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Item Saccharomyces cerevisiae polo-like kinase, Cdc5 exhibits ATP-dependent Mg2+ -enhanced kinase activity in vitro(Elsevier B.V., 2019) Chauhan, Sujata; Samanta, Subhasis; Sharma, Nitin; Thakur, Jitendra K.; Dev, Kamal; Sourirajan, AnuradhaPhosphorylation of proteins on serine/threonine residues represents an important biochemical mechanism to regulate several cellular processes. Polo-like kinases (PLKs) are a family of serine-threonine kinases that play an imminent role in cell cycle regulation in yeast to humans, and thus an important therapeutic target for cancers. The present study provides insights into the enzymatic features of Saccharomyces cerevisiae PLK, Cdc5 using in vitro casein phosphorylation assays. The recombinant yeast PLK, GST-Cdc5 showed maximum casein phosphorylation activity at 30 °C, pH 9 and 45 min of incubation period. GST-Cdc5 exhibited a KM of 1.35 μM for casein, and high affinity for ATP, since addition of non-radioactive ATP chased out casein phosphorylation by radiolabeled ATP. The recombinant enzyme showed maximum kinase activity at 2.7 μM of GST-Cdc5. Casein was found to be the best in vitro substrate of GST-Cdc5 followed by BSA (Bovine Serum Albumin) and MBP (Myelin Basic Protein). Of the metal ions tested, Mg2+ (at 20 mM) was found to enhance GST-Cdc5 kinase activity, while Ca2+ (at 5 mM) and Mn2+ (at 10 mM) inhibited the same. The presence of EDTA, SDS and PMSF inhibited phosphorylation by GST-Cdc5, while DTT had no effect. The recombinant GST-Cdc5 can be used as a tool for deciphering PLKs’ structure and functions, which are still at infancy.Item Production and cytological characterization of a synthetic amphiploid derived from a cross between Oryza sativa and Oryza punctata(NRC Research Press, 2019) Kumar, Kishor; Neelam, Kumari; Singh, Gurpreet; Mathan, Jyotirmaya; Ranjan, Aashish; Brar, Darshan Singh; Singh, KuldeepOryza punctata Kotschy ex Steud. (BB, 2n = 24) is a wild species of rice that has many useful agronomic traits. An interspecific hybrid (AB, 2n = 24) was produced by crossing O. punctata and Oryza sativa variety Punjab Rice 122 (PR122, AA, 2n = 24) to broaden the narrow genetic base of cultivated rice. Cytological analysis of the pollen mother cells (PMCs) of the interspecific hybrids confirmed that they have 24 chromosomes. The F1 hybrids showed the presence of 19–20 univalents and 1–3 bivalents. The interspecific hybrid was treated with colchicine to produce a synthetic amphiploid (AABB, 2n = 48). Pollen fertility of the synthetic amphiploid was found to be greater than 50% and partial seed set was observed. Chromosome numbers in the PMCs of the synthetic amphiploid were 24II, showing normal pairing. Flow cytometric analysis also confirmed doubled genomic content in the synthetic amphiploid. Leaf morphological and anatomical studies of the synthetic amphiploid showed higher chlorophyll content and enlarged bundle sheath cells as compared with both of its parents. The synthetic amphiploid was backcrossed with PR122 to develop a series of addition and substitution lines for the transfer of useful genes from O. punctata with least linkage drag.Item Overexpression of OsPUB41, a rice E3 ubiquitin ligase induced by cell wall degrading enzymes, enhances immune responses in rice and Arabidopsis(BioMed Central Ltd, 2019) Kachewar, Neha Rajendra; Gupta, Vishal; Ranjan, Ashish; Patel, Hitendra Kumar; Sonti, Ramesh V.Background: Cell wall degrading enzymes (CWDEs) induce plant immune responses and E3 ubiquitin ligases are known to play important roles in regulating plant defenses. Expression of the rice E3 ubiquitin ligase, OsPUB41, is enhanced upon treatment of leaves with Xanthomonas oryzae pv. oryzae (Xoo) secreted CWDEs such as Cellulase and Lipase/Esterase. However, it is not reported to have a role in elicitation of immune responses. Results: Expression of the rice E3 ubiquitin ligase, OsPUB41, is induced when rice leaves are treated with either CWDEs, pathogen associated molecular patterns (PAMPs), damage associated molecular patterns (DAMPs) or pathogens. Overexpression of OsPUB41 leads to induction of callose deposition, enhanced tolerance to Xoo and Rhizoctonia solani infection in rice and Arabidopsis respectively. In rice, transient overexpression of OsPUB41 leads to enhanced expression of PR genes and SA as well as JA biosynthetic and response genes. However, in Arabidopsis, ectopic expression of OsPUB41 results in upregulation of only JA biosynthetic and response genes. Transient overexpression of either of the two biochemically inactive mutants (OsPUB41C40A and OsPUB41V51R) of OsPUB41 in rice and stable transgenics in Arabidopsis ectopically expressing OsPUB41C40A failed to elicit immune responses. This indicates that the E3 ligase activity of OsPUB41 protein is essential for induction of plant defense responses. Conclusion: The results presented here suggest that OsPUB41 is possibly involved in elicitation of CWDE triggered immune responses in rice.Item OsWAKL21, a putative receptor of rice cell wall damage activates alternate signaling in rice and Arabidopsis to induce immunity(American Phytopathological Society, 2019) Malukani, K. K.; Ranjan, A.; Hota, S. J.; Patel, H. K.; Sonti, Ramesh V.Xanthomonas oryzae pv. oryzae (Xoo) causes the serious bacterial blight disease of rice. As part of its virulence repertoire, Xoo secretes various cell wall degrading enzymes (CWDEs) such as cellulases, xylanases and a Lipase/esterase (LipA). Conversely, treatment of rice tissues with any of these purified enzymes activates immune responses. Plants sense this cell wall damage as a mark of infection and induce immune responses. Very little information is available about the plant functions that are involved in the elaboration of cell wall damage induced immune responses. Transcriptome analyses revealed a rice cell wall-associated receptor kinase, OsWAKL21 that is upregulated following treatment with either LipA or Xoo. VIGS mediated downregulation of OsWAKL21 attenuates LipA induced immune responses. Overexpression of OsWAKL21 in rice mimics LipA treatment in induction of immune responses, activation of JA pathway and enhanced expression of defence related genes, indicating that it plays an important role in elaboration of LipA induced immune responses. Ectopic expression of OsWAKL21 in Arabidopsis also activates plant immune responses. OsWAKL21 is a moonlighting kinase having in vitro kinase and guanylate cyclase activities. Interestingly, OsWAKL21 needs kinase activity to activate immune responses in rice while in Arabidopsis it needs the guanylate cyclase activity. Thus OsWAKL21 is activating similar immune responses in two different species but via different mechanisms.Item Phase variation in rice pathogen Xanthomonas oryzae pv. oryzae(American Phytopathological Society, 2019) Madhavan, V. N.; Patel, H. K.; Patil, P. B.; Sonti, Ramesh V.Xanthomonas oryzae pv. oryzae (Xoo) is the causal pathogen of bacterial blight of rice. Diverse factors contribute to its virulence, e.g., secreted enzymes, protein involved in adhesion, gene regulation. Two such studied virulence factors are Exopolysaccharide (EPS) and Lipopolysaccharide (LPS). LPS is a structural component of the outer membrane of all gram-negative bacteria. LPS is necessary for the function of many outer membrane proteins and protect the bacteria from antimicrobial plant products. EPS is a complex polymer secreted by Xanthomonas genus and the known functions of EPS include biofilm formation, and suppression of the plant immune responses. In this study, we isolated non-mucoid and virulence compromised Xoo colonies from stationary phase cultures. These spontaneous mutants are called as stationary phase variants (SPV). Our data show that, SPVs arises due to insertion of endogenous Insertion Sequence (IS) elements in EPS or LPS O-antigen biosynthetic clusters or by slipped-strand mispairing (SSM) in wxoA gene of LPS O-antigen biosynthetic cluster. The SPVs reverts to wild-type colony morphology and showed true reversion, i.e. restoration of wild-type genotype. The results suggest that we are observing phase variation in Xoo. We hypothesize that similar phase variation may be a part of Xoo’s life cycle. Which may help the bacteria survive in nutrient limited conditions during late stages of infection and on rice seeds and plant debris.Item Immune response induction in rice due to co-expression of XopQ and XopX(American Phytopathological Society, 2019) Deb, S.; Patel, H. K.; Sonti, Ramesh V.Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Xoo uses the type III secretion system (T3SS) to suppress rice immune responses. The T3SS secreted effectors XopQ and XopX suppress rice immune responses by interaction with different rice 14-3-3 proteins. Sub-cellular localisation of XopQ and XopX mutants that are defective in 14-3-3 binding and suppression of immune responses indicates that, for suppression, XopQ requires a cytoplasmic localisation whereas XopX requires nuclear localisation. Hence, both XopQ as well as XopX individually act as suppressors of rice immune responses, probably by targeting unique pathways in different subcellular compartments. However, we find that when XopQ is delivered through Agrobacterium along with XopX, it becomes an inducer of immune responses and that it now localizes in the nucleus. We also find that XopQ and XopX can interact with each other. This raises the possibility that besides being a suppressor of immune responses, XopQ can under certain circumstances also function as an inducer of immune responses.Item Understanding the molecular intricacies of rice-Rhizoctonia solani interactions(American Phytopathological Society, 2019) Ghosh, S.; Kanwar, P.; Jha, GopaljeeSheath blight disease is a devastating disease in rice that causes huge crop losses worldwide. It is caused by a necrotrophic fungus Rhizoctonia solani. In spite of global efforts till now there are no reports of complete resistance against this pathogen. We observed that R. solani has a brief biotrophic phase wherein mycelial grows parallel to rice veins without showing any morphological and anatomical changes. However, at later stage, i.e. necrotrophic phase, infection cushions along with anatomical changes are observed at the site of disease symptoms. Through transcriptome and metabolome studies we have identified several candidate host susceptibility factors and pathogenicity determinants that might play an important role during pathogenesis of R. solani in rice. Furthermore, using whole genome studies, we have identified several gene/gene families that might contribute to the aggressiveness of the R. solani strains. Although gene manipulation studies in R. solani is difficult we have devised ways wherein we can functionally characterize these pathogenicity determinants. Overall the present study will help in better understanding of the rice-R. solani pathosystem which would eventually be helpful in developing strategies for durable sheath blight disease tolerance in rice.Item Burkholderia gladioli strain NGJ1 deploys a prophage tail-like protein to feed on fungi(American Phytopathological Society, 2019) Swain, D.M.; Yadav, S.; Tyagi, I.; Kumar, R.; Kumar, R.; Ghosh, S.; Das, J.; Jha, GopaljeeFungal pathogens are responsible for approximately two third of the infectious plant diseases. Due to extensive usages, pathogens have evolved resistance against most of the commonly used fungicides. Rice sheath blight disease caused by Rhizoctonia solani remains a serious threat to global sustainable agriculture. The pathogen has a complex biology and has been reported occurring world-wide causing necrosis and dampingoff on numerous host plant species. The pathogen is polymorphic and despite extensive efforts, till today no source of complete disease resistance has been identified against this pathogen. We have recently discovered a novel bacterium Burkholderia gladioli strain NGJ1 which has broad spectrum mycophagus ability. We also demonstrated that the bacteria NGJ1 deploys a prophage tail like protein (Bg_9562) to feed over fungi in a T3SS dependant manner. Moreover we observed that Bg_9562 protein has a broad spectrum antifungal activity on Rhizoctonia solani as well as several other phytopathogenic fungi. Furthermore, being equipped to kill and feed upon fungi, the mycophagous bacteria can serve as an experimental tool box to facilitate discovery of novel anti-fungal molecules. This opens up a new biotechnological application of this prophage tail like protein in controlling fungal diseases in rice as well as in other plants.Item Physiological plasticity to high temperature stress in chickpea: Adaptive responses and variable tolerance(Elsevier B.V., 2019) Pareek, Akanksha; Rathi, Divya; Mishra, Divya; Chakraborty, Subhra; Chakraborty, NiranjanHigh temperature stress (HTS) is one of the most crucial factors that limits plant growth and development, and reduces crop yields worldwide. Cool-season crops, particularly the legumes, are severely affected by increasing ambient temperature associated with global climate change. We characterized the HTS-induced modulations of morpho-physicochemical traits and gene expression of several chickpea genotypes and the metabolic profile of the tolerant cultivar. Higher water use efficiency and photosynthetic capacity, minimal membrane lipid peroxidation in conjunction with increased abundance of osmolytes and secondary metabolites depicted thermotolerance of ICC 1205. The adaptive responses were accompanied by high transcript abundance of heat shock proteins and antioxidant enzymes. To integrate stress-responsive signalling and metabolic networks, the HTS-induced physicochemical analysis was further extended to metabolite profiling of the thermotolerant cultivar. The screening of the metabolome landscape led to the identification of 49 HTS-responsive metabolites that include polycarboxylic acid, sugar acids, sugar alcohols and amino acids which might confer thermotolerance in chickpea. The present study, to our knowledge, is the most comprehensive of its kind in dissecting cultivar-specific differential adaptive responses to HTS in chickpea, which might potentiate the identification of genetic traits extendible to improvement of thermotolerance of crops.Item The PHYTOGLOBIN-NO cycle regulates plant mycorrhizal symbiosis(Elsevier B.V., 2019) Kumari, Aprajita; Pathak, Pradeep Kumar; Loake, Gary J.; Gupta, Kapuganti JagadisThe production of the redox-active signaling molecule, NO, has long been associated with interactions between microbes and their host plants. Emerging evidence now suggests that specific NO signatures and cognate patterns of PHYTOGLOBIN1 (PHYTOGB1) expression, a key regulator of cellular NO homeostasis, may help determine either symbiosis or pathogenicity.