Infection of endemic chub Squalius tenellus with the intestinal tapeworm Caryophyllaeus brachycollis (Cestoda): histopathology and ultrastructural surveys.

The endemic chub Squalius tenellus (Heckel, 1843) was introduced more than 100 years ago to Lake Blidinje (Bosnia-Herzegovina). Only 1 species of enteric helminth was found in a sample of 35 chubs, the tapeworm Caryophyllaeus brachycollis (Janiszewska, 1953). The paper includes histopathological investigation with identification of innate immune cells involved in host reaction and molecular data allowed correct designation of the cestode species. Of 35 specimens of chub examined, 21 (60%) harboured individuals of C. brachycollis and a total of 1619 tapeworms were counted, the intensity of infection ranged from 1 to 390 worms per fish (46.2 ± 15.3, mean ± s.e.). Histopathological and ultrastructural investigations showed strict contact between the worm's body and the epithelia and increase in the number of mucous cells, rodlet cells among the epithelial cells. Within the tunica propria-submucosa, beneath the site of scolex attachment, numerous neutrophils and mast cells were noticed. This is the first study of the occurrence of C. brachycollis in chub from Lake Blidinje and on the response of the innate immune cells of S. tenellus to this tapeworm. Interestingly, in 3 very heavily infected chubs, perforation of the intestinal wall was documented; this is uncommon among cestodes which use fish as a definitive host.

Efficacy and Selectivity of Potassium Bicarbonate Salts against Cacopsylla pyri on Pears.

In recent years, the control of pear psyllid in northern Italy has not been particularly problematic, due to the presence of two insecticides (abamectin and spirotetramat) specifically for this pest, and due to the adoption of integrated pest management. However, the withdrawal of these two specific insecticides is imminent and, therefore, it has become necessary to find alternative control tools. More recently, potassium bicarbonate, known for its fungistatic activity against many phytopathogenic fungi, has also shown some activity against some insect pests. In the present study, the efficacy and possible phytotoxicity of potassium bicarbonate were tested in two field trials on second generation Cacopsylla pyri by spraying two different salt concentrations (5 and 7 kg ha-1), with or without polyethylene glycol as an adjuvant. Spirotetramat was used as a commercial reference. The results showed that potassium bicarbonate could positively control the number of juvenile forms (with a mortality percentage of up to 89% at the infestation peak), even though spirotetramat was still more effective. Therefore, potassium bicarbonate appears to be a sustainable integrated tool for psyllid control, especially in the wake of the imminent withdrawal of spirotetramat and other insecticides currently used on this pest.

Topical delivery of dsRNA in two hemipteran species: Evaluation of RNAi specificity and non-target effects.

Double-stranded (ds) RNA-based technologies could provide novel and potential tool for pest management with efficiency and specificity of action. However, before applying this technique in the field, it is necessary to identify effective delivery methods and evaluate the non-target effects that may occur. In this article, we evaluated the effectiveness of dsRNA by topical delivery on a species of great agricultural interest, Halyomorpha halys. The specificity of action of the dsRNA was also investigated in Rhodnius prolixus, an insect phylogenetically close to H. halys. Of the three investigated genes (putative ATPase N2B, ATPase, serine/threonine-protein phosphatase PP1-ß catalytic subunit, PP1, and IAP repeat-containing protein 7-B-like, IAP), IAP and ATPase were able to induce higher mortality in H. halys nymphs compared to the control, with specific concentrations for each gene targeted. However, when the same RNAs were topically delivered to both R. prolixus 2nd and 3rd instar nymphs, no gene silencing and mortality were observed. For this reason, to assess dsRNA application-mediated non-target effects, we injected both H. halys and R. prolixus specific dsRNA in R. prolixus 5th instar nymphs. When the dsRNA targeting H. halys IAP was microinjected into R. prolixus 5th instar nymphs, no mortality was observed, suggesting a strong RNAi specificity. Together, these data suggest that the topical delivery could be suitable for the dsRNA to control H. halys population. Furthermore, its specificity of action would allow treatments towards single harmful species with limited non-target effects.

Reconstruction of Acinetobacter johnsonii ICE_NC genome using hybrid de novo genome assemblies and identification of the 12α-hydroxysteroid dehydrogenase gene.

AIMS: The role of a Acinetobacter johnsonii strain, isolated from a soil sample, in the biotransformation of bile acids (BAs) was already described but the enzymes responsible for these transformations were only partially purified and molecularly characterized. METHODS AND RESULTS: This study describes the use of hybrid de novo assemblies, that combine long-read Oxford Nanopore and short-read Illumina sequencing strategies, to reconstruct the entire genome of A. johnsonii ICE_NC strain and to identify the coding region for a 12α-hydroxysteroid dehydrogenase (12α-HSDH), involved in BAs metabolism. The de novo assembly of the A. johnsonii ICE_NC genome was generated using Canu and Unicycler, both strategies yielded a circular chromosome of about 3.6 Mb and one 117 kb long plasmid. Gene annotation was performed on the final assemblies and the gene for 12α-HSDH was detected on the plasmid. CONCLUSIONS: Our findings illustrate the added value of long read sequencing in addressing the challenges of whole genome characterization and plasmid reconstruction in bacteria. These approaches also allowed the identification of the A. johnsonii ICE_NC gene for the 12α-HSDH enzyme, whose activity was confirmed at the biochemical level. SIGNIFICANCE AND IMPACT OR THE STUDY: At present, this is the first report on the characterization of a 12α-HSDH gene in an A. johnsonii strain able to biotransform cholic acid into ursodeoxycholic acid, a promising therapeutic agent for several diseases.

Effects of Acibenzolar-S-methyl on the Probing Behaviour and Mortality of Cacopsylla pyri on Pear Plants.

European pear psylla, Cacopsylla pyri, is one of the worst pests of pear in Europe. We investigated whether acibenzolar-S-methyl (ASM) application on pear plants might affect the behaviour in C. pyri. The elicitor was applied on pear potted plants, and after 48 h, we confirmed the ASM-mediated induction of several Pathogenesis-Related protein (PR) coding genes. At the same time, an in-depth analysis was performed on the probing behaviour of adults and nymphs of C. pyri on ASM-treated pear plants by the EPG-DC system, as well as the assessment of young nymphs' survival 7 days after the ASM application. The elicitor application weakly interfered with C. pyri nymphs probing behaviour and survival, while it did not affect adult stages. These data confirm previous observations obtained on C. pyricola and suggest that the elicitor does not represent a viable tool in the control of pear psylla species, especially if used alone, but it might be used in integrated management strategies focused on other plant pathogens such as Erwinia amylovora.

Monarda didyma Hydrolate Affects the Survival and the Behaviour of Drosophila suzukii.

Drosophila suzukii (Matsumara) is an herbivorous pest whose control in the field with conventional chemical is particularly difficult and has important drawbacks. Here, we investigated the insecticidal properties of hydrolate from Monarda didyma, scarlet beebalm, an aromatic herb in the Lamiaceae family. The identification of volatile organic compounds (VOCs) by CG-MS systems revealed that thymol (38%) and carvacrol (59%) were the most abundant VOCs in the hydrolate. M. didyma hydrolate did not show fumigant toxicity. Conversely, in contact assays, M. didyma hydrolate showed a LC50 of 5.03 µL mL-1, 48 h after the application on D. suzukii adults. Expression of detoxification genes increased in flies that survived the LC50 application. Furthermore, toxicity persisted for 7 days after the treatment in the survival evaluation. Artificial diet assays with 100 and 1000 µL mL-1 of M. didyma hydrolate resulted in a significant decrease in total food intake in both male and female D. suzukii adults. In addition, electropenetrography (EPG) showed that the D. suzukii females' feeding behaviour was altered in hydrolate-treated diets. The hydrolate also caused a significant reduction in the number of eggs laid in two different oviposition assays. Overall, our findings provide a new perspective for the improvement of D. suzukii control strategies through M. didyma hydrolate.

Constitutive silencing of LRRK2 kinase activity leads to early glucocerebrosidase deregulation and late impairment of autophagy in vivo.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease. LRRK2 modulates the autophagy-lysosome pathway (ALP), a clearance process subserving the quality control of cellular proteins and organelles. Since dysfunctional ALP might lead to α-synuclein accumulation and, hence, Parkinson's disease, LRRK2 kinase modulation of ALP, its age-dependence and relation with pSer129 α-synuclein inclusions were investigated in vivo. Striatal ALP markers were analyzed by Western blotting in 3, 12 and 20-month-old LRRK2 G2019S knock-in mice (bearing enhanced kinase activity), LRRK2 knock-out mice, LRRK2 D1994S knock-in (kinase-dead) mice and wild-type controls. The lysosomotropic agent chloroquine was used to investigate the autophagic flux in vivo. Quantitative Real-time PCR was used to quantify the transcript levels of key ALP genes. The activity of the lysosomal enzyme glucocerebrosidase was measured using enzymatic assay. Immunohistochemistry was used to co-localize LC3B puncta with pSer129 α-synuclein inclusion in striatal and nigral neurons. No genotype differences in ALP markers were observed at 3 months. Conversely, increase of LC3-I, p62, LAMP2 and GAPDH levels, decrease of p-mTOR levels and downregulation of mTOR and TFEB expression was observed in 12-month-old kinase-dead mice. The LC3-II/I ratio was reduced following administration of chloroquine, suggesting a defective autophagic flux. G2019S knock-in mice showed LAMP2 accumulation and downregulation of ALP key genes MAP1LC3B, LAMP2, mTOR, TFEB and GBA1. Subacute administration of the LRRK2 kinase inhibitor MLi-2 in wild-type and G2019S knock-in mice did not replicate the pattern of kinase-dead mice. Lysosomal glucocerebrosidase activity was increased in 3 and 12-month-old knock-out and kinase-dead mice. LC3B puncta accumulation and pSer129 α-synuclein inclusions were dissociated in striatal neurons of kinase-dead and G2019S knock-in mice. We conclude that constitutive LRRK2 kinase silencing results in early deregulation of GCase activity followed by late impairment of macroautophagy and chaperone-mediated autophagy.

Spatial and Temporal Genetic Diversity and Population Structure of Hemileia vastatrix from Peruvian Coffee Plantations.

Population genetic studies of Hemileia vastatrix have been conducted in order to describe the evolutionary dynamics of the pathogen and the disease epidemiology as consequence of changes in disease management and host distribution occurred in Peru after the 2013 epidemic. These analyses were performed by sequencing the internal transcribed spacers of the nuclear ribosomal DNA (rDNA-ITS) of H. vastatrix collected from two coffee growing areas in 2014 and 2018. H. vastatrix population showed high haplotype diversity (Hd = 0.9373 ± 0.0115) with a low nucleotide diversity (π = 0.00322 ± 0.00018). Likewise, AMOVA indicated that fungus population has behaved as a large population without structuring by geographical origin and sampling years (FST = 0.00180, P = 0.20053 and FST = 0.00241, P = 0.19693, respectively). Additionally, the haplotype network based on intraspecific phylogenetic analysis of H. vastatrix using Peruvian and NCBI sequences revealed that Peruvian ancestral haplotypes, which were maintained in time and space, would correspond to the reported sequences of the races II and XXII. This result suggests that no substantial changes have occurred through time in Peruvian Hemileia vastatrix population.

The Insect Type 1 Tyramine Receptors: From Structure to Behavior.

Tyramine is a neuroactive compound that acts as neurotransmitter, neuromodulator, and neurohormone in insects. Three G protein-coupled receptors, TAR1-3, are responsible for mediating the intracellular pathway in the complex tyraminergic network. TAR1, the prominent player in this system, was initially classified as an octopamine receptor which can also be activated by tyramine, while it later appeared to be a true tyramine receptor. Even though TAR1 is currently considered as a well-defined tyramine receptor and several insect TAR1s have been characterized, a defined nomenclature is still inconsistent. In the last years, our knowledge on the structural, biochemical, and functional properties of TAR1 has substantially increased. This review summarizes the available information on TAR1 from different insect species in terms of basic structure, its regulation and signal transduction mechanisms, and its distribution and functions in the brain and the periphery. A special focus is given to the TAR1-mediated intracellular signaling pathways as well as to their physiological role in regulating behavioral traits. Therefore, this work aims to correlate, for the first time, the physiological relevance of TAR1 functions with the tyraminergic system in insects. In addition, pharmacological studies have shed light on compounds with insecticidal properties having TAR1 as a target and on the emerging trend in the development of novel strategies for pest control.

Characterization of Halyomorpha halys TAR1 reveals its involvement in (E)-2-decenal pheromone perception.

In insects, tyramine receptor 1 (TAR1) has been shown to control several physiological functions, including olfaction. We investigated the molecular and functional profile of the Halyomorpha halys type 1 tyramine receptor gene (HhTAR1) and its role in olfactory functions of this pest. Molecular and pharmacological analyses confirmed that the HhTAR1 gene codes for a true TAR1. RT-qPCR analysis revealed that HhTAR1 is expressed mostly in adult brain and antennae as well as in early development stages (eggs, 1st and 2nd instar nymphs). In particular, among the antennomeres that compose a typical H. halys antenna, HhTAR1 was more expressed in flagellomeres. Scanning electron microscopy investigation revealed the type and distribution of sensilla on adult H. halys antennae: both flagellomeres appear rich in trichoid and grooved sensilla, known to be associated with olfactory functions. Through an RNAi approach, topically delivered HhTAR1 dsRNA induced a 50% downregulation in gene expression after 24 h in H. halys 2nd instar nymphs. An innovative behavioural assay revealed that HhTAR1 RNAi-silenced 2nd instar nymphs were less susceptible to the alarm pheromone component (E)-2 decenal as compared with controls. These results provide critical information concerning the role of TAR1 in olfaction regulation, especially alarm pheromone reception, in H. halys. Furthermore, considering the emerging role of TAR1 as target of biopesticides, this work opens the way for further investigation on innovative methods for controlling H. halys.

Monoterpenes alter TAR1-driven physiology in Drosophila species.

Monoterpenes are molecules with insecticide properties whose mechanism of action is, however, not completely elucidated. Furthermore, they seem to be able to modulate the monoaminergic system and several behavioural aspects in insects. In particular, tyramine (TA) and octopamine (OA) and their associated receptors orchestrate physiological processes such as feeding, locomotion and metabolism. Here, we show that monoterpenes not only act as biopesticides in Drosophila species but also can cause complex behavioural alterations that require functional type 1 tyramine receptors (TAR1s). Variations in metabolic traits as well as locomotory activity were evaluated in both Drosophila suzukii and Drosophila melanogaster after treatment with three monoterpenes. A TAR1-defective D. melanogaster strain (TAR1PL00408) was used to better understand the relationships between the receptor and monoterpene-related behavioural changes. Immunohistochemistry analysis revealed that, in the D. melanogaster brain, TAR1 appeared to be mainly expressed in the pars intercerebralis, lateral horn, olfactory and optic lobes and suboesophageal ganglion lobes. In comparison to wild-type D. melanogaster, the TAR1PL00408 flies showed a phenotype characterized by higher triglyceride levels and food intake as well as lower locomotory activity. The monoterpenes, tested at sublethal concentrations, were able to induce a downregulation of the TAR1 coding gene in both Drosophila species. Furthermore, monoterpenes also altered the behaviour in wild-type D. suzukii and D. melanogaster 24 h after continuous monoterpene exposure. Interestingly, they were ineffective in modifying the physiological performance of TAR1-defective flies. In conclusion, it appears that monoterpenes not only act as biopesticides for Drosophila but also can interfere with Drosophila behaviour and metabolism in a TAR1-dependent fashion.

Every cloud has a silver lining: how abiotic stresses affect gene expression in plant-pathogen interactions.

Current environmental and climate changes are having a pronounced influence on the outcome of plant-pathogen interactions, further highlighting the fact that abiotic stresses strongly affect biotic interactions at various levels. For instance, physiological parameters such as plant architecture and tissue organization together with primary and specialized metabolism are affected by environmental constraints, and these combine to make an individual plant either a more or less suitable host for a given pathogen. In addition, abiotic stresses can affect the timely expression of plant defense and pathogen virulence. Indeed, several studies have shown that variations in temperature, and in water and mineral nutrient availability affect the expression of plant defense genes. The expression of virulence genes, known to be crucial for disease outbreak, is also affected by environmental conditions, potentially modifying existing pathosystems and paving the way for emerging pathogens. In this review, we summarize our current knowledge on the impact of abiotic stress on biotic interactions at the transcriptional level in both the plant and the pathogen side of the interaction. We also perform a metadata analysis of four different combinations of abiotic and biotic stresses, which identifies 197 common modulated genes with strong enrichment in Gene Ontology terms related to defense . We also describe the multistress-specific responses of selected defense-related genes.

Modulation of Drosophila suzukii type 1 tyramine receptor (DsTAR1) by monoterpenes: a potential new target for next generation biopesticides.

This study proposes a biochemical and molecular model for the interaction between the Drosophila suzukii type 1 tyramine receptor (DsTAR1) and monoterpenes. A preliminary molecular and functional characterization of DsTAR1 cDNA revealed that a 1.8 kb long ORF codes for a 600 amino acid polypeptide featuring seven transmembrane domains, as expected for a GPCR. A stable HEK 293 cell line expressing DsTAR1 was tested for responsiveness to tyramine (TA) and octopamine (OA). In intracellular calcium mobilization studies, TA led to a concentration-dependent increase in [Ca2+]i (pEC50 ~ 6.40), completely abolished by pre-incubation with the antagonist yohimbine 1 µM. Besides, in dynamic mass redistribution (DMR) studies, TA evoked a positive DMR signal in a concentration-dependent manner (pEC50 ~ 6.80). The recombinant cell line was then used to test three monoterpenes (thymol, carvacrol and α-terpineol) as putative ligands for DsTAR1. The terpenoids showed no agonist effects in both DMR and calcium mobilization assays, but they increased the potency of the endogenous ligand, TA, acting as positive allosteric modulators. Moreover, expression analysis on adults D. suzukii, exposed for 24, 72 or 120 h to a sublethal concentration of the three monoterpenes, showed a downregulation of DsTAR1. This evidence has led to hypothesize that the downregulation of DsTAR1 might be a compensatory mechanism in response to the positive allosteric modulation of the receptor induced by monoterpenes. Therefore, these findings might be useful for the development of a new generation of biopesticides against Drosophila suzukii, targeting TAR1.

The bile acid deoxycholate elicits defences in Arabidopsis and reduces bacterial infection.

Disease has an effect on crop yields, causing significant losses. As the worldwide demand for agricultural products increases, there is a need to pursue the development of new methods to protect crops from disease. One mechanism of plant protection is through the activation of the plant immune system. By exogenous application, 'plant activator molecules' with elicitor properties can be used to activate the plant immune system. These defence-inducing molecules represent a powerful and often environmentally friendly tool to fight pathogens. We show that the secondary bile acid deoxycholic acid (DCA) induces defence in Arabidopsis and reduces the proliferation of two bacterial phytopathogens: Erwinia amylovora and Pseudomonas syringae pv. tomato. We describe the global defence response triggered by this new plant activator in Arabidopsis at the transcriptional level. Several induced genes were selected for further analysis by quantitative reverse transcription-polymerase chain reaction. We describe the kinetics of their induction and show that abiotic stress, such as moderate drought or nitrogen limitation, does not impede DCA induction of defence. Finally, we investigate the role in the activation of defence by this bile acid of the salicylic acid biosynthesis gene SID2, of the receptor-like kinase family genes WAK1-3 and of the NADPH oxidase-encoding RbohD gene. Altogether, we show that DCA constitutes a promising molecule for plant protection which can induce complementary lines of defence, such as callose deposition, reactive oxygen species accumulation and the jasmonic acid and salicylic acid signalling pathways.

The use of ECAS in plant protection: a green and efficient antimicrobial approach that primes selected defense genes.

The use of highly polluting chemicals for plant and crop protection is one of the components of the negative environmental impact of agricultural activities. In the present paper, an environmentally friendly alternative to pesticide application has been studied, based on the so-called electrochemically activated solutions (ECAS). Experiments have been carried out, by applying ECAS having different contents of active ingredients, on tobacco plants at a laboratory scale and on apple trees at fruit garden scale. The results, accumulated during a couple of years, have shown that properly selected dilute solutions of chlorides, once activated by an electrochemical treatment, exhibit a very effective protecting action of plants, irrespective of their nature. Extension of the research has shown that the observed effect is the result of two distinct factors: the expected anti-microbial action of the electrochemically synthesized oxidants, and an unexpected priming of immune plant defenses, which is clearly due to the treatment with ECAS. Interestingly, the repetition of ECAS application triggers an even stronger activation of defense genes. No oxidative damages, due to the use of the activated solutions, could be detected.

Enzymatic Chemoselective Aldehyde-Ketone Cross-Couplings through the Polarity Reversal of Methylacetoin.

The thiamine diphosphate (ThDP) dependent enzyme acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli. The recombinant enzyme shared close similarities with the acetylacetoin synthase (AAS) partially purified from Bacillus licheniformis suggesting that they could be the same enzyme. The product scope of the recombinant Ao:DCPIP OR was expanded to chiral tertiary α-hydroxy ketones through the rare aldehyde-ketone cross-carboligation reaction. Unprecedented is the use of methylacetoin as the acetyl anion donor in combination with a range of strongly to weakly activated ketones. In some cases, Ao:DCPIP OR produced the desired tertiary alcohols with stereochemistry opposite to that obtained with other ThDP-dependent enzymes. The combination of methylacetoin as acyl anion synthon and novel ThDP-dependent enzymes considerably expands the available range of C-C bond formations in asymmetric synthesis.

Croton lechleri Müll. Arg. (Euphorbiaceae) stem bark essential oil as possible mutagen-protective food ingredient against heterocyclic amines from cooked food.

The Amazonian Croton lechleri stem bark essential oil was tested for its anti-mutagenic potential by performing the Ames test against heterocyclic amines (HCAs), in continuing research on applicative functional profile of this phytocomplex as food ingredient (Rossi et al., 2011). Salmonella typhimurium strain TA98 was used with and without metabolic activation (S9 mix). The anti-mutagenic properties was assayed with the following HCAs: 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo-[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx), the imidazoles 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-aminodipirydo-[1,2-a:3',2'-d]imidazole (Glu-P-2). All HCAs with S9 induced mutagenicity at 10(-10) mol/plate. Without S9, IQ and MeIQ showed mutagenicity at 10(-8) mol/plate, MeIQx and Glu-P-1 at 10(-5) mol/plate, while Glu-P-2 was inactive. In presence of HACs (10(-9) mol/plate), C. lechleri essential oil was tested for mutagen-protective properties (concentration range: 0.01-0.10 mg/plate) taking the Highest Uneffective Dose (HUD) as threshold reference. With S9 mix, C. lechleri essential oil displayed a significant reduction of revertants at 0.05 mg/plate, from 21% to 34%. The essential oil showed mutagen-protective efficacy against IQ and MeIQ tested as direct mutagens (10(-7) mol/plate), with a revertants percentage reduction of 39% and 40%, respectively. No anti-mutagen capacity was noted for MeIQx and Glu-P-1 (10(-5) mol/plate). Since HACs are known as possible colon and liver cancer inducers, C. lechleri essential oil was tested for its cytotoxicity and anti-proliferative capacity against LoVo and HepG2 cancer cell lines showing IC50 of 74.95±0.05 µg/ml (LoVo) and 82.28±0.03 µg/ml (HepG2), displaying a promising role of this essential oil as a functional food ingredient with interesting mutagen preventing properties.

PRMT11: a new Arabidopsis MBD7 protein partner with arginine methyltransferase activity.

Plant methyl-DNA-binding proteins (MBDs), discovered by sequence homology to their animal counterparts, have not been well characterized at the physiological and functional levels. In order better to characterize the Arabidopsis AtMBD7 protein, unique in bearing three MBD domains, we used a yeast two-hybrid system to identify its partners. One of the interacting proteins we cloned is the Arabidopsis arginine methyltransferase 11 (AtPRMT11). Glutathione S-transferase pull-down and co-immunoprecipitation assays confirmed that the two proteins interact with each other and can be co-isolated. Using GFP fluorescence, we show that both AtMBD7 and AtPRMT11 are present in the nucleus. Further analyses revealed that AtPRMT11 acts as an arginine methyltransferase active on both histones and proteins of cellular extracts. The analysis of a T-DNA mutant line lacking AtPRMT11 mRNA revealed reduced levels of proteins with asymmetrically dimethylated arginines, suggesting that AtPRMT11, which is highly similar to mammalian PRMT1, is indeed a type I arginine methyltransferase. Further, AtMBD7 is a substrate for AtPRMT11, which post-translationally modifies the portion of the protein-containing C-terminal methylated DNA-binding domain. These results suggest the existence of a link between DNA methylation and arginine methylation.

Biochemical and molecular responses to water stress in resurrection plants.

A small group of angiosperms, known as resurrection plants, can tolerate extreme dehydration. They survive in arid environments because they are able to dehydrate, remain quiescent during long periods of drought, and then resurrect upon rehydration. Dehydration induces the expression of a large number of transcripts in resurrection plants. Gene products with a putative protective function such as LEA proteins have been identified; they are expressed at high levels in the cytoplasm or in chloroplasts upon dehydration and/or ABA treatment of vegetative tissue. An increase in sugar concentration is usually observed at the onset of desiccation in vegetative tissue of resurrection plants. These sugars may be effective in osmotic adjustment or they may stabilize membrane structures and proteins. Regulatory genes such as a protein translation initiation factor, homeodomain-leucine zipper genes and a gene probably working as a regulatory RNA have been isolated and characterized. The knowledge of the biochemical and molecular responses that occur during the onset of drought may help to improve water stress tolerance in plants of agronomic importance.

Arabidopsis MBD proteins show different binding specificities and nuclear localization.

Recent results in animals and plants have shown a strong link between DNA methylation, chromatin structure and epigenetic control. In plants DNA methylation affects both symmetric and asymmetric cytosines by means of different DNA-methyltransferases. In vertebrates these modifications are interpreted by a group of proteins (methylated DNA-binding domain proteins, MBDs) able to specifically bind methylated CpG. In plants several genes sharing structural homology to mammalian MBD have been identified in Arabidopsis and maize, but their characterization is still to be completed. Here we present the characterization of six different MBDs from Arabidopsis. As judged by semi-quantitative RT-PCR, their expression proved to be differentially modulated in different organs. All the corresponding polypeptides, expressed in Escherichia coli as His-tagged recombinant proteins, have been functionally tested on gel shift experiments but only two of them (namely MBD5, 6) were able to specifically bind methylated CpG oligonucleotides. A third protein, AtMBD11, showed a strong affinity for DNA independently from the level of methylation. Moreover we were able to differentiate MBD5 and 6, despite their high homology, for their ability to recognize methylated asymmetrical sites. The binding specificity of these three AtMBD proteins was tested not only on arbitrarily chosen probes but also on the Arabidopsis E2F recognition sequence containing a single CpG site. Protoplasts transient expression experiments of GFP-fusion proteins showed for AtMBD5 and AtMBD6 a heterochromatic localization which was affected by 5-azacytidine treatment. These data demonstrate that AtMBD5 and AtMBD6 bind methylated DNA in vitro and in vivo with different specificity and might therefore have different roles in methylation-mediated transcriptional silencing.