Competition among Flavescence Dorée Phytoplasma Strains in the Experimental Insect Vector Euscelidius variegatus.

Phytoplasmas are plant pathogenic wall-less bacteria transmitted in a persistent propagative manner by hemipteran insects, mainly belonging to the suborder Auchenorrhyncha (Fulgoromorpha and Cicadomorpha). Flavescence dorée (FD) is a quarantine disease of grapevine, causing great damage to European viticulture and associated with phytoplasmas belonging to 16SrV-C (FD-C) and -D (FD-D) subgroups. FD-C and FD-D strains share similar pathogenicity, but mixed infections are rare in nature. To investigate the competition among FDp strains, specimens of the laboratory vector Euscelidius variegatus (Hemiptera: Cicadellidae) were forced to acquire both phytoplasma haplotypes upon feeding on FD-C- and FD-D-infected plants or after the injection of both strains. The pathogen colonization of insect bodies and heads was monitored with multiplex qPCR, and the efficiencies of phytoplasma transmission were estimated. Single infection, irrespective of strain type, was more frequent than expected, indicating that competition among FD strains occurs. Hypotheses of competition for resources and/or host active sites or the direct antibiosis of one strain against the other are discussed, based on the genetic complexity of FDp populations and on the high genome variability of the FD-D strain. As FD management still mainly relies on insecticides against vectors, the characterization of FDp haplotypes and the description of their epidemiology also have practical implications.

Metatranscriptomic Assessment of the Microbial Community Associated With the Flavescence dorée Phytoplasma Insect Vector Scaphoideus titanus.

Phytoplasmas are insect-borne pathogenic bacteria that cause major economic losses to several crops worldwide. The dynamic microbial community associated with insect vectors influences several aspects of their biology, including their vector competence for pathogens. Unraveling the diversity of the microbiome of phytoplasma insect vectors is gaining increasing importance in the quest to develop novel microbe-based pest control strategies that can minimize the use of insecticides for better environmental quality. The leafhopper Scaphoideus titanus is the primary vector of the Flavescence dorée phytoplasma, a quarantine pest which is dramatically affecting the main grape-growing European countries. In this study, the RNA-Seq data, which were previously used for insect virus discovery, were further explored to assess the composition of the whole microbial community associated with insects caught in the wild in both its native (the United States) and invasive (Europe) areas. The first de novo assembly of the insect transcriptome was used to filter the host sequencing reads. The remaining ones were assembled into contigs and analyzed by blastx to provide the taxonomic identification of the microorganisms associated with S. titanus, including the non-bacterial components. By comparing the transcriptomic libraries, we could differentiate the stable and consistent associations from the more ephemeral and flexible ones. Two species appeared to be universal to the core microbiome of S. titanus: the obligate bacterial symbiont Candidatus Sulcia muelleri and an Ophiocordyceps-allied fungus distantly related to yeast-like symbionts described from other hemipterans. Bacteria of the genus Cardinium have been identified as another dominant member of the microbiome, but only in the European specimens. Although we are yet to witness how the interplay among the microorganisms influences the vector competence of S. titanus, this unbiased in silico characterization of its microbiome is paramount for identifying the naturally occurring targets for new biocontrol strategies to counteract Flavescence dorée spread in Europe.

Silencing of ATP Synthase ß Impairs Egg Development in the Leafhopper Scaphoideus titanus, Vector of the Phytoplasma Associated with Grapevine Flavescence Dorée.

Scaphoideus titanus (Hemiptera: Cicadellidae) is the natural vector of Flavescence dorée phytoplasma, a quarantine pest of grapevine with severe impact on European viticulture. RNA interference (RNAi) machinery components are present in S. titanus transcriptome and injection of ATP synthase ß dsRNAs into adults caused gene silencing, starting three days post injection (dpi) up to 20 dpi, leading to decrease cognate protein. Silencing of this gene in the closely related leafhopper Euscelidiusvariegatus previously showed female sterility and lack of mature eggs in ovaries. Here, alteration of developing egg morphology in S. titanus ovaries as well as overexpression of hexamerin transcript (amino acid storage protein) and cathepsin L protein (lysosome proteinase) were observed in dsATP-injected females. To evaluate RNAi-specificity, E.variegatus was used as dsRNA-receiving model-species. Different doses of two sets of dsRNA-constructs targeting distinct portions of ATP synthase ß gene of both species induced silencing, lack of egg development, and female sterility in E. variegatus, indicating that off-target effects must be evaluated case by case. The effectiveness of RNAi in S. titanus provides a powerful tool for functional genomics of this non-model species and paves the way toward RNAi-based strategies to limit vector population, despite several technical and regulatory constraints that still need to be overcome to allow open field application.

Bacteriophage-Host Association in the Phytoplasma Insect Vector Euscelidius variegatus.

Insect vectors transmit viruses and bacteria that can cause severe diseases in plants and economic losses due to a decrease in crop production. Insect vectors, like all other organisms, are colonized by a community of various microorganisms, which can influence their physiology, ecology, evolution, and also their competence as vectors. The important ecological meaning of bacteriophages in various ecosystems and their role in microbial communities has emerged in the past decade. However, only a few phages have been described so far in insect microbiomes. The leafhopper Euscelidius variegatus is a laboratory vector of the phytoplasma causing Flavescence dorée, a severe grapevine disease that threatens viticulture in Europe. Here, the presence of a temperate bacteriophage in E. variegatus (named Euscelidius variegatus phage 1, EVP-1) was revealed through both insect transcriptome analyses and electron microscopic observations. The bacterial host was isolated in axenic culture and identified as the bacterial endosymbiont of E. variegatus (BEV), recently assigned to the genus Candidatus Symbiopectobacterium. BEV harbors multiple prophages that become active in culture, suggesting that different environments can trigger different mechanisms, finely regulating the interactions among phages. Understanding the complex relationships within insect vector microbiomes may help in revealing possible microbe influences on pathogen transmission, and it is a crucial step toward innovative sustainable strategies for disease management in agriculture.

Silencing of ATP synthase ß reduces phytoplasma multiplication in a leafhopper vector.

The leafhopper Euscelidius variegatus is a natural vector of the chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of the Flavescence dorée phytoplasma (FDp). Previous studies indicated a crucial role for insect ATP synthase α and ß subunits during phytoplasma infection of the vector species. Gene silencing of ATP synthase ß was obtained by injection of specific dsRNAs in E. variegatus. Here we present the long-lasting nature of such silencing, its effects on the small RNA profile, the significant reduction of the corresponding protein expression, and the impact on phytoplasma acquisition capability. The specific transcript expression was silenced at least up to 37 days post injection with an average reduction of 100 times in insects injected with dsRNAs targeting ATP synthase ß (dsATP) compared with those injected with dsRNAs targeting green fluorescent protein (dsGFP), used as negative controls. Specific silencing of this gene was also confirmed at protein level at 15 days after the injection. Total sRNA reads mapping to dsATP and dsGFP sequences in analysed libraries showed in both cases a peak of 21 nt, a length consistent with the generation of dsRNA-derived siRNAs by RNAi pathway. Reads mapped exclusively to the fragment corresponding to the injected dsATPs, probably indicating the absence of a secondary machinery for siRNA synthesis. Insects injected either with dsATP or dsGFP successfully acquired CYp and FDp during feeding on infected plants. However, the average phytoplasma amount in dsATP insects was significantly lower than that measured in dsGFP specimens, indicating a probable reduction of the pathogen multiplication when ATP synthase ß was silenced. The role of the insect ATP synthase ß during phytoplasma infection process is discussed.

Competitive Exclusion of Flavescence dorée Phytoplasma Strains in Catharanthus roseus Plants.

Flavescence dorée phytoplasmas (FDp, 16SrV-C and -D) are plant pathogenic non-cultivable bacteria associated with a severe grapevine disease. The incidence of the two reference strains on cultivated grapevines is unbalanced, and mixed infections are rare. To investigate the interaction between the two strains, Catharanthus roseus plants were graft-infected with both strains, either simultaneously or sequentially. Different combinations of lateral and apical grafting were applied to avoid possible benefits due to graft position. The infection was monitored for four months through a new diagnostic protocol developed for differentiation and relative quantification of the two strains. Regardless of the temporal or spatial advantage at grafting, FD-C generally outcompeted FD-D. The prevalence of FD-C increased over time and, at the end of the experiment, FD-C was the unique strain detected in the aerial part and the roots of 74% and 90% of grafted plants, respectively. These data indicate that the interaction between the two strains results in competitive exclusion. Understanding the bases of the competition between FD-C and FD-D may contribute to explain the biology of the coexistence of different FDp strains under field conditions, aiming at identifying potential suppressor strains, which can provide alternative and environmentally sustainable solutions for FD control.

Prevalence of Flavescence Dorée Phytoplasma-Infected Scaphoideus titanus in Different Vineyard Agroecosystems of Northwestern Italy.

Quantitative estimates of vector populations and their infectivity in the wild and in cultivated compartments of agroecosystems have been carried out to elucidate the role of the wild compartment in the epidemiology of Flavescence dorée (FD). Seven sites were selected for the investigations in the Piedmont Region of Italy. They were characterized by a high variety of agricultural and ecological landscape features, and included a vineyard surrounded by wild vegetation. In order to describe abundance and prevalence of FD-infected vectors in the cultivated and wild compartments of the vineyard agroecosystem, adults of Scaphoideus titanus were collected by yellow sticky traps inside and outside the vineyard over the period July 10th-September 9th, 2015. They were counted and singly analyzed for the presence of FD phytoplasmas by PCR. Multifactorial correlations among vector population level, prevalence of infected insects inside and outside the vineyards, disease prevalence in cultivated and wild Vitis plants, and location of wild Vitis plants with respect to the vineyard were analyzed. Abundance of S. titanus adults significantly decreased from the end of July onwards, particularly inside the vineyard (average range 22.7 ± 2.5 insects/trap). Percentage of FD-positive S. titanus was significantly higher outside the vineyard (up to 48% on average) compared to inside the vineyard (up to 34% on average), and increased during the season in both compartments.

Biological characterization of Euscelidius variegatus iflavirus 1.

Virus-based biocontrol technologies represent sustainable alternatives to pesticides and insecticides. Phytoplasmas are prokaryotic plant pathogens causing severe losses to crops worldwide. Novel approaches are needed since insecticides against their insect vectors and rogueing of infected plants are the only available strategies to counteract phytoplasma diseases. A new iflavirus, named EVV-1, has been described in the leafhopper phytoplasma vector Euscelidius variegatus, raising the potential to use virus-based application strategies against phytoplasma disease. Here transmission routes of EVV-1 are characterized, and localization within the host reveals the mechanism of insect tolerance to virus infection. Both vertical and horizontal transmission of EVV-1 occur and vertical transmission was more efficient. The virus is systemic and occurs in all life-stages, with the highest loads measured in ovaries and first to third instar nymphs. The basic knowledge gained here on the biology of the virus is crucial for possible future application of iflaviruses as biocontrol agents.

Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale.

To study the role of wild areas around the vineyards in the epidemiology of flavescence dorée (FD) and track the origin of new foci, two phytoplasma genetic markers, dnaK and malG, were developed for FD phytoplasma (FDp) characterization. The two genes and the vmpA locus were used to genetically characterize FDp populations at seven agroecosystems of a wine-growing Italian region. Vitis vinifera, "gone-wild" V. vinifera and rootstocks, Clematis spp., and Scaphoideus titanus adults were sampled within and outside the vineyards. A range of genotypes infecting the different hosts of the FDp epidemiological cycle was found. Type FD-C isolates were fairly homogeneous compared to type FD-D ones. Most of the FD-D variability was correlated with the malG sequence, and a duplication of this locus was demonstrated for this strain. Coinfection with FD-C and FD-D strains was rare, suggesting possible competition between the two. Similar levels of FDp genetic variation recorded for grapevines or leafhoppers of cultivated and wild areas and co-occurrence of many FDp genotypes inside and outside the vineyards supported the idea of the importance of wild or abandoned Vitis plants and associated S. titanus insects in the epidemiology of the disease. Genetic profiles of FDp found in Clematis were never found in the other hosts, indicating that this species does not take part in the disease cycle in the area. Due to the robustness of analyses using dnaK for discriminating between FD-C and FD-D strains and the high variability of malG sequences, these are efficient markers to study FDp populations and epidemiology at a small geographical scale.IMPORTANCE Flavescence dorée, a threatening disease of grapevine caused by FD phytoplasma (FDp), is distributed within the most important wine-producing areas of Europe and has severe effects on both vineyard productivity and landscape management. FDp is a quarantine pest in Europe, and despite the efforts to contain the pathogen, the disease is still spreading. In this work, new genetic markers for the fine genetic characterization of FDp at local scale are presented. Our findings improve the knowledge of FDp epidemiological cycle and offer the possibility of tracking the route of the FDp infection. In particular, due to its high genetic variability, one of the newly developed markers could be sufficient to track the origin of new infection foci, either from the wild areas or from nurseries.

RNA interference of muscle actin and ATP synthase beta increases mortality of the phytoplasma vector Euscelidius variegatus.

BACKGROUND: RNA interference (RNAi) techniques have emerged as powerful tools to develop novel management strategies for the control of insect pests. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma and a laboratory vector of Flavescence dorée phytoplasma. Phytoplasmas are insect-borne bacterial plant pathogens that cause economically relevant crop losses worldwide. RESULTS: In this study, we demonstrated that microinjection of muscle actin and ATP synthase ß double-stranded (ds)RNAs into adult insects caused an exponential reduction in the expression of both genes, which began within 72 h of dsRNA administration and lasted for 14 days, leading to almost complete silencing of the target genes. Such silencing effects on muscle actin expression appeared to be both time- and dose-dependent. Our results also showed that the knockdown of both genes caused a significant decrease in survival rates in comparison with green fluorescent protein (GFP) dsRNA-injected control insects. CONCLUSION: The effectiveness of RNAi-based gene silencing in E. variegatus guarantees the availability of a powerful reverse genetic tool for the functional annotation of its genes and the identification of those potentially involved in the interaction with phytoplasmas. In addition, this study demonstrated that muscle actin and ATP synthase ß may represent candidate genes for RNAi-based control of E. variegatus. © 2018 Society of Chemical Industry.

Application of laser microdissection to study phytoplasma site-specific gene expression in the model plant Arabidopsis thaliana.

Many aspects of plant diseases caused by phytoplasmas are still unknown, as these pathogens are phloem restricted, uncultivable wall-less bacteria and must be studied always in association with their host. Phytoplasma transcripts are strongly underrepresented within host tissues and this poses problems for gene expression analyses. In this study, a procedure was established to infect the model plant Arabidopsis thaliana with the phytoplasma Flavescence dorée, a serious threat to European viticulture. Rates of phytoplasma infective insects and transmission efficiency to A. thaliana as well as pathogen loads were measured in different tissues of infected A. thaliana plants, and modification of phloem cell ultrastructure was observed in infected plant tissues at microscopic level. Moreover, a protocol for the application of laser microdissection to analyze plant and phytoplasma gene expression profiles in the specific colonized tissue was designed. The procedure allowed a good preservation of the plant tissue anatomy. Results showed that the extracted RNA was suitable for qualitative and quantitative RT-PCR, since both plant and pathogen transcripts, either abundant or rare ones, could be detected without any pre-amplification step. The combined use of laser microdissection approach and A. thaliana to study phytoplasmas opens the way to exploit biological, molecular and bioinformatic tools available for the model plant and to elucidate key pathways of the infection mechanisms of these important plant pathogen.

miRVIT: A Novel miRNA Database and Its Application to Uncover Vitis Responses to Flavescence dorée Infection.

Micro(mi)RNAs play crucial roles in plant developmental processes and in defense responses to biotic and abiotic stresses. In the last years, many works on small RNAs in grapevine (Vitis spp.) were published, and several conserved and putative novel grapevine-specific miRNAs were identified. In order to reorganize the high quantity of available data, we produced "miRVIT," the first database of all novel grapevine miRNA candidates characterized so far, and still not deposited in miRBase. To this aim, each miRNA accession was renamed, repositioned in the last version of the grapevine genome, and compared with all the novel and conserved miRNAs detected in grapevine. Conserved and novel miRNAs cataloged in miRVIT were then used for analyzing Vitis vinifera plants infected by Flavescence dorée (FD), one of the most severe phytoplasma diseases affecting grapevine. The analysis of small RNAs from healthy, recovered (plants showing spontaneous and stable remission of symptoms), and FD-infected "Barbera" grapevines showed that FD altered the expression profiles of several miRNAs, including those involved in cell development and photosynthesis, jasmonate signaling, and disease resistance response. The application of miRVIT in a biological context confirmed the effectiveness of the followed approach, especially for the identification of novel miRNA candidates in grapevine. miRVIT database is available at http://mirvit.ipsp.cnr.it. Highlights: The application of the newly produced database of grapevine novel miRNAs to the analysis of plants infected by Flavescence dorée reveals key roles of miRNAs in photosynthesis and jasmonate signaling.

Two Phytoplasmas Elicit Different Responses in the Insect Vector Euscelidius variegatus Kirschbaum.

Phytoplasmas are plant-pathogenic bacteria transmitted by hemipteran insects. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of flavescence dorée phytoplasma (FDp). The two phytoplasmas induce different effects on this species: CYp slightly improves whereas FDp negatively affects insect fitness. To investigate the molecular bases of these different responses, transcriptome sequencing (RNA-seq) analysis of E. variegatus infected with either CYp or FDp was performed. The sequencing provided the first de novo transcriptome assembly for a phytoplasma vector and a starting point for further analyses on differentially regulated genes, mainly related to immune system and energy metabolism. Insect phenoloxidase activity, immunocompetence, and body pigmentation were measured to investigate the immune response, while respiration and movement rates were quantified to confirm the effects on energy metabolism. The activation of the insect immune response upon infection with FDp, which is not naturally transmitted by E. variegatus, confirmed that this bacterium is mostly perceived as a potential pathogen. Conversely, the acquisition of CYp, which is naturally transmitted by E. variegatus, seems to increase the insect fitness by inducing a prompt response to stress. This long-term relationship is likely to improve survival and dispersal of the infected insect, thus enhancing the opportunity of phytoplasma transmission.

Genome sequence, prevalence and quantification of the first iflavirus identified in a phytoplasma insect vector.

The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CY) and an efficient vector of flavescence dorée phytoplasma (FD) under laboratory conditions. During a transcriptome sequencing (RNA-seq) project aimed at investigating the interactions between the insect and the two phytoplasmas, a 10,616-nucleotide-long contig with high sequence similarity to known picorna-like viruses was identified among the assembled insect transcripts. The discovery came totally unexpected, because insects from the laboratory colony did not show any evident symptom that could be related to the presence of a virus. The amino acid sequence, the shape and size of viral particles, and the results of phylogenetic analysis suggest that this virus, named Euscelidius variegatus virus 1 (EVV-1), can be considered a new member of a new species in the genus Iflavirus. EVV-1 was detected in all of the tested insects from the laboratory colony used for RNA-seq, both in phytoplasma-exposed and in non-exposed insects, but the viral load measured in FD-exposed samples was significantly lower than that in non-exposed insects. This result suggests the possible existence of an intriguing cross-talk among insects, endogenous bacteria, and viruses. The identification of two other E. variegatus laboratory colonies that were free of EVV-1 could represent the key to addressing some basic virological issues, e.g., viral replication and transmission mechanisms, and offer the opportunity to use infectious clones to express heterologous genes in the leafhopper and manipulate the expression of endogenous genes by promoting virus-induced gene silencing.

Investigation on the partial resistance of Cpkk2 knock out strain of Cryphonectria parasitica to Cryphonectria hypovirus 1 infection in presence of Geneticin and Geneticin resistance gene.

We have recently characterized the central components of the three MAP kinase cascades present in Cryphonectria parasitica : the MEK genes cpkk1, cpkk2 and cpkk3. When we attempted to infect through anastomosis the three knock out strains with Cryphonectria hypovirus 1 (CHV1), only the deletion strain of Cpkk2, the yeast Ste7 homologue, involved in mating and filamentous growth, could not be infected. We then proceeded to attempt virus infection through transformation of Δcpkk2 protoplasts using an infectious cDNA clone able to establish virus infection through transformation. In this case, a very limited number of strains could be recovered as stable transformants compared to the efficiency of control transformations with plasmid carrying only the antibiotic marker. Furthermore, transformants carrying actively replicating virus could be isolated only if the selection marker Geneticin was used during the very initial selection process, and not maintained throughout the growth of the colonies. Moreover, Δcpkk2 isolates that maintained the virus lost Geneticin resistance. We therefore unveiled a specific negative interaction among virus infection, presence of Geneticin in the growth media, and lack of Cpkk2 MEK in the fungal host.

The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense.

The N-terminal region of the Ourmia melon virus (OuMV) coat protein (CP) contains a short lysine/arginine-rich (KR) region. By alanine scanning mutagenesis, we showed that the KR region influences pathogenicity and virulence of OuMV without altering viral particle assembly. A mutant, called OuMV6710, with three basic residue substitutions in the KR region, was impaired in the ability to maintain the initial systemic infection in Nicotiana benthamiana and to infect both cucumber and melon plants systemically. The integrity of this protein region was also crucial for encapsidation of viral genomic RNA; in fact, certain mutations within the KR region partially compromised the RNA encapsidation efficiency of the CP. In Arabidopsis thaliana Col-0, OuMV6710 was impaired in particle accumulation; however, this phenotype was abolished in dcl2/dcl4 and dcl2/dcl3/dcl4 Arabidopsis mutants defective for antiviral silencing. Moreover, in contrast to CPwt, in situ immunolocalization experiments indicated that CP6710 accumulates efficiently in the spongy mesophyll tissue of infected N. benthamiana and A. thaliana leaves but only occasionally infects palisade tissues. These results provided strong evidence of a crucial role for OuMV CP during viral infection and highlighted the relevance of the KR region in determining tissue tropism, host range, pathogenicity, and RNA affinity, which may be all correlated with a possible CP silencing-suppression activity.

Functional characterization of the three mitogen-activated protein kinase kinases (MAP2Ks) present in the Cryphonectria parasitica genome reveals the necessity of Cpkk1 and Cpkk2, but not Cpkk3, for pathogenesis on chestnut (Castanea spp.).

The biological function(s) of the cpkk1, cpkk2 and cpkk3 genes, encoding the three mitogen-activated protein kinase kinases (MAP2Ks) of Cryphonectria parasitica, the causal agent of chestnut blight, were examined through knockout strains. Cpkk1, the Mkk1 orthologue, acts in a phosphorylation cascade essential for cell integrity; Cpkk2 is the Ste7 orthologue involved in the pheromone response pathway; Cpkk3 is the Pbs2 orthologue, the MAP2K activated during the high-osmolarity response. Our analysis confirmed the role of each MAP2K in its respective signalling cascade with some peculiarities: abnormal hyphae with a reduced number of septa and thinner cell walls were observed in Δcpkk1 mutants, and a strong growth defect on solid media was evident in Δcpkk2 mutants, when compared with the controls. Virulence on chestnut was affected in both the Δcpkk1 and Δcpkk2 strains, which were also unable to complete the developmental steps essential for mating. No alterations were reported in Δcpkk3, except under hyperosmotic conditions and in the presence of fludioxonil. Δcpkk2 mutants, however, showed higher sensitivity during growth in medium containing the antibiotic G418 (Geneticin).

Ozone-induced caspase-like activities are dependent on early ion channel regulations and ROS generation in Arabidopsis thaliana cells.

Using A. thaliana cultured cells; we recently reported new insights regarding the effect of acute O3 exposure. This consist in an oxidative dependent controlled cell death process involving cell shrinkage due to an early activation of anion channel (1) and a delayed activation of K(+) outward currents, but also to early events like Ca (2+) influx or singlet oxygen production possibly linked to mitochondrial dysfunction. Here we provide evidence that most of these early events act downstream of caspase-like activities as recently demonstrated for K(+) channel activation.

Post-transcriptional regulation of GORK channels by superoxide anion contributes to increases in outward-rectifying K⁺ currents.

· Ion fluxes are ubiquitous processes in the plant and animal kingdoms, controlled by fine-tuned regulations of ion channel activity. Yet the mechanism that cells employ to achieve the modification of ion homeostasis at the molecular level still remains unclear. This is especially true when it comes to the mechanisms that lead to cell death. · In this study, Arabidopsis thaliana cells were exposed to ozone (O3). Ion flux variations were analyzed by electrophysiological measurements and their transcriptional regulation by RT-PCR. Reactive oxygen species (ROS) generation was quantified by luminescence techniques and caspase-like activities were investigated by laser confocal microscopy. · We highlighted the delayed activation of K(+) outward-rectifying currents after an O3 -induced oxidative stress leading to programmed cell death (PCD). Caspase-like activities are detected under O3 exposure and could be decreased by K(+) channel blocker. Molecular experiments revealed that the sustained activation of K(+) outward current could be the result of an unexpected O2 ·â» post-transcriptional regulation of the guard cell outward-rectifying K(+) (GORK) channels. · This consists of a likely new mode of regulating the processing of the GORK mRNA, in a ROS-dependent manner, to allow sustained K(+) effluxes during PCD. These data provide new mechanistic insights into K(+) channel regulation during an oxidative stress response.

Sequential depolarization of root cortical and stelar cells induced by an acute salt shock - implications for Na(+) and K(+) transport into xylem vessels.

Early events in NaCl-induced root ion and water transport were investigated in maize (Zea mays L) roots using a range of microelectrode and imaging techniques. Addition of 100 mm NaCl to the bath resulted in an exponential drop in root xylem pressure, rapid depolarization of trans-root potential and a transient drop in xylem K(+) activity (A(K+) ) within ∼1 min after stress onset. At this time, no detectable amounts of Na(+) were released into the xylem vessels. The observed drop in A(K+) was unexpected, given the fact that application of the physiologically relevant concentrations of Na(+) to isolated stele has caused rapid plasma membrane depolarization and a subsequent K(+) efflux from the stelar tissues. This controversy was explained by the difference in kinetics of NaCl-induced depolarization between cortical and stelar cells. As root cortical cells are first to be depolarized and lose K(+) to the environment, this is associated with some K(+) shift from the stelar symplast to the cortex, resulting in K(+) being transiently removed from the xylem. Once Na(+) is loaded into the xylem (between 1 and 5 min of root exposure to NaCl), stelar cells become more depolarized, and a gradual recovery in A(K+) occurs.