Kameswara Rao Kottapalli

Physiology and proteomics of the water-deficit stress response in three contrasting peanut genotypes.

Peanut genotypes from the US mini-core collection were analysed for changes in leaf proteins during reproductive stage growth under water-deficit stress. One- and two-dimensional gel electrophoresis (1- and 2-DGE) was performed on soluble protein extracts of selected tolerant and susceptible genotypes. A total of 102 protein bands/spots were analysed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis. Forty-nine non-redundant proteins were identified, implicating a variety of stress response mechanisms in peanut. Lipoxygenase and 1l-myo-inositol-1-phosphate synthase, which aid in inter- and intracellular stress signalling, were more abundant in tolerant genotypes under water-deficit stress. Acetyl-CoA carboxylase, a key enzyme of lipid biosynthesis, increased in relative abundance along with a corresponding increase in epicuticular wax content in the tolerant genotype, suggesting an additional mechanism for water conservation and stress tolerance. Additionally, there was a marked decrease in the abundance of several photosynthetic proteins in the tolerant genotype, along with a concomitant decrease in net photosynthesis in response to water-deficit stress. Differential regulation of leaf proteins involved in a variety of cellular functions (e.g. cell wall strengthening, signal transduction, energy metabolism, cellular detoxification and gene regulation) indicates that these molecules could affect the molecular mechanism of water-deficit stress tolerance in peanut.

Gene expression profiling in peanut using high density oligonucleotide microarrays

BackgroundTranscriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology.ResultsWe have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes.ConclusionThe peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues.

Generation and analysis of expressed sequence tags (ESTs) for marker development in yam (Dioscorea alata L.)

BackgroundAnthracnose (Colletotrichumgloeosporioides) is a major limiting factor in the production of yam (Dioscorea spp.) worldwide. Availability of high quality sequence information is necessary for designing molecular markers associated with resistance. However, very limited sequence information pertaining to yam is available at public genome databases. Therefore, this collaborative project was developed for genetic improvement and germplasm characterization of yams using molecular markers. The current investigation is focused on studying gene expression, by large scale generation of ESTs, from one susceptible (TDa 95-0310) and two resistant yam genotypes (TDa 87-01091, TDa 95-0328) challenged with the fungus. Total RNA was isolated from young leaves of resistant and susceptible genotypes and cDNA libraries were sequenced using Roche 454 technology.ResultsA total of 44,757 EST sequences were generated from the cDNA libraries of the resistant and susceptible genotypes. Greater than 56% of ESTs were annotated using MapMan Mercator tool and Blast2GO search tools. Gene annotations were used to characterize the transcriptome in yam and also perform a differential gene expression analysis between the resistant and susceptible EST datasets. Mining for SSRs in the ESTs revealed 1702 unique sequences containing SSRs and 1705 SSR markers were designed using those sequences.ConclusionWe have developed a comprehensive annotated transcriptome data set in yam to enrich the EST information in public databases. cDNA libraries were constructed from anthracnose fungus challenged leaf tissues for transcriptome characterization, and differential gene expression analysis. Thus, it helped in identifying unique transcripts in each library for disease resistance. These EST resources provide the basis for future microarray development, marker validation, genetic linkage mapping and QTL analysis in Dioscorea species.

Effective strategy for pyramiding three bacterial blight resistance genes into fine grain rice cultivar, Samba Mahsuri, using sequence tagged site markers.

Bacterial leaf blight (BB) of rice is a major disease limiting rice production in several rice growing regions of the world. The pathogen, Xanthomonasoryzae pv oryzae, causing the disease is highly virulent to rice crops and is capable of evolving new races. Breeding efforts to incorporate single BB resistant gene often leads to resistance breakdown within a short period. To overcome such breakdown of resistance and develop germplasm with durable disease resistance, we have introgressed three bacterial blight resistance genes, xa5, xa13, and Xa21 into a fine grain rice variety, Samba Mahsuri, using sequence tagged site (STS) markers linked to these genes. Since the efficiency of the STS markers linked to recessive genes to detect homozygotes is less than 100%, we adopted four different pyramiding schemes to minimize loss of recessive resistance genes in advanced backcross generations. Pyramiding scheme A in which a two-gene Samba Mahsuri pyramid line containing Xa21 and xa5 genes was crossed with the Samba Mahsuri line having xa13 gene alone was found to be most effective in preventing the loss of an important recessive gene xa13. We further demonstrated that there was no yield penalty due to pyramiding of multiple genes into the elite indica rice variety.

Shotgun label-free quantitative proteomics of water-deficit-stressed midmature peanut (Arachis hypogaea L.) seed.

Legume seeds and peanuts, in particular, are an inexpensive source of plant proteins and edible oil. A comprehensive understanding of seed metabolism and the effects of water-deficit stress on the incorporation of the main storage reserves in seeds, such as proteins, fatty acids, starch, and secondary metabolites, will enhance our ability to improve seed quality and yield through molecular breeding programs. In the present study, we employed a label-free quantitative proteomics approach to study the functional proteins altered in the midmature (65-70 days postanthesis) peanut seed grown under water-deficit stress conditions. We created a pod-specific proteome database and identified 93 nonredundant, statistically significant, and differentially expressed proteins between well-watered and drought-stressed seeds. Mapping of these differential proteins revealed three candidate biological pathways (glycolysis, sucrose and starch metabolism, and fatty acid metabolism) that were significantly altered due to water-deficit stress. Differential accumulation of proteins from these pathways provides insight into the molecular mechanisms underlying the observed physiological changes, which include reductions in pod yield and biomass, reduced germination, reduced vigor, decreased seed membrane integrity, increase in storage proteins, and decreased total fatty acid content. Some of the proteins encoding rate limiting enzymes of biosynthetic pathways could be utilized by breeders to improve peanut seed production during water-deficit conditions in the field. The data have been deposited to the ProteomeXchange with identifier PXD000308.

Major Transcriptome Changes Accompany the Growth of Pseudomonas aeruginosa in Blood from Patients with Severe Thermal Injuries.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes serious infections in immunocompromised hosts including severely burned patients. After multiplying within the burn wound, P. aeruginosa translocate into the bloodstream causing bacterial sepsis frequently leading to organ dysfunction and septic shock. Although the pathogenesis of P. aeruginosa infection of thermally-injured wounds has been extensively analyzed, little is known regarding the ability of P. aeruginosa to adapt and survive within the blood of severely burned patients during systemic infection. To identify such adaptations, transcriptome analyses (RNA-seq) were conducted on P. aeruginosa strain PA14 that was grown in whole blood from a healthy volunteer or three severely burned patients. Compared with growth in blood from healthy volunteers, growth of PA14 in the blood from severely burned patients significantly altered the expression of 2596 genes, with expression of 1060 genes enhanced, while that of 1536 genes was reduced. Genes whose expression was significantly reduced included genes related to quorum sensing, quorum sensing-controlled virulence factors and transport of heme, phosphate, and phosphonate. Genes whose expression was significantly enhanced were related to the type III secretion system, the pyochelin iron-acquisition system, flagellum synthesis, and pyocyanin production. We confirmed changes in expression of many of these genes using qRT-PCR. Although severe burns altered the levels of different blood components in each patient, the growth of PA14 in their blood produced similar changes in the expression of each gene. These results suggest that, in response to changes in the blood of severely burned patients and as part of its survival strategy, P. aeruginosa enhances the expression of certain virulence genes and reduces the expression of others.

Sm-p80-based schistosomiasis vaccine mediated epistatic interactions identified potential immune signatures for vaccine efficacy in mice and baboons

Schistosomiasis is a neglected parasitic disease of major public health concern as it affects over 250 million people in developing countries. Currently there is no licensed vaccine available against schistosomiasis. The Schistosoma mansoni calpain protein, Sm-p80, is a leading vaccine candidate now ready to move to clinical trials. In order to better assess Sm-p80 vaccine immunogenicity; here we used a systems biology approach employing RNA-sequencing to identify gene signatures and epistatic interactions following Sm-p80 vaccination in mouse and baboon models that may predict vaccine efficacy. Recombinant Sm-p80 + CpG-oligodeoxynucleotide (ODN) vaccine formulation induced both cellular and humoral immunity genes with a predominant TH1 response as well as TH2 and TH17 gene signatures. Early gene responses and gene-network interactions in mice immunized with rSm-p80 + ODN appear to be initiated through TLR4 signaling. CSF genes, S100A alarmin genes and TNFRSF genes appear to be a signature of vaccine immunogenicity/efficacy as identified by their participation in gene network interactions in both mice and baboons. These gene families may provide a basis for predicting desirable outcomes for vaccines against schistosomiasis leading to a better understanding of the immune system response to vaccination.

Quantitative Proteomics of an Amphibian Pathogen, Batrachochytrium dendrobatidis, following Exposure to Thyroid Hormone.

Batrachochytrium dendrobatidis (Bd), a chytrid fungus, has increasingly been implicated as a major factor in the worldwide decline of amphibian populations. The fungus causes chytridiomycosis in susceptible species leading to massive die-offs of adult amphibians. Although Bd infects the keratinized mouthparts of tadpoles and negatively affects foraging behavior, these infections are non-lethal. An important morphogen controlling amphibian metamorphosis is thyroid hormone (T3). Tadpoles may be infected with Bd and the fungus may be exposed to T3 during metamorphosis. We hypothesize that exposure of Bd to T3 may induce the expression of factors associated with host colonization and pathogenicity. We utilized a proteomics approach to better understand the dynamics of the Bd-T3 interaction. Using liquid chromatography-mass spectrometry (LC-MS), we generated a data set of a large number of cytoplasmic and membrane proteins following exposure of Bd to T3. From these data, we identified a total of 263 proteins whose expression was significantly changed following T3 exposure. We provide evidence for expression of an array of proteins that may play key roles in both genomic and non-genomic actions of T3 in Bd. Additionally, our proteomics study shows an increase in several proteins including proteases and a class of uncommon crinkler and crinkler-like effector proteins suggesting their importance in Bd pathogenicity as well as those involved in metabolism and energy transfer, protein fate, transport and stress responses. This approach provides insights into the mechanistic basis of the Bd-amphibian interaction following T3 exposure.

Sm‐p80‐based vaccine trial in baboons: efficacy when mimicking natural conditions of chronic disease, praziquantel therapy, immunization, and Schistosoma mansoni re‐encounter

Sm‐p80‐based vaccine efficacy for Schistosoma mansoni was evaluated in a baboon model of infection and disease. The study was designed to replicate a human vaccine implementation scenario for endemic regions in which vaccine would be administered following drug treatment of infected individuals. In our study, the Sm‐p80‐based vaccine reduced principal pathology producing hepatic egg burdens by 38.0% and egg load in small and large intestines by 72.2% and 49.4%, respectively, in baboons. Notably, hatching rates of eggs recovered from liver and small and large intestine of vaccinated animals were significantly reduced, by 60.4%, 48.6%, and 82.3%, respectively. Observed reduction in egg maturation/hatching rates was supported by immunofluorescence and confocal microscopy showing unique differences in Sm‐p80 expression in worms of both sexes and matured eggs. Vaccinated baboons had a 64.5% reduction in urine schistosome circulating anodic antigen, a parameter that reflects worm numbers/health status in infected hosts. Preliminary analyses of RNA sequencing revealed unique genes and canonical pathways associated with establishment of chronic disease, praziquantel‐mediated parasite killing, and Sm‐p80‐mediated protection in vaccinated baboons. Overall, our study demonstrated efficacy of the Sm‐p80 vaccine and provides insight into some of the epistatic interactions associated with protection.

Molecular detection and quantification of viable probiotic strains in animal feedstuffs using the commercial direct fed microbial Lactobacillus animalis NP51 as a model

Lactobacillus animalis NP51 is a direct-fed microbial strain (DFM) extensively used as a pre-harvest food safety mitigation in feedlot cattle due to its antagonistic effects against human foodborne pathogens such as Salmonella and Escherichia coli O157:H7. NP51 not only promotes overall gut health but interferes with the ability of these pathogens to colonize the gastrointestinal tract of cattle. As a result, NP51 reduces fecal shedding of Salmonella and E. coli O157:H7 in cattle presented for harvest and the load of these pathogens that enter the human food chain. Cattle are administered a high dose (1 × 109 CFU/head/day) of NP51 to reduce fecal shedding of foodborne pathogens. Ensiled animal feedstuffs naturally contain a high load of lactic acid bacteria (LAB) and it is not possible to detect and quantify the level of a specific LAB strain (e.g., NP51) in this matrix using traditional microbiological culture. The purpose of this study was to develop a molecular method to detect and quantify viable populations of a specific LAB strain (e.g., NP51) in cattle feedstuffs. The NP51 whole genome sequence was aligned with closely related LAB clustering within the same well-supported clade in a LAB phylogeny derived from 30 conserved amino acid encoding sequence to identify orthologs. A sequence encoding recombinational DNA repair protein RecT was found to be unique to NP51 and used to design primers and a probe for molecular detection and quantification of NP51. The primers and probe were confirmed to be specific to NP51 in vitro. Total RNA was extracted from silage samples, including samples naturally inoculated in the field and control samples that were artificially spiked with a range of NP51 concentrations in the laboratory. Reverse-transcriptase quantitative real-time (RT-qRTi) PCR was used to quantify cDNA copies in samples and cycle threshold (Ct) values were compared to a standard curve to estimate NP51 concentrations. Our results indicate this novel molecular method is suitable to confirm the presence and estimate the concentration of a specific LAB strain in animal feedstuffs containing high background levels of LAB.