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Draft genome sequence of Karnal bunt pathogen (Tilletia indica) of wheat provides insights into the pathogenic mechanisms of quarantined fungus.

ABSTRACT: Karnal bunt disease in wheat is caused by hemibiotrophic fungus, Tilletia indica that has been placed as quarantine pest in more than 70 countries. Despite its economic importance, little knowledge about the molecular components of fungal pathogenesis is known. In this study, first time the genome sequence of T. indica has been deciphered for unraveling the effectors' functions of molecular pathogenesis of Karnal bunt disease. The T. indica genome was sequenced employing hybrid approach of PacBio Single Molecule Real Time (SMRT) and Illumina HiSEQ 2000 sequencing platforms. The genome was assembled into 10,957 contigs (N50 contig length 3 kb) with total size of 26.7 Mb and GC content of 53.99%. The number of predicted putative genes were 11,535, which were annotated with Gene Ontology databases. Functional annotation of Karnal bunt pathogen genome and classification of identified effectors into protein families revealed interesting functions related to pathogenesis. Search for effectors' genes using pathogen host interaction database identified 135 genes. The T. indica genome sequence and putative genes involved in molecular pathogenesis would further help in devising novel and effective disease management strategies including development of resistant wheat genotypes, novel biomarkers for pathogen detection and new targets for fungicide development.

SUBMITTER: Kumar A

PROVIDER: S-EPMC5289553 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia indica, the Karnal bunt of wheat fungus.

Project description:Mitochondrial DNA (mtDNA) from five isolates of Tilletia indica was isolated and digested with several restriction enzymes. A 2.3-kb EcoRI fragment was chosen, cloned, and shown to hybridize with total DNA restricted with EcoRI from T. indica and not from a morphologically similar smut fungus, Tilletia barclayana. The clone was partially sequenced, and primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2.3-kb fragment from total DNA of 17 T. indica isolates from India, Pakistan, and Mexico. DNA from 25 isolates of other smut fungi (T. barclayana, Tilletia foetida, Tilletia caries, Tilletia fusca, and Tilletia controversa) did not produce any bands, as detected by ethidium bromide-stained agarose gels and Southern hybridizations. The sensitivity of the assay was determined and increased by using a single nested primer in a second round of amplification, so that 1 pg of total mycelial DNA could be detected. The results indicated that the primers which originated from a cloned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contaminating wheat seeds.

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De novo genome sequencing and secretome analysis of Tilletia indica inciting Karnal bunt of wheat provides pathogenesis-related genes.

Project description:Tilletia indica is an internationally quarantined fungal pathogen causing Karnal bunt of wheat. The present study carried out that the whole genome of T. indica was sequenced and identified transposable elements, pathogenicity-related genes using a comparative genomics approach. The T. indica genome assembly size of 33.7 MB was generated using Illumina and Pac Bio platforms with GC content of 55.0%. A total of 1737 scaffolds were obtained with N50 of 58,667 bp. The ab initio gene prediction was performed using Ustilago maydis as the reference species. A total number of 10,113 genes were predicted with an average gene size of 1945 bp out of which functionally annotated genes were 7262. A total number of 3216 protein-coding genes were assigned in different categories. Out of a total number of 1877 transposable elements, gypsy had the highest count (573). Total 5772 simple sequence repeats were identified in the genome assembly, and the most abundant simple sequence repeat type was trinucleotide having 42% of total SSRs. The comparative genome analysis suggested 3751 proteins of T. indica had orthologs in five fungi, whereas 126 proteins were unique to T. indica. Secretome analysis revealed the presence of 1014 secretory proteins and few carbohydrate-active enzymes in the genome. Some putative candidate pathogenicity-related genes were identified in the genome. The whole genome of T. indica will provide a window to understand the pathogenesis mechanism, fungal life cycle, survival of teliospores, and novel strategies for management of Karnal bunt disease of wheat.

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Isolation and in-silico characterization of Peroxidase isoenzymes from Wheat (Triticum aestivum) against Karnal Bunt (Tilletia indica).

Project description:To investigate the role of Peroxidase and its physiological significance under Karnal Bunt (KB) were determined in resistant (HD-29) and susceptible genotype (WH-542) of wheat during different developmental stages. The enzymes were expressed constitutively in both the susceptible and resistant genotype. In gel assay and differential expression analysis of POD was significantly higher (p >0.05) in Sv and S2, than the S1 and S3 stages. in silico analysis of Peroxidase for eg. physico-chemical properties, secondary structural features and phylogenetic classification for comparative analysis. Motif and Domain analysis of Peroxidase by MEME, to be important for the biological functions, and studies of evolution. Our results clearly indicate that the enhanced expression of POD at the WS2 stage, which reinforces its role in stage dependent immunity against Karnal bunt and role of POD metabolism provides genotype and stage dependant structural barrier resistance in wheat against KB.

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Expression and in silico characterization of Phenylalanine ammonium lyase against karnal bunt (Tilletia indica) in wheat (Triticum aestivum).

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Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in Tilletia indica Isolates Inciting Karnal Bunt of Wheat.

Project description:Karnal bunt of wheat is an internationally quarantined disease affecting trade, quality, and production of wheat. During 2015-2016, a severe outbreak of Karnal bunt disease occurred in north-western plain zone of India. The present study was undertaken to decipher genetic variations in Indian isolates of Tilletia indica collected from different locations. Seven multilocus sequence fragments were selected to differentiate and characterize these T. indica isolates. A phylogenetic tree constructed based on pooled sequences of actin-related protein 2 (ARP2), ?-tubulin (TUB), eukaryotic translation initiation factor 3 subunit A (EIF3A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 2B (H2B), phosphoglycerate kinase (PGK), and serine/threonine-protein kinase (STPK) showed that isolate KB-11 (Kaithal, Haryana) was highly conserved as it was located in cluster 1 and has the maximum sequence similarity with the reference strain. Other isolates in cluster 1 included KB-16 and KB-17, both from Uttar Pradesh, and KB-19 from Haryana. Isolates KB-07 (Jind, Haryana) and KB-18 (Mujaffar Nagar, Uttar Pradesh) were the most diverse and grouped in a subgroup of cluster 2. Maximum numbers of single nucleotide polymorphisms (SNPs) (675) were in the PGK gene across the T. indica isolates. The minimum numbers of SNPs (67) were in KB-11 (Kaithal, Haryana), while the maximum number of SNPs (165) was identified in KB-18, followed by 164 SNPs in KB-14. KB-18 isolate was found to be the most diverse amongst all T. indica isolates. This first study on multilocus sequence typing (MLST) revealed that the population of T. indica was highly diverse.

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Project description:Karnal bunt (KB) of wheat incited by Tilletia indica Mitra is now gaining importance from the last few years due to its increasing incidence. Regular surveys are conducted to collect wheat grains samples from different grain markets of Punjab, India. Since weather plays a significant role in the initiation as well as the development of Karnal bunt. Thus, the variation in Karnal bunt incidence worked out and is being interpreted in relation to the prevailing environmental conditions during the most susceptible stage for the two decades (1991-92 to 2014-15) for the Punjab, India. The incidence of Karnal bunt was correlated with the weather parameters during the February and March of the corresponding year. The correlation analysis revealed the positive role of rainfall, rainy days, evening relative humidity, and Humid thermal index of March and the negative role of sunshine hours of February in the development and incidence of Karnal bunt. By using these parameters, a multiple regression model was developed and validated for forecasting the disease. The regression analysis showed a coefficient of determination of 0.77 and a D.W value of 1.88. The detailed analysis of historical data for more than two decades divulged the amount of total rainfall as well as the number of rainy days of March as the most critical factor for the Karnal bunt development.Supplementary informationThe online version contains supplementary material available at 10.1007/s42360-022-00520-w.

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Project description:Tilletia indica is a quarantine fungal pathogen that poses a serious biosecurity threat to wheat-exporting countries. Acquiring genetic data for the pathogenicity characters of T. indica is still a challenge for wheat breeders and geneticists. In the current study, double digest restriction-site associated-DNA genotyping by sequencing was carried out for 39 T. indica isolates collected from different locations in India. The generated libraries upon sequencing were with 3,346,759 raw reads on average, and 151 x 2 nucleotides read length. The obtained bases per read ranged from 87 Mb in Ti 25 to 1,708 Mb in Ti 39, with 505 Mb on average per read. Trait association mapping was performed using 41,473 SNPs, infection phenotyping data, population structure, and Kinship matrix, to find single nucleotide polymorphisms (SNPs) linked to virulence genes. Population structure analysis divided the T. indica population in India into three subpopulations with genetic mixing in each subpopulation. However, the division was not in accordance with the degree of virulence. Trait association mapping revealed the presence of 13 SNPs associated with virulence. Using sequences analysis tools, one gene (g4132) near a significant SNP was predicted to be an effector, and its relative expression was assessed and found upregulated upon infection.

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Unravelling the Complex Genetics of Karnal Bunt (Tilletia indica) Resistance in Common Wheat (Triticum aestivum) by Genetic Linkage and Genome-Wide Association Analyses.

Project description:Karnal bunt caused by Tilletia indica Mitra [syn. Neovossia indica (Mitra) Mundkur] is a significant biosecurity concern for wheat-exporting countries that are free of the disease. It is a seed-, soil-and air-borne disease with no effective chemical control measures. The current study used data from multi-year field experiments of two bi-parental populations and a genome-wide association (GWA) mapping panel to unravel the genetic basis for resistance in common wheat. Broad-sense heritability for Karnal bunt resistance in the populations varied from 0.52 in the WH542×HD29 population, to 0.61 in the WH542×W485 cross and 0.71 in a GWAS panel. Quantitative trait locus (QTL) analysis with seven years of phenotypic data identified a major locus on chromosome 3B (R2 = 27.8%) and a minor locus on chromosome 1A (R2 = 12.2%), in the WH542×HD29 population, with both parents contributing the high-value alleles. A major locus (R2 = 27.8%) and seven minor loci (R2 = 4.4-15.8%) were detected in the WH542×W485 population. GWA mapping validated QTL regions in the bi-parent populations, but also identified novel loci not previously associated with Karnal bunt resistance. Meta-QTL analysis aligned the results from this study with those reported in wheat over the last two decades. Two major clusters were detected, the first on chromosome 4B, which clustered with Qkb.ksu-4B, QKb.cimmyt-4BL, Qkb.cim-4BL, and the second on chromosome 3B, which clustered with Qkb.cnl-3B, QKb.cimmyt-3BS and Qkb.cim-3BS1 The results provide definitive chromosomal assignments for QTL/genes controlling Karnal bunt resistance in common wheat, and will be useful in pre-emptive breeding against the pathogen in wheat-producing areas that are free of the disease.

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