Manasi Mishra

Interaction of recombinant CanPIs with Helicoverpa armigera gut proteases reveals their processing patterns, stability and efficiency.

Six diverse representative Capsicum annuum (common name: hot pepper; Solanaceae) protease inhibitor genes, viz CanPI‐5, ‐7, ‐13, ‐15, ‐19, and 22 comprising 1–4 inhibitory repeat domains (IRDs), were cloned and expressed in Pichia pastoris. The recombinant proteins were evaluated for their interactions with bovine trypsin, chymotrypsin, and Helicoverpa armigera gut proteases (HGP) using electrophoretic (native and denaturing) and mass spectrometric (MALDI‐TOF‐MS in combination with intensity fading assays) techniques. These techniques allow qualitative and semiquantitative analysis of multiple and processed IRDs of purified recombinant Capsicum annuum proteinase inhibitor (rCanPI) proteins. rCanPIs showed over 90% trypsin inhibition, varying chymotrypsin inhibition depending on the number of respective IRDs and over 60% inhibition of total HGP. rCanPI‐15 that has only one IRD showed exceptionally low inhibition of these proteases. Interaction studies of rCanPIs with proteases using intensity fading‐MALDI‐TOF‐MS revealed gradual processing of multi‐IRD rCanPIs into single IRD forms by the action of HGP at the linker region, unlike their interactions with trypsin and chymotrypsin. Intensity fading‐MALDI‐TOF‐MS assay showed that CanPI‐13 and ‐15, possessing single IRD and expressed predominantly in stem tissue are degraded by HGP; indicating their function other than defense. In vitro and in vivo studies on rCanPI‐5 and ‐7 showed maximum inhibition of HGP isoforms and their processed IRDs were also found to be stable in the presence of HGP. Even single amino acid variations in IRDs were found to change the HGP specificity like in the case of HGP‐8 inhibited only by IRD‐12. The presence of active PI in insect gut might be responsible for changed HGP profile. rCanPI‐5 and ‐7 enhanced HGP‐7, reduced HGP‐4, ‐5, ‐10 expression and new protease isoforms were induced. These results signify isoform complexity in plant PIs and insect proteases.

Complementation of intramolecular interactions for structural–functional stability of plant serine proteinase inhibitors

BACKGROUND Plant protease inhibitors (PIs) constitute a diverse group of proteins capable of inhibiting proteases. Among PIs, serine PIs (SPIs) display stability and conformational restrictions of the reactive site loop by virtue of their compact size, and by the presence of disulfide bonds, hydrogen bonds, and other weak interactions. SCOPE OF REVIEW The significance of various intramolecular interactions contributing to protein folding mechanism and their role in overall stability and activity of SPIs is discussed here. Furthermore, we have reviewed the effect of variation or manipulation of these interactions on the activity/stability of SPIs. MAJOR CONCLUSIONS The selective gain or loss of disulfide bond(s) in SPIs can be associated with their functional differentiation, which is likely to be compensated by non-covalent interactions (hydrogen bonding or electrostatic interactions). Thus, these intramolecular interactions are collectively responsible for the functional activity of SPIs, through the maintenance of scaffold framework, conformational rigidity and shape complementarities of reactive site loop. GENERAL SIGNIFICANCE Structural insight of these interactions will provide an in-depth understanding of kinetic and thermodynamic parameters involved in the folding and stability mechanisms of SPIs. These features can be explored for engineering canonical SPIs for optimizing their overall stability and functionality for various applications.

Stress inducible proteinase inhibitor diversity in Capsicum annuum

BackgroundWound-inducible Pin-II Proteinase inhibitors (PIs) are one of the important plant serine PIs which have been studied extensively for their structural and functional diversity and relevance in plant defense against insect pests. To explore the functional specialization of an array of Capsicum annuum (L.) proteinase inhibitor (CanPIs) genes, we studied their expression, processing and tissue-specific distribution under steady-state and induced conditions. Inductions were performed by subjecting C. annuum leaves to various treatments, namely aphid infestation or mechanical wounding followed by treatment with either oral secretion (OS) of Helicoverpa armigera or water.ResultsThe elicitation treatments regulated the accumulation of CanPIs corresponding to 4-, 3-, and 2-inhibitory repeat domains (IRDs). Fourty seven different CanPI genes composed of 28 unique IRDs were identified in total along with those reported earlier. The CanPI gene pool either from uninduced or induced leaves was dominated by 3-IRD PIs and trypsin inhibitory domains. Also a major contribution by 4-IRD CanPI genes possessing trypsin and chymotrypsin inhibitor domains was specifically revealed in wounded leaves treated with OS. Wounding displayed the highest number of unique CanPIs while wounding with OS treatment resulted in the high accumulation of specifically CanPI-4, -7 and −10. Characterization of the PI protein activity through two dimensional gel electrophoresis revealed tissue and induction specific patterns. Consistent with transcript abundance, wound plus OS or water treated C. annuum leaves exhibited significantly higher PI activity and isoform diversity contributed by 3- and 4-IRD CanPIs. CanPI accumulation and activity was weakly elicited by aphid infestation yet resulted in the higher expression of CanPI-26, -41 and −43.ConclusionsPlants can differentially perceive various kinds of insect attacks and respond appropriately through activating plant defenses including regulation of PIs at transcriptional and post-translational levels. Based on the differentially elicited CanPI accumulation patterns, it is intriguing to speculate that generating sequence diversity in the form of multi-IRD PIs is a part of elaborative plant defense strategy to obtain a diverse pool of functional units to confine insect attack.

Plasticity of protease gene expression in Helicoverpa armigera upon exposure to multi-domain Capsicum annuum protease inhibitor

BACKGROUND A multi-domain Pin-II type protease inhibitor from Capsicum annuum (CanPI-7) is known to be effective against the insect pest, Helicoverpa armigera. The present study is an attempt to investigate the optimal dose of recombinant CanPI-7 (rCanPI-7) for effective antibiosis to H. armigera and further to characterize the responses of digestive proteases upon rCanPI-7 ingestion. METHODS The gut protease activity was assessed biochemically and transcript accumulation pattern for selected trypsin and chymotrypsin genes was analyzed by quantitative Real-Time PCR. RESULTS The growth retardation upon exposure to rCanPI-7 was more prominent in neonates as compared to third instar larvae. Influence of stage and dosage of rCanPI-7 was conspicuous on the expression and regulation of candidate trypsin and chymotrypsin genes in H. armigera. The transcript accumulation pattern correlated with the protease activity in rCanPI-7 exposed larvae. CONCLUSIONS We conclude that early exposure and specific dose of protease inhibitor are essential for effective antibiosis despite the large diversity and plasticity in the expression of protease genes in H. armigera. Moreover, it is also evident that the regulation and expression of H. armigera gut proteases are specific to the stage of PI exposure. GENERAL SIGNIFICANCE These results highlight the requirement of optimal PI concentration for effective growth retardation and for inhibiting the major gut proteases of H. armigera.

The remarkable efficiency of a Pin-II proteinase inhibitor sans two conserved disulfide bonds is due to enhanced flexibility and hydrogen bond density in the reactive site loop

Capsicum annuum (L.) expresses diverse potato type II family proteinase inhibitors comprising of inhibitory repeat domain (IRD) as basic functional unit. Most IRDs contain eight conserved cysteines forming four disulfide bonds, which are indispensible for their stability and activity. We investigated the functional significance of evolutionary variations in IRDs and their role in mediating interaction between the inhibitor and cognate proteinase. Among the 18 IRDs encoded by C. annuum, IRD-7, -9, and -12 were selected for further characterization on the basis of variation in their reactive site loop, number of conserved cysteine residues, and higher theoretical ΔGbind for interaction with Helicoverpa armigera trypsin. Moreover, inhibition kinetics showed that IRD-9, despite loss of some of the disulfide bonds, was a more potent proteinase inhibitor among the three selected IRDs. Molecular dynamic simulations revealed that serine residues in the place of cysteines at seventh and eighth positions of IRD-9 resulted in an increase in the density of intramolecular hydrogen bonds and reactive site loop flexibility. Results of the serine residues chemical modification also supported this observation and provided a possible explanation for the remarkable inhibitory potential of IRD-9. Furthermore, this natural variant among IRDs showed special attributes like stability to proteolysis and synergistic inhibitory effect on other IRDs. It is likely that IRDs have coevolved selective specialization of their structure and function as a response towards specific insect proteases they encountered. Understanding the molecular mechanism of pest protease–plant proteinaceous inhibitor interaction will help in developing effective pest control strategies. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:39

Stress inducible proteomic changes in Capsicum annuum leaves

Herbivore attack induces defense responses in plants, activating several signaling cascades. As a result, molecules deterrent to the herbivores are produced and accumulated in plants. Expression of defense mechanism/traits requires reorganization of the plant metabolism, redirecting the resources otherwise meant for growth. In the present work, protein profile of Capsicum annuum leaves was examined after herbivore attack/induction. Majority of proteins identified as differentially accumulated, were having roles in redox metabolism and photosynthesis. For example, superoxide dismutase and NADP oxidoreductase were upregulated by 10- and 6-fold while carbonic anhydrase and fructose-1,6-bisphosphatase were downregulated by 9- and 4-fold, respectively. Also, superoxide dismutase, NADPH quinone oxidoreductase and NADP dependent isocitrate dehydrogenase transcripts showed a higher accumulation in induced leaf tissues at early time points. In general, proteins having role in defense and damage repair were upregulated while those involved in photosynthesis appeared downregulated. Thus metabolic reconfiguration to balance defense and tolerance was evident in the stress-induced leaves.

Ecological turmoil in evolutionary dynamics of plant–insect interactions: defense to offence

AbstractMain conclusionAvailable history manifests contemporary diversity that exists in plant-insect interactions. A radical thinking is necessary for developing strategies that can co-opt natural insect-plant mutualism, ecology and environmental safety for crop protection since current agricultural practices can reduce species richness and evenness. The global environmental changes, such as increased temperature, CO2and ozone levels, biological invasions, land-use change and habitat fragmentation together play a significant role in re-shaping the plant-insect multi-trophic interactions. Diverse natural products need to be studied and explored for their biological functions as insect pest control agents. In order to assure the success of an integrated pest management strategy, human activities need to be harmonized to minimize the global climate changes. Plant–insect interaction is one of the most primitive and co-evolved associations, often influenced by surrounding changes. In this review, we account the persistence and evolution of plant–insect interactions, with particular focus on the effect of climate change and human interference on these interactions. Plants and insects have been maintaining their existence through a mutual service-resource relationship while defending themselves. We provide a comprehensive catalog of various defense strategies employed by the plants and/or insects. Furthermore, several important factors such as accelerated diversification, imbalance in the mutualism, and chemical arms race between plants and insects as indirect consequences of human practices are highlighted. Inappropriate implementation of several modern agricultural practices has resulted in (i) endangered mutualisms, (ii) pest status and resistance in insects and (iii) ecological instability. Moreover, altered environmental conditions eventually triggered the resetting of plant–insect interactions. Hence, multitrophic approaches that can harmonize human activities and minimize their interference in native plant–insect interactions are needed to maintain natural balance between the existence of plants and insects.

Structural-functional insights of single and multi-domain Capsicum annuum protease inhibitors.

Pin-II protease inhibitors (PIs) are the focus of research interest because of their large structural-functional diversity and relevance in plant defense. Two representative Capsicum annuum PI genes (CanPI-15 and -7) comprising one and four inhibitory repeat domains, respectively, were expressed and recombinant proteins were characterized. β-Sheet and unordered structure was found predominant in CanPI-15 while -7 also displayed the signatures of polyproline fold, as revealed by circular dichroism studies. Inhibition kinetics against bovine trypsin indicated three times higher potency of CanPI-7 (K(i)~57 μM) than -15 (~184 μM). Activity and structural stability of these CanPIs were revealed under various conditions of pH, temperature and denaturing agent. Structure prediction, docking studies with proteases and mass spectroscopy revealed the organization of multiple reactive site loops of multi domain PIs in space as well as the steric hindrances imposed while binding to proteases due to their close proximity.