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.

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.

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.

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.

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.

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.

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.

Genome sequencing of four strains of Phylotype I, II and IV of Ralstonia solanacearum that cause potato bacterial wilt in India

Abstract Ralstonia solanacearum is a heterogeneous species complex causing bacterial wilts in more than 450 plant species distributed in 54 families. The complexity of the genome and the wide diversity existing within the species has led to the concept of R. solanacearum species complex (RsSC). Here we report the genome sequence of the four strains (RS2, RS25, RS48 and RS75) belonging to three of the four phylotypes of R. solanacearum that cause potato bacterial wilt in India. The genome sequence data would be a valuable resource for the evolutionary, epidemiological studies and quarantine of this phytopathogen.

Genome sequencing of four strains of Phylotype I, II and IV of Ralstonia solanacearum that cause potato bacterial wilt in India.

Ralstonia solanacearum is a heterogeneous species complex causing bacterial wilts in more than 450 plant species distributed in 54 families. The complexity of the genome and the wide diversity existing within the species has led to the concept of R. solanacearum species complex (RsSC). Here we report the genome sequence of the four strains (RS2, RS25, RS48 and RS75) belonging to three of the four phylotypes of R. solanacearum that cause potato bacterial wilt in India. The genome sequence data would be a valuable resource for the evolutionary, epidemiological studies and quarantine of this phytopathogen.

Expression of the cry9Aa2 B.t. gene in tobacco chloroplasts confers resistance to potato tuber moth.

We report here the control of potato tuber moth (Phthorimaea operculella) by incorporating a truncated Bacillus thuringiensis cry9Aa2 gene in the plastid genome. Plasmids pSKC84 and pSKC85 are derivatives of a new polycistronic plastid transformation vector, pPRV312L, that carries spectinomycin resistance (aadA) as a selective marker and targets insertions in the trnI-trnA intergenic region. The Cry9Aa2 N-terminal region (82.1 kDa; 734 amino acids) was expressed in a cassette, which consists of 49 nucleotides of the cry9Aa2 leader and the 3'-untranslated region of the plastid rbcL gene (TrbcL), and relies on readthrough transcription from the plastid rRNA operon. In a tobacco leaf bioassay, expression of Cry9Aa2 conferred resistance to potato tuber moth. In accordance, the Cry9Aa2 insecticidal protein accumulated to high levels, approximately 10% of the total soluble cellular protein and approximately 20% in the membrane fraction. However, high-level Cry9Aa2 expression significantly delayed plant development. Thus, a practical system to control potato tuber moth by Cry9Aa2 expression calls for down-regulation of its expression.

Cloning and sequencing of coat protein gene of an Indian potato leaf roll virus (PLRV) isolate and its similarity with other members of Luteoviridae.

An Indian strain of potato leaf roll virus (PLRV) was purified to generate complementary DNA corresponding to the coat protein (CP) gene. Virus cDNA was synthesized from purified viral RNA using oligo (dT)-anchor primer and virus specific primers. The viral sequence encoding the coat protein was specifically amplified by polymerase chain reaction (PCR), using specific primers bordering the CP gene. The unique amplified product thus obtained was A-T cloned into the pGEM-T Easy vector and the authenticity of the cloned gene verified by dot blot hybridization and sequence analysis. Run-way-transcripts of the cloned CP gene could detect PLRV in tissue imprints and tissue dilution. The nucleotide sequences and the deduced amino acid sequences were compared with the other PLRV isolates and found to be 97-99% identical at both the nucleotide and amino acid sequence level of other isolates. Multiple sequence alignment of deduced amino acid sequences revealed considerable homology to other luteoviruses. A nuclear localization signal located close to the N-terminus of the CP gene was predicted. This is the first report of PLRV coat protein sequence from an Indian strain.