Identification and Characterization of Defensin Genes in Brassica Juncea and Camelina Sativa and Analysis of Their Expression in Response to Biotic and Abiotic Stress

ACTA SCIENTIFIC AGRICULTURE (ISSN: 2581-365X) Volume 3 Issue 12 December 2019

Research Article

Brassica juncea Camelina sativa Identification and Characterization of Defensin Genes in and and Analysis of their Expression in Response to Biotic and Abiotic Stress Gamage Dona Gaya Chaturani, Zahoor Ahmad Mir, Prashant Mishra and Anita Grover* National Institute for Plant Biotechnology, New Delhi, India *Corresponding Author:

Received: AnitaPublished: Grover, National Institute for Plant Biotechnology, New Delhi, India DOI: October 28, 2019; November 29, 2019 10.31080/ASAG.2019.03.0732

Abstract

Plants are attacked by various biotic and abiotic factors throughout their lives and how efficient and effective the defense mechanisms are, determine the survival. Defensins, belong to Pathogen Related (PR) protein class-12. They are one of the key modulators in plant defense mechanisms which have diverse functions including inhibitory effect on a broad range of phyto- In-silico C. pathogenic fungi, and are involved in biotic stress response. In depth study on defensins gives an insight into their role in defense sativa B. juncea which further can be used in conferring disease resistance in crop plants. analysis identified 56 putative defensins in (resistant to Alternaria blight) whereas, only six were identified in (sueptible toAlternaria blight). Multiple sequence α β alignment and consensus analysis confirmed the conserved eight cysteine residues, and structural analysis revealed the presence of an -helix and triple strand anti parallel -sheets. Defensin genes identified were having a single intron, and identification of Methyle Brassica Arabidopsis Jasmonate (MeJA) responsive elements in promoter analysis confirm their regulation under Jasmonic Acid (JA) signalling pathway. Alternaria These defensins were phylogenetically related to other species as well as to indicating their close evolutionary relationship. Expression analysis indicated that defensin is responsive to JA, Salicylic Acid (SA), infection, and wounding. The antagonistic effect between SA and JA was observed in defensin gene expression in response to their exogenous application. This study will provide an important foundation for further investigation on defensin as a possible source for creating disease resistance Keywords:in transgenics. Defensin; Brassica; Camelina; JA; SA; Alternaria Blight

Introduction

necessities of adopting biotechnological strategies to improve re- B. juncea Alternaria sistance against this pathogen. Biotechnological approaches can Alternaria blight of cruciferous vegetables, incited by different A. brassicae is successfully be utilized to develop resistant variety of species of , remains an increasing threat to Brassica- [37] A. brassicae provided that molecular mechanism of defense is delineated. ceae crops throughout the world. In oil seed rape, Brassica jun- the dominant invasive species . , is one of the most [14] cea The molecular mechanisms underlying activation of plant de- common and destructive disease of Indian mustard ( [32] fense responses are exceedingly complex and understand- ) and the yield losses have been estimated to range from 35 to B. juncea A. brassicae ing how plants defend themselves against a range of pathogens 46% . Conventional breeding to develop resistant cultivars in is therefore of crucial importance for successful agriculture [1]. against is confounded due to non-availability Once plant defense responses are activated at the site of infection, of suitable resistant sources within the available germplasm of A. brassicae a systemic defense is often triggered in distal plant parts to pro- cultivated species of Brassica [35], though some varieties differ in [40] tect these undamaged tissues against subsequent invasion by the their resistance level. A high degree of resistance to [44] Camelina sativa [21] pathogen . Systemic defense is controlled by a complex signal has been found in the wild relatives of Brassica outside the tribe [19] transduction network that coordinate the overall defense re- Brassicaceae including . Thereby, there are sponses of the plant . Resistance to biotrophic pathogens is

Citation: ., et al. Brassica juncea Camelina sativa Acta Scientific Agriculture Anita Grover “Identification and Characterization of Defensin Genes in and and Analysis of their Expression in Response to Biotic and Abiotic Stress”. 3.12 (2019): 96-112. Identification and Characterization of Defensin Genes in Brassica juncea and Camelina sativa and Analysis of their Expression in Response to Biotic and Abiotic Stress

[40] [41] mediated through phyto-hormones such as salicylic acid (SA) characteristics of defensins from above two genotypes remain to [28] B. juncea and necrotrophic pathogens by jasmonic acid (JA) and ethylene be unclear. Rawat isolated the full-length pathogen-inducible (ET)-signaling pathways . [29] plant defensin gene from and have reported its similar- [09] ity with gamma thionin and knottin families of plant antimicrobial Various novel proteins are induced collectively known as [33] peptides. Efforts are being made in order to have sufficient knowl- PR proteins is a group of the most important inducible de- edge about the genes induced during infection and their regulation B. juncea C. sativa fense-related antifungal proteins . They are associated with measures. Therefore the objective of this study is to characterize the development of systemic acquired resistance in wide range [13] different defensin genes in , and related species Alternaria from cell wall rigidification to signal transduction and antimicrobi- to find their structure, evolution, cellular localization, and studying al activity [26]. They are the key modulators of plant defense their regulation in response to SA, JA and infection. [44] Materials and Methods which cause activation of a different set of defence related genes, including plant defensin genes . Plant defensins which belong Genome wide identification of defensin genes in B. juncea and [03] to PR-12 family of PR proteins, are cysteine rich, highly basic [50] C. sativa prominent cationic peptide in plants with different roles in Arabidopsis thaliana [10] defense [25, 39]. They are one of the largest families of antifungal The available defensin genes were down- peptide molecules . Since the beginning of 1990s, many cat- loaded from TAIR database (https://www.arabidopsis.org/), and ionic plant cysteine rich antimicrobial peptides have been studied their coding sequences were identified using ORFfinder program and plant defensins were first described in the seeds of wheat and (https://www.ncbi.nlm.nih.gov/orffinder/). By using the ExPASy barley [05, 31]. translate tool (https://web.expasy.org/translate/) the coding se- A. thaliana B. The main biological function of plant defensins was found to quences were translated to peptide sequences. The above peptide juncea C. sativa inhibit the growth of broad range of phyto-pathogenic fungi at sequences of defensin genes were queried against B. juncea micro-molecular concentrations. The precise mechanism of ac- and peptide sequences in BRAD database (http:// tion that is employed by plant defensins to inhibit the fungi is not brassicadb.org/brad/) to find putative defensins in ge- [23] completely understood, although it is generally accepted that they nome using blastp. From the blast output the peptide sequences act at the level of the plasma membrane . While some results showing >100 bit score were selected and redundant hits were re- [49] moved to select unique sequences. point to cellular membrane as the point of action, others suggest Protein structure, conserved domain identification and intracellular targets . There are two major hypothesis which computation of physical and chemical parameters explain the mechanism of action of antimicrobial defensin: the car- pet model and the pore model. In both models, defensins are described to interact with the negatively charged molecules present The NCBI-CDD database (https://www.ncbi.nlm.nih.gov/Struc- [22] at the cell membrane of pathogens, causing an increase of its per- ture/cdd/wrpsb.cgi) was used to analyze the conserved domain of miabilization, leading to cell leakage and death by necrosis . In all non-redundant sequence and SMART (http://smart.embl-hei- addition to being antimicrobial, plant defensins are also involved delberg.de/) and Pfam (https://pfam.xfam.org/) were used to con- ment [48] in the biotic stress response, as well as plant growth and develop- firm that identified genes were members of defensin family. Those . protein containing gamma-thionin domain under Knot1 superfamily were defined to be belonging to the defensin family (previously In Brassica only a few defence related genes have been re- known as gamma-thionin family). Full length amino acid sequences studied ported. These include PR proteins including few plant defensins of the selected defensin proteins were compiled and aligned using [15], β-glucanases [36], and Chitinases [55]. Tiwari [53] ClustalW and the alignment was visualized by Bio Edit V 7.0.4.soft- A. brassicae Arabidopsis the effect of methyl jasmonate on disease severity and expression ware to find conserved regions, and the three dimensional second- PDF1.2 of plant defensin gene during infection in ary structure of defensin proteins were constructed by a ProMod3 A. brassicae and he has reported that transcripts of accumulated at a Version 1.3.0. homology modelling method using SWISS-MODEL greater level upon challenge inoculation with along server (https://swissmodel.expasy.org/interactive) to confirm with jasmonic acid compared to treatment containing pathogen as that the selected proteins have stuctures similar to plant defensins, B. juncea α β well as jasmonic acid alone. However no attempts have been un- as they are generally defined by their conserved cysteine scaffold or in Alternaria C. sativa dertaken to find the number of defensin genes present in with -helix and triple strand anti parallel -sheets connected to resistant and the structural and functional the scaffold. Computer analysis of the amino acid sequence to com-

pute chemical and physical parameters were performed with the were treated with conidial suspensions using a hypodermic needle ProtParam tool on the ExPASy server (https://web.expasy.org/ and control plants were treated same way with sterilized distilled protparam/) and possible disulphide bridges were determined water. To study the effect of hormone on defensin gene expression, B. juncea using the DISUFIND server (http://disulfind.dsi.unifi.it/). The plants were sprayed with 100 µM MeJA, and 2 mM SA solutions C. sativa subcellular localization of each defensin protein of and separately and control plants were sprayed with sterilized distilled was predicted using Plant-mPLoc (http://www.csbio.sjtu. water. Small puncture wounds were made on the leaves using a hy- edu.cn/bioinf/plant-multi/). podermic needle filled with sterilized distilled water, to analyze the Gene Structure construction and prediction of cis-acting effect of wounding on the expression of defensin genes. Leaf sam- elements ples were collected at 3, 6, 12, 24, 48, 72 and 96 h post treatment.

Total RNATM was isolated from leaf tissues using TRIzol reagent (In- B. juncea C. sativa The defensin sequences retrieved from BRAD database were vitrogen ) and first strand cDNA synthesis was performed fromTM 2 employed to identify respective genes from and µg total RNA using Superscript III cDNA synthesis kit (Invitrogen ) B. juncea genome using local blast in Bio Edit V 7.0.4.software. The exon- following the manufacturer’s instructions. Quantitative realTM time intron structure of defensin encoding genes were deter- PCR was performed using SYBR Premix Ex Taq Kit (Takara ) run mined based on alignment of coding sequences with correspond- at 95 ᵒC for 10 min followed by 40 cycles of 95 ᵒC for 10 s and 55 ᵒC β-tubulin ing genome sequences and graphical display was created using for 30 s with F-5' CATGAAGCTCTCTATGCG 3'and R-5' CGATGGAT- cis online Gene Structure Display Server (http://gsds.cbi.pku.edu. CAGCGATTTCTGG 3' primers. was used as the reference cn/). Conserved -acting regulatory elements in the promoter gene for mRNA relative expression pattern analysis. The reactions region of the putative defensin genes were analyzed by plant care were performed with three biological replicates and three technical database (http://bioinformatics.psb.ugent.be/webtools/plant- replicates per sample. The relative quantification method (∆∆CT) care/html/). was used to evaluate quantitative variation between the samples. Phylogenetic analysis of defensin proteins of B. juncea, C. Results and Discussion sativa and related species Genome wide identification of defensin genes in B. juncea and C. sativa

To compare the composition of the defensin gene family in in-silico B. rapa B. napus B. oleracea C. A. thaliana Brassica, defensin genes were identified in three other Brassica During analysis, fifteen defensin protein coding sequen- sativa species, , and and also the wild type ces were identified in from the TAIR database. They B. juncea, B. rapa, B. (http://brassicadb.org/brad/) using the same method. To were sub divided into three sub families, which seven belonging to oleracea B. napus A. thaliana study the evolutionary relationships among family I, six to family II and two plant defensins in family III. All the B. and defensins, an unrooted neighbor- coding sequences were converted into their respective amino acid juncea, B. rapa B. oleracea, B. napus C. sativa. B. joining phylogenetic tree was constructed using MEGA 7.0 soft- sequences and used as a reference to find the plant defensins in juncea ware based on the amino acid sequences of the defensin proteins and Availability of B. juncea of above genotype and a bootstrap test with 1000 replicates was genome sequence made it possible to identify all the defensin performed. A. thaliana Expression analysis of defensin genes in B. juncea and C. sativa proteins present in Brassica. When genome was queried in response to SA, JA and A. brassicae infection and wounding against 15 defensin proteins of as described above, only six putative defensin proteins namely BjuB040113, BjuB040109, B. juncea C. sativa BjuB027448, BjuA030410 BjuA025766 and BjuB040111 were B. and plants were raised from seeds in pots identified by the analysis that matches to the criteria of plant de- juncea C. sativa and were maintained at 25 ᵒC for 16 h light/8 h dark in glass house fensins. However comparative to low number of defensins in A. brassicae B. rapa at National Phytotron Facility, Indian Agriculture Research Insti- , 56 defensin proteins were identified in belonging B. oleracea. B. ni- tute (IARI). isolated from disease infected field grown to plant defensins. Twelve defensins were identified both in gra. B. juncea B. nigra plants was cultured on Radish Dextrose Agar medium and was and Surprisingly no defensins were identified in B. rapa B. juncea identified by Indian Type Culture Collection, IARI (ID No. 81651). Since was evolved from cross between and Conidial suspensions were prepared by scraping sporulated myce- B. 3 , identification of only six defensins in can be jus- napus lium from 21-day old cultures and suspending in sterilized distilled tified. However total 42 of defensin proteins were identified in water. Conidial concentration was adjusted to 5x10 conidia/ml indicating the possibility of gene duplications. using hemocytometer. 45 days old healthy plants with six leaves,

Analysis of physical, chemical and structural properties of defensin proteins smaller than 40 is predicted as stable; a value above 40 predicts B. juncea that the protein may be unstable. Instability index indicates that A. thaliana C. sativa Computer analysis of physical and chemical parameters of all four defensin proteins out of six proteins were unstable the defensin proteins was done by ProtParam (Table 1). The length while most defensins in and were stable. Ins- in B. juncea C. sativa of the defensin proteins ranged from 77 to 82 amino acid residues tability index provides an estimate of the stability of the protein in while in it ranged from 72 to 131 amino acid a test tube. Calculations on aliphatic index indicated that it varies iso residues. Relative molecular mass varied from 7.79 kDa to 14.33 with 32.52 to 91.17 in all three genotypes. Grand average of hydro- kDa, while the theoretical -electric point measurement varied pathy (GRAVY) calculations indicted that most of the defensins are from 4.81 to 12.00. Instability index of all the defensin proteins polar. GRAVY is an indication of hydropathy values of amino acids identified was also calculated. A protein whose instability index is in a protein; negative values indicates that the protein is non-polar and positive values indicates that the protein is polar. Number Molecular Theoritial Instability Aliphatic Number of Number of Prot. ID GRAVY of aa Weight (kDa) pI index index -ve residue +ve residue 80 4 7 80 4 NM_106233.4 8.709 8.47 27.49 74.50 0.339 80 4 NM_123809.4 8.518 8.14 27.68 81.88 6 0.454 80 4 NM_128161.2 8.640 8.14 28.23 78.12 6 0.389 80 4 NM_123810.3 8.550 8.14 25.27 75.75 6 0.358 78 8 NM_128160.3 8.580 8.14 25.28 78.25 6 0.436 80 9 8 NM_101817.4 8.840 8.44 55.88 52.69 5 0.113 77 8 NM_104375.2 9.139 5.65 31.07 65.88 0.006 77 3 10 NM_126272.4 8.056 9.02 28.58 91.17 5 0.600 77 3 12 NM_126271.3 8.524 9.37 40.13 63.25 0.201 8 NM_126273.4 8.544 9.63 39.18 59.48 0.004 73 4 9 NM_104788.4 76 8.289 8.52 51.47 80.79 5 0.182 73 3 8 NM_125761.2 8.387 8.92 56.73 54.79 -0.130 122 12 11 NM_126274.3 7.718 8.92 76.05 73.56 0.281 129 12 10 NM_123194.3 13.122 6.05 31.71 67.95 -0.130 80 4 7 NM_119153.2 13.862 5.57 43.91 55.89 -0.154 80 12 BjuB040113 8.734 8.47 30.55 78.25 0.335 77 4 11 BjuB040109 8.862 9.14 29.92 68.50 5 0.101 78 4 9 BjuB027448 8.574 9.33 45.54 58.18 0.031 83 13 BjuA030410 8.814 8.85 57.46 60.13 0.103 82 8 7 BjuA025766 8.592 12.00 89.44 64.34 6 0.200 80 4 7 BjuB040111 8.905 6.24 63.66 68.05 0.170 80 4 7 Csa07g047380.1 8.734 8.47 26.02 74.50 0.225 80 4 7 Csa09g080580.1 8.691 8.47 26.00 75.75 0.291 80 4 Csa16g039940.1 8.744 8.47 27.48 74.50 0.215 80 4 Csa16g039930.1 8.619 8.15 31.90 77.00 6 0.310 80 4 Csa13g034620.1 8.665 8.15 29.77 75.67 6 0.351 80 4 7 Csa09g080550.1 8.707 8.16 32.96 77.01 6 0.348 80 4 Csa09g080530.1 8.676 8.48 28.94 73.38 0.201 80 4 7 Csa09g080540.1 8.709 8.15 31.90 75.74 6 0.324 Csa09g080520.1 8.736 8.48 28.94 73.99 0.246

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80 4 80 3 Csa09g080500.1 8.799 8.48 29.13 75.01 6 0.309 80 4 7 Csa16g039910.1 8.798 8.47 29.11 75.77 6 0.308 80 3 8 Csa16g039920.1 8.742 8.87 25.59 68.50 0.241 80 3 8 Csa09g080480.1 8.792 8.87 22.87 78.00 0.143 80 4 Csa07g047330.1 8.790 8.14 23.93 80.50 0.179 80 4 Csa05g073650.1 8.846 6.00 24.05 64.75 5 0.325 80 3 8 Csa05g073660.1 8.831 6.00 24.05 69.62 5 0416 79 4 7 Csa16g039870.1 8.806 8.87 23.93 85.38 0.246 4 Csa11g046460.1 8.782 8.42 33.48 65.57 0.161 78 4 7 Csa07g047360.1 75 8.195 8.36 28.85 86.00 6 0.281 80 4 Csa12g074210.1 8.693 8.42 33.78 71.41 0.238 82 7 Csa09g080510.1 8.792 8.16 33.11 60.00 6 0.233 82 3 8 Csa07g047340.1 9.064 8.16 35.51 80.98 5 0.580 82 8 Csa16g039900.1 8.873 8.93 30.00 73.90 0.556 82 9 Csa16g039890.1 8.961 8.49 33.50 94.02 5 0.657 78 8 Csa07g047350.1 9.022 8.72 27.38 86.95 5 0.570 78 7 Csa03g023400.1 8.844 8.45 55.04 51.41 5 0.047 78 10 Csa17g025410.1 8.695 8.12 45.86 51.12 5 0.131 77 3 13 Csa11g002160.1 8.934 8.77 35.63 65.13 5 -0.158 77 3 13 Csa04g007620.1 8.671 10.04 42.45 56.88 -0.044 77 4 13 Csa06g002180.1 8.721 10.00 42.55 58.88 -0.053 77 4 12 Csa06g002170.1 8.694 9.69 37.13 61.95 -0.023 112 8 19 Csa04g007610.1 8.720 9.49 41.86 61.90 0.006 74 Csa00456s080.1 12.583 9.77 41.80 63.48 -0.157 73 10 Csa16g052960.1 7.998 7.52 49.61 81.62 5 6 0.408 72 9 Csa00456s090.1 8.240 8.96 69.01 68.08 5 0.047 73 3 8 Csa18g037270.1 8.435 8.74 62.68 55.56 5 -0.133 73 3 8 Csa06g002200.1 7.792 8.92 54.62 73.56 0.281 73 3 8 Csa04g007630.1 7.802 8.92 59.89 73.56 0.270 122 8 12 Csa00456s050.1 7.823 8.91 54.62 73.50 0.255 130 9 11 Csa16g045530.1 12.937 8.26 29.81 58.36 -0.053 131 8 11 Csa10g044380.1 14.004 7.79 41.00 58.38 -0.060 131 10 12 Csa12g017170.1 13.978 8.09 36.48 57.33 -0.145 120 10 11 Csa11g013400.1 14.083 7.81 39.82 58.78 -0.147 124 13 13 Csa12g084420.1 12.842 7.31 57.62 61.67 -0.072 130 14 12 Csa20g039330.1 13.490 6.65 22.24 73.87 0.095 130 14 14 Csa12g017150.1 14.169 5.65 26.82 57.08 -0.204 113 9 14 Csa10g012480.1 14.335 6.67 33.24 36.77 -0.376 111 10 Csa11g073450.1 12.351 8.48 18.48 80.27 0.245 114 8 Csa11g013370.1 12.098 4.81 42.21 32.52 16 -0.601 123 10 9 Csa12g085440.1 12.231 8.28 56.83 66.67 5 -0.112 Csa17g089850.1 13.264 5.21 55.25 68.21 0.004 Table 1: A. thaliana B. juncea C. sativa.

Chemical and physical parameters of defensin proteins in , and

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Controlling the stability of cellular proteins is a fundamental released from internal stores to protect tissues against fungal way by which cells regulate growth, differentiation, survival, and pathogens. development. Measuring the turnover rate of a protein is often the first step in assessing whether or not the function of a protein is The defensin proteins are characterized by specific sequence regulated by proteolysis under specific physiological conditions diversity and structural feature homology. The protein domains [56]. The half-life is a prediction of the time it takes for half of the of these defensin proteins were identified and similar protein amount of protein in a cell to disappear after its synthesis in the structure and domain compositions were found, demonstrating [12] that the protein structure is remarkably conserved within the plant cell. ProtParam estimates the half-life by looking at the N-terminal B. juncea amino acid of the sequence under investigation and it was defensins. All non-redundant sequence were used to confirm that in E-coli. estimated that defensin proteins have more than 10 hrs of half-life identified proteins of were members of defensin family (Figure 1). Those proteins containing gamma-thionin domain under Knot1 superfamily were defined to be belonging to the defensin The subcellular localization prediction revealed that these family [previously known as gamma-thionin family]. Defensin and [8] proteins exhibited cell membrane and vacuolar localization. defensin like peptides are functionally diverse namely disrupting et al [47] According to Donnes and Hoglund , the organelles present microbial membranes and acting as ligands for cellular recognition unique biological conditions to the proteins. It has been observed and signalling. According to Stotz., . , the first members of that proteins from different organelles differ in their overall the family of plant defensins were isolated from wheat and barley amino acid composition and each protein has evolved over time grains in the early 1990s and those proteins were originally called to function optimally in a certain subcellular localization. Plant gamma-thionins because of their size and cysteine content that [38] defensins seem to have specific activities depending on whether were found to be similar to thionins. Subsequently gamma-thionins they are over-expressed by plant tissues or applied externally . homologous proteins were identified and it as noticed that the The subcellular localization of plant defensins thus appears to be a plant peptides’ structural and functional properties resemble those et al [11] crucial point to understand their dual action on plant cells. As all of insect and mammalian defensins, and therefore later termed the plant defensins have a strongly hydrophobic N-terminal sequence family of peptides ‘plant defensins’ in. Gachomo., . also that may serve as a signal peptide and have no obvious internal reported that the plant defensins are structurally similar to their B. juncea [30] retention signal, they are generally predicted to be secreted insect counterparts despite the low amino acid sequence similarity [24] proteins. However, most of the plant defensins are between these two organisms. Maarof and co-workers also Nicotiana alata Capsicum predicted to be localized in vacuole. The same was observed reported the presence of Knot1 domain and gamma-thionin in NaD1 in flowers. However, their internal location domain in their studies on CDef1 in . The Knot1 domain et al is not incompatible with their antimicrobial activity as Sticher., or knottins represent plant antimicrobial peptides, plant amylase . [46] confirmed that when cells are damaged, defensins are inhibitors, plant gamma-thionins and arthropod defensin.

Figure 1: B. juncea C. sativa

The presence of gamma-thionin domain under Knot1 superfamily in, and defensins.

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α β In addition to the conserved domains, other conserved motifs ture as all the defensin proteins were represented by an -helix and α could also be important for the function of plant defensins. Plant triple strand anti parallel -sheets (Figure 2). Despite their struc- β defensins share a common motif consisting of an -helix and triple tural similarity, plant defensins have great diversity in amino acid [11] strand anti parallel -sheets, a feature that is also shared by several sequence. This variation in the primary sequence is associated with toxins from insects, scorpions, honeybees and spider venom . the specificity and diverse biological activities of antimicrobial pep- [23] et al [18] The structural features of above defensins were analyzed in order tides such as antibacterial, antifungal, antioxidant and other vari- to understand the functional characteristics of defensin family. ous antimicrobial activities . Kaewklom., . reported lel β Similar to the domain distribution results, the modeled defensins that recently, a conserved ᵞ-core motif composed of two antiparal- also share similarity with each other in terms of secondary struc- -sheets and an interposed loop has been identified and shown to be important for their functions.

Figure 2: B. juncea C. sativa

Secondary structure analysis of and defensins.

B. juncea C. sativa

Multiple sequence alignment of and defensin dges. The amino acid sequence conservation of defensin proteins proteins revealed a highly conserved eight cysteine residues near is limited to a small number of residues. These conserved residues et al [23] the N-terminal domain of the proteins (Figure 3 and Figure 4). are absolutely restricted to the eight cysteines, a glycine residue, as [11] This observation is consistent with the finding of Lay., . , well as a serine and an aromatic residue (F/W/H/Y) that is always who suggested that the conservation of these residues may be due followed by another glycine . They are the most conserved po- to their roles in providing stability and in the folding mechanism, sitions and essential for structural folding and stabilization of the especially the cysteines involved in the formation of disulfide bri- proteins.

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Figure 3: B. juncea

Sequence comparison between plant defensin proteins in . The invariable cysteine residues are depicted in gray background. The disulfide bond connectivity is shown below by connecting line.

Figure 4: C. sativa.

Sequence comparison between plant defensin proteins in

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Gene Structure construction and prediction of cis-acting elements The overall primary action of plant defensins is to form pores on the membrane of target pathogens and increase membrane B. juncea permeability [52]. They can resist temperatures near the boiling To gain further insight into the structural evolution of the defen- point without losing function and their structures are well main- [11] sin genes in , the exon-intron organization was analyzed tained. Their ultra-stability is generally due to the cysteine scaffold by comparing the corresponding genomic sequences with their of their structures . Conserved amino acid residue analysis of coding sequences through Gene Structure Display software. Based other Brassica members revealed that only eight cysteine residues on the predicted schematic structures, all the genes displayed that are strictly conserved among members of the Brassica family. It a single intron and two exons existed in all the coding sequences was reported that the significance of both basic residues and their in B. juncea [17] (Figure 5). This conserved exon number among all plant defensins et al et al asymmetric distribution over the molecular surface causes the am- revealed their close evolutionary relationship. As de- phiphilic properties of defensins. Hoover., . reported the et al [42] scribed by Heyn., . [16], the overall rate of transcript produc- importance of basic residues for the antimicrobial activity. Accor- tion is determined by transcription and RNA processing rates. Gene ding to Sagehashi., . , the site directed mutational analysis architectural features, primarily the number and length of exons of Rs-AFP2, a defensin isolated from radish seeds showed that ba- and introns have recently emerged as important regulatory play- sic amino acid residues contribute to the antifungal potency of this ers. Several new studies indicated that rapidly cycling cells contain peptide. The substitutions of neutral residues Gly or Val by argini- [23] gene-architecture toward short genes with few introns, allowing ne increased the Rs-AFP2 antifungal activity. However, according efficient expression during short cell cycles and in genes that need to Lay and Anderson , comparison of a series of sequences of rapid regulation during stress. Therefore, the conserved trend to- plant defensins from various plant species reveals that the plant ward shorter gene with fewer introns in plant defensins indicates defensin family shows very limited sequence conservation except their rapid induction following pathogen attack. for eight cysteine, two glycine and one glutamate acid residues.

Figure 5: B. juncea

Gene structure of defensin BjuB040113, BjuB040109, BjuB027448, BjuA030410, BjuA025766 and BjuB040111.

105 cis Phylogenetic analysis of defensin proteins of B. juncea, C. cis sativa and related species In plants, gene transcription is regulated by -acting regulatory elements that bind to target transcription factors. Some - B. juncea regulatory elements are involved in stress responses. To analyze B. juncea B. juncea A phylogenetic tree was constructed on the basis of full length how the expression level of defensins respond to stress cis defensin protein sequences to understand the phyloge- stimuli, 2.0 kb upstream promoter regions of defensin netic relationship among these proteins (Figure 6). According to cis [43] genes were scanned for stress-related -regulatory elements us- the MEGA software, the evolutionary history was inferred using the ing the plant CARE online service. Two -acting regulatory ele- B. juncea Neighbor-Joining method . The optimal tree with the sum of ments involved in MeJA responsiveness, CGTA-motif and TGACG- branch length = 3.45580578 is shown. The tree is drawn to scale, motif were identified in the promoter region of defensin with branch lengths in the same units as those of the evolutionary genes denoting to the possible regulation of plant defensin genes distances used to infer the phylogenetic tree. The evolutionary dis- under JA responsive pathway. Two light responsive elements GTI tances were computed using the Poisson correction method [57] and a G-box, TGA-element in auxin responsiveness were identi- and are in the units of the number of amino acid substitutions per fied indicating that these defensin genes might be involved in the site. The proportion of sites where at least one unambiguous base response to various light stress and hormone treatments via par- common cis is present in at least one sequence for each descendent clade is ticipating in different regulatory mechanisms. Apart from that, 10 shown next to each internal node in the tree. The analysis involved -acting elements in promoter and enhancer region, cis 6 amino acid sequences. All positions containing gaps and missing and 14 core promoter elements around -30 of transcription start B. juncea data were eliminated. There were a total of 77 positions in the final site were identified. The type and number of -acting regulatory B. junea B. juncea dataset. Evolutionary analyses were conducted in MEGA7. The tree elements in the promoter region of each defensin gene indicates that defensins belong to three distinct groups, were discrepant indicating that the members of defensin C. sativa cis majority of defensins clustered under group I. However, the de- gene family might be able to respond to different abiotic stresses. C. sativa fensin protein sequences of used in phylogenetic analysis Similar pattern was observed in -acting regulatory elements in resulting in the categorization into five distinct groups (Figure 7). defensin genes.

Figure 6: B. juncea

The phylogenetic tree of defensin proteins constructed by Neighbor-Joining method.

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0.00 0.00 Csa09g080500.1 0.00 0.00 Csa16g039910.1 0.00 0.00 Csa09g080520.1 0.00 0.00 Csa09g080550.1 0.00 0.00 Csa13g034620.1 0.00 0.00 Csa07g047360.1 0.03 0.00 Csa09g080540.1 0.00 Csa16g039930.1 Group I 0.03 0.00 0.03 Csa09g080530.1 0.03 Csa16g039920.1 0.00 0.12 0.00 Csa09g080510.1 0.00 Csa05g073650.1 0.00 0.15 Csa05g073660.1 0.00 0.00 Csa07g047330.1 0.51 0.00 0.13 Csa16g039870.1 0.03 0.03 Csa09g080480.1 0.06 0.03 Csa11g046460.1 0.10 Csa12g074210.1 0.06 0.00 Csa09g080580.1 0.03 Group II 0.06 0.03 0.38 Csa16g039940.1 0.00 Csa07g047380.1 0.02 0.03 Csa16g039890.1 0.12 Csa16g039900.1 0.63 0.08 0.03 Csa07g047340.1 0.00 1.33 0.00 Csa07g047350.1 0.60 Csa05g060830.1 0.40 Csa00456s090.1 0.03 0.41 Csa03g023400.1 0.00 0.80 Csa17g025410.1 0.90 Csa11g002160.1 0.66 0.98 0.00 Csa00456s050.1 0.00 0.00 Csa06g002200.1 Group III 0.03 0.00 0.60 0.20 Csa04g007630.1 0.00 Csa06g002180.1 0.00 Csa04g007620.1 0.34 0.33 Csa16g052960.1 0.03 0.39 Csa00456s080.1 0.18 0.00 0.06 Csa06g002170.1 0.00 0.00 Csa04g007610.1 2.31 Csa18g037270.1 0.51 0.20 Csa10g044380.1 0.00 0.29 Csa20g039330.1 0.05 Csa11g073450.1 0.38 0.03 0.19 Csa12g017170.1 0.03 0.40 Csa11g013400.1 0.03 Group IV 0.16 Csa12g084420.1 0.08 0.50 Csa12g085440.1 0.15 0.05 Csa16g045530.1 0.06 0.05 Csa12g017150.1 0.02 1.25 Csa10g012480. 1.63 0.62 Csa07g047370.1 1.54 Csa09g080560.1 0.94 0.32 Csa11g013370.1 2.34 Csa07g023910.1 Group V 0.88 1.24 0.47 Csa09g095600.1 3.24 0.55 Csa17g089850.1

Figure 7: C. sativa

The phylogenetic tree of defensin proteins constructed by Neighbor-Joining method.

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B. juncea C. sativa Arabidopsis B. B. juncea To study the evolutionary relationship of and cies together with were clearly divided into five sub- napus, B. oleracea, B. rapa, B. nigra C. sativa to other related species comprehensively, defensin proteins of groups. Most of the defensins belong to subgroup I, while C. and were also obtained from one defensin belong to subgroup IV. defensins were scat- B. juncea B. sativa et al BRAD database. A neighbor-joining tree was constructed with de- tered in every group indicating the evolutionary relationship of napus, B. oleracea, B. rapa A. thaliana . [11] Arabidopsis fensin proteins of and other related species including with Brassica species. As also observed by Gachomo., . and (Figure 8) Phylogenetic , defensins are spread all over the tree suggesting analysis showed that all the identified defensins from Brassica spe- an ancient divergence.

Figure 8: B. juncea, B. rapa, B. oleracea, B. napus, C. sativa A. thaliana The phylogenetic analysis of and defensin proteins by Neighbor-Joining method. Expression analysis of defensin genes in B. juncea and C. sativa in response to SA, JA and A. brasicae infection and wounding isolated from leaf sample at different time intervals. The impor- B. juncea tance of SA, JA and ET as dominant primary signals in local and sys- C. saiva To study the role of plant defensin in defense of and temic induced defense signaling has been well documented. Exog- , induction of defensin genes in response to exogenous ap- enous application of JA induces a set of defense genes that are also plication of SA and JA was studied by performing qRT-PCR on RNA activated upon pathogen infection, among which are genes encod-

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[40] Arabidopsis ing plant defensins and thionins . Expression analysis of defen- of accessions, highlighting the potential significance of B. juncea sin revealed that both the genotypes respond to JA application. In SA/JA crosstalk in nature [6]. Though many reports describe SA/ [34] the expression of defensin gene increase gradually up to JA antagonism, synergistic interaction have been reported as well 12 h and then with a sharp increase, peaked at 48 h after applica- . Emerging data suggests that, there is a more complex signal- C. sativa B. [28] Arabidopsis tion. After 48 h, the expression of the gene was declined (Figure ing network evoking both positive and negative regulatory inter- juncea 9). However, in the expression was early compared to actions . Furthermore, analysis of some mutants et al , revealing a sharp peak at 6 h after JA application, and then with constitutive SA responses reveals a pathway in which JA and cis Arabidopsis down-regulated at each time point after treatment. Presence of ET signaling are required for SA responses [54]. Mur., . in 2006 two -acting regulatory elements involved in MeJA responsive- reported that in treatments with low concentrations et al [4] PDF1.2 PR1 ness in promoter region of defensin genes further support the of JA and SA resulted in a synergistic effect on the JA and SA re- PR3, theory of regulation of defensins by JA signaling. Chamil., . sponsive genes and , respectively. However, at higher PDF1.2 GST Arabidop- also reported that upon JA treatment, increased expression of concentrations the effects were antagonistic, demonstrating that sis PDF1.2 et al [4] , and was observed. This is in agreement with the outcome of the SA-JA interaction is dependent on the relative PR3 PDF1.2 defense signaling pathway that JA generally induces and concentration of each hormone. Chamil., . also reported in Arabidopsis PDF1.2 C. sativa and therefore, those two genes have been extensively used as that although is known to be induced by JA but not by SA LOX2, VPS PDF1.2 markers for JA signaling pathway [51]. , could also be induced by SA in . The JA-responsive genes and contain one or more SA responsive TGACG motifs in their promoters. The occurrence of this [2] motif in defensin gene promoter indicates a cross talk between SA and JA pathways .

Figure 9: C. sativa B. juncea Real-time PCR analysis of defensin genes in Figure 10: and in response to exogenous application C. sativa B. juncea of JA shows differential expression. Real-time PCR analysis of defensin genes in and in response to exogenous application of SA a down-regulation. Howver, unlike JA, there was a significantly low level of defensin Alternaria gene expression in response to exogenous SA application in both A. brassicae the genotypes (Figure 10). The decrease in defensin gene expres- In order to study the expression of defensin gene to sion by SA coincides with the increased expression by JA confirm- infection, plants were infected with a suspension of C. sativa Alternaria ing the antagonistic interaction between the SA and JA response spores. Quantitative real time PCR analysis indicated that, relative PDF1.2 pathways. Past literature revealed that JA responsive genes such as expression of defensin gene in in response to A. brassicae are highly sensitive to suppression by exogenous applica- infection is comparatively very low in spite of the genotypes’ resis- tion of SA [20,40,45]. tancy to (Figure 11). The gene induction was very mar- ginal. This can be explained by studying other defensin genes which Many studies have demonstrated that endogenously accumu- are many [56] in case the gene in consideration in this study may C. sativa lating SA antagonizes JA-dependent defenses, thereby prioritizing not involve in resistance. Also might be important are levels of SA et al [4] PDF1.2 SA-dependent resistance over JA-dependent defense. This antago- and JA in which needs to be explained. However, Chamil., nism between SA and JA signaling was observed in a large number . demonstrated a remarkable induction of in locally

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C. sativa A. brassicae [7] challenged leaves of in response to infection. According to Dong the wound induced octadecanoid path- sion in B. juncea C. sativa However, there was a marked increase in defensin gene expres- way results in the synthesis of the signal molecule JA, and subse- compared to in response to infection. The quent activation of genes in defense including defensin. Wounding et al [40] [39] C. relative expression was highest at 24 h, continue till 48 h, and then not only causes a rapid production of JA but the level of ethylene sativa decreased. According to Pieterse., . , biotrophic pathogens as well . Wounding and JA evoke a very similar response in are generally sensitive to defense responses that are regulated by in our study further strengthening the idea that wounding Arabidop- PLD SA, whereas pathogens with a necrotrophic lifestyle are commonly exerts its effect through JA signaling. There are two distinct path- sis deterred by defenses that are controlled by JA and ET. An ways for synthesis of JA. One starts from (phospholipase D) in- PDF1.2 DAD1 defensin, activated after necrotrophic fungi invasion, is encoded duce by wound, pest, pathogen, elicitors, and the other starts from A. brassicae by gene and is a predominant marker gene for JA pathway (defective anther dehiscence1) in developmental pathway Arabi- [51]. Since is a necrotrophic pathogen, it is obvious lead to sysnthesis of AOC (allene oxide cyclase). Growing evidence dopsis that induction of defensin by the pathogen as defensin gene is in indicates that developmentally regulated JA biosynthesis in JA signaling pathway. is controlled through activation of a JA biosynthetic pathway that differs from, but overlaps with the biosynthetic pathway that Conclusionregulates wound-induced JA biosynthesis [54]. In-silico B. juncea C. sativa analysis and characterization of defensin genes in

and leads to a better understanding of their roles in response to biotic and abiotic stresses. The wild Camelina poses a significantly higher number of defensin genes compared to Bras- sica and the proteins were more stable. This may be a possible cis- Figure 11: B. reason of Camelina’s resistance to Alternaria blight. Presence of juncea C. sativa Alternaria Real-time PCR analysis of defensin genes in acting regulatory elements involved in MeJA responsiveness Alternaria and in response to a infection. indicates their expression in response to stress conditions. Anal- ysis of defensin gene response to SA, JA, infection, and C. sativa wounding revealed a complex and interesting network of pathways Two genotypes responded differentially to wounding. Expres- that function both separately and together to render defense re- sion of defensin gene in wounded leaves increased initial- B. juncea sponses in both the genotypes. The defensin genes can be used for ly, showing maximum fold change at 6h, and then decreased gradu- A. further studies including antimicrobial studies to find effectiveness ally while in there was a marked decline of the expression brassicae of using defensin in transgenic Brassica to confer resistance to from the initial point (Figure 12). Acknowledgments.

Authors wish to acknowledge the support given by National Phytotron Facility for providing glasshouse facilities. Chaturani GDG acknowledge the receipt of PhD fellowship from Indian Coun- Bibliographycil for Agricultural Research.

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