological applications for enhancing the activity of some Bt Cry toxins against specific insect pests.

Wounding and pathogen infection induce a chloroplast-targeted lipoxygenase in the common bean (Phaseolus vulgaris L.).

Chloroplastic LOXs are implicated in the biosynthesis of oxylipins like jasmonic acid and C6 volatiles among others. In this study, we isolated the cDNA of a novel chloroplast-targeted Phaseolus vulgaris LOX, (PvLOX6). This gene is highly induced after wounding, non-host pathogen infection, and by signaling molecules as H2O2, SA, ethylene and MeJA. The phylogenetic analysis of PvLOX6 showed that it is closely related to chloroplast-targeted LOX from potato (H1) and tomato (TomLOXC); both of them are implicated in the biosynthesis of C6 volatiles. Induction of PvLOX6 mRNA by wounding ethylene and jasmonic acid on the one side, and non-host pathogen, salicylic acid on the other indicates that common bean uses the same LOX to synthesize oxylipins in response to different stresses.

AtMCP1b, a chloroplast-localised metacaspase, is induced in vascular tissue after wounding or pathogen infection.

cDNA corresponding to the Arabidopsis type I metacaspase AtMCP1b was isolated from plants infected with Pseudomonas syringae. A positive correlation between AtMCP1b expression and cell death was observed in the presence of staurosporine, a protein kinase inhibitor that induces programmed cell death. The tissue localisation of an AtMCP1b promoter-GUS fusion was observed in the vascular tissue of transgenic plants. GUS activity increased in response to an incompatible DC3000 (avrRpm1) or a compatible DC3000 P. syringae infection, or to wounding. Confocal and immunohistochemical analysis of Arabidopsis thaliana (L.) leaves showed that an AtMCP1b-GFP fusion protein was localised in the chloroplasts. Our data support a positive correlation between AtMCP1b gene expression and cell death in response to wounding or pathogenic interactions. Moreover, the localisation of AtMCP1b gene expression within vascular tissue and cells of abscission regions strongly supports a role for AtMCP1b in programmed cell dismantling events in response to environmental and developmental triggers. The AtMCP1b-GFP subcellular localisation infers a role for the plastid organelles in PCD and, thus, in responses to pathogen attack and development.

A red beet (Beta vulgaris) UDP-glucosyltransferase gene induced by wounding, bacterial infiltration and oxidative stress.

Mechanical wounding, infiltration with P. syringae or A. tumefaciens, and exposure to an H(2)O(2)-generating system (Glc/Glc oxidase) induce betacyanin synthesis in red beet (Beta vulgaris) leaves. These conditions also induced the expression of BvGT, a gene encoding a glucosyltransferase (GT) from Beta vulgaris. BvGT has a high similarity to Dorotheanthus bellidiformis betanidin-5 GT involved in betacyanin synthesis. Furthermore, the transient expression of a BvGT antisense construct resulted in the reduction of BvGT transcript accumulation and betanin synthesis, suggesting a role for this gene product in betacyanin glucosylation. In addition, the NADPH oxidase inhibitor, diphenylene iodonium (DPI), inhibited the accumulation of the BvGT transcript in response to infiltration with Agrobacterium tumefaciens. Hence, this result suggests that ROS produced by a plasma membrane NADPH oxidase may act as a signal to induce BvGT expression, necessary for betanin synthesis after wounding and bacterial infiltration.

Rhizobium etli CFN42 contains at least three plasmids of the repABC family: a structural and evolutionary analysis.

In this paper, we report the identification of replication/partition regions of plasmid p42a and p42b of Rhizobium etli CFN42. Sequence analysis reveals that both replication/partition regions belong to the repABC family. Phylogenetic analysis of all the complete repABC replication/partition regions reported to date, shows that repABC plasmids coexisting in the same strain arose most likely by lateral transfer instead of by duplication followed by divergence. A model explaining how new incompatibility groups originate, is proposed.