Aphid-Responsive Defense Networks in Hybrid Switchgrass.

Aphid herbivory elicits plant defense-related networks that are influenced by host genetics. Plants of the upland switchgrass (Panicum virgatum) cultivar Summer can be a suitable host for greenbug aphids (Schizaphis graminum; GB), and yellow sugarcane aphids (Sipha flava, YSA), whereas the lowland cultivar Kanlow exhibited multi-species resistance that curtails aphid reproduction. However, stabilized hybrids of Summer (♀) x Kanlow (♂) (SxK) with improved agronomics can be damaged by both aphids. Here, hormone and metabolite analyses, coupled with RNA-Seq analysis of plant transcriptomes, were utilized to delineate defense networks induced by aphid feeding in SxK switchgrass and pinpoint plant transcription factors (TFs), such as WRKYs that potentially regulate these responses. Abscisic acid (ABA) levels were significantly higher in GB infested plants at 5 and 10 days after infestation (DAI). ABA levels were highest at 15DAI in YSA infested plants. Jasmonic acid levels were significantly elevated under GB infestation, while salicylic acid levels were signifi40cantly elevated only at 15 DAI in YSA infested plants. Similarly, levels of several metabolites were altered in common or specifically to each aphid. YSA infestation induced a significant enrichment of flavonoids consistent with an upregulation of many genes associated with flavonoid biosynthesis at 15DAI. Gene co-expression modules that responded singly to either aphid or in common to both aphids were differentiated and linked to specific TFs. Together, these data provide important clues into the interplay of metabolism and transcriptional remodeling accompanying defense responses to aphid herbivory in hybrid switchgrass.

Diversity of navel microbiome in young adults.

Introduction. Human skin microbial communities represent a tremendous source of genetic diversity that evolves as a function of human age. Microbiota differs between regions of oily and moist skin, and appears to stabilize with age.Aim. We have a minimal understanding of the time frame required for the stabilization of skin microbiota, and the role played by gender. In the current study, we examined the microbiota present in the navel region of college-attending young adults in the age group of 18-25 years and investigated if diversity is associated with gender (male and female).Method. The study involved 16 female and six male subjects. Isolated DNA samples from navel swabs were processed using the Nextera XT library preparation kit and sequenced using the MiSeq platform. Data were analysed using QIIME and statistical analysis performed in R.Results. Microbiota of navel skin is dominated by Corynebacterium and Staphylococcus and includes opportunistic pathogens like Clostridium and Pseudomonas. Also present as the major component of the flora were the organisms normally associated with the gastrointestinal tract such as Acinetobacter, Campylobacter, Klebsiella and organisms from the Enterobacteriaceae and Moraxellaceae families. Comparison of alpha and beta diversity of the microbiota in the male and female navel regions suggests that the flora is not statistically different (P>0.05). However, pairwise comparison suggests that the abundance of 12 specific genera varied with gender, including higher abundance of Klebsiella and Enterobacter in females.Conclusion. Our findings indicate that the navel skin microbiota of young adults has a core microbiota of Corynebacterium and Staphylococcus. We also noted the presence of a significant number of opportunistic pathogens. A minor gender difference in the abundance of individual organisms was also observed.

Transcriptional analysis of defense mechanisms in upland tetraploid switchgrass to greenbugs.

BACKGROUND: Aphid infestation of switchgrass (Panicum virgatum) has the potential to reduce yields and biomass quality. Although switchgrass-greenbug (Schizaphis graminum; GB) interactions have been studied at the whole plant level, little information is available on plant defense responses at the molecular level. RESULTS: The global transcriptomic response of switchgrass cv Summer to GB was monitored by RNA-Seq in infested and control (uninfested) plants harvested at 5, 10, and 15 days after infestation (DAI). Differentially expressed genes (DEGs) in infested plants were analyzed relative to control uninfested plants at each time point. DEGs in GB-infested plants induced by 5-DAI included an upregulation of reactive burst oxidases and several cell wall receptors. Expression changes in genes linked to redox metabolism, cell wall structure, and hormone biosynthesis were also observed by 5-DAI. At 10-DAI, network analysis indicated a massive upregulation of defense-associated genes, including NAC, WRKY, and MYB classes of transcription factors and potential ancillary signaling molecules such as leucine aminopeptidases. Molecular evidence for loss of chloroplastic functions was also detected at this time point. Supporting these molecular changes, chlorophyll content was significantly decreased, and ROS levels were elevated in infested plants 10-DAI. Total peroxidase and laccase activities were elevated in infested plants at 10-DAI relative to control uninfested plants. The net result appeared to be a broad scale defensive response that led to an apparent reduction in C and N assimilation and a potential redirection of nutrients away from GB and towards the production of defensive compounds, such as pipecolic acid, chlorogenic acid, and trehalose by 10-DAI. By 15-DAI, evidence of recovery in primary metabolism was noted based on transcript abundances for genes associated with carbon, nitrogen, and nutrient assimilation. CONCLUSIONS: Extensive remodeling of the plant transcriptome and the production of ROS and several defensive metabolites in an upland switchgrass cultivar were observed in response to GB feeding. The early loss and apparent recovery in primary metabolism by 15-DAI would suggest that these transcriptional changes in later stages of GB infestation could underlie the recovery response categorized for this switchgrass cultivar. These results can be exploited to develop switchgrass lines with more durable resistance to GB and potentially other aphids.

Identification, characterization, and gene expression analysis of nucleotide binding site (NB)-type resistance gene homologues in switchgrass.

BACKGROUND: Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that can be used as a second generation bioenergy crop. However, foliar fungal pathogens, like switchgrass rust, have the potential to significantly reduce switchgrass biomass yield. Despite its importance as a prominent bioenergy crop, a genome-wide comprehensive analysis of NB-LRR disease resistance genes has yet to be performed in switchgrass. RESULTS: In this study, we used a homology-based computational approach to identify 1011 potential NB-LRR resistance gene homologs (RGHs) in the switchgrass genome (v 1.1). In addition, we identified 40 RGHs that potentially contain unique domains including major sperm protein domain, jacalin-like binding domain, calmodulin-like binding, and thioredoxin. RNA-sequencing analysis of leaf tissue from 'Alamo', a rust-resistant switchgrass cultivar, and 'Dacotah', a rust-susceptible switchgrass cultivar, identified 2634 high quality variants in the RGHs between the two cultivars. RNA-sequencing data from field-grown cultivar 'Summer' plants indicated that the expression of some of these RGHs was developmentally regulated. CONCLUSIONS: Our results provide useful insight into the molecular structure, distribution, and expression patterns of members of the NB-LRR gene family in switchgrass. These results also provide a foundation for future work aimed at elucidating the molecular mechanisms underlying disease resistance in this important bioenergy crop.

Identification of an orthologous clade of peroxidases that respond to feeding by greenbugs (Schizaphis graminum) in C4 grasses.

Knowledge of specific peroxidases that respond to aphid herbivory is limited in C4 grasses, but could provide targets for improving defence against these pests. A sorghum (Sorghum bicolor (L.) Moench) peroxidase (SbPrx-1; Sobic.002G416700) has been previously linked to biotic stress responses, and was the starting point for this study. Genomic analyses indicated that SbPrx-1 was part of a clade of five closely related peroxidase genes occurring within a ~30kb region on chromosome 2 of the sorghum genome. Comparison of this ~30-kb region to syntenic regions in switchgrass (Panicum virgatum L.) and foxtail millet (Setaria italica L.) identified similar related clusters of peroxidases. Infestation of a susceptible sorghum cultivar with greenbugs (Shizaphis graminum Rondani) induced three of the five peroxidases. Greenbug infestation of switchgrass and foxtail millet plants showed similar inductions of peroxidases. SbPrx-1 was also induced in response to aphid herbivory in a greenbug-resistant sorghum line, Cargill 607E. These data indicate that this genomic region of C4 grasses could be valuable as a marker to assess potential insect resistance in C4 grasses.

The WRKY transcription factor family and senescence in switchgrass.

BACKGROUND: Early aerial senescence in switchgrass (Panicum virgatum) can significantly limit biomass yields. WRKY transcription factors that can regulate senescence could be used to reprogram senescence and enhance biomass yields. METHODS: All potential WRKY genes present in the version 1.0 of the switchgrass genome were identified and curated using manual and bioinformatic methods. Expression profiles of WRKY genes in switchgrass flag leaf RNA-Seq datasets were analyzed using clustering and network analyses tools to identify both WRKY and WRKY-associated gene co-expression networks during leaf development and senescence onset. RESULTS: We identified 240 switchgrass WRKY genes including members of the RW5 and RW6 families of resistance proteins. Weighted gene co-expression network analysis of the flag leaf transcriptomes across development readily separated clusters of co-expressed genes into thirteen modules. A visualization highlighted separation of modules associated with the early and senescence-onset phases of flag leaf growth. The senescence-associated module contained 3000 genes including 23 WRKYs. Putative promoter regions of senescence-associated WRKY genes contained several cis-element-like sequences suggestive of responsiveness to both senescence and stress signaling pathways. A phylogenetic comparison of senescence-associated WRKY genes from switchgrass flag leaf with senescence-associated WRKY genes from other plants revealed notable hotspots in Group I, IIb, and IIe of the phylogenetic tree. CONCLUSIONS: We have identified and named 240 WRKY genes in the switchgrass genome. Twenty three of these genes show elevated mRNA levels during the onset of flag leaf senescence. Eleven of the WRKY genes were found in hotspots of related senescence-associated genes from multiple species and thus represent promising targets for future switchgrass genetic improvement. Overall, individual WRKY gene expression profiles could be readily linked to developmental stages of flag leaves.

Morphology and Proteome Characterization of the Salivary Glands of the Western Chinch Bug (Hemiptera: Blissidae).

The western chinch bug, Blissus occiduus Barber, is a serious pest of buffalograss, Buchloe dactyloides (Nuttall) due to physical and chemical damage caused during the feeding process. Although previous work has investigated the feeding behaviors of chinch bugs in the Blissus complex, no study to date has explored salivary gland morphology and the associated salivary complex of this insect. Whole and sectioned B. occiduus salivary glands were visualized using light and scanning electron microscopy to determine overall structure and cell types of the salivary glands and their individual lobes. Microscopy revealed a pair of trilobed principal glands and a pair of tubular accessory glands of differing cellular types. To link structure with function, the salivary gland proteome was characterized using liquid chromatography tandem mass spectrometry. The salivary proteome analysis resulted in B. occiduus sequences matching 228 nonhomologous protein sequences of the pea aphid, Acyrthosiphon pisum (Harris), with many specific to the proteins present in the salivary proteome of A. pisum. A number of sequences were assigned the molecular function of hydrolase and oxido-reductase activity, with one specific protein sequence revealing a peroxidase-like function. This is the first study to characterize the salivary proteome of B. occiduus and the first of any species in the family Blissidae.

Switchgrass (Panicum virgatum L) flag leaf transcriptomes reveal molecular signatures of leaf development, senescence, and mineral dynamics.

Switchgrass flag leaves can be expected to be a source of carbon to the plant, and its senescence is likely to impact the remobilization of nutrients from the shoots to the rhizomes. However, many genes have not been assigned a function in specific stages of leaf development. Here, we characterized gene expression in flag leaves over their development. By merging changes in leaf chlorophyll and the expression of genes for chlorophyll biosynthesis and degradation, a four-phase molecular roadmap for switchgrass flag leaf ontogeny was developed. Genes associated with early leaf development were up-regulated in phase 1. Phase 2 leaves had increased expression of genes for chlorophyll biosynthesis and those needed for full leaf function. Phase 3 coincided with the most active phase for leaf C and N assimilation. Phase 4 was associated with the onset of senescence, as observed by declining leaf chlorophyll content, a significant up-regulation in transcripts coding for enzymes involved with chlorophyll degradation, and in a large number of senescence-associated genes. Of considerable interest were switchgrass NAC transcription factors with significantly higher expression in senescing flag leaves. Two of these transcription factors were closely related to a wheat NAC gene that impacts mineral remobilization. The third switchgrass NAC factor was orthologous to an Arabidopsis gene with a known role in leaf senescence. Other genes coding for nitrogen and mineral utilization, including ureide, ammonium, nitrate, and molybdenum transporters, shared expression profiles that were significantly co-regulated with the expression profiles of the three NAC transcription factors. These data provide a good starting point to link shoot senescence to the onset of dormancy in field-grown switchgrass.

Transcriptional response of soybean to thiamethoxam seed treatment in the presence and absence of drought stress.

BACKGROUND: Neonicotinoid insecticides are widely known for their broad-spectrum control of arthropod pests. Recently, their effects on plant physiological mechanisms have been characterized as producing a stress shield, which is predicted to enhance tolerance to adverse conditions. Here we investigate the molecular underpinnings of the stress shield concept using the neonicotinoid thiamethoxam in two separate experiments that compare gene expression. We hypothesized that the application of a thiamethoxam seed treatment to soybean would alter the expression of genes involved in plant defensive pathways and general stress response in later vegetative growth. First, we used next-generation sequencing to examine the broad scale transcriptional effects of the thiamethoxam seed treatment at three vegetative stages in soybean. Second, we selected ten target genes associated with plant defense pathways in soybean and examined the interactive effects of thiamethoxam seed treatment and drought stress on expression using qRT-PCR. RESULTS: Direct comparison of thiamethoxam-treated and untreated soybeans revealed minor transcriptional differences. However, when examined across vegetative stages, the thiamethoxam seed treatment induced substantial transcriptional changes that were not observed in untreated plants. Genes associated with photosynthesis, carbohydrate and lipid metabolism, development of the cell wall and membrane organization were uniquely upregulated between vegetative stages in thiamethoxam-treated plants. In addition, several genes associated with phytohormone and oxidative stress responses were downregulated between vegetative stages. When we examined the expression of a subset of ten genes associated with plant defense and stress response, the application of thiamethoxam was found to interact with drought stress by enhancing or repressing expression. In drought stressed plants, thiamethoxam induced (upregulated) expression of a thiamine biosynthetic enzyme (THIZ2) and gibberellin regulated protein (GRP), but repressed (downregulated) the expression of an apetala 2 (GmDREB2A;2), lipoxygenase (LIP), and SAM dependent carboxyl methyltransferase (SAM). CONCLUSIONS: We found evidence that a thiamethoxam seed treatment alters the expression soybean genes related to plant defense and stress response both in the presence and absence of drought stress. Consistent with the thiamethoxam stress shield concept, several genes associated with phytohormones showed enhanced expression in drought stressed plants.