Transcriptome profiling and differential gene expression analysis provides insights into Lr24-based resistance in wheat against Puccinia triticina.

Leaf rust caused by Puccinia triticina is an important disease of wheat and Lr24 gene confers resistance to all known pathotypes of P. triticina in India. Transcripts associated with the Lr24 mediated resistance were identified through transcriptome sequencing and further expression analysis of differentially regulated genes was performed using qPCR technique. De novo transcriptome assembly showed 66,415 and 68,688 transcripts in resistant and susceptible genotypes, respectively. The study revealed that 5873 genes unique to resistant; 6782 genes unique to susceptible, while 10,841 genes were common to both. Gene Ontology distribution statistics showed 1030 and 1068 CDS in biological processes; 1234 and 1326 CDS in cellular processes; 1321 and 1352 CDS in molecular functions, respectively. A total of 659 genes were found to be differentially expressed, of which 349 were upregulated and 310 were downregulated in resistant genotype. Pathway analysis of transcripts appeared in resistant genotype revealed that 279 transcripts had homology with genes involved in signal transduction, 18 transcripts in membrane transport, one transcript in signaling molecules. Real-time PCR study showed that most of the up-regulated defense related genes expressed in early hours indicating that a cascade of defense starts early in Lr24 mediated resistance, which successfully inhibited pathogen establishment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02972-9.

Genome-Wide Analysis of Four Pathotypes of Wheat Rust Pathogen (Puccinia graminis) Reveals Structural Variations and Diversifying Selection.

Diseases caused by Puccinia graminis are some of the most devastating diseases of wheat. Extensive genomic understanding of the pathogen has proven helpful not only in understanding host- pathogen interaction but also in finding appropriate control measures. In the present study, whole-genome sequencing of four diverse P. graminis pathotypes was performed to understand the genetic variation and evolution. An average of 63.5 Gb of data per pathotype with about 100× average genomic coverage was achieved with 100-base paired-end sequencing performed with Illumina Hiseq 1000. Genome structural annotations collectively predicted 9273 functional proteins including ~583 extracellular secreted proteins. Approximately 7.4% of the genes showed similarity with the PHI database which is suggestive of their significance in pathogenesis. Genome-wide analysis demonstrated pathotype 117-6 as likely distinct and descended through a different lineage. The 3-6% more SNPs in the regulatory regions and 154 genes under positive selection with their orthologs and under negative selection in the other three pathotypes further supported pathotype 117-6 to be highly diverse in nature. The genomic information generated in the present study could serve as an important source for comparative genomic studies across the genus Puccinia and lead to better rust management in wheat.

Lr80: A new and widely effective source of leaf rust resistance of wheat for enhancing diversity of resistance among modern cultivars.

KEY MESSAGE: A new leaf rust resistance gene Lr80 was identified and closely linked markers were developed for its successful pyramiding with other marker-tagged genes to achieve durable control of leaf rust. Common wheat landrace Hango-2, collected in 2006 from the Himalayan area of Hango, District Kinnaur, in Himachal Pradesh, exhibited a very low infection type (IT;) at the seedling stage to all Indian Puccinia triticina (Pt) pathotypes, except the pathotype 5R9-7 which produced IT 3+. Genetic analysis based on Agra Local/Hango-2-derived F3 families indicated monogenic control of leaf rust resistance, and the underlying locus was temporarily named LrH2. Bulked segregant analysis using 303 simple sequence repeat (SSR) markers located LrH2 in the short arm of chromosome 2D. An additional set of 10 chromosome 2DS-specific markers showed polymorphism between the parents and these were mapped on the entire Agra Local/Hango-2 F3 population. LrH2 was flanked by markers cau96 (distally) and barc124 (proximally). The 90 K Infinium SNP array was used to identify SNP markers linked with LrH2. Markers KASP_17425 and KASP_17148 showed association with LrH2. Comparison of seedling leaf rust response data and marker locations across different maps demonstrated the uniqueness of LrH2 and it was formally named Lr80. The Lr80-linked markers KASP_17425, KASP_17148 and barc124 amplified alleles/products different to Hango-2 in 82 Australian cultivars indicating their robustness for marker-assisted selection of this gene in wheat breeding programs.

Identification of QTLs/Defense Genes Effective at Seedling Stage Against Prevailing Races of Wheat Stripe Rust in India.

Resistance in modern wheat cultivars for stripe rust is not long lasting due to the narrow genetic base and periodical evolution of new pathogenic races. Though nearly 83 Yr genes conferring resistance to stripe rust have been cataloged so far, few of them have been mapped and utilized in breeding programs. Characterization of wheat germplasm for novel sources of resistance and their incorporation into elite cultivars is required to achieve durable resistance and thus to minimize the yield losses. Here, a genome-wide association study (GWAS) was performed on a set of 391 germplasm lines with the aim to identify quantitative trait loci (QTL) using 35K Axiom® array. Phenotypic evaluation disease severity against four stripe rust pathotypes, i.e., 46S119, 110S119, 238S119, and 47S103 (T) at the seedling stage in a greenhouse providing optimal conditions was carried out consecutively for 2 years (2018 and 2019 winter season). We identified, a total of 17 promising QTl which passed FDR criteria. Moreover these 17 QTL identified in the current study were mapped at different genomic locations i.e. 1B, 2A, 2B, 2D, 3A, 3B, 3D, 4B, 5B and 6B. These 17 QTLs identified in the present study might play a key role in marker-assisted breeding for developing stripe rust resistant wheat cultivars.

Identification and characterization of Dicer-like genes in leaf rust pathogen (Puccinia triticina) of wheat.

Puccinia triticina (P. triticina) is one of the most devastating fungal pathogens of wheat which causes significant annual yield loss to the crop. Understanding the gene regulatory mechanism of the biotrophic pathogen is one of the important aspects of host-pathogen interaction studies. Dicer-like genes are considered as important mediators of RNAi-based gene regulation. In this study, we report the presence of three Dicer-like genes (Pt-DCL1, Pt-DCL2, Pt-DCL3) in P. triticina genome identified through computational and biological analyses. Quantitative real-time PCR studies revealed an increase in the expression of these genes in germinating spore stages. Heterologous expression combined with mass spectrometry analysis of Pt-DCL2 confirmed the presence of a canonical Dicer-like gene in P. triticina. Phylogenetic analysis of the Pt-DCLs with the Dicer-like proteins from other organisms showed a distinct cluster of rust pathogens from the order Pucciniales. The results indicated a species-specific duplication of Dicer-like genes within the wheat rust pathogens. This study, for the first time, reports the presence of Dicer-dependent RNAi pathway in P. triticina that may play a role in gene regulatory mechanism of the pathogen during its development. Our study serves as a vital source of information for further RNAi-based molecular studies for better understanding and management of the wheat leaf rust disease.

Discovery and profiling of small RNAs from Puccinia triticina by deep sequencing and identification of their potential targets in wheat.

Cross-kingdom RNAi is a well-documented phenomenon where sRNAs generated by host and pathogens may govern resistance or susceptible phenotypes during host-pathogen interaction. With the first example of the direct involvement of fungal generated sRNAs in virulence of plant pathogenic fungi Botrytis cinerea and recently from Puccinia striiformis f. sp. tritici, we attempted to identify sRNAs in Puccinia triticina (P. triticina). Four sRNA libraries were prepared and sequenced using Illumina sequencing technology and a total of ~ 1-1.28 million potential sRNAs and two microRNA-like small RNA (mil-RNAs) candidates were identified. Computational prediction of targets using a common set of sRNAs and P. triticina mil-RNAs (pt-mil-RNAs) within P. triticina and wheat revealed the majority of the targets as repetitive elements in P. triticina whereas in wheat, the target genes were identified to be involved in many biological processes including defense-related pathways. We found 9 receptor-like kinases (RLKs) and 14 target genes of each related to reactive oxygen species (ROS) pathway and transcription factors respectively, including significant numbers of target genes from various other categories. Expression analysis of twenty selected sRNAs, targeting host genes pertaining to ROS related, disease resistance, metabolic processes, transporter, apoptotic inhibitor, and transcription factors along with two pt-mil-RNAs by qRT-PCR showed distinct patterns of expression of the sRNAs in urediniospore-specific libraries. In this study, for the first time, we report identification of novel sRNAs identified in P. triticina including two pt-mil-RNAs that may play an important role in biotrophic growth and pathogenicity.

Molecular characterization of Indian pathotypes of Puccinia striiformis f. sp. tritici and multigene phylogenetic analysis to establish inter- and intraspecific relationships.

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is one of the most devastating diseases of wheat (Triticum spp.) worldwide. Indian isolates were characterised based on their phenotypic reaction on differential hosts carrying different Yr genes. Based on virulence/avirulence structure, isolates were characterised into ten different pathotypes viz. 70S0-2, 67S64, 70S4, 66S0, 70S64, 66S64-1, 38S102, 47S102, 46S119, and 78S84. These Indian pathotypes of P. striiformis f. sp. tritici and 38 pathotypes of other rust species (P. graminis tritici and P. triticina) were used in this study to analyze their molecular phylogenetic relationship. The nucleotides of rDNA-ITS, partial ß-tubulin and ketopantoate reductase genes of all the pathotypes were sequenced directly after PCR. Based on sequence data of rDNA-ITS and ß-tubulin, three phylogenetic groups corresponding to three different species of Puccinia were obtained. Asian isolates formed a distinct evolutionary lineage than from those derived from USA. The sequence similarity of Indian pathotypes with other Asian (China and Iran) isolates indicated the same origin of pathotypes. The results will allow rapid identification of Indian P.striiformis f. sp. tritici pathotypes causing stripe rust in wheat, assist in making predictions regarding potential rust pathotypes, and identifying sources of resistance to the disease in advance.

Genome Wide Association Mapping of Seedling and Adult Plant Resistance to Barley Stripe Rust (Puccinia striiformis f. sp. hordei) in India.

Barley stripe rust is caused by Puccinia striiformis f.sp. hordei, (Psh), occurs worldwide, and is a major disease in South Asia. The aim of this work was to identify and estimate effects of loci underlying quantitative resistance to rust at seedling and adult plant stages. HI-AM panel of 261 barley genotypes consisting of released cultivars from North and South America, Europe, Australia, advanced breeding lines, and local landraces from ICARDA barley program were screened at seedling and adult plant stages for resistance to Psh. Seedling resistance was evaluated with the five prevalent Psh races in India. Screening for the adult plant stage resistance was also performed in two different locations by inoculating with a mixture of the five races used for seedling screeing. The panel was genotyped using DaRT-Seq high-throughput genotyping platform. The genome-wide association mapping (GWAM) showed a total of 45 QTL located across the seven barley chromosomes for seedling resistance to the five races and 18 QTL for adult plant stage resistance. Common QTL for different races at seedling stage were found on all chromosomes except on chromosome 1H. Four common QTL associated with seedling and adult plant stage resistance were found on chromosomes 2, 5, and 6H. Moreover, one of the QTL located on the long arm of chromosome 5H showed stable effects across environments for adult plant stage resistance. Several QTL identified in this study were also reported before in bi-parental and association mapping populations studies validating current GWAM. However 15 new QTL were found at adult plant stage on all chromosomes except the 4H, explaining up to 36.79% of the variance. The promising QTL detected at both stages, once validated, can be used for MAS in Psh resistance breeding program globally.

Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification.

Leaf rust of wheat caused by Puccinia triticina has significant impact on wheat production worldwide. Effective and quick detection methodologies are required to mitigate yield loss and time constraints associated with monitoring and management of leaf rust of wheat. In the present study, detection of P. triticina has been simplified by developing a rapid, reliable, efficient and visual colorimetric method i.e., loop mediated isothermal amplification of DNA (LAMP). Based on in silico analysis of P. triticina genome, PTS68, a simple sequence repeat was found highly specific to leaf rust fungus. A marker (PtRA68) was developed and its specificity was validated through PCR technique which gave a unique and sharp band of 919 bp in P. triticina pathotypes only. A novel gene amplification method LAMP which enables visual detection of pathogen by naked eye was developed for leaf rust pathogen. A set of six primers was designed from specific region of P. triticina and conditions were optimised to complete the observation process in 60 minutes at 65o C. The assay developed in the study could detect presence of P. triticina on wheat at 24 hpi (pre-symptomatic stage) which was much earlier than PCR without requiring thermal cycler. Sensitivity of LAMP assay developed in the study was 100 fg which was more sensitive than conventional PCR (50 pg) and equivalent to qPCR (100 fg). The protocol developed in the study was utilized for detection of leaf rust infected samples collected from different wheat fields. LAMP based colorimetric detection assay showed sky blue color in positive reaction and violet color in negative reaction after addition of 120 µM hydroxyl napthol blue (HNB) solution to reaction mixture. Similarly, 0.6 mg Ethidium bromide/ml was added to LAMP products, placed on transilluminator to witness full brightness in positive reaction and no such brightness could be seen in negative reaction mixture. Further, LAMP products spread in a ladder like banding pattern in gel electrophoresis. Our assay is significantly faster than the conventional methods used in the identification of P. triticina. The assay developed in the study shall be very much useful in the development of diagnostic kit for monitoring disease, creation of prediction model and efficient management of disease.

Dissection of genomic features and variations of three pathotypes of Puccinia striiformis through whole genome sequencing.

Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici, is one of the important diseases of wheat. We used NGS technologies to generate a draft genome sequence of two highly virulent (46S 119 and 31) and a least virulent (K) pathotypes of P. striiformis from the Indian subcontinent. We generated ~24,000-32,000 sequence contigs (N50;7.4-9.2 kb), which accounted for ~86X-105X sequence depth coverage with an estimated genome size of these pathotypes ranging from 66.2-70.2 Mb. A genome-wide analysis revealed that pathotype 46S 119 might be highly evolved among the three pathotypes in terms of year of detection and prevalence. SNP analysis revealed that ~47% of the gene sets are affected by nonsynonymous mutations. The extracellular secreted (ES) proteins presumably are well conserved among the three pathotypes, and perhaps purifying selection has an important role in differentiating pathotype 46S 119 from pathotypes K and 31. In the present study, we decoded the genomes of three pathotypes, with 81% of the total annotated genes being successfully assigned functional roles. Besides the identification of secretory genes, genes essential for pathogen-host interactions shall prove this study as a huge genomic resource for the management of this disease using host resistance.

Long-term administration of tacrolimus and everolimus prevents high cholesterol-high fructose-induced steatosis in C57BL/6J mice by inhibiting de-novo lipogenesis.

AIM: To investigate the effects of tacrolimus (TC) and everolimus (EV) on non-alcoholic steatohepatitis (NASH) induced by high fat, high cholesterol and fructose (fast food) diet in C57BL/6J mice. MATERIALS AND METHODS: C57BL/6J mice were divided into four groups (n=8). 1) Standard Chow (SC); 2) Fast food (FF) diet; 3) FF + Tacrolimus (TC, 1mg/kg) and; 4) FF + Everolimus (EV, 1mg/kg) and treated for 16 weeks. Serum and tissue samples were analyzed for evidence of inflammation, fibrosis, lipogenesis, and apoptosis. RESULTS: TC and EV treatments significantly reduced the hepatic lipid accumulation, improved liver-body weight ratio, blood biochemistry, and insulin resistance in mice fed with FF diet. However, inflammation, enlarged portal tracts, and fibrosis were pronounced in EV treated group. The lipogenic parameters, Peroxisome proliferator-activated receptor gamma (PPAR-γ), Sterol regulatory element-binding protein 1(SREBP-1), mammalian target of rapamycin (m-TOR), Stearoyl-CoA desaturase-1 (SCD-1) and fatty acid translocase (CD36) were significantly down-regulated in livers of TC and EV treated groups as compared to FF group. TC improved Bcl2/Bax ratio, decreased apoptosis, CYP2E1 protein expression and liver fibrosis levels, however, EV offered no such protection. Further, in an In-vitro model of lipotoxicity using the mouse hepatocyte (AML-12) cell line, treatment with TC and EV significantly reduced lipid accumulation and lipogenic and apoptotic markers induced with palmitic acid. CONCLUSION: In FF diet induced model of NASH, both TC and EV inhibited hepatic lipid accumulation and improved metabolic parameters such as insulin resistance and dyslipidemia. However, mice administered with EV exhibited inflammatory and fibrotic responses despite reduced hepatic steatosis.

Draft Genome of the Wheat Rust Pathogen (Puccinia triticina) Unravels Genome-Wide Structural Variations during Evolution.

Leaf rust is one of the most important diseases of wheat and is caused by Puccinia triticina, a highly variable rust pathogen prevalent worldwide. Decoding the genome of this pathogen will help in unraveling the molecular basis of its evolution and in the identification of genes responsible for its various biological functions. We generated high quality draft genome sequences (approximately 100- 106 Mb) of two races of P. triticina; the variable and virulent Race77 and the old, avirulent Race106. The genomes of races 77 and 106 had 33X and 27X coverage, respectively. We predicted 27678 and 26384 genes, with average lengths of 1,129 and 1,086 bases in races 77 and 106, respectively and found that the genomes consisted of 37.49% and 39.99% repetitive sequences. Genome wide comparative analysis revealed that Race77 differs substantially from Race106 with regard to segmental duplication (SD), repeat element, and SNP/InDel characteristics. Comparative analyses showed that Race 77 is a recent, highly variable and adapted Race compared with Race106. Further sequence analyses of 13 additional pathotypes of Race77 clearly differentiated the recent, active and virulent, from the older pathotypes. Average densities of 2.4 SNPs and 0.32 InDels per kb were obtained for all P. triticina pathotypes. Secretome analysis demonstrated that Race77 has more virulence factors than Race 106, which may be responsible for the greater degree of adaptation of this pathogen. We also found that genes under greater selection pressure were conserved in the genomes of both races, and may affect functions crucial for the higher levels of virulence factors in Race77. This study provides insights into the genome structure, genome organization, molecular basis of variation, and pathogenicity of P. triticina The genome sequence data generated in this study have been submitted to public domain databases and will be an important resource for comparative genomics studies of the more than 4000 existing Puccinia species.

Acteoside-mediates chemoprevention of experimental liver carcinogenesis through STAT-3 regulated oxidative stress and apoptosis.

In the absence of an effective therapy against Hepatocellular Carcinoma (HCC), chemoprevention remains an important strategy to circumvent morbidity and mortality. Here, we examined chemopreventive potential of Acteoside (ACT), a plant derived phenylethanoid glycoside against an environmental and dietary carcinogen, diethylnitrosamine (DEN)-induced rat hepatocarcinogenesis. ACT treatment (0.1 and 0.3% supplemented with diet) started 2 weeks before DEN challenge and continued for 18 weeks thereafter, showed a remarkable chemopreventive activity. ACT treatment resulted in reduced HCC nodules. Histopathology showed progressive tissue damage, necrosis (5 weeks), hepatocytic injury (10 weeks), anisonucleosis with presence of prominent nucleoli, sinusidal dilations, and lymphomono nuclear inflammation (18 weeks). Biochemical analysis showed hepatocytic injury (raised ALT, p < 0.001), inflammation [IL-6, IFN-γ (p < 0.05), and TNF-α (p < 0.001)], apoptosis [elevated Caspase-3 (p < 0.001)]. ACT at 0.1 and 0.3% ameliorated DEN-induced pre-hepatocarcinogenic manifestations. Mechanistic studies of ACT chemoprevention was elucidated using Hep3B cells with an aim to develop an in vitro DEN-induced toxicity model. Hep3B was found to be a reliable and more sensitive towards DEN toxicity compared to HepG2 and HuH7 cells. ACT prevented DEN-induced cytotoxicity (p < 0.001), DNA damage, and genotoxicity (micronuclei test, DNA ladder test, Hoechst staining, cell cycle analysis). ACT significantly (p < 0.001) scavenged DEN-induced reactive oxygen species (ROS) levels and prevented mitochondrial membrane potential (MMP) loss. Immunoblotting showed ACT treatment reversed DEN-induced NF-κB, Bax, Cytochrome C, Bcl-2, and Stat-3 levels. We conclude that chemoprotective effect of ACT is mediated by STAT-3 dependent regulation of oxidative stress and apoptosis and ACT has potential to be developed as a chemopreventive agent. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 782-798, 2016.

Glycyrrhizic acid prevents ultraviolet-B-induced photodamage: a role for mitogen-activated protein kinases, nuclear factor kappa B and mitochondrial apoptotic pathway.

Glycyrrhizic acid (GA), a natural triterpene, has received attention as an agent that has protective effects against chronic diseases including ultraviolet UV-B-induced skin photodamage. However, the mechanism of its protective effect remains elusive. Here, we used an immortalized human keratinocyte cell line (HaCaT) and a small animal model (BALB/c mice), to investigate the protective effects of GA against UV-B-induced oxidative damage, and additionally, delineated the molecular mechanisms involved in the UV-B-mediated inflammatory and apoptotic response. In the HaCaT cells, GA inhibited the UV-B-mediated increase in intracellular reactive oxygen species (ROS) and down-regulated the release of pro-inflammatory cytokines interleukin (IL)-1α, -1ß and -6, tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2). GA inhibited UV-B-mediated activation of p38 and JNK MAP kinases, COX-2 expression and nuclear translocation of NF-κB. Furthermore, GA inhibited UV-B-mediated apoptosis by attenuating translocation of Bax from the cytosol to mitochondria, thus preserving mitochondrial integrity. GA-treated HaCaT cells also exhibited elevated antiapoptotic Bcl-2 protein, concomitant with reduced caspase-3 cleavage and decreased PARP-1 protein. In BALB/c mice, topical application of GA on dorsal skin exposed to UV-B irradiation protected against epidermal hyperplasia, lymphocyte infiltration and expression of several inflammatory proteins, p38, JNK, COX-2, NF-κB and ICAM-1. Based on the above findings, we conclude that GA protects against UV-B-mediated photodamage by inhibiting the signalling cascades triggered by oxidative stress, including MAPK/NF-κB activation, as well as apoptosis. Thus, GA has strong potential to be used as a therapeutic/cosmeceutical agent against photodamage.

Comparative Temporal Transcriptome Profiling of Wheat near Isogenic Line Carrying Lr57 under Compatible and Incompatible Interactions.

Leaf rust caused by Puccinia triticina (Pt) is one of the most important diseases of bread wheat globally. Recent advances in sequencing technologies have provided opportunities to analyse the complete transcriptomes of the host as well as pathogen for studying differential gene expression during infection. Pathogen induced differential gene expression was characterized in a near isogenic line carrying leaf rust resistance gene Lr57 and susceptible recipient genotype WL711. RNA samples were collected at five different time points 0, 12, 24, 48, and 72 h post inoculation (HPI) with Pt 77-5. A total of 3020 transcripts were differentially expressed with 1458 and 2692 transcripts in WL711 and WL711+Lr57, respectively. The highest number of differentially expressed transcripts was detected at 12 HPI. Functional categorization using Blast2GO classified the genes into biological processes, molecular function and cellular components. WL711+Lr57 showed much higher number of differentially expressed nucleotide binding and leucine rich repeat genes and expressed more protein kinases and pathogenesis related proteins such as chitinases, glucanases and other PR proteins as compared to susceptible genotype. Pathway annotation with KEGG categorized genes into 13 major classes with carbohydrate metabolism being the most prominent followed by amino acid, secondary metabolites, and nucleotide metabolism. Gene co-expression network analysis identified four and eight clusters of highly correlated genes in WL711 and WL711+Lr57, respectively. Comparative analysis of the differentially expressed transcripts led to the identification of some transcripts which were specifically expressed only in WL711+Lr57. It was apparent from the whole transcriptome sequencing that the resistance gene Lr57 directed the expression of different genes involved in building the resistance response in the host to combat invading pathogen. The RNAseq data and differentially expressed transcripts identified in present study is a genomic resource which can be used for further studying the host pathogen interaction for Lr57 and wheat transcriptome in general.