Differential expression of structural and functional proteins during bean common mosaic virus-host plant interaction

Microbial Pathogenesis 138 (2020) 103812

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Differential expression of structural and functional proteins during bean common mosaic virus-host plant interaction

T

Sunil Kumara, Chetna Dhemblab, Hariprasad Pc, Monica Sunddb, Ashok Kumar Patela,∗ a

Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, 110016, India National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India c Centre for Rural Development and Technology, Indian Institute of Technology Delhi, New Delhi, 110016, India b

ARTICLE INFO

ABSTRACT

Keywords: Host-virus interaction Proteomics BCMV Common bean

Bean common mosaic virus (BCMV), the most common seed-borne pathogen in Phaseolus vulgaris L. is known to cause severe loss in productivity across the globe. In the present study, proteomic analyses were performed for leaf samples from control (healthy) and susceptible BCMV infected plants. The differential expression of proteins was evaluated using two-dimensional gel electrophoresis (2-DE). Approximately, 1098 proteins were spotted, amongst which 107 proteins were observed to be statistically significant with differential expression. The functional categorization of the differential proteins illustrated that they were involved in biotic/abiotic stress (18%), energy and carbon metabolism (11%), photosynthesis (46%), protein biosynthesis (10%), chaperoning (5%), chlorophyll (5%) and polyunsaturated fatty acid biosynthesis (5%). This is the first report on the comparative proteome study of compatible plant-BCMV interactions in P. vulgaris which contributes largely to the understanding of protein-mediated disease resistance/susceptible mechanisms.

1. Introduction One of the most important legume crops is common bean (Phaseolus vulgaris L.), which is a rich source of proteins, vitamins, carbohydrates, phytonutrients, minerals, antioxidants and fiber [1,2]. Globally, common bean is grown over an area of 18 million hectares with 12 million tons of production per year [3]. Common beans infected by several pathogens are the main concern in its production. Moreover, approximately 140 viral diseases are known to occur in legumes [4]. Bean common mosaic virus (BCMV) has been reported to have the highest percentage of seed transmission of 83% and causes significant yield losses up to 50–100% in common bean [5,6]. Symptoms of BCMV are green-yellow mosaic, leaf curling, chlorosis, plant dwarfing, and in some cases, necrosis is also observed [7,8]. The genome of BCMV encodes a single polyprotein of 340–370 kDa which later cleaves into eight proteins viz. proteinase (P1), helper component proteinase (HC-Pro), proteinase (P3), 6K1, cylindrical inclusion (CI), 6K2, nuclear inclusion a (NIa), nuclear inclusion b (NIb) and CP [9]. The BCMV infects bean plants as a complex of 8 pathogroups which can be differentiated on the basis of virus response in differential bean lines [10]. The differential bean lines were characterized on the basis of the presence/absence of a single resistance gene or complex of resistance (R) genes [11]. Seven resistance alleles ∗

have been recognized in response to BCMV: one dominant allele I, and six recessive alleles (bc-u, bc-1, bc-12, bc-2, bc-22, and bc-3) [12,13]. Various strains of BCMV have been reported; genome of these strains is similar to each other and recombination may take place between them which results change in their pathotype specificity [14]. However, the specific mechanism that facilitates the interaction between resistant genes and BCMV is yet to be elucidated. The mechanism of interaction between resistant alleles and BCMV has been characterized for one allele i.e. “bc-3” (as the gene eIF4E) [15]. Plants stimulate a number of signaling pathways upon the entry of the virus and facilitate suitable defense mechanism against them [16]. Resistance mechanism evokes differential expression of various proteins related to a number of pathways such as metabolism, signal transduction, photosynthesis and other cellular functions [17]. Hence, elucidations of various regulatory factors related to host-pathogen interactions could play a crucial role in the resistance mechanisms by identifying key proteins. The elucidation of potential biological interactions in susceptible host is a foremost prerequisite to understand the epidemiology and to develop viral-resistant plant. The potential enzymatic pathways related to BCMV infection are not completely understood. In earlier reports, transcriptomics has been applied to study the interactions between BCMV and common bean [11]. Moreover, the differently expressed proteins (DEPs) can explain in

Corresponding author. E-mail address: [email protected] (A.K. Patel).

https://doi.org/10.1016/j.micpath.2019.103812 Received 6 September 2019; Received in revised form 15 October 2019; Accepted 18 October 2019 Available online 25 October 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.

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a better way the physiological changes that occur at translational level in response to BCMV infection. The relative and absolute quantification-based quantitative proteomic approach is a well-accepted approach, which has been applied to analyze the interactions of viruses with various vectors/hosts [18]. The present study explains the comparative proteome profiles in control and susceptible common bean plant during BCMV infection through a 2D gel-based proteomic approach. 2. Material and methods

35,000 kV h and hold at 50 V. Further, strips were equilibrated for 15 min in buffer-I (6 M urea, 50 mM Tris-Cl (pH 8.8), 30% w/v glycerol, 1% w/v DTT, 2% w/v SDS, 0.002% w/v bromophenol blue) and further incubated for 15 min in the same buffer with 2.5% w/v iodoacetamide (without DTT). Second dimension electrophoresis was performed for equilibrated strips by 12% SDS-PAGE as mentioned earlier [22]. The gels were stained as explained earlier [23], and destined (10% acetic acid and 35% methanol). The destined gels were documented using Gel Doc™ XR + Gel Documentation System (Bio-Rad, USA) and images were analyzed through Melanie software (GE Healthcare, Sweden).

2.1. Plant material and BCMV infection

2.4. Protein identification

Common bean (P. vulgaris) seed samples were collected from National Seeds Corporation (NSC) New Delhi, private seed companies and local seed traders. Different bean cultivars were screened for seedborne BCMV infection under greenhouse conditions. Seedlings were raised in pots containing sterilized farm yard manure (Soil: sand: Fym, 2:1:1, v/v/v) at a frequency of 6 seeds per pot. Seeds were sown in earthen pots and maintained in an insect-proof screen-house. Seedlings were monitored for disease incidence from primary leaf stage, first trifoliate to third trifoliate stage. The seedlings showed seed-borne BCMV symptoms such as mosaic and leaf rolling. The presence of BCMV was confirmed by reverse transcription polymerase chain reaction (RTPCR) using specific primers. Total RNA was extracted from 100 mg BCMV infected leaves using RNeasy Plant mini kit (Qiagen) by following manufacturer's protocol. The cDNA synthesis was carried out from isolated RNA using ImProm-II™ Reverse Transcription kit (Promega) according to the manufacturer's protocol. The coat protein gene was PCR amplified from synthesized cDNA using gene-specific primers as mentioned earlier [19] (Table S1 in the supplementary information 1). The amplified coat protein gene was confirmed by sequencing.

The differentially expressed proteins bands were excised from the gels as described earlier [18]. The stain was removed from the excised gel using a wash solution (50 mM ammonium bicarbonate (NH4HCO3) and 50% acetonitrile). Further, the wash solution was removed, and gel pieces were vacuum dried using speedVac (Thermo Scientific, USA). The dried gel pieces were rehydrated with 50 mM NH4HCO3 containing 50 ng sequencing grade trypsin (Promega). The digested peptides were extracted in extraction buffer (0.1% trifluoroacetic acid and 50% acetonitrile). The eluted peptides were identified through a 3000 Ultimate nano-LC system interfaced to an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific, USA). 2.5. Bioinformatics analysis The heat map with hierarchical clustering expression profile was generated for the identified differentially expressed proteins using an online server heatmapper [24]. The possible interaction between differentially expressed proteins was carried out through an online web server STRING (http://string-db.org/) [25]. STRING displays the predict association between proteins by physical as well as functional interaction which is based on gene fusion, co-occurrence, gene neighborhood, previous experiments and database.

2.2. Protein extraction Leaf samples were crushed to a fine powder in liquid N2 using prechilled mortar and pestle. Total proteins were extracted using phenol precipitation method with slight modifications as mentioned earlier [20]. Fine powder was resuspended in extraction buffer (50 mM TrisHCl, pH 7.5; 0.7 M sucrose; 100 mM EDTA; 1 mM PMSF; 1% (w/v) CHAPS; 1 mM DTT; Roche protease inhibitor) The suspension was kept on constant shaking condition at 4 °C for 15 min. An equal volume of Tris-saturated phenol was added to the suspension and kept for continuous shaking at 4 °C for 15 min. The phenolic phase was separated by centrifugation for 15 min at 14,000 rpm. Further, an equal volume of extraction buffer was added to the phenolic phase. The suspension was vortexed and phases separation was achieved by centrifugation for 15 min at 14,000 rpm. The phenol layer was collected and protein precipitation was achieved with overnight incubation at −20 °C by adding 0.1 M ammonium acetate in ice cold methanol. The pellet was washed with chilled acetone and was air-dried. Protein pellet was dissolved in rehydration buffer containing 7 M urea, 2 M thiourea, 4% (w/ v) CHAPS and protein quantification was performed as described earlier [21].

3. Result and discussion

2.3. Gel electrophoresis analysis

The proteome analysis of P. vulgaris leaves were carried out to identify the proteins involved in viral-host interaction. The differential protein profiling of the infected and healthy leaves of P. vulgaris plants was studied through 2D-gel analysis. Total 1098 protein spots were observed between 3 and 11 isoelectric point (pI) with molecular mass ranges between 10–250 kDa (Fig. 2). The protein expression of the control leaf samples were compared with virus infected leaf samples by univariate and multivariate statistical analysis. By analyzing the gel using Melaine software, 107 differential expressed proteins were identified, among which 80 were successfully excised from the gel and

3.1. Virus propagation and identification of BCMV infection The BCMV infection in P. vulgaris was observed at 3-leaf stage seedlings of infested seeds. The infested seeds showed green mosaic and vein mosaic symptoms upon BCMV infection (Fig. 1A). The fifteen different cultivars were screened for the BCMV infection, in which 9 cultivars showed disease incidence percentage between 5.19 and 24.66%. The local variety-Mysore cultivar was selected for the study which showed maximum 24.66% disease incidence. Further, the BCMV infection was identified using molecular methods such RT-PCR. The coat protein gene was amplified from RNA isolated from infected leaves. The amplified product of RT-PCR was observed at ~717 bp in agarose gel (Fig. 1B). The sequencing result of amplified PCR product confirmed the presence of BCMV coat protein gene (Genbank MN046209). 3.2. Proteome analysis of BCMV infected plants

Isoelectric focusing was performed using an isoelectric focusing unit (PROTEAN i12 IEF Cell; Bio-Rad). The immobilized pH gradient (IPG) strips (containing pH range of 3–10 with 11 cm length) were rehydrated in 150 μL rehydration buffer (350 μg plant proteins; 0.5% Bio-Lyte pH 3–10) for 12 h at 25 °C. IEF program was carried out for strips with current limit of 50 μA under the following regime: 1 h at 100 V, 1 h at 500 V, 1 h at 1000 V, 1 h at 2000 V, 30 min at 4000 V gradient and 2 h at 4000 V, 30 min at 8000 V gradient and 8000 V with a total of 2

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Fig. 1a. Symptoms of BCMV infection on P. vulgaris; A& B - healthy; C&D -leaf mosaic.

belonged to biotic/abiotic stress regulators, energy and carbon metabolism, photosynthesis, protein biosynthesis related protein, chaperones, and chlorophyll/polyunsaturated fatty acid biosynthesis. A cluster analysis was performed for differentially expressed proteins in the BCMV infected and control leaf samples using pairwise average linkage cluster study (Fig. 3). Plant virus not only overcome the defense mechanism of host but also utilizes host proteins to complete their infection cycle. During the entry of virus, host cells treat virus as foreign material, which activate number of defense pathways such as hypersensitive reaction (HR) and RNA silencing. The virus defeats this defense mechanism by producing suppressor proteins. The interaction of viral proteins leads to differential expression of host proteins. 3.3. Biotic/abiotic stress proteins At initial stage of infection, viruses cause oxidative stress in various organelles in plants which results in the accumulation of reactive oxygen species (ROS) in the tissues and organelles in the form of H2O2 [26,27]. In infected cell, H2O2 could interact with the superoxide (O2 –) radical or metal ions to generate a hydroxyl radical (OH•) that is extremely toxic to pathogens and host cells. This oxidative stress leads to demolished pigments, proteins, and nucleic acids [28]. To reduce the accumulation of H2O2, plants activate several enzymatic and nonenzymatic pathways. These include free radical scavengers, such as superoxide dismutase, catalase (CAT), Glutathione peroxidase (GPx), peroxidases, and the enzymes involved in the ascorbate–glutathione cycle [29]. The level of H2O2 regulated through ascorbate-glutathione pathway where the superoxide dismutase converts the H2O2 into H2O and O2 [30,31]. During BCMV infection, up-regulation of the oxidative stress response enzymes, such as GPx and CAT was observed. The four isoforms of GPx (spot# 102, 103, 104, and 105) were seen to be upregulated (Table 1). These isoforms were observed due to the posttranslational modification of the GPx enzyme. Earlier report shows that, the catalase 3 (CAT3) expressions were enhanced during H2O2

Fig. 1b. Detection of BCMV from P. vulgaris leaf samples by RT-PCR. Amplified cDNA fragments (ca. 717 bp) were analyzed by electrophoresis on 1.0% agarose gel. M: marker separated by 100 bp, lane 1–2: leaf samples subjected to RTPCR.

subjected for LC-MS/MS analysis. Forty five spots were successfully identified as either plant or virus proteins, 15 as unknown, and 20 were unidentified (Fig. 2, Table 1). The differentially expressed proteins 3

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Fig. 2. Profiles of identified proteins after 2D-electrophoresis; (A) Identification of differential protein spots from control leaf of P. vulgaris; (B) Identification of differential protein spots in BCMV infected leaf; the numbers indicate the differentially expressed and identified protein spots.

accumulation in vascular bundles [32,33], which interacted with the 2b viral protein of Cucumber mosaic virus (CMV) [34]. In our study, CAT3 expression was seen to be up-regulated in BCMV infected samples (Spot #12). During the plant-pathogen interaction in tomato, the induced expression of subtilisin-like protease was observed in the infection of citrus exocortis viroid (CEV) [35]. Moreover, it is been reported that, subtilases accumulated in the plant extracellular matrix (ECM) of parenchyma cells during pathogenesis, which was observed to be associated with various function such as engulfed ‘inclusion bodies’, degradation of the large subunit of RuBPCase (ribulose bisphosphate carboxylase) [36]. The defense enzyme subtilisin-like protease was seen to be up-regulated in BCMV infected samples (Spot #0, 01). The subtilases belong to large family of serine peptidases, which is subdivided into five distinct subfamilies involved in specific proteolytic processes during plant pathogenesis [37,38].

3.5. Photosynthesis-related proteins The process of photosynthesis is regulated by a number of enzymes which are abundantly present in the plant cells. Earlier studies reveal that the photosynthesis rate is usually reduced in virus-infected plants. Virus infected plants display characteristic symptoms due to alteration in morphology and physiology of their infected cells. Most viruses affect chloroplast structure and function, which leads to reduced photosynthesis rate. The leaf mosaic symptoms of virus occur due to damaged chloroplast present in the mesophyll cells [44]. The viral coat proteins (CPs) are the main cause of altered chloroplast and symptoms development [45]. The CP of Potyvirus Y interacts with the large subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCo), which leads to the appearance of mosaic and chlorosis symptoms [46]. In the present study, we observed that the enzymes related to both photosynthesis and photorespiration were repressed in response to BCMV infection (Table 1). Several isoforms of RuBisCo (#13,14,15,16,17,19,20,21,77,78,79,80, and 81) were observed to be differentially modulated. Although, these isoforms were of equal molecular weight, yet display different isoelectric point (pI), which could be due to the alterations in their post-translational modification state. These isoforms viz., # 13, 14, 15, 16, and 20 showed a downregulation in BCMV infected samples, whereas some of these isoforms viz. # 17,19,21,77,78,79,80 and 81 were observed to be up-regulated. These results indicate that different isoforms of RuBisCo are differentially affected by BCMV infection. Another photosynthetic enzyme, carbonic anhydrase was found to be up-regulated specifically in BCMV infected leaves. However, the activation of ribulose bisphosphate carboxylase/oxygenase was regulated by a soluble chloroplast protein, RuBisCo activase, which was identified in two different isoforms (#1475, and 1478). The isoforms of RuBisCo activase were observed to modulate the expression in BCMV infected leaves. Phosphoribulokinase (PRKA) enzyme was down-regulated in virus infected samples (#44). The generation of ribulose-1,5-bisphosphate (RuBp) from ribulose 5-phosphate, depends upon the catalytic activity of PRKA [47]. The reduced rate of RuBisCo activity and RuBp production eventually leads to poor photosynthesis rate in infected plants [27]. Further, the light-dependent pathway of photosynthesis contains two consecutive photosystems (photosystem I and photosystem II), which is carried out in thylakoid membrane of the chloroplasts [48]. In the present study, photosystem involving proteins, ferredoxin-NADP

3.4. Chlorophyll biosynthesis The 5-aminolevulinic acid dehydratase (ALAD) plays a vital role in chlorophyll and heme synthesis at an early stage of chlorophyll biosynthesis [39]. The two δ‐aminolevulinic acid (δ‐ALA) molecules are combined by the enzyme ALAD to form the pyrrole molecule and porphobilinogen. The tetrapyrrole is the precursor for chlorophyll biosynthesis, which contains four pyrrole rings. The pyrrole molecules linked together in linear or circular manner to form tetrapyrrol molecules. The insertion of Mg++ into protoporphyrin IX is catalyzed by the enzyme magnesium-protoporphyrin IX chelatase (Mg-chelatase) for chlorophyll biosynthesis [40,41]. The ALAD and Mg-chelatase are essential enzymes and their expression is interconnected at early stage of chlorophyll biosynthesis [40]. In the present study, chlorophyll biosynthesis related proteins ALAD (spot #53) and Mg-chelatase (spot #30) were observed to be down-regulated in response to BCMV infection. Previous report suggested that several proteins related to chlorophyll biosynthesis which include, delta-aminolevulinic acid dehydratase and Mg-chelatase were found to be down-regulated in response to Rice stripe virus (RSV) which infect rice plants [42]. The expression of Mg-chelatase is regulated by Y-satellite RNA of CMV which cause yellow mosaic in tobacco plant during viral infection [43].

4

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Table 1 The list of differentially expressed proteins identified in BCMV infected and control leaves of P. vulgaris. Spot ID

Protein Name

Experimental MW/pI

Fold change

Healthy intensity

Infected Intensity

102 103 104 105 12 0 01 13 14 15 16 17 19 20 21 77 78 79 80 81 44 91 96 27 29 26 11 23 93 08 56 53 30 45 46 51 57 25 90 32 33 38 42 43 99

Glutathione peroxidase Glutathione peroxidase Glutathione peroxidase Glutathione peroxidase Catalase Subtilisin-like protease Subtilisin-like protease RuBisCo RuBisCo RuBisCo RuBisCo RuBisCo RuBisCo RuBisCo RuBisCo RuBisCo RuBisCo RuBisCo RuBisCo RuBisCo Phosphoribulokinase (PRKA) Ferredoxin-NADP reductase Cytochrome f Fructose-bisphosphate aldolase Glyceraldehyde-3-phosphate dehydrogenase Phosphoglycerate kinase ATP synthase subunit beta Eukaryotic elongation factor 1A Cysteine synthase Heat shock 70 kDa protein Peptidyl-prolyl-cis/trans-isomerases Delta-aminolevulinic acid dehydratase Mg-protoporphyrin IX Inositol-1-monophosphatase Inositol-1-monophosphatase Photosystem I reaction centers subunit III Photosystem I reaction centers subunit III Elongation factor Tu Thiamine thiazole synthase Coat Protein Coat Protein Coat Protein Coat Protein Coat Protein Coat Protein

18.5/5.5 17.5/6.0 18.5/6.5 19.0/6.2 57.0/4.7 85.9/6.2 85.9/6.8 52/11.0 52/10.6 52/10.1 51/9.8 52/3.0 50/7.4 52/8.8 50/7.0 51/5.3 52/5.8 50/8.0 50.6.5 52/6.2 24/4.8 34/8.6 34/6.9 41/6.0 37/5.0 40/6.4 55/4.9 48/4.9 36/7.6 70/4.6 22/6.3 21/8.3 35/10.3 24/4.8 24/4.2 21/10.7 21/10.7 42/7.4 33/7.5 27/10.7 27/10.7 27/4.0 27/4.0 27/4.0 27/6.5

1.86957 1.875 2.05 2.5 3.21429 1.25 7 1.88462 2.84 1.37143 1.08475 1.24286 1.17476 1.92982 2.51429 1.48276 1.02247 1.06931 1.48387 1.16327 1.33846 1.8125 1.21918 1.3 1.35065 1.3125 1.25 3.28 1.36207 1.55556 5.25 2.23529 4.33333 1.24561 1.33846 4.13333 3.27586 1.04082 1.46154 4.125 4.70588 5.4 3.4375 5.25 2.04545

46 32 20 22 28 55 91 98 71 48 64 70 103 110 35 58 89 101 62 49 87 58 89 80 77 80 80 25 58 63 16 76 78 71 87 62 95 49 65 65 16 17 15 32 16

86 60 41 55 90 44 13 52 25 35 59 87 121 57 88 86 91 108 92 57 65 32 73 104 104 105 64 82 79 98 84 34 18 57 65 15 29 51 95 95 66 80 81 110 84

reductase (#91) and cytochrome f (#45) were down-regulated in BCMV infected leaves. However, photosystem I reaction centers the subunit III protein which was also shown to be down-regulated during the viral infection (#51, 57). Earlier studies observed that the expression of RuBisCO, oxygen-evolving enhancer and PSII was suppressed due to viral infection in plants [18,27]. During the TMV infection, the coat protein of TMV was found to interact with photosystem II (PSII) complex, which leads to the disruption of PSII machinery of the host [49]. However, susceptible viral infection leads to the disruption in electron transport chains of chloroplasts/mitochondria and reduced expressions of photosynthesis related proteins results in the suppression of plant primary metabolic activities.

(spot #27), phosphoglycerate kinase (spot #26), glyceraldehyde-3phosphate dehydrogenase (spot #29) modulate expression in response to viral stress. It has been shown that glucose/sucrose play a critical role in signaling pathways in plants [52]. Earlier researchers observed that the disturbance in the carbohydrate metabolism could lead to an innate host defense response, as activation of the resistance genes are regulated by the levels of soluble sugars [53]. The soluble sugar content was reduced in Cucurbita pepo leaves due to the infection of zucchini yellow mosaic virus (ZYMV) [54]. Previous studies reports that an increase in sugar level was present in the phloem of cucumber mosaic virus (CMV) infected plants [55]. Additionally, glucose-1-phosphate adenylyltransferase and 6-phosphogluconate dehydrogenase (decarboxylating) enzymes involved in the glycogen metabolism have been shown to be down-regulated in response to virus infection. Interestingly, nucleoside diphosphate kinase was up-regulated in the virus infected samples. The virus utilizes the host de nove biosynthesis of purine/pyrimidine to generate nucleotides required for replication of viral RNA [56]. However, ATP synthase subunit alpha/beta (spot #11) was observed to modulate the expression in response to BCMV infection. The ATP synthase subunit alpha/beta played a crucial role in maintaining the ion and metabolite homeostasis through active transport mechanism [57]. ATP synthase subunit beta was seen to be downregulated in the virus infected samples. Deficiency of ATP synthase

3.6. Energy and carbon metabolism An ATP-generating enzyme, phosphoglycerate kinase (PGK) played crucial role in several pathways such as glycolysis, gluconeogenesis and photosynthesis [50]. The chloroplast-PGK also play roles in viral infection via transport viral RNA from cytoplasm to the chloroplast [51]. The present study shows that enzymes related to the primary metabolism were differentially modulated in BCMV infected samples. The enzymes related to glycolysis and gluconeogenesis pathways were downregulated due to viral infection. The fructose-bisphosphate aldolase 5

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Fig. 3. Heat map representation and hierarchical clustering of differential identified proteins in control leaf and BCMV infected leaf of P. vulgaris.

leads to alteration in ultra-structure of mitochondrial and chloroplast [58].

3.8. Chaperones Heat shock proteins (HSPs), play a crucial role in maintaining the cellular homeostasis by converting the misfolded proteins to their native conformation [68]. The major classes of chaperones are HSP70 class of proteins and the expression of these chaperons were up-regulated in response to viral infection [69]. It has been observed that HSP70 played a crucial role in viral replication during early stage of infection. The HSP70 and other co-chaperones showed interaction with viral proteins such as RNA-dependent RNA polymerase (RDRP) and coat protein (CP) [70,71]. The HSPs inhibit premature assembly to facilitate efficient replication and translation of potyvirus genome [72]. In this study, the expression of HSP70 protein (spot #8) was observed to be up-regulated due to BCMV infection. Earlier reports suggested that the HSP83 and HSC70 were up-regulated in response to TYLCV infection in tomato [67]. Another chaperone activity containing enzyme Peptidyl-prolyl-cis/trans-isomerases (PPIases) played a role in protein folding by catalyzing cis/trans-isomerization of peptide bonds [73]. We observed that the expression of PPIases (spot #56) were up-regulated in BCMV infected samples. It is well known that the plant ribosomal proteins (R-proteins) are involved in RNA chaperone activity to maintain the structure of mature ribosomes [74]. However, earlier researchers suggested that the mRNA expression of R-proteins has been increased in Nicotiana benthamiana due to the infection of potyviruses and tobamovirus [75]. In this study, the expression of 30S ribosomal protein S9 was up-regulated in response to BCMV infection. The 30S ribosomal proteins participated in viral replication and accumulation. The RNAi suppressor 2b protein of CMV interacted with 30S ribosomal protein for viral infection in Nicotiana benthamiana plant [76].

3.7. Protein biosynthesis A group of non-ribosomal proteins named eukaryotic elongation factors (eEFs) are essential for translation elongation in eukaryotes. Eukaryotic elongation factor 1A (eEF1A) is one of the most abundant protein in plant cells, play very important role in viral RNA replication and translation by interacting with viral proteins [59]. Earlier reports hint that eEF1A interact with the replication complex of potyviruses, tobamoviruses and tombusviruses [60]. The binding of the 3′-untranslated region (3′-UTR) of Turnip yellow mosaic virus (TYMV) with eEF1A enhanced the translation of viral RNA [61]. The eEF1A has been shown to associate directly with viral RdRp, VPg-protease (VPg-Pro) of Turnip mosaic virus (TuMV) [62], pseudoknot (PK) structure and methyltransferase (MT) domain of RdRp in Tobacco mosaic virus (TMV) [63,64]. Moreover, mutated eEF1A failed to interact with viral genome, which leads to low viral RNA accumulation [59]. In the present study, the expression of eEF1A (spot #23) and elongation factor Tu (spot #25) proteins were shown to be up-regulated in response to viral infection. The thiamin is essential for plant growth, and plays a role in plant defense in abiotic and biotic stress [65]. The subsequent increase in thiamine and its phosphate esters trigger resistance to diverse pathogens [66]. In the present study, the amino acid biosynthesis enzymes, cysteine synthase (spot #93) and thiamine thiazole synthase (spot #90) were up-regulated in BCMV infected sample. Similar results were observed in tomato where cysteine synthase and serine hydroxymethyltransferase showed up-regulated expression in response to the infection of Tomato yellow leaf curl virus (TYLCV) [67]. 6

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Fig. 4. Protein–protein interaction network as elucidated through STRING online server. The interconnected proteins were XP_007131755.1-aldolase; rbcL; XP_ 007135965.1-Glyceraldehyde-3-phosphate dehydrogenase; XP_007136018.1-Catalase; XP_007137439.1-Delta-aminolevulinic acid dehydratase; XP_007138007.1-Elongation factor Tu; XP_007142995.1-ATP synthase subunit beta; XP_007148073.1-Mg-chelatase; XP_007149208.1-Phosphoglycerate kinase; XP_007155256.1-Photosystem I reaction center subunit III; XP_007158494.1Glutathione peroxidase; petA- Cytochrome f;

XP_007143239.1,XP_007139728.1,XP_007150325.1, XP_007148656.1- Uncharacterized protein; XP_007161788.1-Glyceraldehyde-3-phosphate dehydrogenase.

Fig. 5. Distribution of functional categorizations of differentially expressed proteins presented in BCMV infected leaf sample.

3.9. Polyunsaturated fatty acid biosynthesis

transgenic potato during the susceptible infection of PVY and TMV [82]. In this study, we found that these two fatty acid biosynthesis enzymes were down-regulated in BCMV infected samples (spot #44, 45).

Unsaturated fatty acids are well known to play an important role in the membrane integrity and fluidity [77,78], and is also reported to participate in the plant defense mechanism against various environmental stress and pathogens [79]. Acyl-[acyl-carrier-protein] desaturase enzyme is required for the biosynthesis of oleic acid, where it converts stearoyl-ACP to oleoyl-ACP [80]. Another fatty acid biosynthesis enzyme Inositol-1-monophosphatase plays a crucial role in de novo synthesis of inositol [81]. The inositol hexakisphosphate (InsP6) is a phosphate store in plants and works as a signal molecule during the viral infection. Decreased accumulation of InsP6 was observed in

3.10. Virus coat protein In the present study, different isoforms of BCMV-CP (spots #32, 33, 38, 42, 43 and 99) were observed in the BCMV infected leaves samples, which displayed with a molecular mass of around 25 kDa (Fig. 2). The sufficient amount of CP synthesis is foremost prerequisite for successful viral infection. The regulation of CP gene expression is essential for 7

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Fig. 6. The BCMV infection response network in leaf of P. vulgaris.

viral genome replication, virion assembly and movement. There are several evidences about viral coat protein involved in number of hostpathogen interaction which result in the alteration in cellular environment to developed viral infection [72].

these biological functions could result in the reduced plant growth and low yield in production. The differentially expressed proteins can be used as candidate for advance antiviral research, as these proteins are associated with various biological functions and plant–virus interactions.

3.11. Protein-protein interaction analysis

Declaration of competing interest

Proteins do not function as an isolated element in a living cell, rather, one protein interact with another protein in a networks to constitute a biological pathway. The biological pathways were affected in the presence of BCMV due to altered expression of proteins. The STRING server was used to determine affected interlinked pathways through predicted protein-protein interaction network. STRING network analysis revealed the interaction between cytochrome f- RuBisCOPhotosystem I reaction center subunit III- Elongation factor TuFructose-bisphosphate Aldolase- Glyceraldehyde-3-phosphate dehydrogenase- Phosphoglycerate kinase- ATP synthase subunit beta- Deltaaminolevulinic acid dehydratase-Mg-protoporphyrin IX chelataseCatalase- Glutathione peroxidase (Fig. 4). These inter-connected proteins belong to chlorophyll biosynthesis, photosynthesis, biotic/abiotic stress, energy and carbon metabolism, and protein biosynthesis. Additionally, the connectivity of proteins reflects the key links of biological pathways to support viral infection in a plant cell. In conclusion, this is the first report that analyzed proteomic variation between control and susceptible BCMV infected plants. The results revealed that differentially expressed proteins were related to different biological function, in which 46% proteins belong to photosynthesis, 18% biotic/abiotic stress, 11% energy and carbon metabolism, 10% protein biosynthesis, 5% chaperones, 5% chlorophyll biosynthesis, and 5% polyunsaturated fatty acid biosynthesis (Fig. 5). The key differentially expressed proteins are mentioned in Fig. 6 to understand the effect of these proteins in BCMV infected leaf. The major affected biological function was photosynthesis due to the BCMV infection. The down regulation of photosynthesis related proteins leads to low photosynthesis rate as well as deficient energy production, which might be the reason for stunted growth of the plant. The disturbance in

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