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Label-free Proteomics Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.) Ana L. S. Paiva, Jose T.A. Oliveira, Gustavo Antonio de Souza, and Ilka Maria Vasconcelos J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00211 • Publication Date (Web): 04 Nov 2016 Downloaded from http://pubs.acs.org on November 8, 2016

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Label-free Proteomic Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.)

Ana L. S. Paiva, † Jose T. A. Oliveira, †,* Gustavo A. de Souza,‡ Ilka M. Vasconcelos§

†

Laboratory of Plant Defense Proteins, Department of Biochemistry and Molecular

Biology, Federal University of Ceara, Fortaleza, Brazil ‡

Proteomics Core Facility, Institute of Immunology (IMM), Rikshospitalet, Oslo-

Norway §

Laboratory of Plant Toxins, Department of Biochemistry and Molecular Biology,

Federal University of Ceara, Fortaleza, Brazil

* Corresponding author (J.T.A. Oliveira) address: Laboratory of Plant Defense Proteins, Department of Biochemistry and Molecular Biology, Federal University of Ceara, Fortaleza, Brazil, Av. Mister Hull, P.O. Box: 60451-970 Fortaleza, CE, Brazil. Tel.: +55 (85) 33669823; fax: +55 (85) 33669789.

ACS Paragon Plus Environment


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ABSTRACT: Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

KEYWORDS: Vigna unguiculata, CPSMV, compatible interaction, Label-free proteomic


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INTRODUCTION

Cowpea (Vigna unguiculata L. Walp.) is a nutritious legume crop cultivated in the warm and hot regions of the world.1 Although well adapted to unfavorable environmental conditions, pest and pathogen attacks limit cowpea production.2 Cowpea can be infected by more than 20 different virus species and Cowpea Severe Mosaic Virus (CPSMV) is one of the most devastating, responsible for huge losses in productivity.3, 4 CPSMV is classified into the order picornavirales, family Secoviridae, subfamily Comovirinae and the genus Comovirus. Viruses that belong to Comovirus are non-enveloped, have capsids with icosahedral shapes, a bipartite linear single-stranded positive RNA (ssRNA+) genome composed of RNA-1 (6-8 Kb) and RNA-2 (4 to 7 kb), which are translated into two polyproteins further processed in functional proteins.5, 6 In spite of being continuously confronted with unfavorable environmental conditions plants survive because they have developed efficient biochemical and physiological mechanisms in concert with morphological adaptations to cope with adverse conditions.7 By sensing stresses, plants activate and integrate complex signaling and regulatory networks that control the expression of responsive genes to deal with the unfavorable situation and to re-establish cellular homeostasis.8 Studies on the mechanisms of plant immunity and resistance to viruses are numerous and they attempted to explain how plants perceive viral attack, which virus effectors are recognized by the host, which defense genes, proteins and other molecules are involved in the defense mechanisms that plants use to escape infection or reduce severity of disease. However, research studies dealing with the compatible interactions between viruses and plants are scarce. Unanswered or poorly answered questions on the successful infection strategies that viruses use, what and how viral effectors control the



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gene expression of the host to their own benefit, how viruses efficiently use the host molecular and biochemical machinery to replicate their genetic material, how they spread systemically in the plant and cause symptoms, and how they perpetuate in nature are all major issues that should be addressed toward a better understanding of the virus x plant interaction. Therefore, detailed information of this pathosystem could indicate ways to enhance the defensive systems of plants toward increasing health and productivity. For instance, most of these questions remain unanswered for the compatible relationship between cowpea and CPSMV. As final products of gene expression proteins have fundamental importance in the defense mechanism that plants use to counteract natural enemies and tolerate environmental stresses. In compatible or incompatible interactions between plants and pathogens, induction and/or suppression of gene expression alter protein profiles, but few studies on virus-plant interactions have been conducted9-12 with the objective of investigating the reprogramming of host defense-related genes and their encoded proteins, which are the executor molecules that ultimately control cell activities. Liquid Chromatography–Mass Spectrometry (LC–MS) is currently the preferred method of choice for relative protein quantification in proteomics because it is robust, accurate, reproducible, and can achieve low limits of detection.13, 14 Particularly, labelfree quantification based on LC-MS has being increasingly applied to proteomic measurements in place of gel-based methods as differential abundance of proteins can be detect hypothetically for an unlimited number of samples, although each one analyzed individually.15 Therefore on the basis of these technical premises, in this currently pioneering study a gel-free, mass spectrometry-based label-free quantitative shotgun proteomic approach was used to shed light on the possible mechanisms involved in the cowpea-CPSMV compatible interaction. To achieve such objective, a


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high resolution differential proteomic profiling of the leaf proteins of the highly susceptible cowpea genotype CE-3116 was established after inoculation with a CPSMV isolate as an attempt to find out correlations between the susceptibility trait and marker proteins and/or specific metabolic pathways.

EXPERIMENTAL PROCEDURES

Reagents Ammonium acetate, ammonium bicarbonate, polyvinylpolipirrolidone (PVPP), 2mercaptoethanol (2-ME), Tris–phenol solution was purchased from Sigma-Aldrich, Brazil.

DL-dithiothreitol

(DTT),

ethylenediaminetetraacetic

acid

(EDTA),

phenylmethylsulfonyl fluoride (PMSF), iodoacetamide, sodium dodecyl sulfate (SDS), trichloroacetic acid (TCA), and trifluoracetic acid (TFA) were acquired from GE Healthcare, Brazil. M-MLV Reverse Transcriptase, Trypsin and ProteaseMAX were from Promega, Madison, WI, USA. TRIZOL reagent and 24-polyTV primer were from Invitrogen, UK. Other reagents were of analytical grade and obtained from different companies.

Plant Material and Growth Conditions Cowpea seeds (Vigna unguiculata L. Walp., genotype CE-31 (syn. Pitiuba), highly susceptible to the CPSMV isolate CPSMVCE.16 were obtained from the Brazilian Enterprise for Agricultural Research (EMBRAPA) – Meio-Norte, Teresina, Piaui, Brazil. After surface sterilized with 1% (v/v) hypochlorite (0.05% active chlorine) for 3 min, rinsed exhaustively with and soaked in distilled water for 10 min, the seeds were germinated in Germtest® paper (28 x 38 cm) previously moistened with



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Milli-Q grade water under sterile condition with a volume corresponding twice the paper dry mass and kept near 100% relative humidity in the dark for 3 days. The germinated seeds were sown in 1.3 L plastic pots (three per pot) containing river sand that had previously been washed with tap water (5x) and distilled water (3x) before autoclaving (120 °C, 1.5 × 105 Pa, 30 min). The seedlings used for the experiments were irrigated with 10x, 5x, 3x-diluted and undiluted modified17 sterile Hoagland and Arnon’s nutritive solution from the 1st-3th, 4th-7th, 8th-10th and 11th to the end of the experimental period. Plants were kept in a greenhouse under natural conditions of a semi-arid region of Brazil, with day/night temperatures of 27.0 ± 0.8 °C and 31.0 ± 3.0 °C, respectively, 79.8 ± 10.9% relative humidity, exposed to 12-h natural light with the photosynthetic photon flux density (PPFD) varying from 300-650 µmol m−2 s−1, measured (190SA quantum sensor, LI-COR, USA) at plant canopy.

Preparation of the Virus Inoculums To prepare the virus inoculums, leaves of the cowpea genotype CE-31 infected with the isolate CPSMVCE, from Ceara state, Brazil,16 thereafter referred as CPSMV, showing the typical mosaic symptoms, were macerated (1:10, m/v) with 10 mM sodium phosphate buffer, pH 7.0, containing 0.01% (m/v) sodium bisulphite. The suspension obtained was mixed with 500-600 mesh carborundum powder (1:10, m/v), used as abrasive,18 and kept to infect the plants.

Virus Inoculation The CE-31 cowpea genotype was inoculated in the morning (9:00 to 10:00 AM), 12 days after sowing in the pots, by gentle rubbing the virus suspension on the adaxial and abaxial surfaces of fully expanded cowpea primary leaves that were positioned


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individually between the tips of the index finger and thumb with hands protected with surgical gloves. Control (mock inoculated) plants were treated only with 10 mM Kaphosphate buffer, pH 7.0, containing 0.1% (m/v) sodium sulfite and the carborundum powder (abrasive injury), but virus free. Plantlets were arranged in a completely randomized block design with 3 per block with 3 biological replicates. The experiment was repeated three times under the same conditions. Only the primary leaves were collected in the morning (9:00 to 10:00 AM), 2 and 6 days after mock and virus inoculation (DAI), and kept at -80 ˚C for posterior analysis.

Phenolic, lignin, and hydrogen peroxide measurements The qualitative determination of phenolic compounds was performed as previously described.19 Phenolics were visualized by staining the cowpea secondary leaves with toluidine blue as violet-blue structures. For lignin detection decolorized leaf pieces were immersed in phloroglucinol-HCl reagent.19 Phenolic compounds and lignin leaf accumulation were visualized under an optic microscope (Olympus BX 60 Microscope System). To detect H2O2, cowpea leaves were infiltrated with 3’-3’-diaminobenzidine (DAB).20

Protein Extraction This was performed as previously described21 with modifications. Mock and CPSMV inoculated leaf materials (2 g) were finely ground to a powder with a mortar and pestle under liquid nitrogen and 15.0 mL of 10% (m/v) TCA and 2% (v/v) 2-ME in acetone were added for homogenization. After centrifugation at 15,000 × g, 15 min, 4 °C, the supernatant was discarded and the precipitate thrice washed with cold 2% (v/v) 2-ME in acetone and centrifuged as above. The precipitate obtained was solubilized



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with 6.0 mL of 100 mM Tris–HCl buffer, pH 8.0, containing 30% (m/v) sucrose, 2% (m/v) SDS, 1 mM PMSF (a serine protease inhibitor), and 10% (m/v) PVPP, at 4 °C for 10 min, and centrifuged at 10,000 × g, 4 °C, 10 min. To the resulting supernatant an equal volume of commercial Tris–phenol solution, pH 8.0, was added and the mixture centrifuged as above. The upper phenol phase was withdrawn and 6 volumes of 100 mM ammonium acetate in methanol were added and gently mixed. After incubation for 2 h at −20 °C, the mixture was centrifuged (15,000 × g, 15 min, 4 °C) and the precipitate thrice washed with cold 80% (v/v) acetone, and air-dried.

Mass Spectrometry For in-solution digestion, the protein samples were ressuspended in 20.0 µL of 0.2% (v/v) ProteaseMAX in 50 mM ammonium bicarbonate, pH 8.0. After quantification by infrared resonance (Direct Detect, Millipore), 20 µg of protein extracts were reduced with 1 mM DTT at 57 °C for 1 h, followed by alkylation with 5 mM iodoacetamide at room temperature (23 ± 2 °C) for 45 min in the dark. Trypsin (0.5 µg) in 50 mM ammonium bicarbonate, pH 8.0, was added and incubated overnight at 37 °C. Reaction was stopped by adding 1% (v/v) TFA. The ProteaseMax surfactant was removed by centrifugation at 15,000 x g for 1 min, 4 °C. The peptides present in the resulting supernatant were desalted on C18 Empore 3M resin filters (St. Paul, MN). All experiments were performed on a Dionex Ultimate 3000 nano-LC system (Sunnyvale CA, USA) connected to a quadrupole-Orbitrap mass spectrometer (ThermoElectron, Bremen, Germany) equipped with a nanoelectrospray ion source (Easy Spray/Thermo Scientific). For liquid chromatography separation an in-house reverse phase C18 capillary column from Thermo Scientific (Easy Spray column, 2-µm beads, 100 Å, 75-


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µm inner diameter, and 25-cm bed length) was used. The flow rate was adjusted to 0.3 µL/min, and the column equilibrated with solvent A consisting of aqueous 0.1% (v/v) formic acid in 2% (v/v) acetonitrile. Gradient elution was performed using solvent B composed of aqueous 0.1% (v/v) formic acid and 90% (v/v) acetonitrile. The gradient was 5%-32% of B in 240 min, and 32-80% B in 20 min. The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 300 to 1,750) were acquired in the Orbitrap with resolution R = 70,000 at m/z 200 (after accumulation to a target of 3,000,000 ions in the quadruple or 50 ms injection time). The method used allowed sequential isolation of the most intense multiply-charged ions, up to ten, depending on signal intensity, for fragmentation on the HCD cell using high-energy collision dissociation at a target value of 100,000 charges or maximum acquisition time of 100 ms. MS/MS scans were collected at 17,500 resolution at the Orbitrap cell. Target ions previously selected for MS/MS were dynamically excluded for 30 s. General mass spectrometry conditions were: electrospray voltage, 2.0 kV; no sheath and auxiliary gas flow; heated capillary temperature of 250 °C; and normalized HCD collision energy of 25%. Ion selection threshold was set to 1e4 counts. Isolation width of 3.0 Da was used. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 22 partner repository with the dataset identifier PXD005025.

Protein Identification and Label-free Quantification MS raw files were submitted to MaxQuant software version 1.5.2.8 for protein identification.23 Parameters were set as follow: carbamidomethylation of cysteine was used as fixed modification; protein N-acetylation and methionine oxidation as variable modifications. Mass tolerance for MS1 (First search option) was 20 ppm and main



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search tolerance was 6 ppm after recalibration of data. MS/MS mass tolerance was 20 ppm. Trypsin without proline restriction enzyme option was used, with two allowed miscleavages. Minimal unique peptides were set to 1, and FDR allowed was 0.01 (1%) for peptide and protein identification. The LFQ intensities of each protein per sample from MaxQuant outputs (i.e., normalized quantitative values calculated by MaxQuant) were loaded into Perseus, a statistics script from the MaxQuant package. Those values were log transformed to base 2, categorical groups were created based on sample replicates, and groups were statistically tested using Student t-test. Significant differences was determined under stringent criteria (cut-off value * p≤0.05 or ** p≤0.01). An in-house Uniprot database was built including Uniprot sequences for Glycine max, Phaseolus sp., Vigna sp., and CPSMV (downloaded at September 22, 2016). Generation of reversed sequences was selected to assign FDR rates. The VigGS database24 was used (January 4, 2016) to validate protein identification done with the above databases. MaxQuant also adds a list of common contaminants to the database to avoid false-discoveries from contaminant proteins. Proteins identified from the Reversed or the Contaminant list were manually removed prior to sample comparison and statistics. Only proteins with more than 80% of similarity were considered.

Interaction Network Analysis The protein–protein interaction network was built using the publicly available program STRING 9.05 (http://string-db.org/). Briefly, the protein list was blasted against the Arabidopsis thaliana STRING database that included the physical and

functional

relationships of protein molecules supported by associations derived from

eight

evidences: neighborhood in the genome; gene fusions; co-occurrence across genome;


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co-expression; experimental/biochemical data; databases (associated in curated databases); textmining (co-mentioned in Pubmed abstracts); and homology.

Virus detection by RT-PCR Total RNA was extracted and purified from cowpea fresh leaves of uninfected and virus infected plantlets at 6DAI using the TRIZOL reagent, according to the manufacturer's protocol, and the quality of the resulting RNA was assessed by 1% (m/v) agarose gel electrophoresis. Synthesis of first-strand cDNA was performed by incubating 1 µg of total RNA with the M-MLV Reverse Transcriptase and a 24-polyTV primer. After cDNA synthesis, the samples were diluted 100-fold in sterile water. To confirm the absence and/or the presence of CPSMV in the uninfected and virus infecting cowpea plantlets, the first-strand cDNA product that correspond to a fragment of the viral coat protein gene obtained was amplified by RT-PCR using two degenerated universal

oligonucleotide

primers for

the

Comovirus genus25:

GCATGGTCCACWCAGGT-3’(forward)

and

5’5’-

YTCRAAWCCVYTRTTKGGMCCACA-3’ (reverse). The RT-PCR reaction thermal cycler programmed

was an

initial denaturation step at

95 °C

for 5 min,

40 cycles consisting of denaturation at 94 °C for 20 sec, annealing at 41 °C for 20 sec, and extension at 72 °C for 45 sec, followed by an extension step at 72 °C for 10 min. Upon completion of the reaction, the products were visualized (gel-red staining) after 1% (m/v) agarose gel electrophoresis run carried out on a Pharmacia Biotec electrophoresis unit for 40 min, at 25 °C, 100 V, 50 mA.



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Enzymatic Activity Mock and CPSMV inoculated leaf material (1 g) were immersed in liquid nitrogen, finely ground to a powder with a mortar and pestle, and 3 mL of 100 mM K-phosphate buffer, pH 6.8, containing 0.1 mM EDTA, were added to allow protein extraction. After centrifugation at 16,000 × g, 15 min, 4 °C, the soluble protein content of the supernatant was quantified26 and, subsequently, used to evaluate the enzymatic activities. The activities of guaiacol peroxidase (POX), catalase (CAT), and ascorbate peroxidase (APX) were assayed as previously described.27-29 One activity unit (AU) was defined as the change of 1.0 unit of absorbance per milliliter of the protein extract, and expressed as activity unit per milligram of protein (AU/mgP).

RESULTS AND DISCUSSION

Morphological Changes in Cowpea Associated with the CPSMV Disease Development The virus infection did not influence the plant biomass (data not shown), even 6 days after inoculation (DAI). However, the characteristic mosaic symptoms in the compatible interaction with CPSMV were observed (Figure 1A), from 3 days after inoculation (3DAI) in the highly susceptible cultivar CE-31, with appearance of chlorosis and mosaic on the cowpea leaves. These symptoms were absent in the immune cowpea genotype Macaibo,16 confirming the strong susceptibility30 of the cultivar analyzed in this current work. Other common characteristic of this compatible interaction was the presence of symptoms in young tertiary leaves, showing the rapid accumulation and systemic virus movement in the course of infection (Figure 1B). These symptoms were similar to those found in other cowpea CPSMV-susceptible cultivars.4,16


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Phenolic, H2O2, and Lignin contents in Cowpea Associated with the CPSMV Disease Development Phenolic, H2O2, and lignin accumulated in the cowpea primary leaves infected with CPSMV (Figures 2A and B, respectively). Interestingly, lignin was deposited particularly in the guard cells (arrows). Nevertheless, accumulation of these compounds could be related with the plant defense strategies of cowpea against CPSMV. For example, significant phenol and lignin accumulation in wheat infected with wheat streak mosaic virus (WSMV) compared to the healthy controls was also noticed.31,32 Actually, increased H2O2 contents has also been observed in several plant-virus pathosystems and appears to be related with plant defense and disease symptom development.33-35 However in spite of phenol, H2O2, and lignin accumulation in the cowpea primary leaves they were not efficient to hamper CPSMV colonization as virus particles accumulated from 2 to 6 DAI as verified by RT-PCR (Figure 2C).

Cowpea Proteome Changes in Response to CPSMV Infection Virus-induced changes in the protein profile, resulting from plant gene reprogramming in response to infection, provide important clues regarding to the molecules and metabolic pathways involved. In this current study we analyzed the proteome of the cowpea genotype CE-31 in the compatible interaction with CPSMV. Analysis of virushost interactions is often complicated because the viral infection has a progressive character, in the sense that the relative time of replication in isolated cells can be within few hours, while the time required to induce changes in the plant phenotype may be days or weeks.36,37 Therefore, in our study two different time points after the CPSMV inoculation were chosen to be evaluated: an earlier stage, before the appearance of the



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characteristic symptoms (2 DAI); and a later stage, when symptoms were well established and visible to the naked eyes (6 DAI). More than 3000 proteins were identified in the cowpea primary leaves, but only 75 and 55 of them were differentially represented at 2 and 6 DAI, respectively, as compared with the mock inoculated control (Table 1; Tables S-1 and S-2). At 2 DAI, 57 proteins (76%) out of the 75 differentially represented decreased in amount whereas 18 (24%) increased. Interestingly, at 6 DAI, all the differentially represented proteins (55) were accumulated (100%) in relation to the mock inoculated controls. These quantitative results clearly show a marked difference of the protein profiles between the two time points analyzed after CPSMV infection and indicate that during the early stage of infection (2 DAI), before the characteristic disease symptoms were noticed, the protein synthesis machinery of cowpea was blocked or inhibited, as most of the proteins (76%) decrease in abundance. This shift in the plant protein metabolism might be critical for the virus survival and replication in the cell cytoplasm, ensuring viral particle accumulation (Figure 2C) and systemic movement allowing virus maintenance and spread in the plant tissues. At 6 DAI, however, the protein profile changed dramatically. Indeed, 100% of the host proteins accumulated, but despite an apparent response to cope with CPSMV attack had been set up, at this later stage, actually the plant no longer successfully managed to hinder the negative effects incited by viral infection. Qualitative analysis was also performed and the cowpea leaf proteins were classified according to the metabolic processes they participate. In this compatible interaction between CE-31 x CPSMV the differentially accumulated host proteins encoded in response to gene reprogramming, both at 2 and 6 DAI, are involved in several metabolic processes: 4% related to redox homeostasis; 5% to energetic metabolism; 20% to protein metabolism; 25% to defense/stress; 3% to signaling; 9% to RNA/DNA


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metabolism; 15% to other functions; and 19% to proteins whose functions remain unknown (Figure 3). This wide diversity of functions attributed to these proteins suggests influence on the general metabolism of the CPSMV-infected cowpea. Similar result was also noticed for Arabidopsis thaliana in the compatible interactions with Tobacco mosaic virus (TMV).38 Moreover, the levels of the majority of the cowpea leaf proteins involved with various metabolic functions decreased at 2 DAI, but increased at 6 DAI after CPSMV infection (Figure 3). This finding might be linked to the virus ability to differently recruit host proteins, along the infection stages (penetration, replication, movement, and propagation). In RYMV (Rice yellow mottle virus)-infected rice, some host proteins were also specifically recruited, at the early infection stage (one week after infection), during replication in systemically infected leaves, at the onset of the symptom appearance (2 weeks after infection), and at the end of replication and development of other characteristic symptoms (3 weeks after infection).39

Inhibition of Protein Synthesis in CPSMV Infected Cowpea Expression inhibition of several genes was previously observed in cucumber and pea cotyledons infected with CMV (Cucumber mosaic virus) and PSbMV (Pea seed-borne mosaic virus), respectively, coincident with the virus replication.36,40 In TMV-infected Arabidopsis, microarray analysis revealed that 35 out of the 53 genes regulated during infection were repressed. 38 Nevertheless, the synthesis of some host proteins can escape inhibition as occurred during PSbMV infection of peas in which HSP70 and polyubiquitin have their corresponding mRNAs accumulated while several other genes had their expression completely abolished,41 corroborating with the results showed in this current work, particularly at 2 DAI, when 76% of the 75 differentially represented



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leaf cowpea proteins decreased in amount, but 24% increased (Table 1; Tables S-1 and S-2). The negative effects induced by reduction in protein accumulation depend on whether this inhibition is transient or permanent. Obviously that the decrease in abundance of enzymes will trigger negative consequences for the plant organism because there will be a disturbance in the normal cell development. However, for the virus, it is not advantageous to induce many negative effects on the host, as it needs living plant tissue to stay alive and induction of host cell necrosis and death is not a clever strategy. Thus, it is hypothesized that the transient synthesis inhibition of some protein in CE-31 during the initial phase (2 DAI) of CPSMV infection observed in this current work may be crucial for the establishment of the viral disease and susceptibility of the studied cowpea genotype. Conversely, at 6 DAI, protein inhibition no longer occurs (Figure 3) and the cowpea plants activated defense responses as an attempt to minimize the negative effects of the viral infection, but in a delayed fashion. Indeed, as viral particles were accumulated (Figure 2C) and the systemic infection took place at 6 DAI (Figure 1), host defense was not effective and the compatible interaction was established. This hypothesis complements previous findings from our research group, in which gene shutoff was not observed for a cowpea cultivar (Macaibo) immune to CPSMV, at the early stage of infection.30

Proteins Related to Energetic Metabolism Several studies reported that in virus-infected plants decreased energetic metabolism is usual, suggesting that this process is one of the responses suppressed during host defense.42-44 In the CPSMV-infected CE-31 cowpea genotype the energetic-related proteins corresponded to 5% of the differentially represented proteins identified herein


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(Figure 3). At 2 DAI, 4 proteins of this group decreased in abundance compared with mock-inoculated control plants (Table 1): NADPH-dependent quinone oxidoreductase (-4.25); Chlorophyll a-b binding protein CP26 (-1.53); Putative arsenical pump-driving ATPase (-1.58); and Thylakoid lumenal 17.9 kDa (-4.07). At 6 DAI, 3 proteins increased in abundance: Ferrochelatase (4.07); Fructose-bisphosphate aldolase (1.92); and Succinyl-CoA ligase subunit beta (5.25). Since the virus disease symptoms began to be visible at 3 DAI, suppression of protein synthesis linked to the energetic metabolism in the initial stage of infection may turn the plant less apt to fight against CPSMV infection. Under this scenario biosynthesis repression of proteins involved with energy generation in cowpea, as a result of CPSMV infection, may be advantageous for the virus because it weakens the host metabolism and thus the plant ability to invest more in the generation of defense compounds to block the pathogen action. In A. thaliana, genes related to the functioning of chloroplast, plastid composition, and energy metabolism, for example, are suppressed, resulting in chlorosis.45 In Nicotiana benthamiana infected with PMMoV (Pepper mild mottle virus) and PaMMoV (Paprika mild mottle virus), a significant reduction in electron transport in the photosystem II (PSII) was observed, the thylakoid membranes were seriously affected, and the synthesis of some energetic proteins was suppressed.42 Similarly, in susceptible tomato plants infected with CMV, repression of genes encoding proteins of the primary metabolism and involved in photosynthesis and respiration, also occurred.46

Proteins Related to Protein Metabolism The proteins involved in this class corresponded to 20% of all differentially represented proteins in the CPSMV infected cowpea. This might have serious implication in reducing the host defense capacity whereas, somehow, favors the synthesis of the virus



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proteins. Several proteins involved in protein synthesis were inhibited at 2 DAI (Table 1), as elongation factor 1-alpha (-1.17), aspartate aminotransferase (-2.66), N-acetylgamma-glutamyl-phosphate reductase (-1.94), among others. In addition two proteins related to protein degradation also decreased in abundance (UBX domain-containing protein 1 [-2.49]; Ubiquitin fusion degradation 1 [-3.39]) at 2 DAI. The ubiquitinproteasome system (UPS) has emerged as efficient defense machinery against viruses,47,48 but inhibition of its biosynthesis may favor the virus infection. Indeed, inhibition of the ubiquitin gene expression in cabbage inoculated with the cabbage leaf curl virus (CaLCuV) impairs the host defense.49 Moreover, the 26S proteasome, which makes up the UPS, is engaged in the defense against viruses in almost all stages of the host defense mechanism.47,49

Proteins Related to Redox Homeostasis, and Defense/Stress The leaf proteins of CPSMV-infected cowpea associated with the redox homeostasis that changed in abundance corresponded to 4.6% of all those differentially represented at 2 and 6 DAI (Figure 3). At 2 DAI, ascorbate peroxidase (3.99), increased in amount but monothiol glutaredoxin-S17-like protein (-3.17), superoxide dismutase Cu-Zn (7.09), peroxidase (-2.01), and chloroplast stromal ascorbate peroxidase (-3.20) decreased (Table 1). At 6 DAI, only nucleoredoxin 1-like protein (8.03) increased (Table 1). Inhibition of antioxidant proteins and oxidative burst induction in the host during virus interactions has also been reported in other works, such as the systemic production of ROS in response to successful viral infection in Arabidopsis50 and in the susceptible maize ecotypes infected with sugarcane mosaic virus.51 However, the possible role of ROS accumulation in virus infection of susceptible plants is largely


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unknown, particularly because this could lead to HR and programmed cell death (PCD) in the host plant, which is normally unfavorable to virus survival and spread. The class of defense/stress proteins represents one of the most affected after CPSMV infection. It accounted for 24.6% of all differentially represented proteins at 2 and 6 DAI (Figure 3). At 2 DAI, only 4 proteins increased in abundance, while 13 declined (Figure 3). At 6 DAI, the picture was completely reversed and all 15 affected proteins after the virus infection increased in abundance (Table 1). It was previously commented herein that inhibition of the cowpea defense mechanisms and cellular protection at 2 DAI might constitute a great advantage for the virus to replicate. Amongst the proteins related to stress, identified at 2 DAI, peptidyl-prolyl cis-trans isomerases (-3.70) and the prefolding subunit 6 protein (-3.76), decreased in amount whereas the chaperone HSP70 (1.31) accumulated (Table 1). In general, plants need these proteins to facilitate the correct folding of nascent proteins because some may misfold and aggregate as a consequence of various mutations produced during virus replication.52 HSP70 accumulation at 2 DAI, coincident with the decreased amount of most proteins, is a very interesting result. HSP70 is one of the main classes of chaperone and an essential component of the defense mechanism during HR.53 For instance, Pisum sativum, Nicotiana benthamiana, Cucurbita pepo, Nicotiana tabacum, and Arabidopsis thaliana had the HSP70 synthesis induced after viral infection.36,46,52,54 Although the exact role of HSP70 in response to virus attack is not well understood, it is speculated that they are required during the rapid maturation of the viral proteins and virus cell-to-cell movement. Prokhnevsky et al. (2002) showed that HSP70 aids in the virus movement of members of the Closteroviridae family through plasmodesmata.55 Similarly Aoki et al. (2002) showed that HSP70 is related to the virus intercellular trafficking during infection.56



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Important defense proteins directly related to active responses to pathogens were also differentially accumulated (Figure 3). At 2 DAI, most decreased in amount, as putative glutathione S-transferase (-5.68), huntingtin-interacting protein K (-4.19), bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (-6.44), and others (Table 1). Conversely, glucan endo-1,3-beta-glucosidase (β-1,3-glucanase) was the unique protein that accumulated (6.11). β-1,3-glucanases have a curious role in virus-host interaction. To succeed in infection, plant viruses spread throughout their hosts using a number of pathways, such as cell-to-cell movement through plasmodesmata and systemic movement through phloem.57-58 Often plants try to interrupt the virus movement by accumulating callose, a polysaccharide formed by β-1,3-glucans units, in the plasmodemata channels. As β-1,3-glucanase degrades callose, the increased synthesis of this enzyme promotes viral infection and spread, helping the virus movement. Several studies showed that there is a relationship between the deficiency in β-1,3-glucanase activity and a consequent increase in the deposition of callose and thus viral movement restriction in the plant, such as in tobacco mutant line deficient in β-1,3-glucanase that showed higher susceptibility to viral infection,59,60 and in potato that by increasing β1,3-glucanase activity enhanced the PVTNTN (potato virus) spread.61 All together these above results revealed that the changes of several host biochemical processes during the viral infection may be essential for the establishment of the compatible relationship between CPSMV and the cowpea genotype CE-31, highlighting the importance of the inhibition of several host defense mechanisms and the special role of various proteins, particularly HSP70 and β-1,3-glucanase.


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Proteins Related to RNA/DNA Metabolism The plant proteins related to RNA/DNA metabolism deserve special attention because they can trigger specific defense mechanisms against viruses. In this current study they comprised 9% of the differentially represented proteins identified in cowpea (CE-31) primary leaves after CPSMV inoculation compared to control plants (Figure 3). At 2 DAI, only WD-40 repeat-containing protein MSI4 (2.94) increased in abundance (and 9 decreased), while at 6 DAI only KH domain-containing protein (1.65) accumulated (Table 1). These proteins are associated with cellular functions linked to mRNA metabolism, such as processing, transport, location, and stability.62 Moreover, these proteins are involved with post-transcriptional gene silencing (PGTS), which is used by many plants to recognize and avoid virus infection, and suppress RNA accumulation in a sequence-specific manner.63-65 Such mechanism is the principle of RNA interference, which has been intensively used to promote plant protection against many pathogens, especially viruses.66,67 Despite PGTS has been considered a conserved and general plant mechanism for protection against viruses, this pathogens can induce a counter-defense suppressing PGTS by their rapid replication and movement and induction of a silencing state in the host that modifies translation of plant defense proteins.68 For example, potyvirus encodes two accessory proteins, P1 and HC-Pro, and CMV encodes a protein 2b, to impede the virus gene silencing by plants.68 It is also known that many of these suppressive molecules may be responsible for the virus-induced repression of host protein synthesis.66,67 Thus, our results indicate that the CPSMV-induced repression of protein synthesis involved in cowpea transcriptional gene silencing could be an efficient counter-defense induced by this virus and one of the major factors for the successful disease establishment observed and the strong susceptibility of CE-31 genotype.



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Proteins Related to Signaling and Other Functions The category of signaling-related proteins corresponded to 3% of those that had their relative levels modified in cowpea (CE-31) primary leaves after CPSMV inoculation (Figure 3). At 2 DAI, 1 Phosphoinositide phospholipase C (5.32) increased in abundance (Table 1). Likewise, at 6 DAI, Rho guanine nucleotide exchange factor 6 (4.88), Calcium-transporting ATPase 4 (4.24), and Calcineurin subunit B (3.51). Signaling-associated proteins together with phytohormones and Ca2+ are related to plant defense mechanism such as ROS generation, HR activation, and the biosynthesis of PRproteins.69,70 The signaling mediated by Ca2+ is directly related to ROS signaling and activation of HR and PR-proteins.68-71 In relation to proteins categorized in other functions class, they corresponded to 15% of those differentially accumulated. Comparison of CPSMV infected plants with mock inoculated showed that, at 2 DAI, 4 proteins increased and 6 decreased in abundance, whereas, at 6 DAI, 9 had increased levels. Within this category are proteins related to lipid

biosynthesis,

carbohydrate

metabolism,

cell

wall

synthesis,

cytoplasm

organization, and ion and water transporter, among others. It was previously reported that proteins involved with cell wall modification can act against biotic stresses by stimulating the host defense, either directly or indirectly.72,73 The lipid metabolism in plant-virus interaction can also have key role in relation to both energy supply in catabolic or anabolic reactions, and to plasma membrane. For instance, there is evidence that to move from cell to cell after replication viruses induce changes in the plant membrane lipids by reprogramming the host lipid synthesis, degradation, and compartmentalization.74 In this current work, proteins related to the carbohydrate metabolism were also included in the other function group (Figure 3). Viral infections commonly affect plant


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carbon assimilation and carbohydrate metabolism.74-77 It was previously shown that soluble sugar accumulation occurred in PLRV (potato leafroll virus)- and TMVinfected plants or in transgenic plants expressing viral proteins, as an indirect effect induced by the virus movement proteins (MPs).72-75 MPs could interact with plasmodesmata channels favoring both the viral movement and the phloem loading control in infected plants, which influence the storage, translocation, and sugar partitioning.76,77 Accumulation of sugars may also be involved in gene regulation, leading to the repression of genes involved in photosynthesis, as observed in PLRVinfected tobacco.74

Host Protein-protein Interaction Network We searched for known and predicted interactions for the differentially represented proteins identified by Label-Free-based proteomics in the STRING protein-protein interaction database and constructed a protein-protein interaction network (Figure 4). The network is presented under evidence view, whereby stronger associations are represented by thicker lines or edges and vice-versa. Proteins are represented as nodes. The protein names and gene symbols used in this network are listed in the Tables S-3 and S-4. Figure 4 shows 27 differentially represented proteins identified by proteomic approach involved in 30 interactions (p-value 9.14e-3) at 2 DAI, and 20 proteins implicated in 29 interactions at 6 DAI (p-value 2.82e-2). In both figures (A and B), 10 predicted functional partners were added to enhance the information value of results. These relationships were found to be linked either directly or indirectly through one or more interacting proteins, suggesting the existence of known functional linkages. In both times, 2 and 6 DAI, it was possible to clearly detect the existence of complex protein clusters, mainly associated with protein metabolism, stress response, redox



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homeostasis, and energetic production (represented by the purple, yellow, red, and orange circles, respectively) (Figure 4). This bioinformatic analysis provided information on the physical and functional properties, known and predicted interactions of the corresponding genes and their products, and revealed a relatively complex interaction map. In addition, these biological networks can also give information about relative importance of these proteins to virus infection. The nodes with a high degree of connectivity symbolize central points for virus colonization and have been used to explore many virus-host interactions.78,79

Validation of Selected Differentially Represented Proteins The enzymatic activities of some proteins identified in this current work, which are involved in redox homeostasis class, were analyzed to validate the proteomic results. The use of enzymatic activities to validate proteomic data are often more reliable than other techniques such as RT-qPCR, taking into consideration that changes on mRNA levels do not always will represent changes in protein abundance. Figure 5 shows that peroxidase, ascorbate peroxidase and catalase activities significantly decrease at 2 DAI. These enzymatic activities are a functional validation and are directly correlated with the enzyme protein abundance80 confirming our proteomic results. These assays have been used in many others proteomic studies.81,82

General Interpretation of the Compatible Interaction of Cowpea CE-31 Genotype and CPSMV All together, the results of the present study suggest that during the compatible relationship between cowpea (CE-31 genotype) and CPSMV, a great number of host leaf proteins decreased in amount probably due to inhibition of their synthesis induced


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by the virus to its own benefit at the earlier stage of infection (2 DAI). Importantly, several of these proteins have central role in disease establishment. A general overview of such interaction is represented in Figure 6. The mechanism by which the transient shutoff of cowpea primary leaf proteins is induced by CPSMV is not yet known. Some proteins that favor plant defense were identified probably because the host gene shutoff induced by CPSMV is not very specific, as the virus induced alteration in the profile of several classes of proteins even those that can impair the virus infection. However, apparently, there is a balance between the plant defense proteins toward minimizing the negative effects of infection, but the virus-inducing plant protein shutoff favored the successful establishment of the virus in the host. Often plants are considered susceptible not because they cannot respond actively to a biotic stress, but because they did not respond within an appropriate time interval after the pathogen challenge. There are several examples of susceptible plants to various diseases in which the hosts react to the attack even with stronger intensity compared to the resistant counterparts, but induced in a delayed fashion with no value toward preventing the establishment of the disease.

CONCLUSIONS This pioneering proteomic study was conducted to gain insights into the molecular basis of cowpea (CE-31 genotype) susceptibility to CPSMV. More than 3000 proteins were identified and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased in amount. Thus CPSMV transiently suppresses the synthesis of proteins involved particularly in



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the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone number: +55(85)3366 9823. Fax number: +55 (85) 33669789. Addresses Laboratory of Plant Defense Proteins, Department of Biochemistry and Molecular Biology, Federal University of Ceara, Fortaleza, Brazil

Author Contributions JTAO, IMV, and ALSP designed the experimental project. ALSP, JTAO, GAS, and IMV performed the experimental work: GAS and ALSP conducted the mass spectrometry analysis. JTAO, ALSP, GAS, and IMV wrote the manuscript. JTAO, IMV, and ALSP proofread and edited the manuscript.

CONFLICTS OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGMENTS All authors acknowledge the financial support and ALSP a MS grant from Council for Advanced Professional Training (CAPES). JTAO also acknowledges the financial support from the National Council for Scientific and Technological Development


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(CNPq, grant 308107/2013-6) and the Research Council of the State of Ceara (FUNCAP, grant 2155/PRONEX).

SUPPORTING INFORMATION

Table S-1, differentially accumulated proteins identified by label-free proteomic in cowpea (CE-31 genotype) primary leaves 2 days after CPSMV inoculation (2 DAI);

Table S-2, differentially accumulated proteins identified by label-free proteomic in cowpea (CE-31 genotype) primary leaves 6 days after CPSMV inoculation (6 DAI);

Table S-3, list of proteins analyzed by the String program for protein-protein interaction predictions 2 days after CPSMV inoculation (2 DAI);

Table S-4, list of proteins analyzed by the String program for protein-protein interaction predictions 6 days after CPSMV inoculation (6 DAI)



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Table 1. Summary of differentially accumulated proteins* in the primary leaves of cowpea (CE-31 genotype) identified by label-free proteomics at 2 and 6 days after CPSMV inoculation compared with mock-inoculated control 2 DAI REDOX HOMEOSTASIS Ascorbate peroxidase (3.99) Monothiol glutaredoxin-S17-like protein (-3.17) Superoxide dismutase [Cu-Zn] (-7.09) Peroxidase (-2.01) Chloroplast stromal ascorbate peroxidase (-3.20) DEFENSE/STRESS Glucan endo-1,3-beta-glucosidase 12 (6.11) Intracellular pathogenesis related protein (2.94) Heat shock 70 kDa protein 14 (1.31) Subtilisin-like protease (5.81) GrpE protein homolog (-4.23) Peptidyl-prolyl cis-trans isomerase (-5.95) Putative glutathione S-transferase (-3.45) Aspartic proteinase (-3.83) Prefoldin subunit 6 (-3.76) Peptidyl-prolyl cis-trans isomerase (-3.70) Huntingting-interacting protein K (-4.19) 14 kDa proline-rich protein DC2.15 (-6.13) Bifunctional dihydroflavonol reductase/flavanone 4-reductase (-6.44) S- glutathione S-transferase (-5.68) Peptidyl-prolyl cis-trans isomerase (-1.47) Desiccation protectant protein Lea14 like (-1.43) SAL1 phosphatase (-0.99)

6 DAI

Nucleoredoxin 1-like protein (8.03)

Glutathione S-transferase-like (5.20) Copper chaperone for superoxide dismutase (3.41) Aldehyde dehydrogenase family 2 member B7 (4.94) 18.5 kDa class I heat shock protein (7.73) Apoptotic chromatin condensation inducer (5.73) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (5.36) Peptidyl-prolyl cis-trans isomerase FKBP62 (7.44) Heat shock 70 kDa protein, mitochondrial (1.54) Heat shock 22 kDa protein, mitochondrial (7.02) Intracellular pathogenesis related protein (7.65) Tau class glutathione S-transferase (5.73) Universal stress protein A-like protein (2.86637) Phosphatase IMPL1, chloroplastic (4.34) Heat shock protein 83 (3.88) Subtilisin-like protease (8.81)

RNA/DNA METABOLISM WD-40 repeat-containing protein MSI4 (2.94) RNA and export factor-binding protein 2 (-2.34) RNA and export factor-binding protein 2 (-4.47) Nuclear pore complex protein NUP35 (-3.05) Transcription termination factor family protein (3.83) KH domain-containing protein (1.65) High mobility group B protein (-4.24) Snrnp sm protein (-6.97) HMG-Y-related protein A (-5.21) HMG1/2-like protein (-7.25) Squamosa promoter-binding-like protein 12 (4.17) PROTEIN METABOLISM


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60S ribosomal protein L8-3-like protein (1.90) Threonine--tRNA ligase, mitochondrial (2.50) Alanine--tRNA ligase (3.01778) N-acetyl-gamma-glutamyl-phosphate reductase (3.19) Elongation factor 1-alpha (-1.17) UBX domain-containing protein 1 (-2.49) Aspartate aminotransferase (-2.66) Putative bifunctional methylthioribulose-1phosphate dehydratase/enolase-phosphatase E1 (4.18) Aspartate aminotransferase (-2.06) Ubiquitin fusion degradation (-3.39) Aspartate-semialdehyde dehydrogenase (-2.34) Vesicle transport V-snare protein vti1a (-3.32) N-acetyl-gamma-glutamyl-phosphate reductase (1.94) ENERGETIC METABOLISM NADPH-dependent quinone oxidoreductase (4.25) Chlorophyll a-b binding protein CP26 (-1.53) Putative arsenical pump-driving ATPase (-1.58) Thylakoid lumenal 17.9 kDa (-4.07) SIGNALING Phosphoinositide phospholipase C (5.32) OTHERS FUNCTIONS Glycosyltransferase (8.64) Molybdopterin biosynthesis protein CNX1 (3.56) Alpha-1,4 glucan phosphorylase (2.17) Potassium transporter (1.50) Fasciclin-like arabinogalactan protein 1-like (5.66) Transaldolase (-4.41) Phosphomannomutase/phosphoglucomutase (4.35) Cell division protein FtsZ like 1, chloroplastic (1.59) Triose phosphate/phosphate translocator (-2.89) Formin-like protein (-2.98)

Protein Ycf2 (1.73) UBA and UBX domain-containing protein (4.90) 3-isopropylmalate dehydratase (4.12) Coatomer subunit beta'-1 (4.74) Aspartate-tRNA ligase (3.84) Vacuolar protein sorting-associated protein 2 like 3 (6.25) Mitochondrial-processing peptidase subunit alpha (1.34) Vacuolar sorting receptor (2.99) Putative methionine--tRNA ligase (4.02) 60S ribosomal protein L12, putative (4.67) Ribosomal protein L19 (6.20) 40S ribosomal protein S3-3 (2.41) Eukaryotic translation initiation factor 3 (4.81)

Ferrochelatase (3.37) Fructose-bisphosphate aldolase (1.92) Succinyl-CoA ligase subunit beta (5.25)

Rho guanine nucleotide exchange factor 6 (4.88) Calcium-transporting ATPase 4, endoplasmic reticulum-type (4.24) Calcineurin subunit B (3.51)

Putative aquaporin PIP2;2 (7.62) Histone H2A (5.44) Putative ripening-related protein 2 (6,68) GAMYB-binding protein (3.30) Beta-xylosidase/alpha-L-arabinofuranosidase-like (2.46) Aquaporin TIP2-1 (6.28) 3-ketoacyl-CoA synthase (3.90) Cell division protein FtsZ like 1, chloroplastic (4.75) Acyl-coenzyme A oxidase 4, peroxisomal (4.75)

* The number in parenthesis represents the ratio between the content of the protein in the mock-inoculated control and that in the CPSMV-inoculated plants expressed in base-2 logarithm (log2). Positive values mean that the protein was more abundant in CPSMV-inoculated plants than in control plants. Negative values mean that the protein was more abundant in control as compared with CPSMV-inoculated plants.



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FIGURE LEGENDS

Figure 1. Disease symptoms in cowpea plants 6 days after CPSMV inoculation (6 DAI). (A) Comparison between the susceptible (CE-31) and resistant (Macaibo) genotypes. Symptoms are well visible in CE-31. (B) Symptoms are shown in the secondary leaves of the CPSMV infected cowpea CE-31genotype. Photograph courtesy of Paiva, A.L.S. Copyright 2016.

Figure 2. Phenolic, lignin, hydrogen peroxide, and virus accumulation in cowpea (CE31 genotype) primary leaves of mock- and CPSMV-inoculated plants at 6 DAI. (A) Left panel: phenolic compounds (pink); right panel: lignin (green, arrow). (B) Hydrogen peroxide accumulation (dark spots). (C) CPSMV accumulation assessed by RT-PCR. Photograph courtesy of Paiva, A.L.S. Copyright 2016.

Figure 3. (A) Functional classification of differentially accumulated proteins identified in cowpea (CE-31) primary leaves after CPSMV inoculation. (B) Distribution of differentially represented protein of cowpea (CE-31) primary leaves highlighting the differential accumulation over time. Red bars represent proteins that decreased in amount. Green bars represent proteins that increased in amount after CPSMV inoculation.

Figure 4: Bioinformatic STRING analysis of the differentially represented proteins of cowpea (CE-31) primary leaves identified by label-free proteomic 2 (A) and 6 (B) days after CPSMV inoculation. Stronger associations are represented by thicker lines or edges and vice-versa. Proteins are represented as nodes. The protein names and gene


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symbols used in this network are listed in the Tables S-3 and S-4. In both figures, 10 predicted functional partners were added to enhance the information value of results. Protein clusters associated with protein metabolism, stress response, redox homeostasis, and energetic production are represented by the purple, yellow, red, and orange circles, respectively.

Figure 5. Protein content and enzyme activity of peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT) in cowpea (CE-31) primary leaves 2, 4, and 6 days after CPSMV inoculation. * and ** represent significant difference between means at p ≤0.05 and p ≤0.01, respectively.

Figure 6. Schematic proposal of the molecular events that allow CPSMV infection and spreading in a susceptible cowpea genotype (CE-31), based on the present knowledge of plant-virus interactions and the observations from this current research work. (1) virus entry, virus uncoating, and release of the viral genomic RNA followed by induction of protein synthesis repression; (2) reduction in energy production and photosynthesis; (3) repression of protein synthesis related to plant post-transcriptional silence (PGTS) mechanism; (4) HSP70 over-accumulation and ROS generation; (5) β-1,3-glucanase accumulation and callose degradation at plasmodesmata, allowing;; (6) viral particles accumulation into cell cytoplasm virus and cell-to-cell movement.



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Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5


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Figure 6



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