Publications of NIPGR Scientists
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Item Magnaporthe oryzae MoNdt80 is a transcriptional regulator of GlcNAc catabolic pathway involved in pathogenesis(Elsevier B.V., 2020) Bhatt, Dharmendra Nath; Ansari, Sekhu; Kumar, Anil; Ghosh, Sumit; Narula, Alka; Datta, AsisAvailability and efficient utilization of host-derived nutrients by pathogens decide the fate of host-pathogen interaction. In Magnaporthe oryzae, N-acetylglucosamine (GlcNAc) catabolic pathway was found essential for successful host colonization and pathogenicity. GlcNAc catabolic enzymes hexokinase, GlcNAc-6-phosphate deacetylase (MoDac) and GlcN-6-phosphate deaminase (MoDeam) are encoded in a genomic cluster in M. oryzae and several phytopathogenic fungi. However, transcriptional regulation of GlcNAc catabolic pathway was not understood. We identified a conserved Ndt80/PhoG-like transcriptional regulator as a part of the GlcNAc catabolic gene cluster in M. oryzae and other fungi. We found that MoNdt80 is essential for GlcNAc utilization and pathogenicity of M. oryzae. Unlike WT, ΔMoNdt80 failed to induce transcription of GlcNAc catabolic pathway genes in response to GlcNAc. MoNdt80 could bind to a specific cis-acting consensus sequence GNCRCAAA[AT], present in the promoter of MoDac, MoDeam and β-hexosaminidase (MoHex). Further, comparative RNA-sequencing analysis using WT and ΔMoNdt80 revealed a large set of GlcNAc responsive genes that are under the transcriptional control of MoNdt80. These genes encoded GlcNAc catabolic enzymes, transporters and cell wall degrading enzymes which are required for hyphal growth expansion during host colonization. Overall, these results suggest MoNdt80 mediated transcriptional regulation of GlcNAc catabolic pathway is essential for successful host colonization and pathogenesis.Item Fruit ripening regulation of α-Mannosidase expression by the MADS box transcription factor RIPENING INHIBITOR and ethylene(Frontiers Media S.A., 2016) Irfan, Mohammad; Ghosh, Sumit; Meli, Vijaykumar S.; Kumar, Anil; Kumar, Vinay; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asisα-Mannosidase (α-Man), a fruit ripening-specific N-glycan processing enzyme, is involved in ripening-associated fruit softening process. However, the regulation of fruit-ripening specific expression of α-Man is not well understood. We have identified and functionally characterized the promoter of tomato (Solanum lycopersicum) α-Man to provide molecular insights into its transcriptional regulation during fruit ripening. Fruit ripening-specific activation of the α-Man promoter was revealed by analysing promoter driven expression of beta-glucuronidase (GUS) reporter in transgenic tomato. We found that RIPENING INHIBITOR (RIN), a MADS box family transcription factor acts as positive transcriptional regulator of α-Man during fruit ripening. RIN directly bound to the α-Man promoter sequence and promoter activation/α-Man expression was compromised in rin mutant fruit. Deletion analysis revealed that a promoter fragment (567 bp upstream of translational start site) that contained three CArG boxes (binding sites for RIN) was sufficient to drive GUS expression in fruits. In addition, α-Man expression was down-regulated in fruits of Nr mutant which is impaired in ethylene perception and promoter activation/α-Man expression was induced in wild type following treatment with a precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC). Although, α-Man expression was induced in rin mutant after ACC treatment, the transcript level was less as compared to ACC-treated wild type. Taken together, these results suggest RIN-mediated direct transcriptional regulation of α-Man during fruit ripening and ethylene may acts in RIN-dependent and -independent ways to regulate α-Man expression.Item Magnaporthe oryzae aminosugar metabolism is essential for successful host colonization(John Wiley & Sons, 2016) Kumar, Anil; Ghosh, Sumit; Bhatt, Dharmendra Nath; Narula, Alka; Datta, AsisPathogens encounter and metabolize a range of host-derived metabolites while proliferating inside the host. Our understanding of these metabolites and their metabolic processes has remained largely incomplete. We investigated the role of the Magnaporthe oryzae N-acetylglucosamine (GlcNAc) catabolic pathway during rice infection. The catabolic pathway is composed of a GlcNAc transporter (MoNgt1), hexokinase(s), a GlcNAc-6-phosphate deacetylase (MoDac) and a GlcN-6-phosphate deaminase (MoDeam). A detailed characterization of the Δmongt1, Δmodac and Δmodeam null mutants revealed that a defect in GlcNAc catabolism impairs the pathogenicity of M. oryzae. These mutants showed severely reduced virulence in susceptible rice cultivar due to their inability to neutralize host-derived reactive oxygen species and their failure to develop invasive hyphal growth within the host tissue. Interestingly, during oxidative stress, M. oryzae proliferated efficiently in GlcNAc-containing media compared to other sugars, and the expression of fungal antioxidant genes was up-regulated following GlcNAc treatment. However, GlcNAc inhibited the growth of the Δmodac and Δmodeam mutants, and this growth inhibition was enhanced during oxidative stress. These results suggest that GlcNAc helps fungus to overcome oxidative stress inside its host, perhaps by activating an anti-oxidant defence. In the absence of a functional catabolic pathway, GlcNAc becomes toxic to the cells.Item Induction of senescence and identification of differentially expressed genes in tomato in response to monoterpene(PLOS, 2013) Ghosh, Sumit; Singh, Upendra Kumar; Meli, Vijaykumar S.; Kumar, Vinay; Kumar, Anil; Irfan, Mohammad; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, AsisMonoterpenes, which are among the major components of plant essential oils, are known for their ecological roles as well for pharmaceutical properties. Geraniol, an acyclic monoterpene induces cell cycle arrest and apoptosis/senescence in various cancer cells and plants; however, the genes involved in the process and the underlying molecular mechanisms are not well understood. In this study, we demonstrate that treatment of tomato plants with geraniol results in induction of senescence due to a substantial alteration in transcriptome. We have identified several geraniol-responsive protein encoding genes in tomato using suppression subtractive hybridization (SSH) approach. These genes comprise of various components of signal transduction, cellular metabolism, reactive oxygen species (ROS), ethylene signalling, apoptosis and DNA damage response. Upregulation of NADPH oxidase and antioxidant genes, and increase in ROS level after geraniol treatment point towards the involvement of ROS in geraniol-mediated senescence. The delayed onset of seedling death and induced expression of geraniol-responsive genes in geraniol-treated ethylene receptor mutant (Nr) suggest that geraniol-mediated senescence involves both ethylene dependent and independent pathways. Moreover, expression analysis during tomato ripening revealed that geraniol-responsive genes are also associated with the natural organ senescence process.Item A physiologically regulated multidomain cystatin of wheat shows stage-dependent immunity against karnal bunt (Tilletia indica)(Springer, 2012) Purwar, Shalini; Sundaram, Shanthy; Verma, Praveen K.; Srivastava, Shaili; Kumar, AnilTo identify novel components of basal resistance against the Tellitia indica of wheat, breeding for disease resistance was carried out on resistant and susceptible genotype of Karnal Bunt. The different members of wheat cystatin gene families were cloned, and their role in triggering differential resistance through co-expression was analyzed in our lab. The multidomain wheat cystatin (WCM) is a proteinase inhibitor characterized by cloning the gene from susceptible (WH542) and resistant genotype (HD 29). A WCM cDNA was isolated from both genotypes and sequenced. The WCM had a highly conserved N-terminal cystatin domain and a long C-terminal extension containing a second region, which exhibited similarity to the cystatin domain. The expression level was significantly (P > 0.001) higher in resistant compared to susceptible genotype at all the physiological stages of wheat spikes. In order to characterize the biochemical properties of WCM, the coding sequence was expressed in Escherichia coli using pET expression vector. The recombinant WCM was purified from soluble fraction of the cell extract by using affinity chromatography. WCM, with 23 KDa molecular mass, showed cysteine proteinase inhibitory activity against papain (Ki 3.039 × 10−7 M) as determined by using BAPNA as substrate. Furthermore, it was able to arrest the fungal mycelial growth of T. indica. Hyphae growth was inhibited, and morphological changes such as swelling and fragmentation of the fungus were observed. Overall, these observations suggest an endogenous high expression of cystatin, possibly associated with the resistance of wheat against Karnal bunt.Item The N-glycan processing enzymes {alpha}-mannosidase and {beta}-D-N-acetylhexosaminidase are involved in ripening-associated softening in the non-climacteric fruits of Capsicum(Oxford University Press, 2011) Ghosh, Sumit; Meli, Vijaykumar S.; Kumar, Anil; Thakur, Archana; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, AsisExcessive softening of fruits during the ripening process leads to deterioration. This is of significant global importance as softening-mediated deterioration leads to huge postharvest losses. N-glycan processing enzymes are reported to play an important role during climacteric fruit softening: however, to date these enzymes have not been characterized in non-climacteric fruit. Two ripening-specific N-glycan processing enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex), have been identified and targeted to enhance the shelf life in non-climacteric fruits such as capsicum (Capsicum annuum). The purification, cloning, and functional characterization of α-Man and β-Hex from capsicum, which belong to glycosyl hydrolase (GH) families 38 and 20, respectively, are described here. α-Man and β-Hex are cell wall glycoproteins that are able to cleave terminal α-mannose and β-D-N-acetylglucosamine residues of N-glycans, respectively. α-Man and β-Hex transcripts as well as enzyme activity increase with the ripening and/or softening of capsicum. The function of α-Man and β-Hex in capsicum softening is investigated through RNA interference (RNAi) in fruits. α-Man and β-Hex RNAi fruits were approximately two times firmer compared with the control and fruit deterioration was delayed by approximately 7 d. It is shown that silencing of α-Man and β-Hex enhances fruit shelf life due to the reduced degradation of N-glycoproteins which resulted in delayed softening. Altogether, the results provide evidence for the involvement of N-glycan processing in non-climacteric fruit softening. In conclusion, genetic engineering of N-glycan processing can be a common strategy in both climacteric and non-climacteric species to reduce the post-harvest crop losses.Item Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum(Springer, 2009) Mishra, Raghvendra Kumar; Chaudhary, Swati; Kumar, Anil; Kumar, SushilIn order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.Item Mapping of the multifoliate pinna (mfp) leaf-blade morphology mutation in grain pea Pisum sativum(Indian Academy of Sciences, 2009) Mishra, Raghvendra Kumar; Kumar, Anil; Chaudhary, Swati; Kumar, SushilThe multifoliate pinna (mfp) mutation alters the leaf-blade architecture of pea, such that simple tendril pinnae of distal domain are replaced by compound pinna blades of tendrilled leaflets in mfp homozygotes. The MFP locus was mapped with reference to DNA markers using F2 and F2:5 RIL as mapping populations. Among 205 RAPD, 27 ISSR and 35 SSR markers that demonstrated polymorphism between the parents of mapping populations, three RAPD markers were found linked to the MFP locus by bulk segregant analyses on mfp/mfp and MFP/MFP bulks assembled from the F2:5 population. The segregational analysis of mfp and 267 DNA markers on 96 F2 plants allowed placement of 26 DNA markers with reference to MFP on a linkage group. The existence of common markers on reference genetic maps and MFP linkage group developed here showed that MFP is located on linkage group IV of the consensus genetic map of pea.Item Regulation of stipule development by COCHLEATA and STIPULE-REDUCED genes in pea Pisum sativum(Springer, 2009) Kumar, Sushil; Mishra, Raghvendra Kumar; Kumar, Anil; Srivastava, Suchi; Chaudhary, SwatiPisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.