RNA-seq analysis reveals an early defense response to tomato leaf curl New Delhi virus in potato cultivar Kufri Bahar.

Potatoes in India are very susceptible to apical leaf curl disease, which causes severe symptoms and greater yield losses. Because the majority of potato cultivars are susceptible to the virus, it is crucial to discover sources of resistance and investigate the mechanism of resistance/susceptibility in potato cultivars. In this study, the gene expression profile of two potato cultivars, Kufri Bahar (resistant) and Kufri Pukhraj (susceptible), varying in their level of resistance to ToLCNDV, was analyzed using RNA-Seq. The Ion ProtonTM system was used to sequence eight RiboMinus RNA libraries from inoculated and uninoculated potato plants at 15 and 20 days after inoculation (DAI). The findings indicated that the majority of differentially expressed genes (DEGs) were cultivar-or time-specific. These DEGs included genes for proteins that interact with viruses, genes linked with the cell cycle, genes for proteins involved in defense, transcription and translation initiation factors, and plant hormone signaling pathway genes. Interestingly, defense responses were generated early in Kufri Bahar, at 15 DAI, which may have impeded the replication and spread of ToLCNDV. This research provides a genome-wide transcriptional analysis of two potato cultivars with variable levels of ToLCNDV resistance. At an early stage, we observed suppression of genes that interact with viral proteins, induction of genes associated with restriction of cell division, genes encoding defense proteins, AP2/ERF transcription factors, and altered expression of zinc finger protein genes, HSPs, JA, and SA pathway-related genes. Our findings add to a greater comprehension of the molecular basis of potato resistance to ToLCNDV and may aid in the development of more effective disease management techniques.

Genome sequence analysis provides insights on genomic variation and late blight resistance genes in potato somatic hybrid (parents and progeny).

Wild Solanum species are the important resources for potato improvement. With the availability of potato genome and sequencing progress, knowledge about genomic resources is essential for novel genes discovery. Hence, the aim of this study was to decipher draft genome sequences of unique potato genotypes i.e. somatic hybrid P8 (J1), wild species S. pinnatisectum (J2), progeny MSH/14-112 (P8 × cv. Kufri Jyoti) (J3), and S. tuberosum dihaploid C-13 (J4). Draft genome sequencing using Illumina platform and reference-based assemblies with the potato genome yielded genome assembly size of 725.01 Mb (J1), 724.95 Mb (J2), 725.01 Mb (J3), and 809.59 Mb (J4). Further, 39,260 (J1), 25,711 (J2), 39,730 (J3) and 30,241 (J4) genes were identified and 17,411 genes were found common in the genotypes particularly late blight resistance genes (R3a, RGA2, RGA3, R1B-16, Rpi-blb2, Rpi and Rpi-vnt1). Gene ontology (GO) analysis showed that molecular function was predominant and signal transduction was major KEGG pathways. Further, gene enrichment analysis revealed dominance of metabolic process (GO: 0008152) in all the samples. Phylogeny analysis showed relatedness with potato and other plant species. Heterozygous single nucleotide polymorphism (SNP) was more than homozygous, and SNP in genic region was more than inter-genic region. Copy number variation (CNV) analysis indicated greater number of deletions than duplications. Sequence diversity and conserved motifs analysis revealed variation for late blight resistance genes. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed differential expression of late blight resistance genes. Our study provides insights on genome sequence, structural variation and late blight resistance genes in potato somatic hybrid (parents and progeny) for future research.

First Report of Collar and Stem Rot Caused by Rhizoctonia solani AG1-IA on Sesbania sesban in India.

Sesbania sesban (L.) Merr., (family Fabaceae) commonly called as "dhaincha" in India, is a multi-purpose crop used as a cover crop, as green manure, in the paper industry as well as animal fodder. The leaves of Sesbania contain high amounts of pinitol, which acts as an anti-diabetic agent (Misra and Siddiqi 2004). During July/August from 2017 to 2019, Sesbania plants exhibiting typical Rhizoctonia-like symptoms, including collar rot, wilting, and necrotic lesions on stems were regularly observed at ICAR-Central Potato Research Institute Regional Station, Meerut, Uttar Pradesh. The disease incidence ranged between 5 and 10% in a Sesbania crop being grown on 25 ha in Sesbania-potato rotation. Ten diseased plants were collected from different fields and brought to the laboratory for diagnosis. Affected stem pieces approximately 5 mm in size were surface sterilized with 2% sodium hypochlorite, washed twice in sterilized water and air dried. Four diseased pieces per plate were inoculated on 2% water agar amended with 2% streptomycin sulfate and incubated at 28±1℃ in the dark. All four affected pieces began to produce Rhizoctonia-like colonies after 48 h of incubation and in total eight isolates were purified and stored at 4℃ for further use. The colonies of eight isolates were evaluated and all were whitish during early growth and became light brown after 72 h. Dark-brown sclerotia appeared in the random pattern on PDA after 120 h. Microscopic observations showed that all isolates had hyphal branching at right angles with slight constriction at the base of the branch, presence of dolipore septum near the branching and multinucleate individual hyphae compartments (Sneh 1991). Based on these morphological characteristics, the fungus was identified as Rhizoctonia solani. All isolates were further characterized to determine anastomosis group (AG) by pairing with a known AG tester of R. solani AG-1-IA (ITCC 7650), AG-1-IB (ITCC 5650), and AG-3 (RS-20) procured from Indian Type Culture Collection, Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi and ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh, India, respectively. All eight isolates showed positive anastomosis with a known AG-1-IA tester isolate while no anastomosis was observed with other known tester isolates (Carling 1996). Furthermore, a single ~265 bp amplicon was amplified with AG-specific primer, which was specific to R. solani AG1-IA group; confirms the AG-specific identity of the isolates (Matsumoto 2002). Amplification was not observed with AG1-IB, AG3 and AG2 specific primers (Khodayari et al. 2009). The selected four isolates were molecularly characterized by amplifying the internal transcribed spacer (ITS) and ribosomal DNA (rDNA) 5.8s regions by polymerase chain reaction using ITS1 and ITS4 primer pairs (White et al. 1990). The nucleotide BLAST (BLASTn) analysis of the resulting four sequences i.e. GenBank acc. no. MT105386, MT105387, MT105388, and MT105389 supported the identification of the isolates as AG-1-IA sub-group and showed 95.12%, 98.93%, 96.79%, and 98.04%, respectively, sequence homology with known cultures of R. solani AG1-IA isolated from rice in China (KC285893), and India (MK481078). To confirm pathogenicity, Sesbania plants were grown in pots and maintained in the greenhouse at 25℃ with a 12-h-light/dark photoperiod. After 35 to 40 days of growth, the stems of ten Sesbania plant were artificially inoculated with PDA plugs containing R. solani mycelia (Jia et al. 2007) and covered with aluminium foil. Plants inoculated with noncolonized agar plugs served as control. After 96 h of incubation, all the plants inoculated presented the typical collar and stem rot symptoms. No symptoms were observed in the control plants. R. solani was re-isolated cent percent from these ten infected plants fulfilling Koch's postulates. R. solani AG1-IA has been reported to cause sheath blight and banded leaf and sheath blight diseases of rice and maize, respectively (Ogoshi 1987). To our knowledge, this is the first report of Sesbania sesban infected by R. solani AG1-IA, and serve as a host for the pathogen. The result from our findings will be helpful for planning of crop rotations in an agro-ecosystem.

MiRNA160 is associated with local defense and systemic acquired resistance against Phytophthora infestans infection in potato.

To combat pathogen infection, plants employ local defenses in infected sites and elicit systemic acquired resistance (SAR) in distant tissues. MicroRNAs have been shown to play a significant role in local defense, but their association with SAR is unknown. In addition, no such studies of the interaction between potato and Phytophthora infestans have been reported. We investigated the role of miR160 in local and SAR responses to P. infestans infection in potato. Expression analysis revealed induced levels of miR160 in both local and systemic leaves of infected wild-type plants. miR160 overexpression and knockdown plants exhibited increased susceptibility to infection, suggesting that miR160 levels equivalent to those of wild-type plants may be necessary for mounting local defense responses. Additionally, miR160 knockdown lines failed to elicit SAR, and grafting assays indicated that miR160 is required in both local and systemic leaves to trigger SAR. Consistently, SAR-associated signals and genes were dysregulated in miR160 knockdown lines. Furthermore, analysis of the expression of defense and auxin pathway genes and direct regulation of StGH3.6, a mediator of salicylic acid-auxin cross-talk, by the miR160 target StARF10 revealed the involvement of miR160 in antagonistic cross-talk between salicylic acid-mediated defense and auxin-mediated growth pathways. Overall, our study demonstrates that miR160 plays a crucial role in local defense and SAR responses during the interaction between potato and P. infestans.

Genotypic background of the recipient plant is crucial for conferring RB gene mediated late blight resistance in potato.

BACKGROUND: Late blight, caused by oomycetes pathogen Phytophthora infestans (Mont.) de Bary, is the most devastating potato disease in the world. RB gene from Solanum bulbocastanum has been shown to impart broad spectrum resistance against P. infestans races. In this study Katahdin transgenic event SP951 was used as male parent to cross with the popular Indian potato cultivars viz., Kufri Bahar (KB) and Kufri Jyoti (KJ) to enhance the late blight resistance. RESULTS: Populations of 271 F1seedlings from the crosses KB × SP951 (87) and KJ × SP951 (184) were screened for inheritance of RB transgene through PCR and bioassay. Disease response based on AUDPC of different hybrid lines varied from immunity to complete susceptibility. High degree of resistance (<25% infection) was observed in KJ × SP951 derived seedlings (85.2%), whereas level of resistance in KB × SP951 (36.4% infection) derived seedlings was of low order. CONCLUSION: This study provides valuable genetic materials for development of potentially durable late blight resistant potato varieties. Besides, it also corroborates the fact that efficacy of R gene is not solely dependent on its presence in the variety but largely depends on the genetic background of the recipient genotype.

Association of proinflammatory cytokines and islet resident leucocytes with islet dysfunction in type 2 diabetes.

AIMS/HYPOTHESIS: Chronic inflammation in type 2 diabetes is proposed to affect islets as well as insulin target organs. However, the nature of islet inflammation and its effects on islet function in type 2 diabetes remain unclear. Moreover, the immune cell profiles of human islets in healthy and type 2 diabetic conditions are undefined. We aimed to investigate the correlation between proinflammatory cytokine expression, islet leucocyte composition and insulin secretion in type 2 diabetic human islets. METHODS: Human islets from organ donors with or without type 2 diabetes were studied. First and second phases of glucose-stimulated insulin secretion were determined by perifusion. The expression of inflammatory markers was obtained by quantitative PCR. Immune cells within human islets were analysed by FACS. RESULTS: Type 2 diabetic islets, especially those without first-phase insulin secretion, displayed higher CCL2 and TNFa expression than healthy islets. CD45(+) leucocytes were elevated in type 2 diabetic islets, to a greater extent in moderately functional type 2 diabetic islets compared with poorly functional ones, and corresponded with elevated ALOX12 but not with CCL2 or TNFa expression. T and B lymphocytes and CD11c(+) cells were detectable within both non-diabetic and type 2 diabetic islet leucocytes. Importantly, the proportion of B cells was significantly elevated within type 2 diabetic islets. CONCLUSIONS/INTERPRETATION: Elevated total islet leucocyte content and proinflammatory mediators correlated with islet dysfunction, suggesting that heterogeneous insulitis occurs during the development of islet dysfunction in type 2 diabetes. In addition, the altered B cell content highlights a potential role for the adaptive immune response in islet dysfunction.

12/15-Lipoxygenase signaling in the endoplasmic reticulum stress response.

Central obesity is associated with chronic inflammation, insulin resistance, ß-cell dysfunction, and endoplasmic reticulum (ER) stress. The 12/15-lipoxygenase enzyme (12/15-LO) promotes inflammation and insulin resistance in adipose and peripheral tissues. Given that obesity is associated with ER stress and 12/15-LO is expressed in adipose tissue, we determined whether 12/15-LO could mediate ER stress signals. Addition of 12/15-LO lipid products 12(S)-HETE and 12(S)-HPETE to differentiated 3T3-L1 adipocytes induced expression and activation of ER stress markers, including BiP, XBP-1, p-PERK, and p-IRE1α. The ER stress inducer, tunicamycin, upregulated ER stress markers in adipocytes with concomitant 12/15-LO activation. Addition of a 12/15-LO inhibitor, CDC, to tunicamycin-treated adipocytes attenuated the ER stress response. Furthermore, 12/15-LO-deficient adipocytes exhibited significantly decreased tunicamycin-induced ER stress. 12/15-LO action involves upregulation of interleukin-12 (IL-12) expression. Tunicamycin significantly upregulated IL-12p40 expression in adipocytes, and IL-12 addition increased ER stress gene expression; conversely, LSF, an IL-12 signaling inhibitor, and an IL-12p40-neutralizing antibody attenuated tunicamycin-induced ER stress. Isolated adipocytes and liver from 12/15-LO-deficient mice fed a high-fat diet revealed a decrease in spliced XBP-1 expression compared with wild-type C57BL/6 mice on a high-fat diet. Furthermore, pancreatic islets from 12/15-LO-deficient mice showed reduced high-fat diet-induced ER stress genes compared with wild-type mice. These data suggest that 12/15-LO activity participates in ER stress in adipocytes, pancreatic islets, and liver. Therefore, reduction of 12/15-LO activity or expression could provide a new therapeutic target to reduce ER stress and downstream inflammation linked to obesity.

Evidence for activation of inflammatory lipoxygenase pathways in visceral adipose tissue of obese Zucker rats.

Central obesity is associated with low-grade inflammation that promotes type 2 diabetes and cardiovascular disease in obese individuals. The 12- and 5-lipoxygenase (12-LO and 5-LO) enzymes have been linked to inflammatory changes, leading to the development of atherosclerosis. 12-LO has also been linked recently to inflammation and insulin resistance in adipocytes. We analyzed the expression of LO and proinflammatory cytokines in adipose tissue and adipocytes in obese Zucker rats, a widely studied genetic model of obesity, insulin resistance, and the metabolic syndrome. mRNA expression of 12-LO, 5-LO, and 5-LO-activating protein (FLAP) was upregulated in adipocytes and adipose tissue from obese Zucker rats compared with those from lean rats. Concomitant with increased LO gene expression, the 12-LO product 12-HETE and the 5-LO products 5-HETE and leukotriene B4 (LTB4) were also increased in adipocytes. Furthermore, upregulation of key proinflammatory markers interleukin (IL)-6, TNFα, and monocyte chemoattractant protein-1 were observed in adipocytes isolated from obese Zucker rats. Immunohistochemistry indicated that the positive 12-LO staining in adipose tissue represents cells in addition to adipocytes. This was confirmed by Western blotting in stromal vascular fractions. These changes were in part reversed by the novel anti-inflammatory drug lisofylline (LSF). LSF also reduced p-STAT4 in visceral adipose tissue from obese Zucker rats and improved the metabolic profile, reducing fasting plasma glucose and increasing insulin sensitivity in obese Zucker rats. In 3T3-L1 adipocytes, LSF abrogated the inflammatory response induced by LO products. Thus, therapeutic agents reducing LO or STAT4 activation may provide novel tools to reduce obesity-induced inflammation.

Molecular characterization and infectivity analysis of tomato leaf curl New Delhi virus isolates infecting potato.

Nucleotide sequence of complete genome of a new isolate (KAN-6) of tomato leaf curl New Delhi virus (ToLCNDV) from Kanpur, Uttar Pradesh, India was determined. Sequence analysis indicated that it shared maximum identity to ToLCNDV isolates from pumpkin and ashgourd. Infectious clones of isolate KAN-6 along with two other ToLCNDV isolates (MOD-21 & FAI-19) obtained from potato fields of Modipuram and Faizabad, India were produced and used in symptom expression studies in N. benthamiana and potato plants through agro-inoculation. These isolates produced different symptoms both in N. benthamiana and potato. Severe symptoms of yellow mottling, downward curling and stunted growth were observed in N. benthamiana plants inoculated with KAN-6. MOD-21-inoculated plants also showed downward curling, stunted growth, but yellow mottling was observed only in older leaves whereas FAI-19-inoculated plants produced only downward curling symptoms. In case of potato, typical symptoms of apical leaf curl disease were observed in cultivar Kufri Pukhraj inoculated with MOD-21 and KAN-6 that are similar to those produced by virus-infected plants in the field. However, MOD-21 produced more prominent yellow mosaic symptoms as compared to KAN-6. FAI-19 produced only restricted yellow spots in Kufri Pukhraj. Only mild symptoms appeared in KAN-6 and no symptoms were observed in MOD-21- and FAI-19-inoculated Kufri Bahar plants which is known to show lowest seed degeneration under field conditions. Analysis of genomic components indicated that these isolates had 94.8-94.9% and 87.9-97.3% identity among them in DNA A and DNA B, respectively. The results of the study indicate the association of ToLCNDV isolates of different symptomatology with apical leaf curl disease of potato. This is also a first experimental demonstration of Koch's postulate for a begomovirus associated with apical leaf curl disease of potato.Author names: Please confirm if the author names (Swarup Kumar Chakrabarti) are presented accurately and in the correct sequence (given name, middle name/initial, family name).Yes. It is correct. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02752-5.

Differential expression and localization of 12/15 lipoxygenases in adipose tissue in human obese subjects.

Adipose tissue inflammation in obesity is a major factor leading to cardiovascular disease and type 2 diabetes.12/15 lipoxygenases (ALOX) play an important role in the generation of inflammatory mediators, insulin resistance and downstream immune activation in animal models of obesity. However, the expression and roles of 12/15ALOX isoforms, and their cellular sources in human subcutaneous (sc) and omental (om) fat in obesity is unknown. The objective of this study was to examine the gene expression and localization of ALOX isoforms and relevant downstream cytokines in subcutaneous (sc) and omental (om) adipose tissue in obese humans. Paired biopsies of sc and om fat were obtained during bariatric surgeries from 24 morbidly obese patients. Gene and protein expression for ALOX15a, ALOX15b and ALOX 12 were measured by real-time PCR and western blotting in adipocytes and stromal vascular fractions (SVF) from om and sc adipose tissue along with the mRNA expression of the downstream cytokines IL-12a, IL-12b, IL-6, IFNγ and the chemokine CXCL10. In a paired analysis, all ALOX isoforms, IL-6, IL-12a and CXCL10 were significantly higher in om vs. sc fat. ALOX15a mRNA and protein expression was found exclusively in om fat. All of the ALOX isoforms were expressed solely in the SVF. Further fractionation of the SVF in CD34+ and CD34- cells indicated that ALOX15a is predominantly expressed in the CD34+ fraction including vascular and progenitor cells, while ALOX15B is mostly expressed in the CD34- cells containing various leucocytes and myeloid cells. This result was confirmed by immunohistochemistry showing exclusive localization of ALOX15a in the om fat and predominantly in the vasculature and non-adipocyte cells. Our finding is identifying selective expression of ALOX15a in human om but not sc fat. This is a study showing a major inflammatory gene exclusively expressed in visceral fat in humans.

Genome-wide identification and characterization of microRNAs by small RNA sequencing for low nitrogen stress in potato.

Nitrogen is an important nutrient for plant growth and tuber quality of potato. Since potato crop requires high dose of N, improving nitrogen use efficiency (NUE) of plant is an inevitable approach to minimize N fertilization. The aim of this study was to identify and characterize microRNAs (miRNAs) by small RNA sequencing in potato plants grown in aeroponic under two contrasting N (high and low) regimes. A total of 119 conserved miRNAs belonging to 41 miRNAs families, and 1002 putative novel miRNAs were identified. From total, 52 and 54 conserved miRNAs, and 404 and 628 putative novel miRNAs were differentially expressed in roots and shoots, respectively under low N stress. Of total 34,135 predicted targets, the gene ontology (GO) analysis indicated that maximum targets belong to biological process followed by molecular function and cellular component. Eexpression levels of the selected miRNAs and targets were validated by real time-quantitative polymerase chain reaction (RT-qPCR) analysis. Two predicted targets of potential miRNAs (miR397 and miR398) were validated by 5' RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). In general, predicted targets are associated with stress-related, kinase, transporters and transcription factors such as universal stress protein, heat shock protein, salt-tolerance protein, calmodulin binding protein, serine-threonine protein kinsae, Cdk10/11- cyclin dependent kinase, amino acid transporter, nitrate transporter, sugar transporter, transcription factor, F-box family protein, and zinc finger protein etc. Our study highlights that miR397 and miR398 play crucial role in potato during low N stress management. Moreover, study provides insights to modulate miRNAs and their predicted targets to develop N-use efficient potato using transgenic/genome-editing tools in future.

Transcriptome analysis of potato shoots, roots and stolons under nitrogen stress.

Potato crop requires high dose of nitrogen (N) to produce high tuber yield. Excessive application of N causes environmental pollution and increases cost of production. Hence, knowledge about genes and regulatory elements is essential to strengthen research on N metabolism in this crop. In this study, we analysed transcriptomes (RNA-seq) in potato tissues (shoot, root and stolon) collected from plants grown in aeroponic culture under controlled conditions with varied N supplies i.e. low N (0.2 milli molar N) and high N (4 milli molar N). High quality data ranging between 3.25 to 4.93 Gb per sample were generated using Illumina NextSeq500 that resulted in 83.60-86.50% mapping of the reads to the reference potato genome. Differentially expressed genes (DEGs) were observed in the tissues based on statistically significance (p ≤ 0.05) and up-regulation with ≥ 2 log2 fold change (FC) and down-regulation with ≤ -2 log2 FC values. In shoots, of total 19730 DEGs, 761 up-regulated and 280 down-regulated significant DEGs were identified. Of total 20736 DEGs in roots, 572 (up-regulated) and 292 (down-regulated) were significant DEGs. In stolons, of total 21494 DEG, 688 and 230 DEGs were significantly up-regulated and down-regulated, respectively. Venn diagram analysis showed tissue specific and common genes. The DEGs were functionally assigned with the GO terms, in which molecular function domain was predominant in all the tissues. Further, DEGs were classified into 24 KEGG pathways, in which 5385, 5572 and 5594 DEGs were annotated in shoots, roots and stolons, respectively. The RT-qPCR analysis validated gene expression of RNA-seq data for selected genes. We identified a few potential DEGs responsive to N deficiency in potato such as glutaredoxin, Myb-like DNA-binding protein, WRKY transcription factor 16 and FLOWERING LOCUS T in shoots; high-affinity nitrate transporter, protein phosphatase-2c, glutaredoxin family protein, malate synthase, CLE7, 2-oxoglutarate-dependent dioxygenase and transcription factor in roots; and glucose-6-phosphate/phosphate translocator 2, BTB/POZ domain-containing protein, F-box family protein and aquaporin TIP1;3 in stolons, and many genes of unknown function. Our study highlights that these potential genes play very crucial roles in N stress tolerance, which could be useful in augmenting research on N metabolism in potato.

Physiological and genome-wide RNA-sequencing analyses identify candidate genes in a nitrogen-use efficient potato cv. Kufri Gaurav.

Nitrogen (N) is an important nutrient for plant growth. However, its excess application leads to environmental damage. Hence, improving nitrogen use efficiency (NUE) of plant is one of the plausible options to solve the problems. Aim of this study was to identify candidate genes involved in enhancing NUE in potato cv. Kufri Gaurav (N efficient). Plants were grown in aeroponic with two contrasting N regimes (low N: 0.75 mM, and high N: 7.5 mM). Higher NUE in Kufri Gaurav was observed in low N based on the parameters like NUE, NUpE (N uptake efficiency), NUtE (N utilization efficiency) and AgNUE (agronomic NUE). Further, global gene expression profiles in root, leaf and stolon tissues were analyzed by RNA-sequencing using Ion Proton™ System. Quality data (≥Q20) of 2.04-2.73 Gb per sample were mapped with the potato genome. Statistically significant (P ≤ 0.05) differentially expressed genes (DEGs) were identified such as 176 (up-regulated) and 30 (down-regulated) in leaves, 39 (up-regulated) and 105 (down-regulated) in roots, and 81 (up-regulated) and 694 (down-regulated) in stolons. The gene ontology (GO) terms like metabolic process, cellular process and catalytic activity were predominant. Our RT-qPCR analysis confirmed the gene expression profiles of RNA-seq. Overall, we identified candidate genes associated with improving NUE such as superoxide dismutase, GDSL esterase lipase, probable phosphatase 2C, high affinity nitrate transporters, sugar transporter, proline rich proteins, transcription factors (VQ motif, SPX domain, bHLH) etc. Our findings suggest that these candidate genes probably play crucial roles in enhancing NUE in potato.

Curcumin Downregulates GSK3 and Cdk5 in Scopolamine-Induced Alzheimer's Disease Rats Abrogating Aß40/42 and Tau Hyperphosphorylation.

Alzheimer's disease (AD) is the most common form of dementia. Extracellular amyloid-ß (Aß) aggregation and tau hyperphosphorylation are the key drivers of AD. Glycogen synthase kinase 3 (GSK3) and cyclin dependent kinase 5 (Cdk5) have been known as leading applicants arbitrating abnormal tau hyperphosphorylation. Thus, we evaluated the efficacy and underlying mechanism of action of curcumin in scopolamine-induced AD rats in our study. We found that curcumin-treated AD rats markedly reduced the levels of Aß40 and Aß42 in the brain and in the plasma in comparison to untreated AD rats. Moreover, the levels of phosphorylated tau at Ser396 (PHF13), Ser202/Thr205 (AT8), and Aß40/42 (MOAB2) were decreased significantly in AD rats treated with curcumin. Phospho-GSK3ß (Tyr216), the active form of GSK3ß, and total GSK3ß were significantly decreased in AD rats treated with curcumin. Furthermore, Cdk5 and its activators p35 and p25 were significantly decreased in curcumin-treated AD rats. The reduced levels of Cdk5, p35, p25, and GSK3ß in curcumin-treated AD rats may result decreased Aß aggregation and tau hyperphosphorylation, thus ameliorating AD. Impaired spatial memory and locomotor activity in AD rats were partially reversed by curcumin. Therefore, curcumin, as a natural compound present in turmeric, may be a more effective therapeutic agent in the treatment of AD in humans.

Development and application of fluorescent loop mediated isothermal amplification technique to detect Phytophthora infestans from potato tubers targeting ITS-1 region.

The goal of this study was to develop a fluorescent based loop-mediated isothermal amplification (LAMP) assay for a simple, sensitive and visual detection of P. infestans from tubers targeting a novel internal transcribed spacer 1 (ITS-1) region of ribosomal DNA. The ITS-1 LAMP primers were designed using the Primer Explorer V4 software. The optimization of LAMP reaction conditions and reagents concentrations were carried out with time, temperature, MgSO4, dNTPs and WarmStart Bst DNA polymerase. The amplified products were analysed using SYBR Green I dye and by agarose gel electrophoresis. We optimized reaction conditions included reagent mix, incubated at 65 °C for 60 min. The target specificity of primers was assessed with PCR, restriction digestion and sequence analysis. The developed LAMP assay was evaluated for its analytical specificity, sensitivity and validation in field tuber samples. The analytical specificity of LAMP primers indicates positive reaction with P. infestans and closely related species except P. erythosepctica. We were able to detect down to 1 pg/µl of DNA using the newly developed LAMP primers whereas the minimal amount detectable for conventional PCR was 0.1 ng/µl. Further, the samples with positive reaction developed a characteristic fluorescent green color. The detection of LAMP assay for inoculum of P. infestans was determined in the artificially inoculated leaves and tubers. In 98 field tuber samples, 54 (55.10%) were confirmed as positive by LAMP while 39 (39.79%) positive by PCR. The LAMP assay developed in this study has a potential to be a beneficial tool in early detection of P. infestans in low cost laboratory. Because the LAMP assay performed well in aspects of sensitivity, repeatability, target specificity, reliability, and visibility, it is suitable for detection of P. infestans in infected potato tubers.

Curcumin Ameliorates the Impaired Insulin Signaling Involved in the Pathogenesis of Alzheimer's Disease in Rats.

To date, dysregulation of the insulin signaling pathway in the brain has not been demonstrated unequivocally in Alzheimer's disease (AD). The purpose of the study was to examine the possible dysregulation of insulin signaling pathway in an AD rat model. Furthermore, the present study investigated the effect of Donepezil and Curcumin on insulin signaling, insulin, and glucose levels in AD rat brain. The rats were induced to develop AD by intraperitoneal administration of Scopolamine. We found that glucose levels in plasma and brain were decreased in AD rats, whereas the insulin levels was increased in plasma but decreased in brain in AD rats. In addition, insulin signaling proteins IR-ß, IGF-1, IRS-1, IRS-2 p-Akt (Ser473), and Akt were markedly reduced in the AD rats. Furthermore, GLUT3 and GLUT4 levels in the brain were markedly reduced in AD rats. All these data were compared to Saline-treated control rats. Curcumin significantly increased glucose levels in plasma and in brain. However, insulin levels was decreased in plasma and was increased in AD rats' brain. Moreover, GLUT3 and GLUT4 levels were significantly increased in Curcumin-treated AD rats. All these data were compared to Scopolamine- induced AD rats. Thus amelioration of impaired insulin signaling and improved glucose regulation in AD rats by Curcumin may be beneficial in the management of AD.

Analysis of allelic variation in wild potato (Solanum) species by simple sequence repeat (SSR) markers.

Allelic variation in wild potato (Solanum) species was analysed using 14 simple sequence repeat (SSR) markers. SSR allelic profiles showed high polymorphism and distinctness among the wild species. A total of 109 alleles of 14 polymorphic SSR markers were scored in 82 accessions belonging to 22 wild potato species. Allele size ranged from a minimum of 104 bp (STI0030) to a maximum of 304 bp (STM5114). Number of SSR alleles per marker ranged from 4 (STM5127/STM1053) to 13 (STM0019), whereas PIC value varied between 0.66 (STM1053) and 0.91 (STM0019). Cluster analysis using SSR allelic profiles of 82 accessions grouped showed 5 major clusters (I-V) based on the Dice similarity coefficient using neighbour-joining clustering method. Distinct allelic variations were observed among the accessions irrespective of the origin country, series and species. Our study suggests that SSR-based molecular characterization of wild potato species is accession specific and development of an allelic dataset for all the accessions would strengthen their utilization in potato research in future.

Integrated genomics, physiology and breeding approaches for improving nitrogen use efficiency in potato: translating knowledge from other crops.

Potato plays a key role in global food and nutritional security. Potato is an N fertiliser-responsive crop, producing high tuber yields. However, excessive use of N can result in environmental damage and high production costs, hence improving nitrogen use efficiency (NUE) of potato plants is one of the sustainable options to address these issues and increase yield. Advanced efforts have been undertaken to improve NUE in other plants like Arabidopsis, rice, wheat and maize through molecular and physiological approaches. Conversely, in potato, NUE studies have predominantly focussed on agronomy or soil management, except for a few researchers who have measured gene expression and proteins relevant to N uptake or metabolism. The focus of this review is to adapt knowledge gained from other plants to inform investigation of N metabolism and associated traits in potato with the aim of improving potato NUE using integrated genomics, physiology and breeding methods.

An insight into the downstream analysis of RB gene in F1 RB potato lines imparting field resistance to late blight.

Earlier studies have shown that level of late blight resistance conferred by the classical R gene (RB Rpi-blb1) is dependent on genetic background of the recipient genotype. This was revealed in the analysis of late blight response that belonged to a group of F1 progeny obtained from the cross between Kufri Jyoti and SP951, which showed wide variation in late blight resistance response in spite of possessing the same RB gene. The global gene expression pattern in the RB potato lines was studied in response to late blight infection using cDNA microarray analysis to reveal the background effect. Leaf samples were collected at 0, 24, 72 and 120h post inoculation (hpi) with Phytophthora infestans for gene expression analysis using 61031 gene sequences. Significantly upregulated (1477) and downregulated (4245) genes common in the RB-transgenic F1 lines at 24 and 72 hpi were classified into several categories based on GO identifiers and majority of genes were assigned putative biological functions. Highest expression of an NBS-LRR along with protease, pectin esterase inhibitors, chaperones and reactive oxygen species genes were observed which affirmed a significant role of these categories in the defence response of RB-KJ lines. Results suggest that the immune priming of plant receptors are likely to be involved in stability and functionality of RB to induce resistance against P. infestans. This study is important for effective deployment of RB gene in the host background and contributes immensely to scientific understanding of R gene interaction with host protein complexes to regulate defence system in plants.

The role of 12/15-lipoxygenase in the expression of interleukin-6 and tumor necrosis factor-alpha in macrophages.

12/15-lipoxygenase (12/15-LO) enzyme and products have been associated with inflammation and atherosclerosis. However, the mechanism of effects of the 12/15-LO products has not been fully clarified. To study the role of 12/15-LO in cytokine expression, experiments with direct additions of the12/15-LO products, 12(S)-hydroxyeicosa tetraenoic acid or 12(S)-hydroperoxyeicosa-5Z, 8Z, 10E, or 14Z-tetraenoic acid to macrophages were first carried out, and results showed that the 12/15-LO products stimulated mRNA and protein expression of IL-6 and TNF-alpha in a dose-dependent manner. In contrast, an inactive analogue of 12(S)-hydroxyeicosa tetraenoic acid had no effect. To further explore the role of endogenous 12/15-LO in cytokine expression, we used an in vitro and in vivo model to test the effect of 12/15-LO overexpression. The models included Plox-86 cells, a J774A.1 cell line that stably overexpresses leukocyte-type 12/15-LO and primary mouse peritoneal macrophages (MPMs) from 12/15-LO transgenic mice. The results showed a clear increase in IL-6 and TNF-alpha expression in Plox-86 cells and MPMs from 12/15-LO transgenic mice, compared with mock-transfected J774A.1 cells and MPMs from control C57BL6 mice. IL-1beta, IL-12, and monocyte chemoattractant protein (MCP)-1 mRNA were also increased in Plox-86 cells. These data clearly suggest a clear role of 12/15-LO pathway in cytokine production. We also demonstrated that signaling pathways including protein kinase C, p38 MAPK (p38), c-jun NH(2)-terminal kinase as well as nicotinamide adenine dinucleotide phosphate oxidase are important for 12-(S)-hydroxyeicosatetraenoic acid-induced increases in IL-6 and TNF-alpha gene expression. These results suggest a potentially important mechanism linking 12/15-LO activation to chronic inflammation and atherosclerosis.