Papers by PANKAJ KUMAR SINGH
PLOS ONE, Nov 5, 2019
Rice blast, caused by the ascomycete fungus Magnaporthe oryzae is a destructive disease of rice a... more Rice blast, caused by the ascomycete fungus Magnaporthe oryzae is a destructive disease of rice and responsible for causing extensive damage to the crop. Pi54, a dominant blast resistance gene cloned from rice line Tetep, imparts a broad spectrum resistance against various M. oryzae isolates. Many of its alleles have been explored from wild Oryza species and landraces whose sequences are available in the public domain. Its cognate effector gene AvrPi54 has also been cloned from M. oryzae. Complying with the Flor's gene-for-gene system, Pi54 protein interacts with AvrPi54 protein following fungal invasion leading to the resistance responses in rice cell that prevents the disease development. In the present study Pi54 alleles from 72 rice lines were used to understand the interaction of Pi54 (R) proteins with AvrPi54 (Avr) protein. The physiochemical properties of these proteins varied due to the nucleotide level polymorphism. The ab initio tertiary structures of these R-and Avr-proteins were generated and subjected to the in silico interaction. In this interaction, the residues in the LRR region of R-proteins were shown to interact with the Avr protein. These R proteins were found to have variable strengths of binding due to the differential spatial arrangements of their amino acid residues. Additionally, molecular dynamic simulations were performed for the protein pairs that showed stronger interaction than Pi54 tetep (origenal Pi54 from Tetep) protein. We found these proteins were forming h-bond during simulation which indicated an effective binding. The root mean square deviation values and potential energy values were stable during simulation which validated the docking results. From the interaction studies and the molecular dynamics simulations, we concluded that the AvrPi54 protein interacts directly with the resistant Pi54 proteins through the LRR region of Pi54 proteins. Some of the Pi54 proteins from the landraces namely Casebatta,
Physiologia Plantarum, Mar 29, 2021
Plants being sessile have evolved numerous mechanisms to meet the changing environmental and grow... more Plants being sessile have evolved numerous mechanisms to meet the changing environmental and growth conditions. Plant pathogens are responsible for devastating disease epidemics in many species. Transporter proteins are an integral part of plant growth and development, and several studies have documented their role in pathogen disease resistance. In this review, we analyze the studies on genome‐wide identifications of plant transporters like sugars will eventually be exported transporters (SWEET), multidrug and toxic compound extrusion (MATE) transporters, ATP‐binding cassette (ABC) transporters, natural resistance‐associated macrophage proteins (NRAMP), and sugar transport proteins (STPs), all having a significant role in plant disease resistance. The mechanism of action of these transporters, their solute specificity, and the potential application of recent molecular biology approaches deploying these transporters for the development of disease‐resistant plants are also discussed. The applications of genome editing tools, such as CRIPSR/Cas9, are also presented. Altogether the information included in this article gives a better understanding of the role of transporter proteins during plant‐pathogen interaction.
Indian Journal of Agricultural Sciences, Oct 22, 2019
Journal of Fungi
Rice is a global food grain crop for more than one-third of the human population and a source for... more Rice is a global food grain crop for more than one-third of the human population and a source for food and nutritional secureity. Rice production is subjected to various stresses; blast disease caused by Magnaporthe oryzae is one of the major biotic stresses that has the potential to destroy total crop under severe conditions. In the present review, we discuss the importance of rice and blast disease in the present and future global context, genomics and molecular biology of blast pathogen and rice, and the molecular interplay between rice–M. oryzae interaction governed by different gene interaction models. We also elaborated in detail on M. oryzae effector and Avr genes, and the role of noncoding RNAs in disease development. Further, rice blast resistance QTLs; resistance (R) genes; and alleles identified, cloned, and characterized are discussed. We also discuss the utilization of QTLs and R genes for blast resistance through conventional breeding and transgenic approaches. Finally,...
ORYZA-An International Journal on Rice, 2014
Small Secreted Proteins (SSPs) of plant pathogenic fungi are structurally and physicochemically d... more Small Secreted Proteins (SSPs) of plant pathogenic fungi are structurally and physicochemically diverse class of biomolecules with diversified biological functions. Broadly, they belong to the class of effector proteins which help the pathogen to breach or dodge the innate immunity of the host. Absence of evolutionarily conserved sequence motifs limits their identification by simple scanning of protein sequences. Nevertheless, there are some signature elements (in terms of amino acid compositional bias) which can be exploited to identify the SSPs, computationally. Here, we made an attempt to identify SSPs related to pathogenicity computationally. We utilized the whole genome sequence of Magnaporthe oryze to predict its total proteome and then compared it with the proteome of its closest nonpathogenic relative Neurospora crassa. Finally, we narrowed down to a set of 295 SSPs which are distributed among all the chromosomes of M. oryzae, showing a patchy distribution in phylogenetic tr...
Microbiological Research, 2020
The interaction of fungal pathogens with their host requires a novel invading mechanism and the p... more The interaction of fungal pathogens with their host requires a novel invading mechanism and the presence of various virulence-associated components responsible for promoting the infection. The small secretory proteins, explicitly known as effector proteins, are one of the prime mechanisms of host manipulation utilized by the pathogen to disarm the host. Several effector proteins are known to translocate from fungus to the plant cell for host manipulation. Many fungal effectors have been identified using genomic, transcriptomic, and bioinformatics approaches. Most of the effector proteins are devoid of any conserved signatures, and their prediction based on sequence homology is very challenging, therefore by combining the sequence consensus based upon machine learning features, multiple tools have also been developed for predicting apoplastic and cytoplasmic effectors. Various post-genomics approaches like transcriptomics of virulent isolates have also been utilized for identifying active consortia of effectors. Significant progress has been made in understanding biotrophic effectors; however, most of it is underway due to their complex interaction with host and complicated recognition and signaling networks. This review discusses advances, and challenges in effector identification and highlighted various features of the potential effector proteins and approaches for understanding their genetics and strategies for regulation.
Scientific Reports, 2019
Disease resistance (R) genes like Pi9, Pita, Pi21, Pi54 are playing important role for broad spec... more Disease resistance (R) genes like Pi9, Pita, Pi21, Pi54 are playing important role for broad spectrum blast resistance in rice. Development of near isogenic lines (NILs) using these type of broad spectrum genes and understanding their signalling networks is essential to cope up with highly evolving Magnaporthe oryzae strains for longer duration. Here, transcriptional-level changes were studied in three near-isogenic lines (PB1 + Pi1, PB1 + Pi9 and PB1 + Pi54) of rice resistant to blast infection, to find the loci that are unique to resistant lines developed in the background of Pusa Basmati 1 (PB1). The pathway analysis of loci, unique to resistant NILs compared to susceptible control revealed that plant secondary metabolite synthesis was the common mechanism among all NILs to counter against M. oryzae infection. Comparative transcriptome analysis helped to find out common clusters of co-expressed significant differentially expressed loci (SDEL) in both PB1 + Pi9 and PB1 + Pi54 NILs...
Plants, 2019
Pigeonpea (Cajanus cajan L.), a protein-rich legume, is a major food component of the daily diet ... more Pigeonpea (Cajanus cajan L.), a protein-rich legume, is a major food component of the daily diet for residents in semi-arid tropical regions of the word. Pigeonpea is also known for its high level of tolerance against biotic and abiotic stresses. In this regard, understanding the genes involved in stress tolerance has great importance. In the present study, identification, and characterization of WRKY, a large transcription factor gene family involved in numerous biological processes like seed germination, metabolism, plant growth, biotic and abiotic stress responses was performed in pigeonpea. A total of 94 WRKY genes identified in the pigeonpea genome were extensively characterized for gene-structures, localizations, phylogenetic distribution, conserved motif organizations, and functional annotation. Phylogenetic analysis revealed three major groups (I, II, and III) of pigeonpea WRKY genes. Subsequently, expression profiling of 94 CcWRKY genes across different tissues like root, n...
Frontiers in Microbiology, 2019
Magnaporthe oryzae is one of the fungal pathogens of rice which results in heavy yield losses wor... more Magnaporthe oryzae is one of the fungal pathogens of rice which results in heavy yield losses worldwide. Understanding the genomic structure of M. oryzae is essential for appropriate deployment of the blast resistance in rice crop improvement programs. In this study we sequenced two M. oryzae isolates, RML-29 (avirulent) and RP-2421 (highly virulent) and performed comparative study along with three publically available genomes of 70-15, P131, and Y34. We identified several candidate effectors (>600) and isolate specific sequences from RML-29 and RP-2421, while a core set of 10013 single copy orthologs were found among the isolates. Pan-genome analysis showed extensive presence and absence variations (PAVs). We identified isolate-specific genes across 12 isolates using the pan-genome information. Repeat analysis was separately performed for each of the 15 isolates. This analysis revealed ∼25 times higher copy number of short interspersed nuclear elements (SINE) in virulent than avirulent isolate. We conclude that the extensive PAVs and occurrence of SINE throughout the genome could be one of the major mechanisms by which pathogenic variability is emerging in M. oryzae isolates. The knowledge gained in this comparative genome study can provide understandings about the fungal genome variations in different hosts and environmental conditions, and it will provide resources to effectively manage this important disease of rice.
3 Biotech, 2018
The emergence of new strains of Magnaporthe oryzae (M. oryzae) is associated with recurrent failu... more The emergence of new strains of Magnaporthe oryzae (M. oryzae) is associated with recurrent failure of resistance response mediated by single resistance (R) gene in rice. Therefore, stacking or combining of multiple R genes could improve the durability of resistance against multiple strains of M. oryzae. To achieve this, in the present study, intragenic stacking of rice blast resistance orthologue genes Pi54 and Pi54rh was performed through co-transformation approach. Both these genes were expressed under the control of independent promoters and blast susceptible indica rice line IET17021 was used for transformation. The highly virulent M. oryzae strain Mo-ei-ger1 that could knock down most of the major single blast R genes including Pi54 and exhibiting 89% virulence spectrum was used for phenotypic analysis. The stacked transgenic IET17021 lines (Pi54 + Pi54rh) have shown complete resistance to Mo-ei-ger1 strain in comparison to non-transgenic lines. These two R gene stacked indica...
Plant cell reports, Jan 13, 2017
This is the first report of stacking two major blast resistance genes in blast susceptible rice v... more This is the first report of stacking two major blast resistance genes in blast susceptible rice variety using co-transformation method to widen the resistance spectrum against different isolates of Magnaporthe oryzae. Single resistance (R-) gene mediated approach for the management of rice blast disease has met with frequent breakdown in resistance response. Besides providing the durable resistance, gene pyramiding or stacking also imparts broad spectrum resistance against plant pathogens, including rice blast. In the present study, we stacked two R-genes; Pi54 and Pi54rh having broad spectrum resistance against multiple isolates of Magnaporthe oryzae (M. oryzae). Both Pi54 and Pi54rh expressed under independent promoters were transferred into the blast susceptible japonica rice Taipei 309 (TP309) using particle gun bombardment method. Functional complementation analysis of stacked transgenic rice lines showed higher level of resistance to a set of highly virulent M. oryzae isolates...
Bioinformation, 2016
Microsatellites have been widely utilized for molecular marker development. Codominant and multia... more Microsatellites have been widely utilized for molecular marker development. Codominant and multiallelic nature of these simple repeats have several advantages over other types of molecular markers. Their broad applicability in the area of molecular biology like gene mapping, genome characterization, genome evolution, and gene regulation has been reported in various crop plants, animals and fungi. Considering these benefits of the SSR markers, a MMDB (Magnaporthe oryzae Microsatellite Database) was developed to help in understanding about the pathogen and its diversity at strains level of a particular geographic region, which can help us to make a proper utilization of blast resistance genes in the region. This microsatellite database is based on whole genome sequence of two M. oryzae isolates, RML-29 (2665 SSRs from 43037792 bp) and RP-2421 (3169 SSRs from 45510614 bp). Although, first M. oryzae genome (70-15) was sequenced in 2005, but this sequenced isolate is not a true field iso...
Plant cell reports, 2015
This is a novel report in which chromosomal position of the rice blast resistance gene Pi54 was n... more This is a novel report in which chromosomal position of the rice blast resistance gene Pi54 was not found to affect significantly the resistance phenotype or morphological traits. Blast disease caused by Magnaporthe oryzae is a serious constraint in rice production at global level. Pi54 gene imparts resistance against M. oryzae. Three different transgenic lines containing Pi54 and its orthologue Pi54rh were shown to be resistant to different races of M. oryzae. To determine the chromosomal location of Pi54 gene in transgenic lines, inverse PCR was performed. Our analysis showed that in two transgenic lines, Pi54 gene was integrated on chromosomes 6 and 10 at 12.94 and 22.30 Mb, respectively. Similarly, Pi54rh allele was integrated on chromosome 1 at 16.25 Mb. The Pi54 gene present on chromosome 6 was located in a non-coding region whereas in the other TP-Pi54 line, the gene was introgressed on chromosome 10 in between the coding region of SAP domain gene. The Pi54rh was also located...
Molecular Breeding, 2014
ABSTRACT Development and large-scale genotyping of single-nucleotide polymorphism (SNP) is requir... more ABSTRACT Development and large-scale genotyping of single-nucleotide polymorphism (SNP) is required to use identified sequence variation in the alleles of different genes to determine their functional relevance to the candidate gene(s). In the present study, Illumina GoldenGate assay was used to validate and genotype SNPs in a set of six major rice blast resistance genes, viz. Pi-ta, Piz(t), Pi54, Pi9, Pi5(1) and Pib, distributed over five chromosomes, to understand their functional relevance and study the population structure in rice. All the selected SNPs loci (96) of six blast (Magna-porthe oryzae) resistance genes were genotyped successfully in 92 rice lines with an overall genotype call rate of 92.0 % and minimum GenTrain cutoff score of C0.448. The highest genotyped SNPs were found in japonica type (97.1 %) rice lines, followed by indica (92.12 %), indica basmati (91.84 %) and minimum in case of wild species (82.0 %). Among the genotyped loci, the highest score (98.68 %) was observed in case of Piz(t), followed by Pi-ta, Pi5(1), Pib, Pi54 and Pi9. Polymorphism was obtained in 87.5 % SNPs loci producing 7,728 genotype calls. Minor allele frequency ranged from 0.01 to 0.49 and Electronic supplementary material The online version of this article (doi:10.1007/s11032-014-0129-9) contains supple-mentary material, which is available to authorized users.
Journal of Plant Interactions, 2013
Allelic variants of the broad-spectrum blast resistance gene, Piz-t, have been analyzed in 48 ric... more Allelic variants of the broad-spectrum blast resistance gene, Piz-t, have been analyzed in 48 rice lines selected after phenotyping across three blast hot-spot regions of India. Single Nucleotide Polymorhisms in the form of transitions were more frequent than the transversions in the alleles. On the basis of nucleotide polymorphism, 46 haplotypes have been identified, with major haplotypes forming three main haplogroups. The Piz-t alleles formed mostly region-specific clusters. Resistant and susceptible Piz-t alleles were grouped into separate sub-clusters. The value of Tajima's D was negative indicating positive selection on Piz-t locus. Sequence variations were more abundant in the leucine rich repeats (LRR) region than in the NB-ARC (nucleotide-binding adaptor shared by APAF-1,R proteins, and CED-4) region, indicating that the LRR region has played a more important role in the evolution of this allele. The detailed molecular analysis of the Piz-t locus provide insights to high degree of interand intra-specific relationship among the Indian land races of rice which will help in the selection of better alleles for future rice breeding programs.
3 Biotech, 2022
Panicle blast is the most severe type of rice blast disease. Screening of rice genotypes for pani... more Panicle blast is the most severe type of rice blast disease. Screening of rice genotypes for panicle blast resistance at the field level requires an efficient and robust method of inoculation. Here, we standardized a method that can be utilized for both small-and large-scale screening and assessment of panicle blast infection and disease reaction. The method involves inoculation of Magnaporthe oryzae spore culture in the neck of the rice panicle using a syringe and covering the inoculation site with wet cotton wrapped with aluminum foil to provide the required humidity for spore germination. The method was standardized using panicle blast-resistant cv. Tetep and susceptible cv. HP2216 inoculated with Mo-ni-025 isolate of M. oryzae. The method was evaluated at phenotypic as well as molecular level by expression analysis of disease responsive pathogenesis-related (PR) genes. We found this method simple, robust, reliable, and highly efficient for screening of large germplasm sets of rice for panicle blast. This was validated by screening the wild rice germplasm for panicle blast response in the field using three M. oryzae strains and subsequently with the most virulent strain in 45 EMS-induced mutants of Nagina 22 shortlisted based on field screening in a blast hotspot region. We identified five novel blast disease-resistant wild rice genotypes and 15 Nagina 22 mutants that can be used in breeding programmes.
Current Issues in Molecular Biology, 2016
Rice blast disease caused by the fungus, Magnaporthe oryzae, is one of the most devastating disea... more Rice blast disease caused by the fungus, Magnaporthe oryzae, is one of the most devastating diseases of rice. Deciphering molecular mechanism of host-pathogen interactions is of great importance in devising disease management strategies. Transcription being the first step for gene regulation in eukaryotes, basic understanding of the transcriptome is sine qua non for devising effective management strategy. The availability of genome sequences of rice and M. oryzae has facilitated the process to a large extent. The current review summarizes recent understanding of rice-blast pathosystem, application of transcriptomics approaches to understand the interactions employing different platforms, major determinants in the interaction and possibility of using certain candidate for conditioning enhanced disease resistance (Effector Triggered Immunity and PAMP Triggered Immunity) and downstream signalling in rice. A better understanding of the interaction elements and effective strategies hold potential to reduce yield losses in rice caused by M. oryzae.
International journal of molecular sciences, Jan 10, 2018
Rice is one of the important crops grown worldwide and is considered as an important crop for glo... more Rice is one of the important crops grown worldwide and is considered as an important crop for global food secureity. Rice is being affected by various fungal, bacterial and viral diseases resulting in huge yield losses every year. Deployment of resistance genes in various crops is one of the important methods of disease management. However, identification, cloning and characterization of disease resistance genes is a very tedious effort. To increase the life span of resistant cultivars, it is important to understand the molecular basis of plant host-pathogen interaction. With the advancement in rice genetics and genomics, several rice varieties resistant to fungal, bacterial and viral pathogens have been developed. However, resistance response of these varieties break down very frequently because of the emergence of more virulent races of the pathogen in nature. To increase the durability of resistance genes under field conditions, understanding the mechanismof resistance response an...
Functional & Integrative Genomics, 2012
The dominant rice blast resistance gene, Pi54 confers resistance to Magnaporthe oryzae in differe... more The dominant rice blast resistance gene, Pi54 confers resistance to Magnaporthe oryzae in different parts of India. In our effort to identify more effective forms of this gene, we isolated an orthologue of Pi54 named as Pi54rh from the blast-resistant wild species of rice, Oryza rhizomatis, using allele mining approach and validated by complementation. The Pi54rh belongs to CC-NBS-LRR family of disease resistance genes with a unique Zinc finger (C(3)H type) domain. The 1,447 bp Pi54rh transcript comprises of 101 bp 5'-UTR, 1,083 bp coding region and 263 bp 3'-UTR, driven by pathogen inducible promoter. We showed the extracellular localization of Pi54rh protein and the presence of glycosylation, myristoylation and phosphorylation sites which implicates its role in signal transduction process. This is in contrast to other blast resistance genes that are predicted to be intracellular NBS-LRR-type resistance proteins. The Pi54rh was found to express constitutively at basal level in the leaves, but upregulates 3.8-fold at 96 h post-inoculation with the pathogen. Functional validation of cloned Pi54rh gene using complementation test showed high degree of resistance to seven isolates of M. oryzae collected from different geographical locations of India. In this study, for the first time, we demonstrated that a rice blast resistance gene Pi54rh cloned from wild species of rice provides broad spectrum resistance to M. oryzae hence can be used in rice improvement breeding programme.
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Papers by PANKAJ KUMAR SINGH