Immunomodulatory Potential of Non-Classical HLA-G in Infections including COVID-19 and Parasitic Diseases.

Human Leukocyte Antigen-G (HLA-G), a polymorphic non-classical HLA (HLA-Ib) with immune-regulatory properties in cancers and infectious diseases, presents both membrane-bound and soluble (sHLA-G) isoforms. Polymorphism has implications in host responses to pathogen infections and in pathogenesis. Differential expression patterns of HLA-G/sHLA-G or its polymorphism seem to be related to different pathological conditions, potentially acting as a disease progression biomarker. Pathogen antigens might be involved in the regulation of both membrane-bound and sHLA-G levels and impact immune responses during co-infections. The upregulation of HLA-G in viral and bacterial infections induce tolerance to infection. Recently, sHLA-G was found useful to identify the prognosis of Coronavirus disease 2019 (COVID-19) among patients and it was observed that the high levels of sHLA-G are associated with worse prognosis. The use of pathogens, such as Plasmodium falciparum, as immune modulators for other infections could be extended for the modulation of membrane-bound HLA-G in COVID-19-infected tissues. Overall, such information might open new avenues concerning the effect of some pathogens such as parasites in decreasing the expression level of HLA-G to restrict pathogenesis in some infections or to influence the immune responses after vaccination among others.

Cyanide produced with ethylene by ACS and its incomplete detoxification by ß-CAS in mango inflorescence leads to malformation.

Malformation of mango inflorescences (MMI) disease causes severe economic losses worldwide. Present research investigates the underlying causes of MMI. Results revealed significantly higher levels of cyanide, a by-product of ethylene biosynthesis, in malformed inflorescences (MI) of mango cultivars. There was a significant rise in ACS transcripts, ACS enzyme activity and cyanide and ethylene levels in MI as compared to healthy inflorescences (HI). Significant differences in levels of methionine, phosphate, S-adenosyl-L-methionine, S-adenosyl-L-homocysteine, ascorbate and glutathione, and activities of dehydroascorbate reductase and glutathione reductase were seen in MI over HI. Further, a lower expression of ß-cyanoalanine synthase (ß-CAS) transcript was associated with decreased cellular ß-CAS activity in MI, indicating accumulation of unmetabolized cyanide. TEM studies showed increased gum-resinosis and necrotic cell organelles, which might be attributed to unmetabolized cyanide. In field trials, increased malformed-necrotic-inflorescence (MNI) by spraying ethrel and decreased MNI by treating with ethylene inhibitors (silver and cobalt ions) further confirmed the involvement of cyanide in MMI. Implying a role for cyanide in MMI at the physiological and molecular level, this study will contribute to better understanding of the etiology of mango inflorescence malformation, and also help manipulate mango varieties genetically for resistance to malformation.

Plasmodium falciparum specific helicase 2 is a dual, bipolar helicase and is crucial for parasite growth.

Human malaria infection is a major challenge across the globe and is responsible for millions of deaths annually. Rapidly emerging drug resistant strains against the new class of anti-malarial drugs are major threat to control the disease burden worldwide. Helicases are present in every organism and have important role in various nucleic acid metabolic processes. Previously we have reported the presence of three parasite specific helicases (PSH) in Plasmodium falciparum 3D7 strain. Here we present the detailed biochemical characterization of PfPSH2. PfPSH2 is DNA and RNA stimulated ATPase and is able to unwind partially duplex DNA and RNA substrates. It can translocate in both 3' to 5' and 5' to 3' directions. PfPSH2 is expressed in all the stages of intraerythrocytic development and it is localized in cytoplasm in P. falciparum 3D7 strain. The dsRNA mediated inhibition study suggests that PfPSH2 is important for the growth and survival of the parasite. This study presents the detailed characterization of PfPSH2 and lays the foundation for future development of PfPSH2 as drug target.

Stress-induced Oryza sativa RuvBL1a is DNA-independent ATPase and unwinds DNA duplex in 3' to 5' direction.

RuvB, a member of AAA+ (ATPases Associated with diverse cellular Activities) superfamily of proteins, is essential, highly conserved and multifunctional in nature as it is involved in DNA damage repair, mitotic assembly, switching of histone variants and assembly of telomerase core complex. RuvB family is widely studied in various systems such as Escherichia coli, yeast, human, Drosophila, Plasmodium falciparum and mouse, but not well studied in plants. We have studied the transcript level of rice homologue of RuvB gene (OsRuvBL1a) under various abiotic stress conditions, and the results suggest that it is upregulated under salinity, cold and heat stress. Therefore, the OsRuvBL1a protein was characterized using in silico and biochemical approaches. In silico study confirmed the presence of all the four characteristic motifs of AAA+ superfamily-Walker A, Walker B, Sensor I and Sensor II. Structurally, OsRuvBL1a is similar to RuvB1 from Chaetomium thermophilum. The purified recombinant OsRuvBL1a protein shows unique DNA-independent ATPase activity. Using site-directed mutagenesis, the importance of two conserved motifs (Walker B and Sensor I) in ATPase activity has been also reported with mutants D302N and N332H. The OsRuvBL1a protein unwinds the duplex DNA in the 3' to 5' direction. The presence of unique DNA-independent ATPase and DNA unwinding activities of OsRuvBL1a protein and upregulation of its transcript under abiotic stress conditions suggest its involvement in multiple cellular pathways. The first detailed characterization of plant RuvBL1a in this study may provide important contribution in exploiting the role of RuvB for developing the stress tolerant plants of agricultural importance.

Simultaneous Expression of PDH45 with EPSPS Gene Improves Salinity and Herbicide Tolerance in Transgenic Tobacco Plants.

To cope with the problem of salinity- and weed-induced crop losses, a multi-stress tolerant trait is need of the hour but a combinatorial view of such traits is not yet explored. The overexpression of PDH45 (pea DNA helicase 45) and EPSPS (5-enoylpruvyl shikimate-3-phosphate synthase) genes have been reported to impart salinity and herbicide tolerance. Further, the understanding of mechanism and pathways utilized by PDH45 and EPSPS for salinity and herbicide tolerance will help to improve the crops of economical importance. In the present study, we have performed a comparative analysis of salinity and herbicide tolerance to check the biochemical parameters and antioxidant status of tobacco transgenic plants. Collectively, the results showed that PDH45 overexpressing transgenic lines display efficient tolerance to salinity stress, while PDH45+EPSPS transgenics showed tolerance to both the salinity and herbicide as compared to the control [wild type (WT) and vector control (VC)] plants. The activities of the components of enzymatic antioxidant machinery were observed to be higher in the transgenic plants indicating the presence of an efficient antioxidant defense system which helps to cope with the stress-induced oxidative-damages. Photosynthetic parameters also showed significant increase in PDH45 and PDH45+EPSPS overexpressing transgenic plants in comparison to WT, VC and EPSPS transgenic plants under salinity stress. Furthermore, PDH45 and PDH45+EPSPS synergistically modulate the jasmonic acid and salicylic acid mediated signaling pathways for combating salinity stress. The findings of our study suggest that pyramiding of the PDH45 gene with EPSPS gene renders host plants tolerant to salinity and herbicide by enhancing the antioxidant machinery thus photosynthesis.

Genome Wide In silico Analysis of the Mismatch Repair Components of Plasmodium falciparum and Their Comparison with Human Host.

Malaria a major parasitic infection globally particularly in tropical and sub-tropical regions of the world is responsible for about 198 million cases and estimated deaths due to this disease are about 0.6 million. The emergence of drug resistance in the malaria parasite is alarming and it is necessary to understand its underlying cause and molecular mechanisms. It has been established that drug resistant malaria parasites have defective mismatch repair (MMR) therefore it is essential to study this pathway and its components in detail. Recently a number of non-synonymous Single Nucleotide Polymorphisms have been reported in genes involved in MMR pathways. PfMLH is an endonuclease essential to restore the MMR in drug resistant strains of Plasmodium falciparum. Considering all these facts about the role of MMR in emergence of drug resistant parasite, in this manuscript we report a genome wide analysis of the components of the MMR pathway such as MLH, Pms1, MSH2-1, MSH2-2, MSH6, and UvrD using in silico bioinformatics based approaches. The phylogenetic analysis revealed evolutionary closeness with the MMR components of various organisms. It is noteworthy that P. falciparum contains two homologs of MSH2, which are located on different chromosomes. The structural modeling of these components showed their similarity with the human/yeast MMR components. The docking studies reveal that PfUvrD and PfMLH interact with each other. The in silico identification of interacting partners of the major MMR components identified numerous P. falciparum specific proteins. In line with our previous studies the present study will also contribute significantly to understand the MMR pathway of malaria parasite.

ATPase activity of Plasmodium falciparum MLH is inhibited by DNA-interacting ligands and dsRNAs of MLH along with UvrD curtail malaria parasite growth.

Malaria caused by Plasmodium falciparum is the major disease burden all over the world. Recently, the situation has deteriorated because the malarial parasites are becoming progressively more resistant to numerous commonly used antimalarial drugs. Thus, there is a critical requirement to find other means to restrict and eliminate malaria. The mismatch repair (MMR) machinery of parasite is quite unique in several ways, and it can be exploited for finding new drug targets. MutL homolog (MLH) is one of the major components of MMR machinery, and along with UvrD, it helps in unwinding the DNA. We have screened several DNA-interacting ligands for their effect on intrinsic ATPase activity of PfMLH protein. This screening suggested that several ligands such as daunorubicin, etoposide, ethidium bromide, netropsin, and nogalamycin are inhibitors of the ATPase activity of PfMLH, and their apparent IC50 values range from 2.1 to 9.35 µM. In the presence of nogalamycin and netropsin, the effect was significant because in their presence, the V max value dropped from 1.024 µM of hydrolyzed ATP/min to 0.596 and 0.643 µM of hydrolyzed ATP/min, respectively. The effect of double-stranded RNAs of PfMLH and PfUvrD on growth of P. falciparum 3D7 strain was studied. The parasite growth was significantly inhibited suggesting that these components belonging to MMR pathway are crucial for the survival of the parasite.

Plasmodium falciparum Bloom homologue, a nucleocytoplasmic protein, translocates in 3' to 5' direction and is essential for parasite growth.

Malaria caused by Plasmodium, particularly Plasmodium falciparum, is the most serious and widespread parasitic disease of humans. RecQ helicase family members are essential in homologous recombination-based error-free DNA repair processes in all domains of life. RecQ helicases present in each organism differ and several homologues have been identified in various multicellular organisms. These proteins are involved in various pathways of DNA metabolism by providing duplex unwinding function. Five members of RecQ family are present in Homo sapiens but P. falciparum contains only two members of this family. Here we report the detailed biochemical and functional characterization of the Bloom (Blm) homologue (PfBlm) from P. falciparum 3D7 strain. Purified PfBlm exhibits ATPase and 3' to 5' direction specific DNA helicase activity. The calculated average reaction rate of ATPase was ~13 pmol of ATP hydrolyzed/min/pmol of enzyme. The immunofluorescence assay results show that PfBlm is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain. In some stages of development in addition to nucleus PfBlm also localizes in the cytoplasm. The gene disruption studies of PfBlm by dsRNA showed that it is required for the ex-vivo intraerythrocytic development of the parasite P. falciparum 3D7 strain. The dsRNA mediated inhibition of parasite growth suggests that a variety of pathways are affected resulting in curtailing of the parasite growth. This study will be helpful in unravelling the basic mechanism of DNA transaction in the malaria parasite and additionally it may provide leads to understand the parasite specific characteristics of this protein.

Ectopic expression of phloem motor protein pea forisome PsSEO-F1 enhances salinity stress tolerance in tobacco.

KEY MESSAGE: PsSEOF-1 binds to calcium and its expression is upregulated by salinity treatment. PsSEOF - 1 -overexpressing transgenic tobacco showed enhanced salinity stress tolerance by maintaining cellular ion homeostasis and modulating ROS-scavenging pathway. Calcium (Ca(2+)) plays important role in growth, development and stress tolerance in plants. Cellular Ca(2+) homeostasis is achieved by the collective action of channels, pumps, antiporters and by Ca(2+) chelators present in the cell like calcium-binding proteins. Forisomes are ATP-independent mechanically active motor proteins known to function in wound sealing of injured sieve elements of phloem tissue. The Ca(2+)-binding activity of forisome and its role in abiotic stress signaling were largely unknown. Here we report the Ca(2+)-binding activity of pea forisome (PsSEO-F1) and its novel function in promoting salinity tolerance in transgenic tobacco. Native PsSEO-F1 promoter positively responded in salinity stress as confirmed using GUS reporter. Overexpression of PsSEO-F1 tobacco plants confers salinity tolerance by alleviating ionic toxicity and increased ROS scavenging activity which probably results in reduced membrane damage and improved yield under salinity stress. Evaluation of several physiological indices shows an increase in relative water content, electrolyte leakage, proline accumulation and chlorophyll content in transgenic lines as compared with null-segregant control. Expression of several genes involved in cellular homeostasis is perturbed by PsSEO-F1 overexpression. These findings suggest that PsSEO-F1 provides salinity tolerance through cellular Ca(2+) homeostasis which in turn modulates ROS machinery providing indirect link between Ca(2+) and ROS signaling under salinity-induced perturbation. PsSEO-F1 most likely functions in salinity stress tolerance by improving antioxidant machinery and mitigating ion toxicity in transgenic lines. This finding should make an important contribution in our better understanding of the significance of calcium signaling in phloem tissue leading to salinity stress tolerance.

Plasmodium falciparum Werner homologue is a nuclear protein and its biochemical activities reside in the N-terminal region.

RecQ helicases, also addressed as a gatekeeper of genome, are an inevitable family of genome scrutiny proteins conserved from prokaryotes to eukaryotes and play a vital role in DNA metabolism. The deficiencies of three RecQ proteins out of five are involved in genetic abnormalities like Bloom syndrome (BS), Werner syndrome (WS), and Rothmund-Thomson syndrome (RTS). It is noteworthy that Plasmodium falciparum contains only two members of the RecQ family as opposed to five members present in the host Homo sapiens. In the present study, we report the biochemical characterization of the homologue of Werner (Wrn) helicase from P. falciparum 3D7 strain. Although there are significant sequence conservations between Wrn helicases of both H. sapiens and P. falciparum as well as among all the other Plasmodium species, they contain some peculiar differences also. In silico studies reveal that PfWrn is evolutionarily close to the bacterial RecQ protein. The N-terminal fragment (PfWrnN) contains all the helicase motifs along with all the functional domains and the predicted structure resembles with the human RecQ1 protein, whereas the C-terminal fragment (PfWrnC) contains no significant domain. Biochemical characterization further revealed that purified recombinant PfWrnN shows ATPase and DNA helicase activity in 3' to 5' direction, but PfWrnC lacks the ATPase and helicase activities. Immunofluorescence study shows that PfWrn is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain and localizes distinctly in the nucleus. This study can be used for further characterization of RecQ helicases that will aid in understanding the physiological significance of these helicases in the malaria parasite.

Plasmodium falciparum XPD translocates in 5' to 3' direction, is expressed throughout the blood stages, and interacts with p44.

XPD helicase, a TFIIH subunit, is essential for several processes including transcription, NER, cell cycle regulation, and apoptosis in eukaryotes. Another component of TFIIH, namely p44, is among the well-known interacting partners of XPD and is vital in regulating the helicase activities of latter. However, none of the above mentioned proteins have been functionally characterized in Plasmodium falciparum. Consequently, in this study, we performed detailed studies on XPD and its interacting partner, p44, from P. falciparum 3D7 strain. Accordingly, we expressed and purified recombinant PfXPD and its fragments and Pfp44 proteins and characterized the enzymatic activities of PfXPD and its fragments. The in vivo stage-specific expression and subcellular localizations of PfXPD and Pfp44 proteins were studied using the specific antibodies in the intraerythrocytic developmental stages of P. falciparum 3D7 strain. Our results suggest that PfXPD displays the characteristic ssDNA-dependent ATPase and 5'-3' DNA helicase activities. We also report the existence of two high molecular weight forms of p44 in P. falciparum 3D7 strain. Both PfXPD and Pfp44 colocalize in the nucleus and interact with each other, which suggest that they are most likely components of the same complex apparently, TFIIH. Furthermore, during trophozoite and schizont stages, both proteins exhibit a distinct cytoplasmic distribution pattern which implies that PfXPD and Pfp44 might also be involved in other functions. These studies will aid in understanding the basic biology of malaria parasite.

Identification of inhibitors of Plasmodium falciparum RuvB1 helicase using biochemical assays.

Human malaria is a major parasitic infection, and the situation has worsened mainly due to the emergence of resistant malaria parasites to several anti-malarial drugs. Thus, an urgent need to find suitable drug targets has led to the development of newer classes of anti-malarial drugs. Helicases have been targeted to develop therapeutics for viral, bacterial, and other microorganism infections. Recently, Plasmodium falciparum RuvB ATPases/helicases have been characterized and proposed as a suitable antimalarial drug target. In the present study, the screening of various compounds was done and the results suggest that PfRuvB1 ATPase activity is inhibited considerably by the novobiocin and partially by cisplatin and ciprofloxacin. Helicase assay of PfRuvB1 in the presence of various compounds suggest novobiocin, actinomycin, and ethidium bromide as potent inhibitors. Novobiocin inhibits the helicase activity of PfRuvB1 possibly by blocking the ATPase activity of PfRuvB1. This study is unique in respect to the identification of novobiocin as inhibitor of PfRuvB1, partially by competing with ATP binding at its active site and provides evidence for PfRuvB1 as target of novobiocin after DNA gyrase-B and HSP90. These studies will certainly help the pharmacologist to design and develop some novel inhibitor specific to PfRuvB1, which may serve as suitable chemotherapeutics to target malaria.

Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses.

Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression.

Rice SUV3 is a bidirectional helicase that binds both DNA and RNA.

BACKGROUND: Helicases play crucial role in almost all the nucleic acid metabolism including replication, repair, recombination, transcription, translation, ribosome biogenesis and splicing and these processes regulate plant growth and development. It is suggested that helicases play essential roles in stabilizing growth in plants under stress because their presence in the stress-induced ORFs has been identified. Moreover in a recent study we have reported that SUV3 helicase from Oryza sativa (OsSUV3) functions in salinity stress tolerance in transgenic rice by improving the antioxidant machinery. SUV3 helicase has been identified and characterized from yeast and human systems but the properties and functions of plant SUV3 are poorly understood. RESULTS: In this study, the purification and extensive characterization of recombinant OsSUV3 protein (67 kDa) is presented. OsSUV3 binds to DNA and RNA and exhibits DNA as well as RNA-dependent ATPase activities. It also contains the characteristic DNA and RNA helicase activity. OsSUV3 can use mainly ATP or dATP as energy source for the unwinding activity and it cannot unwind the blunt-end duplex DNA substrate. It is interesting to note that OsSUV3 unwinds DNA in both the 5'-3' and 3'-5 directions and thus its activity is bipolar in vitro. The Km values of OsSUV3 are 0.51 nM and 0.95 nM for DNA helicase and RNA helicase, respectively. CONCLUSIONS: This study is the first direct evidence to show the bipolar DNA helicase activity of OsSUV3 protein. The unique properties of OsSUV3 including its dual helicase activity imply that it could be a multifunctional protein involved in biologically significant process of DNA and RNA metabolisms. These results should make significant contribution towards better understanding of SUV3 protein in plants.

OsACA6, a P-type 2B Ca(2+) ATPase functions in cadmium stress tolerance in tobacco by reducing the oxidative stress load.

MAIN CONCLUSION: The present study demonstrates the first direct evidence of the novel role of OsACA6 in providing Cd (2+) stress tolerance in transgenic tobacco by maintaining cellular ion homeostasis and modulating ROS-scavenging pathway. Cadmium, a non-essential toxic heavy metal, interferes with the plant growth and development. It reaches the leaves through xylem and may become part of the food chain, thus causing detrimental effects to human health. Therefore, there is an urgent need to develop strategies for engineering plants for Cd(2+) tolerance and less accumulation. The members of P-type ATPases family transport metal ions including Cd(2+), and thus play important role an ion homeostasis. The present study elucidates the role of P-type 2B Ca(2+) ATPase (OsACA6) in Cd(2+) stress tolerance. The transcript levels of OsACA6 were up-regulated upon Cd(2+), Zn(2+) and Mn(2+) exposure. Transgenic tobacco expressing OsACA6 showed tolerance towards Cd(2+) stress as demonstrated by several physiological indices including root length, biomass, chlorophyll, malondialdehyde and hydrogen peroxide content. The roots of the transgenic lines accumulated more Cd(2+) as compared to shoot. Further, confocal laser scanning microscopy showed that Cd(2+) exposure altered Ca(2+) uptake in OsACA6 transgenic plants. OsACA6 expression in tobacco also protected the transgenic plants from oxidative stress by enhancing the activity of enzymatic (SOD, CAT, APX, GR) and non-enzymatic (GSH and AsA) antioxidant machinery. Transgenic lines also tolerated Zn(2+) and Mn(2+) stress; however, tolerance for these ions was not as significant as observed for Cd(2+) exposure. Thus, overexpression of OsACA6 confers Cd(2+) stress tolerance in transgenic lines by maintaining cellular ion homeostasis and modulating reactive oxygen species (ROS)-scavenging pathway. The results of the present study will help to develop strategies for engineering Cd(2+) stress tolerance in economically important crop plants.

Salinity and drought tolerant OsACA6 enhances cold tolerance in transgenic tobacco by interacting with stress-inducible proteins.

Plant Ca(2+)ATPases regulate many signalling pathways which are important for plant growth, development and abiotic stress responses. Our previous work identified that overexpression of OsACA6 confers salinity and drought tolerance in tobacco. In the present work we report, the function of OsACA6 in cold stress tolerance in transgenic tobacco plants. The expression of OsACA6 was induced by cold stress. The promoter-GUS fusion analyses in the different tissues of transgenic tobacoco confirmed that OsACA6 promoter is cold stress-inducible. Transgenic tobacco plants overexpressing OsACA6 exhibited cold tolerance compared to the wild type (WT) controls. The enhanced tolerance was confirmed by phenotypic analyses as well as by measuring germination, survival rate, chlorophyll content, cell membrane stability, malondialdehyde and proline content. Compared to the WT, the expression of catalase, ascorbate peroxidase and superoxide dismutase increased in the OsACA6 overexpressing plants, which was inversely correlated with the levels of H2O2 in the transgenic lines. We also identified interacting proteins of OsACA6 by using yeast two-hybrid screening assay. Most of the interacting partners of OsACA6 are associated with the widespread biological processes including plant growth, development, signalling and stress adaptation. Furthermore, we also confirmed that OsACA6 is able to self-interact. Overall, these results suggest that OsACA6 plays an important role in cold tolerance at least in part, by regulating antioxidants-mediated removal of reactive oxygen species or by interacting with different calcium signal decoders including calmodulin-like proteins (CaM) calcium/calmodulin dependent protein kinases (CDPKs) and receptor-like protein kinases (RLKs).

Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery.

BACKGROUND: The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum) and its novel function in salinity stress tolerance in plant. RESULTS: The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS) accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities. CONCLUSIONS: To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth.

BACKGROUND: Human malaria parasite infection and its control is a global challenge which is responsible for ~0.65 million deaths every year globally. The emergence of drug resistant malaria parasite is another challenge to fight with malaria. Enormous efforts are being made to identify suitable drug targets in order to develop newer classes of drug. Helicases play crucial roles in DNA metabolism and have been proposed as therapeutic targets for cancer therapy as well as viral and parasitic infections. Genome wide analysis revealed that Plasmodium falciparum possesses UvrD helicase, which is absent in the human host. RESULTS: Recently the biochemical characterization of P. falciparum UvrD helicase revealed that N-terminal UvrD (PfUDN) hydrolyses ATP, translocates in 3' to 5' direction and interacts with MLH to modulate each other's activity. In this follow up study, further characterization of P. falciparum UvrD helicase is presented. Here, we screened the effect of various DNA interacting compounds on the ATPase and helicase activity of PfUDN. This study resulted into the identification of daunorubicin (daunomycin), netropsin, nogalamycin, and ethidium bromide as the potential inhibitor molecules for the biochemical activities of PfUDN with IC50 values ranging from ~3.0 to ~5.0 µM. Interestingly etoposide did not inhibit the ATPase activity but considerable inhibition of unwinding activity was observed at 20 µM. Further study for analyzing the importance of PfUvrD enzyme in parasite growth revealed that PfUvrD is crucial/important for its growth ex-vivo. CONCLUSIONS: As PfUvrD is absent in human hence on the basis of this study we propose PfUvrD as suitable drug target to control malaria. Some of the PfUvrD inhibitors identified in the present study can be utilized to further design novel and specific inhibitor molecules.

Genetically engineered synthetic miniaturized versions of Plasmodium falciparum UvrD helicase are catalytically active.

Helicases catalyze unwinding of double stranded nucleic acids in an energy-dependent manner. We have reported characterization of UvrD helicase from Plasmodium falciparum. We reported that the N-terminal and C-terminal fragments of PfUvrD contain characteristic ATPase and DNA helicase activities. Here we report the generation and characterization of a genetically engineered version of PfUvrD and its derivatives. This synthetic UvrD (sUD) contains all the conserved domains of PfUvrD but only the intervening linker sequences are shortened. sUD (∼ 45 kDa) and one of its smallest derivative sUDN1N2 (∼ 22 kDa) contain ATPase and DNA helicase activities. sUD and sUDN1N2 can utilize hydrolysis of all the NTPs and dNTPs, can also unwind blunt end duplex DNA substrate and unwind DNA duplex in 3 to 5 direction only. Some of the properties of sUD are similar to the PfUvrD helicase. Mutagenesis in the conserved motif Ia indicate that the mutants sUDM and sUDN1N2M lose all the enzyme activities, which further confirms that these activities are intrinsic to the synthesized proteins. These studies show that for helicase activity only the conserved domains are essentially required and intervening sequences have almost no role. These observations will aid in understanding the unwinding mechanism by a helicase.

Structure of RNA-interacting cyclophilin A-like protein from Piriformospora indica that provides salinity-stress tolerance in plants.

Soil salinity problems are widespread around the globe with increased risk of spreading over the years. The fungus Piriformospora indica, identified in Indian Thar desert, colonizes the roots of monocotyledon plants and provides resistance towards biotic as well as abiotic stress conditions. We have identified a cyclophilin A-like protein from P. indica (PiCypA), which shows higher expression levels during salinity stress. The transgenic tobacco plants overexpressing PiCypA develop osmotic tolerance and exhibit normal growth under osmotic stress conditions. The crystal structure and NMR spectroscopy of PiCypA show a canonical cyclophilin like fold exhibiting a novel RNA binding activity. The RNA binding activity of the protein and identification of the key residues involved in the RNA recognition is unique for this class of protein. Here, we demonstrate for the first time a direct evidence of countering osmotic stress tolerance in plant by genetic modification using a P. indica gene.