Abstract
Multigene family pathogenesis-related-10 (PR-10) proteins are indispensable for initiation of plant defense reactions upon pathogen attack. Here, we report the isolation and differential induction of Cicer arietinum L. ABR18 (CaABR18) gene in susceptible and resistant chickpea upon exposure to Fusarium oxysporum f. sp. ciceri Race1 (Foc1). Further, sequence analysis and structural studies confirmed that CaABR18 protein possesses conserved glycine-rich P-loop motif and Betv 1 domain, which are common to many PR-10 family proteins. CaABR18 gene was found to be expressed in all the developing organs, with higher abundance in the mature leaves. Foc1 inoculation resulted in higher expression of CaABR18 gene in the resistant chickpea compared with susceptible one. CaABR18 protein induction was also observed by salicylic acid (SA) or abscisic acid (ABA) treatment. Biochemical analysis was performed using in vitro purified histidine-tagged recombinant ABR18 protein. Purified recombinant protein exhibits in vitro RNase and DNase activities. Application of recombinant ABR18 protein increases PI/SYTOX green uptake and nuclear disintegration and suppresses the growth of Foc1 hyphae in vitro. Agrobacterium-mediated transient expression of ABR18-YFP triggers reactive oxygen species (ROS) formation and cell death in Nicotiana benthamiana leaves. The fusion protein is shown to be targeted to the host nucleus. Taken together, our results revealed that CaABR18 imparts Fusarium resistance in chickpea by RNA/DNA degradation within host cells leading to programmed cell death (PCD) and also shows antifungal activity through its proper internalization, increasing membrane permeability and nuclear disintegration of Foc1.
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References
Agrawal GK, Jwa NS, Han KS, Agrawal VP, Rakwal R (2003) Isolation of a novel rice PR4 type gene whose mRNA expression is modulated by blast pathogen attack and signaling components. Plant Physiol Biochem 41(1):81–90. https://doi.org/10.1016/S0981-9428(02)00012-8
Bahramnejad B, Goodwin PH, Zhang J, Atnaseo C, Erickson LR (2010) A comparison of two class 10 pathogenesis-related genes from alfalfa and their activation by multiple stresses and stress-related signaling molecules. Plant Cell Rep 29:1235–1250. https://doi.org/10.1007/s00299-010-0909-6
Banerjee N, Sengupta S, Roy A, Ghosh P, Das K, Das S (2011) Functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status. PLoS One 6(4):e18593. https://doi.org/10.1371/journal.pone.0018593
Bhar A, Chatterjee M, Gupta S, Das S (2018) Salicylic acid regulates systemic defense signaling in chickpea during Fusarium oxysporum f. sp. ciceri Race1 infection. Plant Mol Biol Report 36:162–175. https://doi.org/10.1007/s11105-018-1067-1
Bowles DJ (1990) Defense-related proteins in higher plants. Annu Rev Biochem 59:873–907. https://doi.org/10.1146/annurev.bi.59.070190.004301
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Breiteneder H, Pettenburger K, Bito A, Valenta R, Kraft D, Rumpold H, Scheiner O, Breitenbach M (1989) The gene coding for the major birch pollen allergen Betv1, is highly homologous to a pea disease resistance response gene. EMBO J 8:1935–1938
Chadha P, Das RH (2006) A pathogenesis related protein, AhPR10b from peanut: an insight of its mode of antifungal activity. Planta 225:213–222. https://doi.org/10.1007/s00425-006-0344-7
Chatterjee M, Gupta S, Bhar A, Chakraborti D, Basu D, Das S (2014) Analysis of root proteome unravels differential molecular responses during compatible and incompatible interaction between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp. ciceri Race1 (Foc1). BMC Genomics 15(1):949. https://doi.org/10.1186/1471-2164-15-949
Choi D, Hwang BK (2011) Proteomics and functional analyses of pepper abscisic acid–responsive 1 (ABR1), which is involved in cell death and defense signaling. Plant Cell 23:823–842. https://doi.org/10.1105/tpc.110.082081
Choi SD, Hwang SI, Hwang KB (2012) Requirement of the cytosolic interaction between pathogenesis-related protein 10 and leucine-rich repeat protein 1 for cell death and defense signaling in pepper. Plant Cell 24:1675–1690. https://doi.org/10.1105/tpc.112.095869
Colditz F, Braun HP, Jacquet C, Niehaus K, Krajinski F (2005) Proteomic profiling unravels insights into the molecular background underlying increased Aphanomyces euteiches-tolerance of Medicago truncatula. Plant Mol Biol 59:387–406. https://doi.org/10.1007/s11103-005-0184-z
Coll NS, Epple P, Dangl JL (2011) Programmed cell death in the plant immune system. Cell Death Differ 18:1247–1256. https://doi.org/10.1038/cdd.2011.37
Darvill AG, Albersheim P (1984) Phytoalexins and their elicitors-a defense against microbial infection in plants. Annu Rev Plant Physiol 35:243–275. https://doi.org/10.1146/annurev.pp.35.060184.001331
Flors V, Ton J, Doorn RV, Jakab G, Garcia-Agustin P, Mauch-Mani B (2008) Interplay between JA, SA and ABA signalling during basal and induced resistance against Pseudomonas syringae and Alternaria brassicicola. Plant J 54:81–92. https://doi.org/10.1111/j.1365-313X.2007.03397.x
Garg R, Sahoo A, Tyagi AK, Jain M (2010) Validation of internal control genes for quantitative gene expression studies in chickpea (Cicer arietinum L.). Biochem Biophys Res Commun 396:283–288. https://doi.org/10.1016/j.bbrc.2010.04.079
Ghosh P, Roy A, Hess D, Ghosh A, Das S (2015) Deciphering the mode of action of a mutant Allium sativum Leaf Agglutinin (mASAL), a potent antifungal protein on Rhizoctonia solani. BMC Microbiol 15(237):237. https://doi.org/10.1186/s12866-015-0549-7
Gupta S, Chakraborti D, Rangi RK, Basu D, Das S (2009) A molecular insight into the early events of chickpea (Cicer arietinum) and Fusarium oxysporum f. sp. ciceri (Race1) interaction through cDNA-AFLP analysis. Phytopathology 99:1245–1257. https://doi.org/10.1094/PHYTO-99-11-1245
Gupta S, Chakraborti D, Sengupta A, Basu D, Das S (2010) Primary metabolism of chickpea is the initial target of wound inducing early sensed Fusarium oxysporum f. sp. ciceri race 1. PLoS One 5:e9030. https://doi.org/10.1371/journal.pone.0009030
Gupta S, Bhar A, Chatterjee M, Das S (2013) Fusarium oxysporum f. sp. ciceri race 1 induced redox state alterations are coupled to downstream defense signaling in root tissues of chickpea (Cicer arietinum L.). PLoS One 8(9):e73163. https://doi.org/10.1371/journal.pone.0073163
Gupta S, Bhar A, Chatterjee M, Ghosh A, Das S (2017) Transcriptomic dissection reveals wide spread differential expression in chickpea during early time points of Fusarium oxysporum f. sp. ciceri race 1 attack. PLoS One 12(5):e0178164. https://doi.org/10.1371/journal.pone.0178164
He MY, Xu Y, Cao JL, Zhu ZG, Jiao YT, Wang Y, Guan X, Yang Y, Xu W, Fu Z (2013) Subcellular localization and functional analyses of a PR10 protein gene from Vitis pseudoreticulata in response to Plasmopara viticola infection. Protoplasma 250:129–140. https://doi.org/10.1007/s00709-012-0384-8
Jimenez-Díaz RM, Castillo P, Jimenez-Gasco MM, Landa BB, Navas-Cortes JA (2015) Fusarium wilt of chickpeas: biology, ecology and management. Crop Prot 73:16–27. https://doi.org/10.1016/j.cropro.2015.02.023
Kim SG, Kim ST, Wang Y, Yu S, Choi IS, Kim YC, Kim WT, Agrawal GK, Rakwal R, Kang KY (2011) The RNase activity of rice probenazole-induced protein1 (PBZ1) plays a key role in cell death in plants. Mol Cells 31:25–31. https://doi.org/10.1007/s10059-011-0004-z
Liu JJ, Ekramoddoullah AKM (2006) The family 10 of plant pathogenesis-related proteins: their structure, regulation, and function in response to biotic and abiotic stresses. Physiol Mol Plant Pathol 68:3–13. https://doi.org/10.1016/j.pmpp.2006.06.004
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔct method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lo SC, Hipskind JD, Nicholson RL (1999) cDNA cloning of a sorghum pathogenesis-related protein (PR-10) and differential expression of defense-related genes following inoculation with Cochliobolus heterostrophus or Colletotrichum sublineolum. Mol Plant-Microbe Interact 12:479–489. https://doi.org/10.1094/MPMI.1999.12.6.479
Lusso M, Kuc J (1995) Increased activities of ribonuclease and protease after challenge in tobacco plants with induced systemic resistance. Physiol Mol Plant Pathol 47:419–428. https://doi.org/10.1006/pmpp.1995.1069
Mackey D, Holt BF, Wiig A, Dangl JL (2002) RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 108:743–754
Mauch F, Mauch-Mani B, Boller T (1988) Antifungal hydrolases in pea tissue. Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. Plant Physiol 88:936–942
McGee JD, Hamer JE, Hodges TK (2001) Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthe grisea. Mol Plant-Microbe Interact 14:877–886. https://doi.org/10.1094/MPMI.2001.14.7.877
Mogensen JE, Wimmer R, Larsen JN, Spangfort MD, Otzen DE (2002) The major birch allergen, Betv1, shows affinity for abroad spectrum of physiological ligands. J Biol Chem 277:23684–23692. https://doi.org/10.1074/jbc.M202065200
Mohammadi M, Anoop V, Gleddie S, Harris LJ (2011) Proteomic profiling of two maize inbreeds during early gibberella ear rot infection. Proteomics 11:3675–3684. https://doi.org/10.1002/pmic.201100177
Park CB, Kim HS, Kim SC (1998) Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochem Biophys Res Commun 244:253–257. https://doi.org/10.1006/bbrc.1998.8159
Park SW, Stevens NM, Vivanco JM (2002) Enzymatic specificity of three ribosome in activating proteins against fungal ribosome, and correlation with antifungal activity. Planta 216:227–234. https://doi.org/10.1007/s00425-002-0851-0
Park CJ, Kim KJ, Shin R, Park JM, Shin YC, Paek KH (2004) Pathogenesis-related protein 10 isolated from hot pepper functions as a ribonuclease in an antiviral pathway. Plant J 37:186–198. https://doi.org/10.1046/j.1365-313X.2003.01951
Puhringer H, Moll D, Hoffmann-Sommergruber K, Watillon B, Katinger H, Machado MLD (2000) The promoter of an apple Ypr10 gene, encoding the major allergen Mald 1, is stress- and pathogen inducible. Plant Sci 152:35–50. https://doi.org/10.1016/S0168-9452(99)00222-8
Rakwal R, Agrawal GK, Yonekura M (2001) Light dependent induction of OsPR10 in rice (Oryza sativa L.) seedlings by the global stress signaling molecule jasmonic acid and protein phosphatase2A inhibitors. Plant Sci 161:477–487. https://doi.org/10.1016/S0168-9452(01)00433-2
Robert N, Ferran J, Breda C, Coutos-Thevenot P, Boulay M et al (2001) Molecular characterization of the incompatible interaction of Vitis vinifera leaves with Pseudomonas syringae pv pisi: expression of genes coding for stilbene synthase and class 10 PR protein. Eur J Plant Pathol 107:249–261
Sen S, Chakraborty J, Ghosh P, Basu D, Das S (2017) Chickpea WRKY70 regulates the expression of a homeodomain-leucine zipper (HD-zip) I transcription factor CaHDZ12, which confers abiotic stress tolerance in transgenic tobacco and chickpea. Plant Cell Physiol 58(11):1934–1952. https://doi.org/10.1093/pcp/pcx126
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599. https://doi.org/10.1093/molbev/msm092
Tomar PPS, Kumar N, Singh A, Selvakumar P, Roy P, Sharma AK (2014) Characterization of anticancer, DNase and antifungal activity of pumpkin 2S albumin. Biochem Biophys Res Comm 448:349–354. https://doi.org/10.1016/j.bbrc.2014.04.158
Troskie AM, de Beer A, Vosloo JA, Jacobs K, Rautenbach M (2014) Inhibition of agronomically relevant fungal phytopathogens by tyrocidines, cyclic antimicrobial peptides isolated from Bacillus aneurinolyticus. Microbiology 160:2089–2101. https://doi.org/10.1099/mic.0.078840-0
Van Loon LC, Van Stien EA (1999) The families of pathogenesis related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol Mol Plant Pathol 55:85–97. https://doi.org/10.1006/pmpp.1999.0213
Walter MH, Liu JW, Wunn J, Hess D (1996) Bean ribonuclease-like pathogenesis-related protein genes (Ypr10) display complex patterns of developmental, dark-induced and exogenous-stimulus-dependent expression. Eur J Biochem 239:281–293
Wang L, Wei J, Zou Y, Xu K, Wang Y, Cui L, Xu Y (2014) Molecular characteristics and biochemical functions of VpPR10s from Vitis pseudoreticulata associated with biotic and abiotic stresses. Int J Mol Sci 15:19162–19182. https://doi.org/10.3390/ijms151019162
Wu F, Yan M, Li Y, Chang S, Song X, Zhou Z, Gong W (2003) cDNA cloning, expression, and mutagenesis of a PR-10 protein SPE-16 from the seeds of Pachyrrhizus erosus. Biochem Biophys Res Commun 312:761–766. https://doi.org/10.1016/j.bbrc.2003.10.181
Xu K, Huang X, Wu M, Wang Y, Chang Y, Liu K, Zhang J, Zhang Y, Zhang F, Yi L, Li T, Wang R, Tan G, Li C (2014a) A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis. PLoS One 9:e83556. https://doi.org/10.1371/journal.pone.0083556
Xu P, Jiang L, Wu J, Li W, Fan S, Zhang S (2014b) Isolation and characterization of a pathogenesis-related protein 10 gene (GmPR10) with induced expression in soybean (Glycine max) during infection with Phytophthora sojae. Mol Biol Rep 41:4899–4909. https://doi.org/10.1007/s11033-014-3356-6
Xu FT, Zhao CX, Jiao TY, Wei YJ, Wang L, Xu Y (2014c) A pathogenesis related protein, VpPR-10.1, from Vitis pseudoreticulata: an insight of its mode of antifungal activity. PLoS One 9(4):e95102. https://doi.org/10.1371/journal.pone.0095102
Yang C, Liang S, Wang H, Han L, Wang F, Cheng H, Wu X, Qu Z, Wu J, Xia G (2015) Cotton major latex protein 28 functions as a positive regulator of the ethylene responsive factor 6 in defense against Verticillium dahlia. Mol Plant 8:399–411. https://doi.org/10.1016/j.molp.2014.11.023
Acknowledgments
Authors are indebted to the Director, Bose Institute, for providing infrastructural facilities. SD acknowledges Indian National Science Academy for providing Senior Scientist Fellowship. MC and JC are thankful to Bose Institute for financial assistance. Authors are thankful to Dr. Suresh C Pande (ICRISAT, Patancheru), Dr. Kiran Kumar Sharma, and Dr. Pooja Bhatnagar (ICRISAT, Patancheru) for providing fungal culture and chickpea seeds. Special thanks are offered to Prof. Shubho Chaudhuri for providing pEarlyGate 101 vector. Sincere thanks are extended to Mr. Swarnava Das for his help during laboratory experiments. Authors also thank Mr. Surojit Maity and Mr. Sudipta Basu for technical support.
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MC and SD conceived the research idea and designed the experiments. MC and JC performed the experiments. MC, JC, and SD analyzed data and wrote the manuscript. All authors have read and approved the manuscript.
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Chickpea ABR18 protein demonstrates RNase/DNase functions, cell death, and antifungal activity that suppresses the growth of the Fusarium oxysporum f. sp. ciceri Race1 hyphae.
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Chatterjee, M., Chakraborty, J. & Das, S. Abscisic Acid–Responsive 18 (CaABR18) Protein from Chickpea Inhibits the Growth of the Wilt-Causing Fusarium oxysporum f. sp. ciceri Race1. Plant Mol Biol Rep 37, 170–185 (2019). https://doi.org/10.1007/s11105-019-01146-5
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DOI: https://doi.org/10.1007/s11105-019-01146-5