Gene silencing of cathepsins B and L using CTV-based, plant-mediated RNAi interferes with ovarial development in Asian citrus psyllid (ACP), Diaphorina citri.

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibacter americanus (CLam) and Candidatus Liberibacter asiaticus (CLas), which are phloem-restricted and associated with the most important and destructive worldwide citrus disease, Huanglongbing (HLB). Currently, no cure for HLB has been described. Therefore, measures have focused on reducing D. citri populations. In these insects, cathepsin B (DCcathB) and L (DCcathL) enzymes play an important role in digestion, and are involved in embryogenesis, immune defense, and ecdysis. In this study, we used a CTV-based vector to deliver dsRNA (CTV-dsRNA) into Citrus macrophylla plants targeting DCcathB and DCcathL genes in D. citri that fed on the phloem of these CTV-RNAi infected plants. Subsequently, we evaluated expression of DCcathB and DCcathL genes as well as the Vitellogenin (Vg) gene by RT-qPCR in D. citri fed on CTV-dsRNA occurring in plant phloem. It was found that a defective phenotype in D. citri females as a result of knockdown of DCcathB and DCcathL genes mediated by CTV dsRNA. These results showed that Psyllids fed on plants treated with the CTV-dsRNA exhibited downregulation of the Vg gene, one of the most important genes associated with embryogenic and female development, which was associated with dsRNA-mediated silencing of the two cathepsin genes. Based on our findings, a CTV-based strategy for delivering RNAi via plants that targets DCcathB and DCcathL genes may represent a suitable avenue for development of dsRNA-based tools to manage D. citri that limits the spread of HLB.

Bacterial Pesticidal Protein Mpp51Aa1 Delivered via Transgenic Citrus Severely Impacts the Fecundity of Asian Citrus Psyllid, Diaphorina citri.

The Asian citrus psyllid (ACP) Diaphorina citri vectors the causative agent of citrus greening disease that has the capacity to decimate citrus production. As an alternative and more sustainable approach to manage D. citri than repeated application of chemical insecticides, we investigated the potential use of the bacteria-derived pesticidal protein, Mpp51Aa1, when delivered by transgenic Citrus sinensis cv. Valencia sweet orange or Citrus paradisi cv. Duncan grapefruit. Following confirmation of transcription and translation of mpp51aa1 by transgenic plants, no impact of Mpp51Aa1 expression was seen on D. citri host plant choice between transgenic and control Duncan grapefruit plants. A slight but significant drop in survival of adult psyllids fed on these transgenic plants was noted relative to those fed on control plants. In line with this result, damage to the gut epithelium consistent with that caused by pore-forming proteins was only observed in a minority of adult D. citri fed on the transgenic Duncan grapefruit. However, greater impacts were observed on nymphs than on adults, with a 40% drop in the survival of nymphs fed on transgenic Duncan grapefruit relative to those fed on control plants. For Valencia sweet orange, a 70% decrease in the number of eggs laid by adult D. citri on transgenic plants was noted relative to those on control plants, with a 90% drop in emergence of progeny. These impacts that contrast with those associated with other bacterial pesticidal proteins and the potential for use of Mpp51Aa1-expressing transgenic plants for suppression of D. citri populations are discussed. IMPORTANCE Pesticidal proteins derived from bacteria such as Bacillus thuringiensis are valuable tools for management of agricultural insect pests and provide a sustainable alternative to the application of chemical insecticides. However, relatively few bacterial pesticidal proteins have been used for suppression of hemipteran or sap-sucking insects such as the Asian citrus psyllid, Diaphorina citri. This insect is particularly important as the vector of the causative agent of citrus greening, or huanglongbing disease, which severely impacts global citrus production. In this study, we investigated the potential of transgenic citrus plants that produce the pesticidal protein Mpp51Aa1. While adult psyllid mortality on transgenic plants was modest, the reduced number of eggs laid by exposed adults and the decreased survival of progeny was such that psyllid populations dropped by more than 90%. These results provide valuable insight for potential deployment of Mpp51Aa1 in combination with other control agents for the management of D. citri.

Identification and Characterization of Potato Zebra Chip Resistance Among Wild Solanum Species.

Potato zebra chip (ZC) disease, associated with the uncultured phloem-limited bacterium, Candidatus Liberibacter solanacearum (CLso), is transmitted by the potato psyllid Bactericera cockerelli. Potato ZC disease poses a significant threat to potato production worldwide. Current management practices mainly rely on the control of the psyllid to limit the spread of CLso. The present study investigated new sources of ZC resistance among wild Solanum species. A taxonomically diverse collection of tuber-bearing Solanum species was screened; one ZC-resistant accession and three ZC-tolerant accessions were identified among the 52 screened accessions. Further characterization of the resistant accession showed that the resistance was primarily associated with antibiosis effects due to differences in leaf trichome density and morphology of the wild accession, which could limit the psyllid feeding and oviposition. This germplasm offers a good resource for further understanding ZC and psyllid resistance mechanisms, contributing to potato breeding efforts to develop ZC resistance cultivars. Alternatively, it could be used as a potential trap crop to manage psyllid and control ZC disease.

Salicylic acid mediated immune response of Citrus sinensis to varying frequencies of herbivory and pathogen inoculation.

BACKGROUND: Plant immunity against pathogens and pests is comprised of complex mechanisms orchestrated by signaling pathways regulated by plant hormones [Salicylic acid (SA) and Jasmonic acid (JA)]. Investigations of plant immune response to phytopathogens and phloem-feeders have revealed that SA plays a critical role in reprogramming of the activity and/or localization of transcriptional regulators via post-translational modifications. We explored the contributing effects of herbivory by a phytopathogen vector [Asian citrus psyllid, Diaphorina citri] and pathogen [Candidatus Liberibacter asiaticus (CaLas)] infection on response of sweet orange [Citrus sinensis (L.) Osbeck] using manipulative treatments designed to mimic the types of infestations/infections that citrus growers experience when cultivating citrus in the face of Huanglongbing (HLB) disease. RESULTS: A one-time (7 days) inoculation access period with CaLas-infected vectors caused SA-associated upregulation of PR-1, stimulating defense response after a long period of infection without herbivory (270 and 360 days). In contrast, while repeated (monthly) 'pulses' of 7 day feeding injury by psyllids stimulated immunity in CaLas-infected citrus by increasing SA in leaves initially (up to 120 days), long-term (270 and 360 days) repeated herbivory caused SA to decrease coincident with upregulation of genes associated with SA metabolism (BMST and DMR6). Similarly, transcriptional responses and metabolite (SA and its analytes) accumulation in citrus leaves exposed to a continuously reproducing population of D. citri exhibited a transitory upregulation of genes associated with SA signaling at 120 days and a posterior downregulation after long-term psyllid (adults and nymphs) feeding (270 and 360 days). CONCLUSIONS: Herbivory played an important role in regulation of SA accumulation in mature leaves of C. sinensis, whether or not those trees were coincidentally infected with CaLas. Our results indicate that prevention of feeding injury inflicted by D. citri from the tritrophic interaction may allow citrus plants to better cope with the consequences of CaLas infection, highlighting the importance of vector suppression as a component of managing this cosmopolitan disease.

Potato Zebra Chip: An Overview of the Disease, Control Strategies, and Prospects.

Potato (Solanum tuberosum L.) is an important food crop worldwide. As the demand for fresh and processed potato products is increasing globally, there is a need to manage and control devastating diseases such as zebra chip (ZC). ZC disease causes major yield losses in many potato-growing regions and is associated with the fastidious, phloem-limited bacterium Candidatus Liberibacter solanacearum (CLso) that is vectored by the potato-tomato psyllid (Bactericera cockerelli Sulc). Current management measures for ZC disease mainly focus on chemical control and integrated pest management strategies of the psyllid vector to limit the spread of CLso, however, they add to the costs of potato production. Identification and deployment of CLso and/or the psyllid resistant cultivars, in combination with integrated pest management, may provide a sustainable long-term strategy to control ZC. In this review, we provide a brief overview of the ZC disease, epidemiology, current management strategies, and potential new approaches to manage ZC disease in the future.

Concanavalin A Toxicity Towards Potato Psyllid and Apoptosis Induction in Midgut Cells.

Concanavalin A (ConA), a legume lectin, has been drawing increasing attention in recent years concerning its toxicity against insects and its potential application in pest management. In an attempt to evaluate the effect of ConA on potato psyllid (Bactericera cockerelli), an economically important pest of solanaceous crops, the effect of ConA on potato psyllid survival, psyllid gut nuclear morphology, and expression of psyllid caspase genes were evaluated. Our results determined that artificial diet-feeding assays using ConA had deleterious effects on potato psyllids, resulting in significant psyllid mortality following ingestion. We also found that an apoptotic response was induced by ConA in psyllid midgut cells, which was demonstrated by the DNA fragmentation and abnormal nuclear architecture in the midgut cells. Following ConA ingestion, there was also upregulation of caspase genes in the psyllid midguts. Therefore, a key mechanism behind ConA toxicity towards potato psyllid probably involves the induction of apoptosis in midgut cells. This study could provide a better understanding of the mechanisms underlying ConA toxicity in insects and be a stepping stone towards the development of new psyllid control strategies based on plant lectins.

Quenching autofluorescence in the alimentary canal tissues of Bactericera cockerelli (Hemiptera: Triozidae) for immunofluorescence labeling.

Immunofluorescence has been widely used to localize microbes or specific molecules in insect tissues or cells. However, significant autofluorescence is frequently observed in tissues which can interfere with the fluorescent identification of target antigens, leading to inaccurate or even false positive fluorescent labeling. The alimentary canal of the potato psyllid, Bactericera cockerelli Sulc, exhibits intense autofluorescence, hindering the application of immunolocalization for the detection and localization of the economically important pathogen transmitted by this insect, "Candidatus Liberibacter solanacearum" (Lso). In the present study, we tested the use of irradiation, hydrogen peroxide (H2 O2 ) and Sudan black B (SBB) treatments to reduce the autofluorescence in the B. cockerelli alimentary canal tissues. Furthermore, we assessed the compatibility of the above-mentioned treatments with Lso immunolocalization and actin staining using phalloidin. Our results showed that the autofluorescence in the alimentary canal was reduced by irradiation, H2 O2 , or SBB treatments. The compatibility assays indicated that irradiation and H2 O2 treatment both greatly reduced the fluorescent signal associated with Lso and actin. However, the SBB incubation preserved those target signals, while efficiently eliminating autofluorescence in the psyllid alimentary canal. Therefore, herein we propose a robust method for reducing the autofluorescence in the B. cockerelli alimentary canal with SBB treatment, which may improve the use of immunofluorescence labeling in this organism. This method may also have a wide range of uses by reducing the autofluorescence in other arthropod species.

Effects of "Candidatus Liberibacter solanacearum" (haplotype B) on Bactericera cockerelli fitness and vitellogenesis.

"Candidatus Liberibacter solanacearum" (Lso) are phloem-restricted and unculturable Gram-negative bacteria. Presently five haplotypes have been identified worldwide; but only haplotypes A and B are associated with the vector Bactericera cockerelli (Sulc.) in the Americas. Previous studies showed that Lso-infection reduces B. cockerelli reproductive output and that Lso haplotype B is more pathogenic than Lso haplotype A. To understand the interaction of Lso haplotype B and B. cockerelli, the fitness of Lso-free and Lso B-infected insects, and the expression of vitellogenin (BcVg1-like), a gene involved directly in the insect reproduction were analyzed. Statistical differences in the number of eggs oviposited, and the total number of progeny nymphs and adults were found among crosses of insects with or without Lso. Significant differences in sex proportions were found between Lso B-infected and Lso-free crosses: a higher proportion of F1 adult females were obtained from Lso B-infected mothers. A significant reduction of BcVg1-like was observed in crosses performed with Lso B-infected females compared to the Lso-free insects. In female cohorts of different age, a significant reduction of BcVg1-like expression was measured in 7-d-old Lso B-infected females (virgin and mated) compared with 7-d-old Lso-free females (virgin and mated), respectively. The reduction of BcVg1-like transcript was associated with a lower number of developing oocytes observed in female's reproductive systems. Overall, this study represents the first step to understand the interaction of Lso B with B. cockerelli, highlighting the effect of Lso B infection on egg production, BcVg1-like expression, and oocyte development.

Temporal Dynamics of Candidatus Liberibacter asiaticus Titer in Mature Leaves from Citrus sinensis cv Valencia Are Associated with Vegetative Growth.

Huanglongbing, a highly destructive disease of citrus species, is associated with a fastidious, gram-negative, phloem-limited bacteria (Candidatus Liberibacter spp.). In Florida, the causative agent of Huanglongbing (HLB) is C. Liberibacter asiaticus (CLas) and it is transmitted by the insect vector, Asian citrus psyllid (Diaphorina citri Kuwayama). Previous investigations have revealed systemic infection of CLas with an erratic and uneven distribution of pathogen in tree phloem. However, previous investigations did not consider the potential impact of plant vegetative growth on presence/absence of CLas in planta. Our objectives were to determine: 1) the effect of vegetative growth of Citrus sinensis (L.) Osbeck cv Valencia on detection of CLas in mature leaves, and 2) the impact of CLas inoculation frequency on progression of CLas titer in citrus leaves through the first year of infection. Temporal dynamics of CLas detection were associated with vegetative flush growth. Surprisingly, there was no difference in CLas titer detected between plants exposed to infected vectors for a one-time 7 d inoculation access period, as compared with plants exposed to continuously breeding CLas-infected insects over the course of an entire year of plant infection. Our results suggest that the CLas bacterium is transported through phloem during annual movement of carbon compounds needed for vegetative plant growth, including transportation from roots to mature leaves. These results highlight the importance of vegetative growth on temporal dynamics of CLas in citrus, and suggest a critical role of the sink-source interaction on presence/absence of CLas in leaves.

Long-term, sustained feeding by Asian citrus psyllid disrupts salicylic acid homeostasis in sweet orange.

BACKGROUND: Phloem-feeding insects are known to modulate the salicylic acid (SA) signaling pathway in various plant-insect interaction models. Diaphorina citri is a phloem feeding vector of the deadly phytopathogens, Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, and the interactions of D. citri with its host that may modulate plant defenses are not well understood. The objectives of this study were to investigate the molecular mechanisms involved in transcriptional regulation of SA modification and activation of defense-associated responses in sweet orange (Citrus sinensis) exposed to various durations (7-, 14- and 150- days) of continuous feeding by D. citri. RESULTS: We quantified expression of genes involved in SA pathway activation and subsequent modification, as well as, associated SA metabolites (SA methyl ester, 2,3-DHBA, and SA 2-O-ß-D-glucoside). NPR1 and PR-1 expression was upregulated in plants exposed to continuous feeding by D. citri for 14 days. Expression of BSMT-like, MES1-like and DMR6-like oxygenase, as well as, accumulation of their respective SA metabolites (SA methyl ester, 2,3-DHBA) was significantly higher in plants exposed to continuous feeding by D. citri for 150 days than in those without D. citri infestation. Concomitantly, expression of UGT74F2-like was significantly downregulated and its metabolite, SA 2-ß-D-glucoside, was highly accumulated in trees exposed to 150 d of feeding compared to control trees without D. citri. CONCLUSIONS: D. citri herbivory differentially regulated transcription and SA-metabolite accumulation in citrus leaves, depending on duration of insect feeding. Our results suggest that prolonged and uninterrupted exposure (150 d) of citrus to D. citri feeding suppressed plant immunity and inhibited growth, which may highlight the importance of vector suppression as part of huanglongbing (HLB) management in citrus.

Reproductive performance among color morphs of Diaphorina citri Kuwayama, vector of citrus greening pathogens.

Diaphorina citri Kuwayama, commonly known as Asian citrus psyllid (ACP), transmits the bacterium Candidatus Liberibacter asiaticus (CLas), one of the causative agents of Huanglongbing (HLB) to citrus trees. Within ACP populations, three adult abdominal color polymorphisms: gray/brown, blue/green, and orange/yellow have been described. Despite the economic importance of this insect, the molecular mechanisms governing reproduction and vitellogenesis are not well understood. The current results describe the expression patterns of VgA1-like and Kr-h1, ovary morphology, and oviposition behavior in two different ACP morphotypes. Our results showed that VgA1-like exhibited female sex-specific expression, was upregulated more in blue/green than gray/brown females, and increased in expression with age only in blue/green morphs. The transcription factor Kr-h1, associated with reproduction in some insect species, was expressed in both sexes, was upregulated in 1 and 7 days old blue/green compared to gray/brown females, and exhibited reduced expression by 14 days of age in both morphotypes. Our results demonstrated an association between VgA1-like expression, oocyte development, and the blue green abdominal color of D. citri, which were linked to higher reproductive output than observed in gray/brown females. Overall, this study described the importance of the genes VgA1-like and Kr-h1 in D. citri vitellogenesis, and explained the mechanisms underlying differential reproductive performance among D. citri abdominal color morphs.

Infection by Candidatus Liberibacter solanacearum' haplotypes A and B in Solanum lycopersicum 'Moneymaker'.

'Candidatus Liberibacter solanacearum' is a plant pathogen associated with diseases affecting several crops of the Solanaceae and Apiaceae families. Two 'Ca. L. solanacearum' haplotypes (LsoA and LsoB) infect solanaceous crops in North America and are transmitted by the tomato psyllid Bactericera cockerelli. Although both 'Ca. L. solanacearum' haplotypes cause zebra chip in potato, the diseases associated with each haplotype in tomato (Solanum lycopersicum) have not been described. 'Ca. L. solanacearum'-infected tomato plants exhibit symptoms resembling those of permanent yellowing disease (known in Mexico as "permanente del tomate") and sometimes called psyllid yellows. In this study, the symptoms associated with each 'Ca. L. solanacearum' haplotype in tomato were compared, and the bacterial abundance in different nodes of the plants was measured by quantitative polymerase chain reaction. Surprisingly, both plant phenotype and bacterium distribution were different between LsoA- and LsoB-infected plants. Plants infected with LsoB died prematurely, whereas those infected with LsoA did not. Across the measured time points, LsoB abundance in infected plants was consistent with previous reports describing a sink to source gradient, while such gradient was only observed in LsoA-infected plants early after infection. This is the first report describing the differences in symptoms in tomato associated with two 'Ca. L. solanacearum' haplotypes, LsoA and LsoB.

The influence of bacteria on multitrophic interactions among plants, psyllids, and pathogen.

The recent emergence of several plant diseases caused by psyllid-borne bacterial pathogens worldwide (Candidatus Liberibacter spp.) has created renewed interest on the interaction between psyllids and bacteria. In spite of these efforts to understand psyllid association with bacteria, many aspects of their interactions remain poorly understood. As more organisms are studied, subtleties on the molecular interactions as well as on the effects of the bacteria on the psyllid host are being uncovered. Additionally, psyllid-borne bacterial phytopathogens can also affect the host plant, which in turn can impact psyllid physiology and behavior. Here, we review the current literature on different aspects of the influence of bacteria on multitrophic interactions among plants, psyllids, and pathogens. We then highlight gaps that need to be addressed to advance this field, which can have significant implications for controlling these newly emergent and other plant diseases.

Identification and expression analyses of vitellogenin in Bactericera cockerelli (Sulc).

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a phloem-feeding insect with preference for Solanaceae. This insect species is vector of the pathogenic bacteria 'Candidatus Liberibacter solanacearum' the causative agent of zebra chip, an important disease of commercial potatoes in several countries worldwide. The recent classification of psyllids among the most dangerous vectors has promoted their study, but still many biological processes such as reproduction and vitellogenesis need to be investigated. As a first step towards the elucidation of vitellogenesis in B. cockerelli, one candidate vitellogenin transcript (6622 bases long) was identified and the expression of the transcript and the protein were analyzed in virgin and mated females between 1 and 7days old. In virgin females, Vg expression increased up to 5days old; while mating significantly up-regulated Vg expression in 5- and 7-day-old females. To determine the role of juvenile hormone in B. cockerelli Vg expression, topical applications of juvenile hormone III were performed on virgin females, resulting in an up-regulation of Vg expression and an increase in the number of mature oocytes present in female reproductive organs. Overall, this study represents the first step to understand vitellogenesis of B. cockerelli and it highlights the role of JH III in the hormonal regulation of Vg expression and oocyte development.

Interactions "Candidatus Liberibacter solanacearum"-Bactericera cockerelli: Haplotype Effect on Vector Fitness and Gene Expression Analyses.

"Candidatus Liberibacter solanacearum" (Lso) has emerged as a serious threat world-wide. Five Lso haplotypes have been identified so far. Haplotypes A and B are present in the Americas and/or New Zealand, where they are vectored to solanaceous plants by the potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae). The fastidious nature of these pathogens has hindered the study of the interactions with their eukaryotic hosts (vector and plant). To understand the strategies used by these pathogens to infect their vector, the effects of each Lso haplotype (A or B) on psyllid fitness was investigated, and genome-wide transcriptomic and RT-qPCR analyses were performed to evaluate Lso gene expression in association with its vector. Results showed that psyllids infected with haplotype B had significantly lower percentage of nymphal survival compared to psyllids infected with haplotype A. Although overall gene expression across Lso genome was similar between the two Lso haplotypes, differences in the expression of key candidate genes were found. Among the 16 putative type IV effector genes tested, four of them were differentially expressed between Lso haplotypes, while no differences in gene expression were measured by qPCR or transcriptomic analysis for the rest of the genes. This study provides new information regarding the pathogenesis of Lso haplotypes in their insect vector.

Long-term effects of abscisic acid (ABA) on the grape berry phenylpropanoid pathway: Gene expression and metabolite content.

ABA has been proposed as the main signal triggering the onset of the ripening process in grapes, and modulating the secondary metabolism in grape berry skins. To determine the effect of ABA on secondary metabolism in berries, clusters of Carménère were sprayed with 0 µLL(-1) ABA; 50 µLL(-1) ABA and 100 µLL(-1) ABA during pre-véraison, and the gene expression of the transcription factors and enzymes of the phenylpropanoid pathway were assessed from véraison to 70 days after véraison (DAV). Additionally, flavonols, tannins and anthocyanins were assessed from véraison until harvest (110 DAV). ABA accelerated sugar and anthocyanin accumulation at véraison. The grape transcript abundance of VvDFR, VvANS, VvUFGT and VvMybA1, all peaking around véraison mimicked the concentration of ABA throughout the season. The highest anthocyanin concentration occurred 35 DAV for all treatments, but higher pigment concentrations were observed in ABA-treated berries at véraison and from 60 to 70 DAV to harvest. VvPAL was also increased by treatment at the higher concentration of ABA from véraison to 40 DAV. Regarding flavanol synthesis, VvLAR2 and VvMyb4A decreased from véraison until 40 DAV and then increased again until 70 DAV. Compared to the control, both ABA treatments resulted in a less-than-proportional reduction of the expression of both genes compared to the control and, after 40 DAV, in a more-than-proportional increase compared to the control, suggesting a long-term effect of the pre-véraison ABA spray on the berries. A concomitant increase in flavanols was observed in berries after 40 DAV, and this occurred at a higher extent in berries treated with the highest ABA concentration.

Identification and expression analysis of aquaporins in the potato psyllid, Bactericera cockerelli.

Aquaporin (AQPs) proteins transport water and uncharged low molecular-weight solutes across biological membranes. Six to 8 AQP genes have been identified in many insect species, but presently only three aquaporins have been characterized in phloem feeding insects. The objective of this study was to identify candidate AQPs in the potato psyllid, Bactericera cockerelli. Herein, we identified four candidate aquaporin cDNAs in B. cockerelli transcriptome. Phylogenetic analysis showed that candidate BcAQP2-like had high similarity to PRIP aquaporins; while candidates BcAQP4-like, BcAQP5-like and BcAQP9-like clustered within clade B. In particular, candidates BcAQP4-like and BcAQP5-like clustered with functionally validated insect aquaglyceroporin proteins. Expression analyses using RT-qPCR showed that all candidates were expressed in all life stages and tissues. Candidates BcAQP4-like and BcAQP5-like were highly expressed in bacteriocytes, while BcAQP9-like appeared to be expressed at high levels in whole body but not in the assayed tissues. This study is the first global attempt to identify putative aquaporins in a phloem feeding insect.

Transcriptome analysis of "Candidatus Liberibacter solanacearum" in its psyllid vector, Bactericera cockerelli.

"Candidatus Liberibacter solanacearum" (Lso) is an emergent pathogen of carrots in Europe and solanaceous plants in North and Central America and New Zealand. This bacterium is closely related to other pathogenic Candidatus Liberibacter spp., all vectored by psyllids. In order to understand the molecular interaction of this pathogen and its psyllid vector, Bactericera cockerelli, Illumina sequencing of psyllid harboring Lso was performed to determine if this approach could be used to assess the bacterial transcriptome in this association. Prior to sequencing, psyllid RNA was purified and insect and bacterial rRNA were removed. Mapping of reads to Lso genome revealed that over 92% of the bacterial genes were expressed in the vector, and that the COG categories Translation and Post-translational modification, protein turnover, chaperone functions were the most expressed functional categories. Expression levels of selected Lso genes were confirmed by RT-qPCR. The transcriptomic analysis also helped correct Lso genome annotation by identifying the expression of genes that were not predicted in the genome sequencing effort.

Impact of cadmium exposure during pregnancy on hepatic glucocorticoid receptor methylation and expression in rat fetus.

Adverse fetal environment due to maternal undernutrition or exposure to environmental chemicals alters glucocorticoid (GC) metabolism increasing the risk of metabolic disorders in adulthood. In this study, we investigated the effects of maternal exposure to cadmium (Cd, 50 ppm) during pregnancy in the methylation of fetal hepatic glucocorticoid receptor promoter (GR) and the correlation with its expression and that of the DNA methyltransferases (DNMT1a and 3a). We also studied the expression of liver phosphoenolpyruvate carboxykinase (PEPCK) and acyl-CoA oxidase (AOX), two enzymes involved in the metabolism of carbohydrates and lipids respectively. The methylation of the rat GR gene exon 1(10) (GR1(10)) in nucleotides -2536 to -2361 was analyzed by pyrosequencing. Quantitative real time PCR was used to assess hepatic GR, PEPCK and AOX mRNA, and their protein levels using Western blotting analysis. Differential methylation was noted across groups at all CpG sites in the GR exon 1(10) in a sex-dependent manner. In males, CpG were more methylated than the controls (185 ± 21%, p<0.001) but only CpG sites 1,6,7 and 9 showed a significantly different extent of methylation. In addition, a lower expression of GR (mRNA and protein) was found. On the contrary, in females, CpG were less methylated than the controls (62 ± 11%, p<0.05) and overexpressed, affecting PEPCK and AOX expression, which did not change in males. The GR methylation profile correlates with DNMT3a expression which may explain epigenetic sex-dependent changes on GR1(10) promoter induced by Cd treatment. In conclusion, Cd exposure during pregnancy affects fetal liver DNMT3a resulting in sex-dependent changes in methylation and expression of GR1(10). Although these effects do not seem to be directly involved in the low birth weight and height, they may have relevant implications for long-term health.

Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis.

BACKGROUND: Morphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis. RESULTS: Microarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction. CONCLUSION: Our data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we recovered a substantial number of unknown genes encoding putative secreted and transmembrane proteins, suggesting new components of signaling pathways that might be incorporated within the existing regulatory networks controlling D. melanogaster embryogenesis. These genes are also good candidates for additional targeted functional analyses similar to those we conducted for CG6234.See related minireview by Vichas and Zallen: http://www.jbiol.com/content/8/8/76.