03016nas a2200277 4500008004100000022001400041245013800055210006900193260007700262300001400339490000700353520197800360653001002338653003002348653003402378653001102412653002102423653002902444100001902473700002802492700002102520700002502541700002102566700002002587856013102607 2010 eng d a1615-985300aInteraction of recombinant CanPIs with helicoverpa armigera gut proteases reveals their processing patterns, stability and efficiency0 aInteraction of recombinant CanPIs with helicoverpa armigera gut  aPO BOX 10 11 61, D-69451 WEINHEIM, GERMANYbWILEY-V C H VERLAG GMBHcJUL a2845-28570 v103 a

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.

10aCanPI10aH. armigera gut proteases10aIntensity fading MALDI-TOF-MS10aPin-II10aPlant proteomics10aPlant-insect interaction1 aMishra, Manasi1 aTamhane, Vaijayanti, A.1 aKhandelwal, Neha1 aKulkarni, Mahesh, J.1 aGupta, Vidya, S.1 aGiri, Ashok, P. uhttp://library.ncl.res.in/content/interaction-recombinant-canpis-helicoverpa-armigera-gut-proteases-reveals-their-processing-003295nas a2200277 4500008004100000022001400041245007100055210006900126260008100195300000800276490000700284520235500291653001002646653001402656653002002670653003802690653002902728100001902757700002202776700002802798700002502826700002102851700002102872700002002893856010402913 2012 eng d a1471-222900aStress inducible proteinase inhibitor diversity in capsicum annuum0 aStress inducible proteinase inhibitor diversity in capsicum annu a236 GRAYS INN RD, FLOOR 6, LONDON WC1X 8HL, ENGLANDbBIOMED CENTRAL LTDcNOV a2170 v123 a

Background: Wound-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. Results: The 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. Conclusions: Plants 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.

10aCanPI10aHerbivory10aOral secretions10aPin-II type proteinase inhibitors10aPlant-insect interaction1 aMishra, Manasi1 aMahajan, Neha, S.1 aTamhane, Vaijayanti, A.1 aKulkarni, Mahesh, J.1 aBaldwin, Ian, T.1 aGupta, Vidya, S.1 aGiri, Ashok, P. uhttp://library.ncl.res.in/content/stress-inducible-proteinase-inhibitor-diversity-capsicum-annuum-002053nas a2200253 4500008004100000022001400041245009800055210006900153260009500222300001400317490000800331520110600339653001001445653002301455653002201478653003901500653002201539100001901561700002201580700002501602700002101627700002001648856013101668 2013 eng d a0006-291X00aStructural-functional insights of single and multi-domain capsicum annuum protease inhibitors0 aStructuralfunctional insights of single and multidomain capsicum a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcJAN a1060-10650 v4303 a

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. beta-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 typsin indicated three times higher potency of CanPI-7 (K-i similar to 57 mu M) than -15 (similar to 184 mu M). 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. (C) 2012 Elsevier Inc. All rights reserved.

10aCanPI10aCircular dichroism10aDisulfide bonding10aPotato type-II protease inhibitors10aProtein stability1 aMishra, Manasi1 aJoshi, Rakesh, S.1 aGaikwad, Sushama, M.1 aGupta, Vidya, S.1 aGiri, Ashok, P. uhttp://library.ncl.res.in/content/structural-functional-insights-single-and-multi-domain-capsicum-annuum-protease-inhibitors-002855nas a2200277 4500008004100000022001400041245009000055210006900145260005700214300001200271490000800283520193300291653001002224653001302234653002202247653001302269653002502282100002202307700002002329700002802349700002202377700001902399700002102418700002002439856011802459 2015 eng d a0032-093500aStructural features of diverse Pin-II proteinase inhibitor genes from capsicum annuum0 aStructural features of diverse PinII proteinase inhibitor genes  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcFEB a319-3310 v2413 a

The proteinase inhibitor (PI) genes from Capsicum annuum were characterized with respect to their UTR, introns and promoter elements. The occurrence of PIs with circularly permuted domain organization was evident. Several potato inhibitor II (Pin-II) type proteinase inhibitor (PI) genes have been analyzed from Capsicum annuum (L.) with respect to their differential expression during plant defense response. However, complete gene characterization of any of these C. annuum PIs (CanPIs) has not been carried out so far. Complete gene architectures of a previously identified CanPI-7 (Beads-on-string, Type A) and a member of newly isolated Bracelet type B, CanPI-69 are reported in this study. The 5' UTR (untranslated region), 3'UTR, and intronic sequences of both the CanPI genes were obtained. The genomic sequence of CanPI-7 exhibited, exon 1 (49 base pair, bp) and exon 2 (740 bp) interrupted by a 294-bp long type I intron. We noted the occurrence of three multi-domain PIs (CanPI-69, 70, 71) with circularly permuted domain organization. CanPI-69 was found to possess exon 1 (49 bp), exon 2 (551 bp) and a 584-bp long type I intron. The upstream sequence analysis of CanPI-7 and CanPI-69 predicted various transcription factor-binding sites including TATA and CAAT boxes, hormone-responsive elements (ABRELATERD1, DOFCOREZM, ERELEE4), and a defense-responsive element (WRKY71OS). Binding of transcription factors such as zinc finger motif MADS-box and MYB to the promoter regions was confirmed using electrophoretic mobility shift assay followed by mass spectrometric identification. The 3' UTR analysis for 25 CanPI genes revealed unique/distinct 3' UTR sequence for each gene. Structures of three domain CanPIs of type A and B were predicted and further analyzed for their attributes. This investigation of CanPI gene architecture will enable the better understanding of the genetic elements present in CanPIs.

10aCanPI10aCapsicum10aGene architecture10aPromoter10aProteinase inhibitor1 aMahajan, Neha, S.1 aDewangan, Veena1 aLomate, Purushottam, R.1 aJoshi, Rakesh, S.1 aMishra, Manasi1 aGupta, Vidya, S.1 aGiri, Ashok, P. uhttp://library.ncl.res.in/content/structural-features-diverse-pin-ii-proteinase-inhibitor-genes-capsicum-annuum-0