Insights on Carica papaya L. proteomic, ultrastructural and physiological changes associated with pre-flowering-related tolerance to papaya sticky disease

Insights on Carica papaya L. proteomic, ultrastructural and physiological changes associated with pre-flowering-related tolerance to papaya sticky disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Insights on Carica papaya L. proteomic, ultrastructural and physiological changes associated with pre-flowering-related tolerance to papaya sticky disease Silas P. Rodrigues, Eduardo de A. Soares, Tathiana F. Sá-Antunes, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4523827/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The development of Papaya Sticky Disease (PSD), caused by the papaya meleira virus (PMeV) complex, only occurs after flowering, suggesting the presence of tolerance mechanisms during the transition from juvenile to adult papaya plants ( C. papaya ). In this study, 1,609 leaf proteins of C. papaya were quantified using a label-free strategy. Differentially accumulated proteins—38, 130, 160, and 17 at 3, 4, 7, and 9 months post-germination, respectively—indicated modulation of biological processes at each development phase, mainly involving photosynthesis and cell wall remodeling. Juvenile C. papaya plants infected with the PMeV complex showed an accumulation of photosynthetic proteins. Correspondingly, chlorophyll fluorescence results suggested enhanced efficiency in photosystem (PS) II and PSI energy flux in these plants. In parallel, pre-flowering plants exhibited a reduction in cell wall-degrading enzymes, followed by an accumulation of proteins involved in the synthesis of wall precursors post-flowering. These findings, combined with ultrastructural data on laticifers, suggest that C. papaya struggles to maintain the integrity of laticifer walls, ultimately failing to do so after the juvenile-adult transition and resulting in latex exudation, thereby supporting initiatives for the genetic improvement of C. papaya to enhance resistance against the PMeV complex. Label-free quantitative proteomics mass spectrometry papaya meleira virus photosynthesis laticifer Figures Figure 1 Figure 2 Figure 3 Key Message The proteomic analysis of PMeV/PMeV2-infected C. papaya unveiled proteins undergoing modulation during the plant's development. The infection notably impacted processes related to photosynthesis and cell wall dynamics. Introduction The global production of papaya ( Carica papaya L. ) fruit is over 13 million tons and the major papaya fruit producers are India, Brazil, Nigeria, Mexico, and Indonesia (FAOSTAT 2018 ). However, papaya cultivation is vulnerable to a "virus complex", composed of papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2) (Sá Antunes et al. 2016 ; Sá Antunes et al. 2020 ). PMeV is a fusagra-like double-stranded RNA virus (dsRNA), which naturally infects C. papaya by unknown routes (Sá Antunes et al. 2020 ). PMeV shows synergism with the umbra-like single-stranded (ssRNA) PMeV2, resulting in P apaya S ticky D isease (PSD) (Sá Antunes et al. 2016 ). The PMeV complex infects C. papaya laticifers, cells specialized in the production and storage of latex, which contain cardenolides, alkaloids, isoprene polymers, enzymes, etc. (Hagel et al. 2008 ). The infection is known to induce changes in the latex structure and composition, and to increase the production of H 2 O 2 and the accumulation of potassium, phosphorus, and water (Rodrigues et al. 2009 ). The main PSD symptom is a spontaneous exudation of watery latex, mostly on fruits and leaves. The latex is oxidized after contact with the air and accumulates on the plant as a sticky and brownish substance, which makes the fruit unfit for marketing (Sá Antunes et al. 2016 ; Sá Antunes et al. 2020 ). Previous literature on the morphophysiological, biochemical and molecular changes undergone by sticky-diseased C. papaya points out that the control of protein turnover is due to a decrease in microRNA (miR162, miR398, and miR408) expression, which is predicted to target proteasome-related proteins (Sá Antunes et al. 2020 ). The levels of proteasome-related proteins increase in leaf samples collected from diseased plants (Rodrigues et al. 2011 ). In the same study, symptomatic C. papaya showed an accumulation of calreticulin, 20S proteasome b subunit, and pathogenesis related proteins (PRs) ( e.g. endochitinase and PR-4) (Rodrigues et al. 2011 ). In parallel, the proteomic analysis of diseased C. papaya latex samples showed reduced serine proteinase inhibitor and chymopapain cysteine protease levels (Rodrigues et al. 2012 ). The first PSD symptoms, small necrotic lesions on the tips of young leaves, appear three to four months post-germination (mpg). This period coincides with the beginning of floral bud development, blooming, and the early development of fruits (Cosmi et al. 2017 ). Our group previously analyzed PMeV complex-infected C. papaya at the pre-flowering stage using proteomics, identifying 111 differential proteins (57 up- and 54 down-regulated) (Soares et al. 2016 ). An increase in photosynthesis-related proteins and a decrease in 26S-proteasome and cell wall remodeling proteins were observed. In parallel, field observations revealed that PSD symptoms seem mostly associated with fruit development (Cosmi et al. 2017 ). Thus, the analysis of PMeV complex-infected C. papaya plants throughout different development phases will help understand the mechanisms involved in PSD symptoms progress. In this study, PMeV complex-infected and non-infected C. papaya plants were grown in the field until they reached 3, 4, 7, and 9 months post-germination (mpg). Leaf samples were collected, and their proteins were identified and quantified via LC-MS/MS-based proteomics. Using a label-free approach, a total of 1,609 proteins were quantified. The differential proteins (38, 130, 160, and 17 proteins at 3, 4, 7, and 9 mpg, respectively) represent a more complete view of the different biological processes modulated in C. papaya during PSD symptoms development, in which photosynthesis, cell wall, protein and RNA metabolism, and response to stress are mainly involved. The results obtained through proteomics were complemented by physiological and microscopy analyses. Material and Methods Plant Material One-month-old C. papaya cv. Golden seedlings (n = 6) were planted at the INCAPER experimental farm, in Sooretama-ES, Brazil. They were cultivated for two months. At that point, the plants were injected at the youngest leaf petiole either with 1 mL of latex from C. papaya sticky-diseased plants diluted (1:1, v/v) in 50 mM sodium phosphate buffer, pH 7.0 (PMeV complex), or with buffer only. After the treatments, the second fully expanded leaf of each plant was immediately collected and frozen in liquid nitrogen (3 mpg samples). The tissues were freeze-dried and stored at -80°C until use. Subsequent sampling was performed at 4 mpg (30 days post-infection, dpi), 7 mpg (120 dpi), and 9 mpg (180 dpi) (Supplemental Table S1 ). Protein Extraction The leaf tissue was mechanically homogenized in liquid nitrogen and 10 mg of tissue powder was submitted to total protein extraction using phenol (Wang et al. 2012 ). The extracted proteins were pelleted after the addition of 4 mL of 0.1 M ammonium acetate in methanol. The mixture was then incubated for 10 hours at -20°C and centrifuged (10 min, 20,000 × g, 4°C). The pellets were washed twice with 1.5 mL of 0.1 M ammonium acetate in methanol, once with 1.5 mL of 80% acetone, and once with 1.5 mL of 70% methanol. The proteins were resuspended in 180 µL of 50 mM Tris-HCl pH 8.0, 8 M urea, and 2 M thiourea solution and assayed using CB-X protein assay (Genotech, St. Louis, MO). Protein Digestion The proteins were incubated for 45 min with 5 mM dithiothreitol (DTT) at 37°C. They were then incubated with 100 mM iodoacetamide for 40 min at 25°C in darkness. Then, 1 M urea with 50 mM Tris-HCl, pH 8.8 was added to the samples. Protein digestion was performed at 37°C for 16 h and 800 rpm using a trypsin solution (Sigma, St. Louis, MO) at 1:50 enzyme/substrate ratio, which was stopped by adding 2% formic acid final concentration. PepClean C18 spin columns (Thermo Scientific, Rockford, lL) were used to desalt the resulting peptides which were resuspended in 150 µL of 0.1% formic acid (FA)/5% acetonitrile (ACN). LC-MS/MS Analysis 1 µg of peptide mixture was loaded onto a trap column (NanoAcquity UPLC 2G-W/M Trap 5 µm Symmetry C18, 180 µm × 20 mm) in a NanoAcquity UPLC system (Waters, Milford, MA) coupled to a TripleTOF 5600 MS/MS (AB SCIEX, Framingham, MA) for 3 min at 5 µL/min. A 5–40% linear gradient of solvent B was used to separate the peptides in a C18 capillary column (NanoAcquity UPLC 1.8 µm HSS T3, 75 µm × 250 mm) at 300 nL/min. Solvent A consisted of 0.1% FA in water and solvent B consisted of 0.1% FA in acetonitrile (ACN). After each sample analysis, a column cleaning was performed (5 min from 40–85% of solvent B and 85% of solvent B for 10 min) and re-equilibration (2 min from 85–5% of solvent B and 5% of solvent B for 13 min). The mass spectrometer was operated in positive ionization and high sensitivity mode. The features were selected for Information Dependent Acquisition (IDA) MS/MS experiments based on an MS survey. The spectrum was accumulated from 350 to 1600 m/z for 250 ms, of which the first 20 features with a charge state of + 2 to + 5 and exceeding a 150 counts threshold were selected. The same features were included on an 8 s dynamic exclusion list prior to fractionation using ± 5% rolling collision energy. As a reference sample, a homogeneous mixture of equivalent peptide amounts from all replicates was analyzed and used for label-free protein quantification. To assure the high mass accuracy in both MS and MS/MS acquisition, the instrument was automatically calibrated after every three samples (6h). Protein identification and label-free quantification Progenesis QI for proteomics v2.0 (NonLinear Dynamics) was used to create two-dimensional ion intensity maps of features extracted from the TripleTOF 5600 raw files (.wiff). The peak picking, reference assignment, and spectra alignment (≥ 80% score) parameters were performed as automatic for the features eluting between 25 and 105 min. The Mascot server v.2.2.2 (Matrix Science Inc., Boston, MA) was used for protein identification by interrogating the peak list file (.mgf) from Progenesis QI against a custom database (27,898 sequences total, May 2015) containing all C. papaya protein entries available on Phytozome 10.2 (27,775 sequences, May 2015) (Souza et al. 2017 ; Guzha et al. 2019 ) combined with NCBI C. papaya organelle (123 sequences, May 2015). The parameters of + 2 to + 4 charge state, two missed cleavages, and a mass tolerance of ± 20 ppm for precursor ions and ± 0.05 Da for fragment ions were considered for protein identification. Additionally, deamidation at asparagine or glutamine, cysteine carbamidomethylation, methionine oxidation, and acetylation at peptide N-term were considered as variable modifications. Mascot percolator algorithm was used, providing a False Discovery Rate (FDR) < 1% prior to XML file exportation and Progenesis QI reimportation for peptide quantification and identification. The protein quantification was performed using the normalized abundances of Hi-3 (up to 3) peptides (Silva et al. 2006 ) filtering for Mascot peptide scores ≥ 13. The protein abundances occurring in all three control and PMeV complex-infected biological replicates were compared by one-way ANOVA test and the protein list was filtered based on p ≤ 0.05 and a Log 2 fold change (FC) of ± 0.58. Differential abundance analysis and protein functional classification Gene ontology (GO) analysis of all identified proteins was performed in Blast2GO ( www.blast2go.org ) by blasting the identified protein sequences against the NCBI non-redundant (nr) database with an expected E-Value threshold of 10 − 10 and the first ranked hit was further used. The GO enrichment for up- and down-accumulated protein sets used a Fisher's Exact test with the multiple testing correction FDR option selected (Benjamini et al. 1995). Additionally, the differential proteins (p ≤ 0.05; FC ± 0.58) were submitted to C. papaya overview metabolic pathway mapping using the MapMan software ( http://mapman.gabipd.org/web/guest/mapman ). Chlorophyll a fluorescence parameter analysis In a greenhouse, 2 mpg plants were either injected with 20 µL latex (n = 5) from sticky-diseased plants diluted with buffer or injected with buffer only (n = 5), similarly to 2.1. Thus, the chlorophyll (Chl) fluorescence parameters were collected weekly from 1–49 days after injection (DAI) at fully developed and 40-min dark-adapted leaves. The measurements were taken from 7:00 to 9:00 AM (UTC-3) using a portable fluorometer (Handy PEA+, Hansatech, UK). The kinetics of Chl a fluorescence was measured using a saturating light pulse of 3,000 µmol (photon) m –2 s –1 at 650 nm for 1 s to generate a true fluorescence intensity of maximum value (FM) using Pea Plus software v. 1.13. Fluorescence parameters were derived from the fast Chl a fluorescence induction (OJIP) according to JIP-test equations (Strasser, 1977 ). The measurements were performed at least in triplicate at six leaves of each plant. The terms and formulas for calculating the JIP-test parameters are described in Supplemental Table S2 . After exporting the data as Excel spreadsheets, it was normalized, compared using the Tukey test and presented as radar plots. Scanning and Transmission Electron Microscopy For scanning electron microscopy (SEM), 1 x 1 cm asymptomatic and symptomatic leaf fragments were fixed in Karnovsky solution (2.5% glutaraldehyde and 4% paraformaldehyde in 0.1M sodium cacodylate (EMS, USA) pH7.2 for 4 h at room temperature. The fragments were post-fixed in a solution containing 1% osmium tetroxide in water, for 4 h at room temperature in the dark. Subsequently, the fragments were dehydrated in an increasing series of ethanol/water solutions (30%, 50%, 70% and 90%, v/v) for 30 minutes, followed by 4 additional dehydration steps with 100% ethanol. In the end, the fragments were dried using the critical point method, cross-sectioned into 2 mm x 1 cm fragments, mounted on stubs and metalized with gold. The samples were observed using a VEGA 3 LMU scanning electron microscope (TESCAN, Czech Republic) operating at 20 kV. For transmission electron microscopy (TEM), symptomatic and asymptomatic leaves were sectioned into 3 x 5 mm. The fragments were fixed as described above and post-fixed in a solution containing 1% osmium tetroxide, 1.25% potassium ferrocyanide and 5 mM calcium chloride in water, for 4 h at room temperature in the dark. The fragments were then dehydrated in an increasing series (30 min each) of acetone/water (30%, 50% and 70% v/v) solutions, followed by 4 additional steps using 90% and 100% acetone. The fragments were then infiltrated with low viscosity epoxy resin (SPURR, Electron Microscopy Science) using different acetone/resin (v/v) solutions (3:1, 2:1, 1:1, 1:2 and 1:3) for 4 h under seesaw agitation. Finally, the fragments were infiltrated with 100% resin for 24 h under seesaw agitation. Ultrathin sections were obtained using a PT-PC PowerTome ultramicrotome (RMC Boeckeler, USA), stained with uranyl acetate and lead citrate, and observed using a TEM FEI TECNAI SPIRIT operating at 120 kV. Optical Microscopy Optical microscopy analyzes were performed using 300 nm semi-thin slices obtained from materials processed for TEM, as described above. Thus, the sections were collected and placed on glass slides previously heated to 70°C. After fixing the sections on the slides, they were stained using a 1% toluidine blue and 1% boric acid solution in water. After drying, the slides were washed with distilled water, and the samples were observed using an optical microscope DM 2500 (LEICA, Germany). Results Proteomic analysis of C. papaya leaf A total of 1,609 proteins (99%) were quantified based on Progenesis QI analysis of 34,624 ions (Supplemental Tables S3-S6). This dataset represents the most extensive proteomic coverage reported for C. papaya to date. The proteins were annotated, and the Gene Ontology (GO) profiles for biological processes, molecular function, and cellular component are shown in Supplemental Figures S1 and S2. The 1,609 quantified proteins consisted of 1,242 proteins from 3 mpg samples (pre-flowering), 1,454 from 4 mpg samples (flowering), 1,493 from 7 mpg samples (fruiting), and 1,442 from 9 mpg samples (ripening). These proteins exhibited an average coefficient of variance (CV) of 28% (23% CV median) (Supplemental Table S3 , Supplemental Figure S3 ). They were utilized to correlate the protein abundances of PMeV complex-infected C. papaya leaf and control samples for each plant age group. A total of 1,533 protein sequences (94%) were assigned to at least one Gene Ontology identification number (GO ID) (Supplemental Table S4 ). The third-level GO term grouping by biological process (Supplemental Figure S4 ), molecular function (Supplemental Figure S5 ), and cellular component (Supplemental Figure S6 ) are presented. Differential proteome of pre-flowering PMeV complex-infected C. papaya vs. control plants At 3 mpg, 38 proteins exhibited significant abundance changes (p ≤ 0.05), with an FC of ± 0.58 or greater (Supplemental Table S7 , Supplemental Figure S7 ). Notably, lipid transfer protein 4 (6,1 FC) and AMP-dependent synthetase and ligase family protein (4,1 FC) showed the highest change in abundance levels, while a putative uricase/urate oxidase/nodulin 35 (-10 FC) and calcium-dependent lipid-binding plant phosphoribosyltransferase family protein (-10 FC) displayed the lowest accumulation levels. The 3 mpg group did not exhibit statistically significant (p ≤ 0.05) enrichment of GO term proteins (Supplemental Table S8 ). At 4 mpg, 130 proteins demonstrated significant changes in abundance among the total quantified proteins (Supplemental Table S10 , Supplemental Figure S7 ). The proteins with the most notable change in abundance levels were haloacid dehalogenase-like hydrolase family protein (3 FC) and sucrose phosphate synthase 3F (2,675 FC), while the proteins with the lowest accumulation levels were p-loop containing nucleoside triphosphate hydrolases superfamily protein (-10 FC) and subtilase family protein (-10 FC). Fifteen GO terms were enriched (p ≤ 0.05) within the 4 mpg C. papaya leaf proteins. Among these, eight were most represented among the up-regulated proteins, primarily associated with photosynthesis and oxidoreductase activity. Conversely, seven GO terms were most represented among the down-accumulated proteins, including RNA binding, catabolic process, and membranous cellular components (Supplemental Table S8 ). The MapMan overview metabolic pathway analysis indicated an up-accumulation of proteins associated with photosynthesis, carbohydrate metabolism, organic acid transformations, and amino acid metabolism. Conversely, there was a down-accumulation of proteins related to cell wall structure, lipid metabolism, stress response, RNA metabolism, protein metabolism, and cell organization during the pre-flowering (Fig. 1 , Supplemental Tables S9 and S11). Differential proteome of post-flowering PMeV complex-infected C. papaya vs. control plants The 7 mpg plant group contained the largest subset of the quantified proteins, with 160 proteins showing significant changes in abundance (p ≤ 0.05, FC ± 0.58) (Supplemental Figure S7 , Supplemental Table S12 ). Only photosynthesis was identified as an enriched GO term (p ≤ 0.05) among the down-accumulated proteins, while plastids, thylakoids, and generation of precursor metabolites and energy were the most represented enriched GO terms among the up-accumulated proteins (Supplemental Table S8 ). The proteins with the highest change in abundance levels were alpha/beta-Hydrolases superfamily protein (4,1 FC) and DEA(D/H)-box RNA helicase family protein (3,1 FC), while the lowest accumulation levels were observed for Kunitz trypsin inhibitor 1 (-3,4 FC) and non-photochemical quenching 1 (-2,9 FC). At 9 mpg, 11 up- and 6 down-accumulated proteins were identified among the 1,442 proteins representing the plant group (p ≤ 0.05, FC ± 0.58, Supplemental Table S14, Supplemental Figure S7 ). The 9 mpg phase had no statistically significant (p ≤ 0.05) enriched GO terms (Supplemental Table S8 ). The proteins with the highest change in abundance levels were ATP-dependent caseinolytic (Clp) protease/crotonase family protein (2,1 FC) and Cyclophilin-like peptidyl-prolyl cis-trans isomerase family protein (2,1 FC), while the lowest accumulation levels were observed for PLC-like phosphodiesterase family protein (-1,8 FC) and P-loop containing nucleoside triphosphate hydrolases superfamily protein (-1,5 FC). The overview metabolic pathway revealed proteins up-accumulated in categories including carbohydrate metabolism, mitochondrial proteins, cell wall, lipid metabolism, amino acid metabolism, stress, nucleotide metabolism, protein metabolism, and signaling. Conversely, among the down-accumulated proteins, only photosynthesis was highlighted at post-flowering phases (Fig. 1 and Supplemental Tables S13 and S15). Chlorophyll fluorescence analysis of PMeV complex-infected C. papaya vs. control plants For these experiments, greenhouse-grown plants were used to simulate the pre-flowering and asymptomatic phase of PMeV-complex-infected C. papaya . Consistent with the up-accumulation of photosynthesis-related proteins in infected plants at 3 and 4 mpg, the quantification of chlorophyll a fluorescence parameter (Supplementary Table S2 ) revealed an increased Performance Index (PI) ABS at 35 and 42 DPI in PMeV-complex-infected C. papaya (Fig. 2 ). PI ABS reflects the energy absorption by PSII antenna pigments, derived from terms expressing energy bifurcations from absorption to the reduction of the electron transport chain (Tsimill-Michael, 2020). This energy cascade involves light absorption (ABS), trapping (TR) (primary photochemistry), reduction of pheophytin (Phe) and quinone A (QA), electron transport (ET) after Qa- to intersystem electron acceptors, and energy dissipation (DI) (Tsimill-Michael, 2020). In the PMeV-complex-infected leaf cross-section (CS), the plants exhibited a reduction in ABS (ABS/CS), DI (DI/CS), and TR (TR/CS) at 28 and 35 DPI (Fig. 2 ). ET (ET/CS) was reduced only at 28 DPI (Fig. 2 ). Additionally, compared to the control, there was a significant increase in oxygen-evolving complex (OEC) activity in infected C. papaya after 28 and 35 DPI. OEC activity returned to normal at 42 and 49 DPI (Fig. 2 ). OEC in photosystem II catalyzes the oxidation of water into dioxygen, protons, and electrons (Ferreira et al., 2004 ). PI total extends beyond PI ABS by incorporating the γ reaction center (γRC) parameter, reflecting the energy bifurcation until the reduction of PSI end electron acceptors (Tsimill-Michael, 2020). In infected plants, both PItotal and γRC/(1 – γRC) increased at 35, 42, and 49 DPI compared to the control (Fig. 2 ). Additionally, the indicator of reduction of end acceptors at the PSI electron acceptor side, RE/CS, significantly increased at 35, 42, and 49 DPI (Supplemental Figure S8 ). These findings collectively suggest a heightened efficiency in PSII and PSI energy flux in PMeV-infected juvenile C. papaya . Bright-field optical microscopy (BFM) and scanning electron microscopy (SEM) Bright-field microscopy (BFM) and scanning electron microscopy (SEM) were employed to examine potential morphological and ultrastructural changes in PMeV-infected cells. BFM of semi-thin sections was additionally conducted to facilitate the selection of sections for transmission electron microscopy analysis. Initial differences between asymptomatic and symptomatic samples were noted via BFM (Supplemental Figure S9 ). In healthy plant leaves, numerous laticifer cells filled with intracellular content were observed (Supplemental Figure S9 C, arrows), some displaying anastomosis. Conversely, diseased samples exhibited empty laticifer cells (Supplemental Figure S9 D, arrow). This observation was supported by SEM imaging (Supplemental Figure S10 ), wherein laticifers in asymptomatic plants appeared replete with intracellular content, while those in symptomatic plants appeared empty. Possible cellular alterations were also discerned (Supplemental Figure S10 ). Transmission electron microscopy (TEM) TEM analysis revealed that laticifer cells in asymptomatic leaves exhibited numerous granules with varying electron densities (Fig. 3 C). Conversely, examination of laticifers in symptomatic plants indicated a notable difference in intracellular contents compared to healthy plants. As depicted in Fig. 3 D, diseased laticifer cells displayed a reduced number of granules, signifying a substantial alteration in cytoplasmic content relative to healthy laticifers (Fig. 3 C). At higher magnification, the ultrastructure of laticifer cell walls appeared well-defined and preserved in asymptomatic plants, encompassing regions of the primary wall, secondary wall, and median lamella akin to those observed in healthy plants. In the inset of Fig. 3 B, the parallel organization of cellulose microfibrils in the laticifer wall of asymptomatic plants is discernible. Conversely, symptomatic plants (Fig. 3 D) exhibited several differences, including disorganization of the laticifer cell wall (thin arrow) and vacuolization in the region of the secondary wall (arrowheads). In the inset of Fig. 3 D, notable vacuolization of the cell wall is evident, indicating signs of cellulose microfibril degradation and loss of ultrastructural organization. Morphometric analysis was conducted to quantify changes in the cell wall (Fig. 3 E). Cell wall thickness was measured across different cells and regions, with a total sample size (N) of 25 measurements. The analysis revealed an average wall thickness of approximately 662 nm in symptomatic laticifers, whereas asymptomatic laticifers exhibited an average thickness of around 300 nm. This twofold increase underscores the degree of organization of cellulose microfibrils, which were observed as juxtaposed and parallel in asymptomatic cells. Conversely, microfibrils in symptomatic cells appeared more loosened, occasionally degraded, and disorganized (Fig. 3 ). Transmission electron microscopy provided insight into the presence of numerous viral particles within the laticifer cells of symptomatic plants (Supplemental Figure S11 , indicated by arrows), often found in proximity to membrane structures, potentially the endoplasmic reticulum. Further magnified images enabled the determination of viral particle size, estimated to be around 50 nm, thereby confirming the observed structures as PMeV. Discussion In a preliminary investigation (Soares et al., 2016 ), the proteome of PMeV complex-infected C. papaya leaf samples during pre-flowering was examined. Expanding upon this research, the current study extends the proteomic analysis to samples collected at four distinct phenological phases (i.e., 3-, 4-, 7-, and 9-mpg) to elucidate the underlying mechanisms contributing to PSD symptom development. Notably, symptoms of PMeV infection manifest post-flowering initiation, specifically at the 7 and 9 mpg time points. Therefore, the 3 and 4 mpg time points represent the plant pre-flowering, despite being infected, and are characterized as asymptomatic. To our knowledge, the protein dataset presented in this study represents the most extensive proteomic coverage of C. papaya to date. Combined with complementary physiological and microscopy experiments, this dataset enhances the understanding of the interaction between PMeV-complex and C. papaya . In the following sections, particular attention is given to proteomic changes associated with photosynthesis and cell wall remodeling. Changes in other areas will be explored in subsequent publications. Changes in photosynthesis-related proteins The reduction of photosynthetic activity is a characteristic feature of symptom development in virus-infected plants (Li et al., 2016 ). In our study, a significant accumulation of photosynthesis-related proteins in PMeV-complex infected C. papaya at 3 and 4 mpg, corresponding to the pre-flowering phase, compared to controls was observed. Notably, PsbQ-like2, a crucial component of the oxygen evolving complex (OEC), exhibited up-accumulation in asymptomatic plants at 4 mpg but down-accumulation in symptomatic plants at 7 mpg. Alongside PsbP (up-accumulated at 4 and 7 mpg), PsbO, and PsbR, PsbQ-like2 positively influences photosystem II (PSII) efficiency (Sasi et al., 2018 ). Moreover, physiological experiments measuring chlorophyll a fluorescence revealed an increased OEC activity in PMeV-infected juvenile C. papaya leaves. PsbQ is known to play a critical role in maintaining and enhancing PSII function and stability under stress conditions (Ifuku et al., 2005 ; Yi et al., 2006 ). These findings suggest that the enhanced accumulation and activity of PSII components may contribute to the inhibition of PSD symptom development by juvenile, pre-flowering, and infected plants. Some chloroplast proteins have been reported to directly interact with viruses, influencing both virus accumulation and symptom development. For instance, PsbP from Arabidopsis was found to interact with the coat protein of Alfalfa mosaic virus (AMV), leading to the inhibition of reactive oxygen species (ROS) production in the chloroplast and facilitating virus accumulation (Balasubramaniam et al., 2014 ). Similarly, the disease-specific protein (SP) encoded by rice stripe virus (RSV) interacts with and reduces the accumulation of PsbP in the chloroplast of Nicotiana benthamiana protoplasts, thereby enhancing virus accumulation and RSV-induced symptoms. Conversely, PsbP overexpression has been shown to suppress AMV replication (Li et al., 2016 ), while overexpression of PsbP reduced chlorosis in leaves caused by RSV (Kong et al., 2014 ). In our study, the accumulation of PsbQ-like 1, PsbQ-like 2, and PsbP proteins in asymptomatic PMeV complex-infected plants was observed. PsbQ protein stabilizes PsbP binding to PSII (Kakiuchi et al. 2012 ), and their increased levels may potentially interfere with PMeV-complex replication. Consistent with this hypothesis, a reduction in PsbQ-like accumulation was associated with the onset of PSD symptoms. Several chloroplast proteins, including those crucial for photosynthesis, have been implicated in plant immunity processes (Järvi et al., 2016 ). For instance, in the compatible interaction between mungbean yellow mosaic India virus (MYMIV) and Vigna mungo , there is a decrease in the photosynthetic rate and chlorophyll content of plants (Kundu et al., 2013 ). Conversely, incompatible interactions lead to an increase in photosynthetic proteins such as rubisco activase, PSII OEC protein, and ribulose-1,5-bisphosphate carboxylase small subunit (Kundu et al., 2013 ). This suggests that successful plant resistance against virus infection involves an upregulation of photosynthetic proteins. In line with this concept, our study revealed the accumulation of rubisco activase, light-harvesting complex photosystem II, and photosystem II subunit proteins in PMeV complex-infected, but symptomless, plants. This accumulation mirrors findings from previous studies (Yoshioka et al., 2011 ; Sánchez-Vicente et al., 2019 ; Caplan et al., 2015 ), supporting the notion that chloroplasts play a pivotal role in PSD symptom development. This role could be attributed to the accumulation of reactive oxygen species (ROS), which contribute to programmed cell death (PCD) (Madroñero et al., 2018 ), and/or the production of salicylic acid (SA), a key regulator of systemic acquired resistance (SAR) (Spoel et al., 2012). The light-driven reactions of photosynthesis, particularly at the level of PSI acceptors, play a crucial role in regulating reactive oxygen species (ROS) production and are highly sensitive to stress conditions. Among physiological parameters, the photosynthesis performance indexes (PI abs and PIt otal ) have emerged as efficient tools for quantifying stress levels (Strasser et al., 2010 ; Huther et al., 2016). PI total , which reflects the performance of PSI end electron acceptors through the δRo / (1 − δRo) parameter, provides insights into the efficiency of electron transfer processes (Tsimilli-Michael & Strasser, 2008 ). In this study, juvenile PMeV-complex infected C. papaya leaves exhibited significant increases in PSI activity and end acceptors reduction, as evidenced by elevated PIt otal , δRo / (1 − δRo), and REo/CSo values. These findings suggest that the accumulation of proteins related to PSI, PSII, and plastocyanin (which were up-accumulated in infected pre-flowering C. papaya ) enhances maximum energy flow and electron transfer efficiency (Strasser et al., 2010 ). Interestingly, infected but asymptomatic plants appeared to modulate light absorption, energy dissipation, and trapping, as indicated by reduced ABS/CS0, DI0/CS0, and TR0/CS0 parameters. The accumulation of two antioxidant enzymes, thioredoxin M-type 4 and NADPH-dependent thioredoxin reductase C, at 4 mpg in PMeV complex-infected C. papaya compared to controls suggests a potential role for redox balance in the chloroplast of juvenile infected plants, contributing to symptom development tolerance. Interestingly, previous studies have shown that the abundance of maize thioredoxin (ZmTrxh) transcripts strongly correlates with the latency of sugarcane mosaic virus (SCMV) symptoms (Liu et al., 2017 ). Moreover, ZmTrxh overexpression has been demonstrated to suppress SCMV RNA replication and accumulation, indicating its involvement in plant resistance against SCMV at early infection stages (Liu et al., 2017 ). Changes in cell wall-related proteins Microscopic examination of leaf sections post-flowering initiation revealed distinct disparities between asymptomatic and symptomatic plants. In bright-field microscopy, laticifers in asymptomatic leaves were visibly filled with latex, while those in symptomatic leaves appeared mostly empty, consistent with 'meleira' symptoms. This observation was further confirmed through scanning electron microscopy. Additionally, transmission electron microscopy revealed noticeable variations in the structure of laticifer cell walls. Healthy laticifer cell walls exhibited well-defined primary, secondary, and median lamella regions, with cellulose microfibrils organized in parallel. In contrast, sections from symptomatic leaves displayed significant disorganization of the laticifer wall, including degradation of cellulose microfibrils and loss of ultrastructural organization, leading to vacuolization within the secondary cell wall. This structural deterioration, coupled with the high osmotic pressure within infected laticifers (Rodrigues et al., 2009 ), likely contributes to latex leakage. Terrestrial plants exhibit a primary cell wall composition that shares similarities across species, primarily comprised of cellulose microfibrils, hemicellulose, and pectic substances (Höfte & Voxeur 2017 ). Cellulose microfibrils form the backbone of the wall and are intertwined with hemicellulose xyloglucans, which act as non-covalent connectors. Xyloglucans feature a (1,4)-β-linked glucan backbone regularly substituted with (1–6)-α-xylosyl residues. Together, cellulose and xyloglucans constitute two-thirds of the dry mass of the cell wall, forming a network crucial for withstanding tensile forces. However, the cleavage of xyloglucan chains, without simultaneous synthesis, can weaken the cell wall, potentially leading to rupture. This process can be facilitated by endo β-1,4 glucanases, which modify the viscosity and porosity of the wall, enabling the action of other enzymes such as expansins (Scheller & Ulvskov 2010 ). In our study, PMeV-complex infected pre-flowering C. papaya exhibited reduced accumulation of several cell-wall degrading enzymes involved in cellulose and hemicellulose degradation, including β-1,4 glucanases, β-D-xylosidase, β-glucosidase, and β-galactosidase, compared to uninfected plants. This reduction in enzyme levels is noteworthy considering their role in host resistance to various pathogens in other plant species. For instance, in tomato plants, endo-β-1,4-glucanase has been shown to influence host resistance by modulating the expression of PR1 and callose deposition. Decreased levels of endo-β-1,4-glucanase are associated with enhanced resistance to Botrytis cinerea but increased susceptibility to Pseudomonas syringae (Flors et al. 2007 ). Similarly, infected C. papaya exhibited elevated levels of PR1 mRNA and protein, suggesting potential activation of defense responses. Additionally, upregulation of callose synthases mRNA and downregulation of the callose-degrading enzyme glucan endo-1,3-beta-glucosidase indicate possible callose accumulation, which could restrict PMeV-complex movement within the host. Furthermore, the decreased accumulation of β-1,3 glucanase, a member of the PR2 family involved in callose degradation and symplastic trafficking regulation, further supports the notion of host defense mechanisms limiting virus spread. In addition to the changes in cell-wall degrading enzymes, infected juvenile plants exhibited increased accumulation of pectin lyase-like protein compared to controls. This protein is involved in the production of 4,5-unsaturated oligogalacturonides (OGs), which serve as important elicitors of plant defense responses against various pathogens (Côté et al. 1994). Studies in Arabidopsis thaliana have shown that OGs accumulate prior to symptom development caused by fungal infection. However, the onset of symptoms coincides with increased OG oxidation, reducing their elicitor activity. This oxidation is mediated by OG oxidases, primarily belonging to the Berberine Bridge Enzyme-like (BBE-like) protein family. Interestingly, our study identified a BBE-like enzyme, methyl esterase 10, which accumulated in PMeV/PMeV2-infected C. papaya plants at 4 and 7 mpg. This finding suggests a potential role for OGs and their oxidation products in modulating the plant's response to PMeV infection. The balance between the accumulation and processing of cell-wall degrading enzymes' products, such as OGs, may contribute to the tolerance of pre-flowering C. papaya plants to PSD symptoms by preserving the integrity of the cell wall structure. The onset of PSD symptom development during the plant post-flowering coincides with disruptions in the laticifer cell wall, as observed in our study. Interestingly, despite these changes, there was no significant alteration in the abundance of wall degradation enzymes between infected and control plants at 7 mpg, suggesting a progressive accumulation of these enzymes over the course of symptom development. Concurrently, infected plants exhibited an induction of several enzymes involved in the synthesis of cell wall precursors, such as Glucose-1-phosphate adenylyl transferase (GPA), UDP-glucose 6-dehydrogenase (UGD), NAD(P)-binding Rossmann-fold superfamily protein, nucleotide-rhamnose synthase, and phosphomannomutase. GPA catalyzes the initial step in starch synthesis, while UGD plays a crucial role in the biosynthesis of nucleotide sugars, including UDP-glucuronic acid, a precursor for various cell wall components (Reboul et al. 2011 ). Notably, mutants lacking UGD activity display swollen cell walls, indicating the importance of this enzyme in maintaining cell wall integrity. One plausible hypothesis is that infected plants respond to the increasing osmotic pressure within laticifers by upregulating wall synthesis enzymes and remodeling enzymes. However, this compensatory response becomes inadequate by the flowering phase, leading to cell wall disruption and leakage. Interaction with jasmonic and salicylic acid-mediated pathways Salicylic acid (SA) is known to be a key regulator in systemic acquired resistance (SAR), a defense mechanism against pathogens in plants (Spoel et al., 2012). Previous studies (Madroñero et al., 2018 ) have demonstrated the induction of several SA-activated genes, including pathogenesis-related proteins (PRs), WRKY transcription factors, reactive oxygen species (ROS), and callose genes in PMeV-infected pre-flowering C. papaya plants. This suggests the involvement of SA signaling in the delayed onset of symptoms observed in these plants. Moreover, treatment of pre-flowering C. papaya with exogenous SA led to a reduction in PMeV and PMeV2 loads compared to control infected plants. SA has been shown to induce the expression of PR proteins, which are known to reduce virus loads in plants (Spoel et al., 2012). This effect is likely mediated through the oxidation of NPR1 (nonexpressor of pathogenesis-related genes 1), a master immune coactivator (Kinkema et al., 2000 ; Tada et al., 2008 ; Fu & Dong, 2013 ). Consistent with this, activated forms of NPR1 were observed in PMeV-infected C. papaya plants at 4 mpg, coinciding with the upregulation of SA/NPR1-dependent transcripts during this developmental phase (Madroñero et al., 2018 ). In our study, an accumulation of photosynthesis-related proteins in infected plants at 3 and 4 mpg, accompanied by an increase in chlorophyll a fluorescence parameter was observed. This finding is consistent with previous research (Yoshioka et al., 2011 ; Sánchez-Vicente et al., 2019 ; Caplan et al., 2015 ), suggesting a crucial role of chloroplasts in conferring viral tolerance. It is likely that this tolerance mechanism involves the accumulation of reactive oxygen species (ROS), which have been shown to contribute to programmed cell death (PSD) in infected plants (Madroñero et al., 2018 ), as well as the production of salicylic acid (SA). Supporting this hypothesis, an accumulation of antioxidant proteins at 4 mpg, indicating a potential defense response against viral infection was observed. The dynamics of the jasmonic acid (JA) pathway in PMeV-infected C. papaya before flowering are intricate. Previous research has demonstrated the accumulation of transcripts encoding a candidate NPR1-inhibitor (NPR1-I/NIM1-I), UDP-glycosyltransferases (UGTs), and genes involved in the ethylene pathway in infected plants (Madroñero et al., 2018 ), all of which are known negative regulators of salicylic acid (SA) signaling. Additionally, at 4 mpg, infected plants accumulate a pectin-hydrolytic enzyme that can enhance the release of cell-wall defense response triggers, potentially leading to increased biosynthesis of JA (Gouveia et al., 2017 ). However, our findings reveal lower levels of UDP-glucosyltransferase proteins than controls before flowering, but higher levels post-flowering (7 mpg). Furthermore, it is noteworthy that one of the most down-regulated proteins in infected plants at 3 mpg is urate oxidase, which produces allantoin, an activator of the JA pathway (Kaur et al., 2023 ). Adding to the complexity is the role of oligogalacturonides (OGs). An accumulation of a protein associated with OG production, pectin lyase-like protein, in juvenile infected plants compared to controls was observed. Conversely, an OG oxidation protein, methyl esterase 10, accumulated in infected plants at 4 to 7 mpg. This mirrors findings from A. thaliana , where OGs were found to accumulate before symptom development but were oxidized just before symptoms appeared during fungal infection (Benedetti et al., 2018 ). Notably, OGs have been shown to induce both the salicylic acid (SA) and jasmonic acid (JA) pathways in A. thaliana (Howlader et al., 2020 ). In post-flowering, characterized by symptom development, we observed a reduction in the overproduction of photosynthesis-related proteins compared to controls. This is reminiscent of findings in symptomatic pecan plants infected with the Badnavirus Pecan Virus, where inhibited SA biosynthesis and reduced photosynthesis were reported (Zhang et al., 2022 ). Conversely, contrasting responses have been observed in soybean plants infected with different forms of the Soybean Mosaic Virus. The virulent form induced the jasmonic acid pathway, while the avirulent form stimulated callose production and upregulated photosynthesis genes, resulting in rapid elimination (Alazem et al., 2018 ). Taken together, these responses suggest that SA signaling likely dominates at the pre-flowering phase of PMeV complex-infected C. papaya , inhibiting the development of PSD symptoms. However, the induction of negative regulators of SA and concurrent activation of the JA pathway prevent full-scale and long-lasting tolerance. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding P.M.B. Fernandes and J.A. Ventura acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq for their research productivity award (# 303432/2018-7 and # 307905/2020-9). This work was supported by FAPES grants # 76437906/16 and # 80598609/17 awarded to P.M.B.F., and E. Soares and thank the Fundação de Amparo à Pesquisa do Espírito Santo, FAPES (# 76437906/16), and T. F. Sá- Antunes, and M. Maurastoni thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES for their scholarships (# 88882.315885/2019-01 and, 88887.467501/2019-00). Author Contributions S.P.R., J.A.V., and P.M.B.F. conceived and designed the study. E.A.S., T.S.A., M.M., S.G.B., L.E.C.N., and B.R.F.V conducted the experiments. All authors analyzed and discussed the results. S.P.R., E.A.S., T.S.A., M.M., D.B., and P.M.B.F. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Acknowledgments The authors extend their gratitude to Emily G. Werth and Leslie M. Hicks from the Department of Chemistry, University of North Carolina at Chapel Hill, for their invaluable technical and scientific assistance with proteomic analysis. 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The numbers indicate the number of sequences grouped in each GO term(s) SupplFigS2.tif Supplemental Fig. S2 Gene Ontology (GO) grouping of PMeV complex-infected C. papaya leaf proteins according to their associated molecular function (A) and cellular component (B) using Blast2GO software. The numbers indicate the number of sequences grouped in each GO term(s) SupplFigS3.tif Supplemental Fig. S3 Protein abundance reproducibility. The coefficient of variation (CV) obtained for the experimentally obtained peptide abundances using %𝐶𝑉=(𝑆𝑇𝐷𝐸𝑉𝑃∕𝐴𝑉𝐸𝑅𝐴𝐺𝐸)×100 SupplFigS4.tif Supplemental Fig. S4 Gene Ontology (GO) bar chart displaying the third level GO terms percentage in up-accumulated (red) and down-accumulated (green) proteins. The proteins were grouped by their predicted GO Biological Process in 3 (A), 4 (B), 7 (C), 9 (D) months post-germination (mpg) SupplFigS5.tif Supplemental Fig. S5 Gene Ontology (GO) bar chart displaying the third level GO terms percentage in up-accumulated (red) and down-accumulated (green) proteins. The proteins were grouped by their predicted GO Molecular Function in 3 (A), 4 (B), 7 (C), 9 (D) months post-germination (mpg) SupplFigS6.tif Supplemental Fig. S6 Gene Ontology (GO) bar chart displaying the third level GO terms percentage in up-accumulated (red) and down-accumulated (green) proteins. The proteins were grouped by their predicted GO Cellular Component in 3 (A), 4 (B), 7 (C), 9 (D) months post-germination (mpg) SupplFigS7.tif Supplemental Fig. S7 Label-free quantitative time course (months post-germination, mpg) PMeV+PMeV2-infected C. papaya proteome coverage. The quantified proteins were present in all three replicates, and were found differential after filtering using p<0.05 and ±0.58 fold change Log2 (Infected/control) SupplFigS8v02.tif Supplemental Fig. S8 Chlorophyll a fluorescence parameters, OEC, ABS/CS0, D10/CS0, TRO/CS0, ET0/CS0, RE0/CS0, RC/CS0. Control (black) and PMeV2 (red) samples, at 1, 7, 14, 21, 28, 35, 42, 49 dpi. SupplFigS9.tif Supplemental Fig. S9 Bright-field optical microscopy. (A) Cross-section of the vascular bundle of asymptomatic leaves of Carica papaya . (C) High magnification of the asymptomatic leaf showing the presence of laticifers (arrows) distributed throughout the vascular bundle. (B) Cross-section of the vascular bundle of symptomatic leaves of Carica papaya . (D) High magnification of the symptomatic leaf showing empty cells. It is difficult to identify the presence of laticifers in this image. The arrow points to one laticifer cell in anastomosis, identifiable by its lack of content. SupplFigS10.tif Supplemental Fig. S10 (A-B) Cross-section of the vascular bundle of asymptomatic leaves of Carica papaya observed by scanning electron microscopy. (B) High magnification of the cross-section of an asymptomatic leaf shows the presence of laticifer cells (arrow) with their vesicular content. (C-D) In contrast, a cross-section of the vascular bundle of symptomatic leaves shows several empty cells where it is difficult to identify the presence of laticifers. Upon closer inspection, alterations in the cell wall can be observed compared to the symptomatic leaves (Fig. 4). SupplFigS11.tif Supplemental Fig. S11 (A-B) Transmission electron microscopy showing various viral particles within laticifer cells of symptomatic plants (arrows), sometimes close to membrane structures, possibly endoplasmic reticulum profiles. (B) High magnification allows determination of the size of the viral particles to 50 nm, suggesting that such structures are PMeV Rodrigueset.al.2024SupplementalTablesS1S15v01.xlsx Supplemental Table Captions Supplemental Table S1 Description of the plant development phases and sampling times used in this study Supplemental Table S2 Term description and calculation formulas for the JIP-test Supplemental Table S3 Proteomic coverage of PMeV complex-infected and control C. papaya leaf samples Supplemental Table S4 Compiled list of proteins identified in PMeV complex-infected and control C. papaya leaf samples Supplemental Table S5 Peptides identified from PMeV complex-infected C. papaya leaf samples Supplemental Table S6 Label-free quantification of PMeV complex-infected C. papaya leaf proteins Supplemental Table S7 Proteins differently modulated in 3 months post-germination (0 days post-inoculation) PMeV complex-infected C. papaya leaf Supplemental Table S8 Gene Ontology terms enrichment analyses of up- vs. down-accumulated PMeV complex-infected C. papaya modulated proteins Supplemental Table S9 Metabolic pathway overview of differential proteins in 3 months post-germination (0 days post-inoculation) PMeV complex-infected and control C. papaya leaf samples Supplemental Table S10 Proteins differently modulated in 4 months post-germination (30 days post-inoculation) PMeV complex-infected C. papaya leaf Supplemental Table S11 Metabolic pathway overview of differential proteins in 4 months post-germination (30 days post-inoculation) PMeV complex-infected and control C. papaya leaf samples Supplemental Table S12 Proteins differently modulated in 7 months post-germination (120 days post-inoculation) PMeV complex-infected C. papaya leaf Supplemental Table S13 Metabolic pathway overview of differential proteins in 7 months post-germination (120 days post-inoculation) PMeV complex-infected and control C. papaya leaf samples Supplemental Table S14 Proteins differently modulated in 9 months post-germination (180 days post-inoculation) PMeV complex-infected C. papaya leaf Supplemental Table S15 Metabolic pathway overview of abundance changed proteins in 9 months post-germination (180 days post-inoculation) PMeV complex-infected and control C. papaya leaf samples Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4523827","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":316157005,"identity":"fa796f94-6414-4be1-9715-0e6914507aec","order_by":0,"name":"Silas P. 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Sá-Antunes","email":"","orcid":"","institution":"UFES: Universidade Federal do Espirito Santo","correspondingAuthor":false,"prefix":"","firstName":"Tathiana","middleName":"F.","lastName":"Sá-Antunes","suffix":""},{"id":316157008,"identity":"ab73a6ee-f8f5-48be-a1e3-32e0eb9363ce","order_by":3,"name":"Marlonni Maurastoni","email":"","orcid":"","institution":"UFES: Universidade Federal do Espirito Santo","correspondingAuthor":false,"prefix":"","firstName":"Marlonni","middleName":"","lastName":"Maurastoni","suffix":""},{"id":316157009,"identity":"89646222-bb08-4365-9afa-b76b9bfbf9ec","order_by":4,"name":"Sabrina G. Broetto","email":"","orcid":"","institution":"UFES: Universidade Federal do Espirito Santo","correspondingAuthor":false,"prefix":"","firstName":"Sabrina","middleName":"G.","lastName":"Broetto","suffix":""},{"id":316157010,"identity":"e0d14fa2-f3ea-4e30-84bf-19d71c85cc1b","order_by":5,"name":"Lucas E. C. Nunes","email":"","orcid":"","institution":"UFRJ: Universidade Federal do Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Lucas","middleName":"E. C.","lastName":"Nunes","suffix":""},{"id":316157011,"identity":"a795559f-8b77-4d14-a763-d87c30be7ce4","order_by":6,"name":"Brunno R. F. Verçoza","email":"","orcid":"","institution":"UFRJ: Universidade Federal do Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Brunno","middleName":"R. F.","lastName":"Verçoza","suffix":""},{"id":316157012,"identity":"b1ab1e55-80a4-4328-8bc4-f29bbccd8153","order_by":7,"name":"David Buss","email":"","orcid":"","institution":"UFES: Universidade Federal do Espirito Santo","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Buss","suffix":""},{"id":316157013,"identity":"e466fed8-6254-48aa-b92a-51309889397b","order_by":8,"name":"Diolina M. Silva","email":"","orcid":"","institution":"UFES: Universidade Federal do Espirito Santo","correspondingAuthor":false,"prefix":"","firstName":"Diolina","middleName":"M.","lastName":"Silva","suffix":""},{"id":316157014,"identity":"fb5f7f82-1992-426c-a1d3-f505dc3e0255","order_by":9,"name":"Juliany C. F. Rodrigues","email":"","orcid":"","institution":"UFRJ: Universidade Federal do Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Juliany","middleName":"C. F.","lastName":"Rodrigues","suffix":""},{"id":316157015,"identity":"af04fc91-72ca-49d7-8c0f-892de9abbd02","order_by":10,"name":"José A. Ventura","email":"","orcid":"","institution":"UFES: Universidade Federal do Espirito Santo","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"A.","lastName":"Ventura","suffix":""},{"id":316157016,"identity":"084892c3-a361-4bef-9dd3-24479df35445","order_by":11,"name":"Patricia Machado Bueno Fernandes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIie2RMUvDQBTHX3jwXK5kPSi1XyGSIc0gfpWEgJOCY4cQbsoUmjXil3Dq6pWDTIGsGRyUQGelUJyKMTUIkqurw/2mP7z78d67B2Aw/FcC4AAo+2yJr7TskqN9j98KBYPSpeovpedHYc5JxTury7eXeDEHwnJ3Fz/Pcrvac7lMwJvKUcXPIiyCkl8IouihKLfufXG75rJS4K+CUcWREUIouCXmmYuMVPjYTNbT91SCU40P5tRtr1wJsnfIDip8qqst3xwSvdIcu4SCGOIk7brADfGNQK3iF60L3S5RSuTiZKXcorl2F7JUzM80P2aHrfURJ5c5YYtsr2Z5rl4bGSfnHtMMNgT6XdEIp05sMBgMhoFPHVlbddungBkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2695-3638","institution":"Universidade Federal do Espirito Santo","correspondingAuthor":true,"prefix":"","firstName":"Patricia","middleName":"Machado Bueno","lastName":"Fernandes","suffix":""}],"badges":[],"createdAt":"2024-06-03 19:49:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4523827/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4523827/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60170252,"identity":"b5dc2ac0-d512-42e5-8ec4-243096d9f3ea","added_by":"auto","created_at":"2024-07-12 15:05:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":917327,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of abundance changed proteins \u0026nbsp;(p ≤0.05; FC ±0.58), displaying the number of up- (red) and down-accumulated \u0026nbsp;(green) proteins mapped in each \u003cem\u003eC. papaya\u003c/em\u003eoverview metabolic pathway using MapMan software\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/59699b481bac14e28e60a7fd.png"},{"id":60170253,"identity":"a1d4b5da-bdd7-40b9-a515-c3de1730824f","added_by":"auto","created_at":"2024-07-12 15:05:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":724056,"visible":true,"origin":"","legend":"\u003cp\u003eChlorophyll a fluorescence parameter, PI\u003csub\u003etotal\u003c/sub\u003e, PI\u003csub\u003eabs\u003c/sub\u003e, yRC/(1-yRC), ϕPo/(1-ϕPo), ψEo/(1-ψEo), δRo/(1- δRo). Control (black) and PMeV2 (red) samples, at 1, 7, 14, 21, 28, 35, 42, 49 dpi\u003c/p\u003e","description":"","filename":"Figure2v02.png","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/371020fd8dc2e1d2ea4e32da.png"},{"id":60170260,"identity":"44ec68af-d070-4e27-bb19-109e7d4f4ea0","added_by":"auto","created_at":"2024-07-12 15:05:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17390574,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscopy. (A) A healthy laticifer cell in a cross-section of a vascular bundle of asymptomatic leaves of \u003cem\u003eCarica papaya\u003c/em\u003e. (C) High magnification of the healthy laticifer cell wall showing a well-defined and preserved primary wall, secondary wall, and median lamella regions. The inset of this image shows the parallel organization of cellulose microfibrils in the laticifer wall of asymptomatic plants. (B) A diseased laticifer in a cross-section of a vascular bundle of symptomatic leaves, showing significant differences in the latex content compared to healthy laticifers. (D) High magnification of the diseased laticifer cell wall showing: (1) disorganization of the laticifer cell wall (thin arrow); and (2) vacuolization in the region of the secondary wall. In the inset, it is possible to observe regions of wall vacuolization where signs of degradation of cellulose microfibrils and loss of ultrastructural organization of the cell wall are observed\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/dc33cb40d2888de19663e07b.png"},{"id":62903760,"identity":"f6f07b97-e02c-44a1-bd0f-6eeee64c0246","added_by":"auto","created_at":"2024-08-21 00:08:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20979887,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/b70e69fa-34a4-48ae-ba3f-7a5c61e9c6c0.pdf"},{"id":60170259,"identity":"b979238b-6750-4a20-a764-4e0053f0e3f3","added_by":"auto","created_at":"2024-07-12 15:05:25","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27698084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. S1\u003c/strong\u003e Gene Ontology (GO) grouping of \u003cem\u003eC. papaya\u003c/em\u003e leaf proteins according to their associated biological process using Blast2GO software. The numbers indicate the number of sequences grouped in each GO term(s)\u003c/p\u003e","description":"","filename":"SupplFigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/5004bdf571303b8db16a9611.tif"},{"id":60170262,"identity":"cef6481c-6b92-4dec-b477-26848497ee13","added_by":"auto","created_at":"2024-07-12 15:05:25","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":73606868,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. S2 \u003c/strong\u003eGene Ontology (GO) grouping of PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003eleaf proteins according to their associated molecular function (A) and cellular component (B) using Blast2GO software. The numbers indicate the number of sequences grouped in each GO term(s)\u003c/p\u003e","description":"","filename":"SupplFigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/02e4c6649e22940a49d4320c.tif"},{"id":60170255,"identity":"8015cdb6-e5a7-4d8f-b495-9e72f9cd5938","added_by":"auto","created_at":"2024-07-12 15:05:24","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":25517948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. S3 \u003c/strong\u003eProtein abundance reproducibility. The coefficient of variation (CV) obtained for the experimentally obtained peptide abundances using %𝐶𝑉=(𝑆𝑇𝐷𝐸𝑉𝑃∕𝐴𝑉𝐸𝑅𝐴𝐺𝐸)×100\u003c/p\u003e","description":"","filename":"SupplFigS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/39c49c7f105e92f6d29f831f.tif"},{"id":60170263,"identity":"6789d24d-e024-4f7d-ad29-9ad35b8657b0","added_by":"auto","created_at":"2024-07-12 15:05:25","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":60945352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. S4 \u003c/strong\u003eGene Ontology (GO) bar chart displaying the third level GO terms percentage in up-accumulated (red) and down-accumulated (green) proteins. The proteins were grouped by their predicted GO Biological Process in 3 (A), 4 (B), 7 (C), 9 (D) months post-germination (mpg)\u003c/p\u003e","description":"","filename":"SupplFigS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/badfc8e3306145b32e26838c.tif"},{"id":60170254,"identity":"4032d17a-2b3c-4102-9f89-3f3c6f2f4107","added_by":"auto","created_at":"2024-07-12 15:05:24","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":31526520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. S5 \u003c/strong\u003eGene Ontology (GO) bar chart displaying the third level GO terms percentage in up-accumulated (red) and down-accumulated (green) proteins. The proteins were grouped by their predicted GO Molecular Function in 3 (A), 4 (B), 7 (C), 9 (D) months post-germination (mpg)\u003c/p\u003e","description":"","filename":"SupplFigS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/80fcfdde420e1bf751e8b23a.tif"},{"id":60170264,"identity":"6d6116f8-3b9f-4507-9901-9c1c694e440f","added_by":"auto","created_at":"2024-07-12 15:05:26","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":36490632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. S6\u003c/strong\u003e Gene Ontology (GO) bar chart displaying the third level GO terms percentage in up-accumulated (red) and down-accumulated (green) proteins. The proteins were grouped by their predicted GO Cellular Component in 3 (A), 4 (B), 7 (C), 9 (D) months post-germination (mpg)\u003c/p\u003e","description":"","filename":"SupplFigS6.tif","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/a98d061288a43de89954ec84.tif"},{"id":60171856,"identity":"e8d63aa9-3745-4ff6-9581-4c1072c03118","added_by":"auto","created_at":"2024-07-12 15:13:25","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":34838240,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. S7 \u003c/strong\u003eLabel-free quantitative time course (months post-germination, mpg) PMeV+PMeV2-infected \u003cem\u003eC. papaya\u003c/em\u003e proteome coverage. The quantified proteins were present in all three replicates, and were found differential after filtering using p\u0026lt;0.05 and ±0.58 fold change Log2 (Infected/control)\u003c/p\u003e","description":"","filename":"SupplFigS7.tif","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/8cbc61bfb4a925c64cf25e1a.tif"},{"id":60171857,"identity":"d94648dc-992c-4fa2-ab41-f784d7d7a050","added_by":"auto","created_at":"2024-07-12 15:13:25","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":32753290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. S8 \u003c/strong\u003eChlorophyll a fluorescence parameters, OEC, ABS/CS0, D10/CS0, TRO/CS0, ET0/CS0, RE0/CS0, RC/CS0. Control (black) and PMeV2 (red) samples, at 1, 7, 14, 21, 28, 35, 42, 49 dpi.\u003c/p\u003e","description":"","filename":"SupplFigS8v02.tif","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/67c1f7ba402ea45d93d97f31.tif"},{"id":60170258,"identity":"c1f3c506-cd75-4e4e-b8c3-2dc80e290de2","added_by":"auto","created_at":"2024-07-12 15:05:25","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":34516300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. S9 \u003c/strong\u003eBright-field optical microscopy. (A) Cross-section of the vascular bundle of asymptomatic leaves of \u003cem\u003eCarica papaya\u003c/em\u003e. (C) High magnification of the asymptomatic leaf showing the presence of laticifers (arrows) distributed throughout the vascular bundle. (B) Cross-section of the vascular bundle of symptomatic leaves of \u003cem\u003eCarica papaya\u003c/em\u003e. (D) High magnification of the symptomatic leaf showing empty cells. It is difficult to identify the presence of laticifers in this image. The arrow points to one laticifer cell in anastomosis, identifiable by its lack of content.\u003c/p\u003e","description":"","filename":"SupplFigS9.tif","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/8029ff7f786ec7979544d17f.tif"},{"id":60170256,"identity":"3da4c184-4ae1-44c4-b74d-38cd9d1b72e4","added_by":"auto","created_at":"2024-07-12 15:05:25","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":58358672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. S10 \u003c/strong\u003e(A-B) Cross-section of the vascular bundle of asymptomatic leaves of Carica papaya observed by scanning electron microscopy. (B) High magnification of the cross-section of an asymptomatic leaf shows the presence of laticifer cells (arrow) with their vesicular content. (C-D) In contrast, a cross-section of the vascular bundle of symptomatic leaves shows several empty cells where it is difficult to identify the presence of laticifers. Upon closer inspection, alterations in the cell wall can be observed compared to the symptomatic leaves (Fig. 4).\u003c/p\u003e","description":"","filename":"SupplFigS10.tif","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/7ce3773b5750dddc94fd9cce.tif"},{"id":60170266,"identity":"fa2e58c1-49c7-48f6-9430-b785b3fa7604","added_by":"auto","created_at":"2024-07-12 15:05:26","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":52432336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. S11 \u003c/strong\u003e(A-B) Transmission electron microscopy showing various viral particles within laticifer cells of symptomatic plants (arrows), sometimes close to membrane structures, possibly endoplasmic reticulum profiles. (B) High magnification allows determination of the size of the viral particles to 50 nm, suggesting that such structures are PMeV\u003c/p\u003e","description":"","filename":"SupplFigS11.tif","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/e0590bad9f36765a8404ce7a.tif"},{"id":60170265,"identity":"91bcd632-d85f-4fe3-864e-aa38874c6dc9","added_by":"auto","created_at":"2024-07-12 15:05:26","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":32011086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table Captions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S1 \u003c/strong\u003eDescription of the plant development phases and sampling times used in this study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S2 \u003c/strong\u003eTerm description and calculation formulas for the JIP-test\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S3\u003c/strong\u003e Proteomic coverage of PMeV complex-infected and control \u003cem\u003eC. papaya\u003c/em\u003eleaf samples\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S4\u003c/strong\u003e Compiled list of proteins identified in PMeV complex-infected and control \u003cem\u003eC. papaya\u003c/em\u003e leaf samples\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S5\u003c/strong\u003e Peptides identified from PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e leaf samples\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S6\u003c/strong\u003e Label-free quantification of PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e leaf proteins\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S7\u003c/strong\u003e Proteins differently modulated in 3 months post-germination (0 days post-inoculation) PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e leaf\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S8\u003c/strong\u003e Gene Ontology terms enrichment analyses of up- vs. down-accumulated PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e modulated proteins\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S9 \u003c/strong\u003eMetabolic pathway overview of differential proteins in 3 months post-germination (0 days post-inoculation) PMeV complex-infected and control \u003cem\u003eC. papaya\u003c/em\u003e leaf samples\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S10\u003c/strong\u003e Proteins differently modulated in 4 months post-germination (30 days post-inoculation) PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e leaf\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S11\u003c/strong\u003e Metabolic pathway overview of differential proteins in 4 months post-germination (30 days post-inoculation) PMeV complex-infected and control \u003cem\u003eC. papaya\u003c/em\u003e leaf samples\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S12\u003c/strong\u003e Proteins differently modulated in 7 months post-germination (120 days post-inoculation) PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e leaf\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S13\u003c/strong\u003e Metabolic pathway overview of differential proteins in 7 months post-germination (120 days post-inoculation) PMeV complex-infected and control \u003cem\u003eC. papaya\u003c/em\u003e leaf samples\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S14\u003c/strong\u003e Proteins differently modulated in 9 months post-germination (180 days post-inoculation) PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e leaf\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table S15\u003c/strong\u003e Metabolic pathway overview of abundance changed proteins in 9 months post-germination (180 days post-inoculation) PMeV complex-infected and control \u003cem\u003eC. papaya\u003c/em\u003e leaf samples\u003c/p\u003e","description":"","filename":"Rodrigueset.al.2024SupplementalTablesS1S15v01.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4523827/v1/2e27911687daf0324702f33c.xlsx"}],"financialInterests":"","formattedTitle":"Insights on Carica papaya L. proteomic, ultrastructural and physiological changes associated with pre-flowering-related tolerance to papaya sticky disease","fulltext":[{"header":"Key Message","content":"\u003cp\u003eThe proteomic analysis of PMeV/PMeV2-infected \u003cem\u003eC. papaya\u003c/em\u003e unveiled proteins undergoing modulation during the plant\u0026apos;s development. The infection notably impacted processes related to photosynthesis and cell wall dynamics.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe global production of papaya (\u003cem\u003eCarica papaya L.\u003c/em\u003e) fruit is over 13\u0026nbsp;million tons and the major papaya fruit producers are India, Brazil, Nigeria, Mexico, and Indonesia (FAOSTAT \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, papaya cultivation is vulnerable to a \"virus complex\", composed of papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2) (S\u0026aacute; Antunes et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; S\u0026aacute; Antunes et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). PMeV is a fusagra-like double-stranded RNA virus (dsRNA), which naturally infects \u003cem\u003eC. papaya\u003c/em\u003e by unknown routes (S\u0026aacute; Antunes et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). PMeV shows synergism with the umbra-like single-stranded (ssRNA) PMeV2, resulting in \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eP\u003c/span\u003eapaya \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS\u003c/span\u003eticky \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eD\u003c/span\u003eisease (PSD) (S\u0026aacute; Antunes et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe PMeV complex infects \u003cem\u003eC. papaya\u003c/em\u003e laticifers, cells specialized in the production and storage of latex, which contain cardenolides, alkaloids, isoprene polymers, enzymes, etc. (Hagel et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The infection is known to induce changes in the latex structure and composition, and to increase the production of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and the accumulation of potassium, phosphorus, and water (Rodrigues et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The main PSD symptom is a spontaneous exudation of watery latex, mostly on fruits and leaves. The latex is oxidized after contact with the air and accumulates on the plant as a sticky and brownish substance, which makes the fruit unfit for marketing (S\u0026aacute; Antunes et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; S\u0026aacute; Antunes et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious literature on the morphophysiological, biochemical and molecular changes undergone by sticky-diseased \u003cem\u003eC. papaya\u003c/em\u003e points out that the control of protein turnover is due to a decrease in microRNA (miR162, miR398, and miR408) expression, which is predicted to target proteasome-related proteins (S\u0026aacute; Antunes et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The levels of proteasome-related proteins increase in leaf samples collected from diseased plants (Rodrigues et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In the same study, symptomatic \u003cem\u003eC. papaya\u003c/em\u003e showed an accumulation of calreticulin, 20S proteasome b subunit, and pathogenesis related proteins (PRs) (\u003cem\u003ee.g.\u003c/em\u003e endochitinase and PR-4) (Rodrigues et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In parallel, the proteomic analysis of diseased \u003cem\u003eC. papaya\u003c/em\u003e latex samples showed reduced serine proteinase inhibitor and chymopapain cysteine protease levels (Rodrigues et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe first PSD symptoms, small necrotic lesions on the tips of young leaves, appear three to four months post-germination (mpg). This period coincides with the beginning of floral bud development, blooming, and the early development of fruits (Cosmi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our group previously analyzed PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e at the pre-flowering stage using proteomics, identifying 111 differential proteins (57 up- and 54 down-regulated) (Soares et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). An increase in photosynthesis-related proteins and a decrease in 26S-proteasome and cell wall remodeling proteins were observed. In parallel, field observations revealed that PSD symptoms seem mostly associated with fruit development (Cosmi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thus, the analysis of PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e plants throughout different development phases will help understand the mechanisms involved in PSD symptoms progress.\u003c/p\u003e \u003cp\u003eIn this study, PMeV complex-infected and non-infected \u003cem\u003eC. papaya\u003c/em\u003e plants were grown in the field until they reached 3, 4, 7, and 9 months post-germination (mpg). Leaf samples were collected, and their proteins were identified and quantified via LC-MS/MS-based proteomics. Using a label-free approach, a total of 1,609 proteins were quantified. The differential proteins (38, 130, 160, and 17 proteins at 3, 4, 7, and 9 mpg, respectively) represent a more complete view of the different biological processes modulated in \u003cem\u003eC. papaya\u003c/em\u003e during PSD symptoms development, in which photosynthesis, cell wall, protein and RNA metabolism, and response to stress are mainly involved. The results obtained through proteomics were complemented by physiological and microscopy analyses.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Material\u003c/h2\u003e \u003cp\u003eOne-month-old \u003cem\u003eC. papaya\u003c/em\u003e cv. Golden seedlings (n\u0026thinsp;=\u0026thinsp;6) were planted at the INCAPER experimental farm, in Sooretama-ES, Brazil. They were cultivated for two months. At that point, the plants were injected at the youngest leaf petiole either with 1 mL of latex from \u003cem\u003eC. papaya\u003c/em\u003e sticky-diseased plants diluted (1:1, v/v) in 50 mM sodium phosphate buffer, pH 7.0 (PMeV complex), or with buffer only. After the treatments, the second fully expanded leaf of each plant was immediately collected and frozen in liquid nitrogen (3 mpg samples). The tissues were freeze-dried and stored at -80\u0026deg;C until use. Subsequent sampling was performed at 4 mpg (30 days post-infection, dpi), 7 mpg (120 dpi), and 9 mpg (180 dpi) (Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eProtein Extraction\u003c/h2\u003e \u003cp\u003eThe leaf tissue was mechanically homogenized in liquid nitrogen and 10 mg of tissue powder was submitted to total protein extraction using phenol (Wang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The extracted proteins were pelleted after the addition of 4 mL of 0.1 M ammonium acetate in methanol. The mixture was then incubated for 10 hours at -20\u0026deg;C and centrifuged (10 min, 20,000 \u0026times; g, 4\u0026deg;C). The pellets were washed twice with 1.5 mL of 0.1 M ammonium acetate in methanol, once with 1.5 mL of 80% acetone, and once with 1.5 mL of 70% methanol. The proteins were resuspended in 180 \u0026micro;L of 50 mM Tris-HCl pH 8.0, 8 M urea, and 2 M thiourea solution and assayed using CB-X protein assay (Genotech, St. Louis, MO).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProtein Digestion\u003c/h2\u003e \u003cp\u003eThe proteins were incubated for 45 min with 5 mM dithiothreitol (DTT) at 37\u0026deg;C. They were then incubated with 100 mM iodoacetamide for 40 min at 25\u0026deg;C in darkness. Then, 1 M urea with 50 mM Tris-HCl, pH 8.8 was added to the samples. Protein digestion was performed at 37\u0026deg;C for 16 h and 800 rpm using a trypsin solution (Sigma, St. Louis, MO) at 1:50 enzyme/substrate ratio, which was stopped by adding 2% formic acid final concentration. PepClean C18 spin columns (Thermo Scientific, Rockford, lL) were used to desalt the resulting peptides which were resuspended in 150 \u0026micro;L of 0.1% formic acid (FA)/5% acetonitrile (ACN).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS Analysis\u003c/h2\u003e \u003cp\u003e1 \u0026micro;g of peptide mixture was loaded onto a trap column (NanoAcquity UPLC 2G-W/M Trap 5 \u0026micro;m Symmetry C18, 180 \u0026micro;m \u0026times; 20 mm) in a NanoAcquity UPLC system (Waters, Milford, MA) coupled to a TripleTOF 5600 MS/MS (AB SCIEX, Framingham, MA) for 3 min at 5 \u0026micro;L/min. A 5\u0026ndash;40% linear gradient of solvent B was used to separate the peptides in a C18 capillary column (NanoAcquity UPLC 1.8 \u0026micro;m HSS T3, 75 \u0026micro;m \u0026times; 250 mm) at 300 nL/min. Solvent A consisted of 0.1% FA in water and solvent B consisted of 0.1% FA in acetonitrile (ACN). After each sample analysis, a column cleaning was performed (5 min from 40\u0026ndash;85% of solvent B and 85% of solvent B for 10 min) and re-equilibration (2 min from 85\u0026ndash;5% of solvent B and 5% of solvent B for 13 min). The mass spectrometer was operated in positive ionization and high sensitivity mode. The features were selected for Information Dependent Acquisition (IDA) MS/MS experiments based on an MS survey. The spectrum was accumulated from 350 to 1600 \u003cem\u003em/z\u003c/em\u003e for 250 ms, of which the first 20 features with a charge state of +\u0026thinsp;2 to +\u0026thinsp;5 and exceeding a 150 counts threshold were selected. The same features were included on an 8 s dynamic exclusion list prior to fractionation using\u0026thinsp;\u0026plusmn;\u0026thinsp;5% rolling collision energy. As a reference sample, a homogeneous mixture of equivalent peptide amounts from all replicates was analyzed and used for label-free protein quantification. To assure the high mass accuracy in both MS and MS/MS acquisition, the instrument was automatically calibrated after every three samples (6h).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eProtein identification and label-free quantification\u003c/h2\u003e \u003cp\u003eProgenesis QI for proteomics v2.0 (NonLinear Dynamics) was used to create two-dimensional ion intensity maps of features extracted from the TripleTOF 5600 raw files (.wiff). The peak picking, reference assignment, and spectra alignment (\u0026ge;\u0026thinsp;80% score) parameters were performed as automatic for the features eluting between 25 and 105 min. The Mascot server v.2.2.2 (Matrix Science Inc., Boston, MA) was used for protein identification by interrogating the peak list file (.mgf) from Progenesis QI against a custom database (27,898 sequences total, May 2015) containing all \u003cem\u003eC. papaya\u003c/em\u003e protein entries available on Phytozome 10.2 (27,775 sequences, May 2015) (Souza et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Guzha et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) combined with NCBI \u003cem\u003eC. papaya\u003c/em\u003e organelle (123 sequences, May 2015). The parameters of +\u0026thinsp;2 to +\u0026thinsp;4 charge state, two missed cleavages, and a mass tolerance of \u0026plusmn;\u0026thinsp;20 ppm for precursor ions and \u0026plusmn;\u0026thinsp;0.05 Da for fragment ions were considered for protein identification. Additionally, deamidation at asparagine or glutamine, cysteine carbamidomethylation, methionine oxidation, and acetylation at peptide N-term were considered as variable modifications. Mascot percolator algorithm was used, providing a False Discovery Rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;1% prior to XML file exportation and Progenesis QI reimportation for peptide quantification and identification. The protein quantification was performed using the normalized abundances of Hi-3 (up to 3) peptides (Silva et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) filtering for Mascot peptide scores\u0026thinsp;\u0026ge;\u0026thinsp;13. The protein abundances occurring in all three control and PMeV complex-infected biological replicates were compared by one-way ANOVA test and the protein list was filtered based on p\u0026thinsp;\u0026le;\u0026thinsp;0.05 and a Log\u003csub\u003e2\u003c/sub\u003e fold change (FC) of \u0026plusmn;\u0026thinsp;0.58.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDifferential abundance analysis and protein functional classification\u003c/h2\u003e \u003cp\u003eGene ontology (GO) analysis of all identified proteins was performed in Blast2GO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.blast2go.org\" target=\"_blank\"\u003ewww.blast2go.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.blast2go.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) by blasting the identified protein sequences against the NCBI non-redundant (nr) database with an expected E-Value threshold of 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e and the first ranked hit was further used. The GO enrichment for up- and down-accumulated protein sets used a Fisher's Exact test with the multiple testing correction FDR option selected (Benjamini et al. 1995). Additionally, the differential proteins (p\u0026thinsp;\u0026le;\u0026thinsp;0.05; FC\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58) were submitted to \u003cem\u003eC. papaya\u003c/em\u003e overview metabolic pathway mapping using the MapMan software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mapman.gabipd.org/web/guest/mapman\u003c/span\u003e\u003cspan address=\"http://mapman.gabipd.org/web/guest/mapman\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eChlorophyll a fluorescence parameter analysis\u003c/h2\u003e \u003cp\u003eIn a greenhouse, 2 mpg plants were either injected with 20 \u0026micro;L latex (n\u0026thinsp;=\u0026thinsp;5) from sticky-diseased plants diluted with buffer or injected with buffer only (n\u0026thinsp;=\u0026thinsp;5), similarly to 2.1. Thus, the chlorophyll (Chl) fluorescence parameters were collected weekly from 1\u0026ndash;49 days after injection (DAI) at fully developed and 40-min dark-adapted leaves. The measurements were taken from 7:00 to 9:00 AM (UTC-3) using a portable fluorometer (Handy PEA+, Hansatech, UK). The kinetics of Chl a fluorescence was measured using a saturating light pulse of 3,000 \u0026micro;mol (photon) m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e s\u003csup\u003e\u0026ndash;1\u003c/sup\u003e at 650 nm for 1 s to generate a true fluorescence intensity of maximum value (FM) using Pea Plus software v. 1.13. Fluorescence parameters were derived from the fast Chl a fluorescence induction (OJIP) according to JIP-test equations (Strasser, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). The measurements were performed at least in triplicate at six leaves of each plant. The terms and formulas for calculating the JIP-test parameters are described in Supplemental Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. After exporting the data as Excel spreadsheets, it was normalized, compared using the Tukey test and presented as radar plots.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eScanning and Transmission Electron Microscopy\u003c/h2\u003e \u003cp\u003eFor scanning electron microscopy (SEM), 1 x 1 cm asymptomatic and symptomatic leaf fragments were fixed in Karnovsky solution (2.5% glutaraldehyde and 4% paraformaldehyde in 0.1M sodium cacodylate (EMS, USA) pH7.2 for 4 h at room temperature. The fragments were post-fixed in a solution containing 1% osmium tetroxide in water, for 4 h at room temperature in the dark. Subsequently, the fragments were dehydrated in an increasing series of ethanol/water solutions (30%, 50%, 70% and 90%, v/v) for 30 minutes, followed by 4 additional dehydration steps with 100% ethanol. In the end, the fragments were dried using the critical point method, cross-sectioned into 2 mm x 1 cm fragments, mounted on stubs and metalized with gold. The samples were observed using a VEGA 3 LMU scanning electron microscope (TESCAN, Czech Republic) operating at 20 kV.\u003c/p\u003e \u003cp\u003eFor transmission electron microscopy (TEM), symptomatic and asymptomatic leaves were sectioned into 3 x 5 mm. The fragments were fixed as described above and post-fixed in a solution containing 1% osmium tetroxide, 1.25% potassium ferrocyanide and 5 mM calcium chloride in water, for 4 h at room temperature in the dark. The fragments were then dehydrated in an increasing series (30 min each) of acetone/water (30%, 50% and 70% v/v) solutions, followed by 4 additional steps using 90% and 100% acetone. The fragments were then infiltrated with low viscosity epoxy resin (SPURR, Electron Microscopy Science) using different acetone/resin (v/v) solutions (3:1, 2:1, 1:1, 1:2 and 1:3) for 4 h under seesaw agitation. Finally, the fragments were infiltrated with 100% resin for 24 h under seesaw agitation. Ultrathin sections were obtained using a PT-PC PowerTome ultramicrotome (RMC Boeckeler, USA), stained with uranyl acetate and lead citrate, and observed using a TEM FEI TECNAI SPIRIT operating at 120 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eOptical Microscopy\u003c/h2\u003e \u003cp\u003eOptical microscopy analyzes were performed using 300 nm semi-thin slices obtained from materials processed for TEM, as described above. Thus, the sections were collected and placed on glass slides previously heated to 70\u0026deg;C. After fixing the sections on the slides, they were stained using a 1% toluidine blue and 1% boric acid solution in water. After drying, the slides were washed with distilled water, and the samples were observed using an optical microscope DM 2500 (LEICA, Germany).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eProteomic analysis of\u003c/b\u003e \u003cb\u003eC. papaya\u003c/b\u003e \u003cb\u003eleaf\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 1,609 proteins (99%) were quantified based on Progenesis QI analysis of 34,624 ions (Supplemental Tables S3-S6). This dataset represents the most extensive proteomic coverage reported for \u003cem\u003eC. papaya\u003c/em\u003e to date. The proteins were annotated, and the Gene Ontology (GO) profiles for biological processes, molecular function, and cellular component are shown in Supplemental Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2. The 1,609 quantified proteins consisted of 1,242 proteins from 3 mpg samples (pre-flowering), 1,454 from 4 mpg samples (flowering), 1,493 from 7 mpg samples (fruiting), and 1,442 from 9 mpg samples (ripening). These proteins exhibited an average coefficient of variance (CV) of 28% (23% CV median) (Supplemental Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, Supplemental Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). They were utilized to correlate the protein abundances of PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e leaf and control samples for each plant age group. A total of 1,533 protein sequences (94%) were assigned to at least one Gene Ontology identification number (GO ID) (Supplemental Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). The third-level GO term grouping by biological process (Supplemental Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), molecular function (Supplemental Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e), and cellular component (Supplemental Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e) are presented.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferential proteome of pre-flowering PMeV complex-infected\u003c/b\u003e \u003cb\u003eC. papaya\u003c/b\u003e \u003cb\u003evs. control plants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAt 3 mpg, 38 proteins exhibited significant abundance changes (p\u0026thinsp;\u0026le;\u0026thinsp;0.05), with an FC of \u0026plusmn;\u0026thinsp;0.58 or greater (Supplemental Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e, Supplemental Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). Notably, lipid transfer protein 4 (6,1 FC) and AMP-dependent synthetase and ligase family protein (4,1 FC) showed the highest change in abundance levels, while a putative uricase/urate oxidase/nodulin 35 (-10 FC) and calcium-dependent lipid-binding plant phosphoribosyltransferase family protein (-10 FC) displayed the lowest accumulation levels. The 3 mpg group did not exhibit statistically significant (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) enrichment of GO term proteins (Supplemental Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt 4 mpg, 130 proteins demonstrated significant changes in abundance among the total quantified proteins (Supplemental Table \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e, Supplemental Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). The proteins with the most notable change in abundance levels were haloacid dehalogenase-like hydrolase family protein (3 FC) and sucrose phosphate synthase 3F (2,675 FC), while the proteins with the lowest accumulation levels were p-loop containing nucleoside triphosphate hydrolases superfamily protein (-10 FC) and subtilase family protein (-10 FC). Fifteen GO terms were enriched (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) within the 4 mpg \u003cem\u003eC. papaya\u003c/em\u003e leaf proteins. Among these, eight were most represented among the up-regulated proteins, primarily associated with photosynthesis and oxidoreductase activity. Conversely, seven GO terms were most represented among the down-accumulated proteins, including RNA binding, catabolic process, and membranous cellular components (Supplemental Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe MapMan overview metabolic pathway analysis indicated an up-accumulation of proteins associated with photosynthesis, carbohydrate metabolism, organic acid transformations, and amino acid metabolism. Conversely, there was a down-accumulation of proteins related to cell wall structure, lipid metabolism, stress response, RNA metabolism, protein metabolism, and cell organization during the pre-flowering (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplemental Tables S9 and S11).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferential proteome of post-flowering PMeV complex-infected\u003c/b\u003e \u003cb\u003eC. papaya\u003c/b\u003e \u003cb\u003evs. control plants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 7 mpg plant group contained the largest subset of the quantified proteins, with 160 proteins showing significant changes in abundance (p\u0026thinsp;\u0026le;\u0026thinsp;0.05, FC\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58) (Supplemental Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e, Supplemental Table \u003cspan refid=\"MOESM12\" class=\"InternalRef\"\u003eS12\u003c/span\u003e). Only photosynthesis was identified as an enriched GO term (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) among the down-accumulated proteins, while plastids, thylakoids, and generation of precursor metabolites and energy were the most represented enriched GO terms among the up-accumulated proteins (Supplemental Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e). The proteins with the highest change in abundance levels were alpha/beta-Hydrolases superfamily protein (4,1 FC) and DEA(D/H)-box RNA helicase family protein (3,1 FC), while the lowest accumulation levels were observed for Kunitz trypsin inhibitor 1 (-3,4 FC) and non-photochemical quenching 1 (-2,9 FC).\u003c/p\u003e \u003cp\u003eAt 9 mpg, 11 up- and 6 down-accumulated proteins were identified among the 1,442 proteins representing the plant group (p\u0026thinsp;\u0026le;\u0026thinsp;0.05, FC\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58, Supplemental Table S14, Supplemental Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). The 9 mpg phase had no statistically significant (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) enriched GO terms (Supplemental Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e). The proteins with the highest change in abundance levels were ATP-dependent caseinolytic (Clp) protease/crotonase family protein (2,1 FC) and Cyclophilin-like peptidyl-prolyl cis-trans isomerase family protein (2,1 FC), while the lowest accumulation levels were observed for PLC-like phosphodiesterase family protein (-1,8 FC) and P-loop containing nucleoside triphosphate hydrolases superfamily protein (-1,5 FC).\u003c/p\u003e \u003cp\u003eThe overview metabolic pathway revealed proteins up-accumulated in categories including carbohydrate metabolism, mitochondrial proteins, cell wall, lipid metabolism, amino acid metabolism, stress, nucleotide metabolism, protein metabolism, and signaling. Conversely, among the down-accumulated proteins, only photosynthesis was highlighted at post-flowering phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplemental Tables S13 and S15).\u003c/p\u003e \u003cp\u003e \u003cb\u003eChlorophyll fluorescence analysis of PMeV complex-infected\u003c/b\u003e \u003cb\u003eC. papaya\u003c/b\u003e \u003cb\u003evs. control plants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor these experiments, greenhouse-grown plants were used to simulate the pre-flowering and asymptomatic phase of PMeV-complex-infected \u003cem\u003eC. papaya\u003c/em\u003e. Consistent with the up-accumulation of photosynthesis-related proteins in infected plants at 3 and 4 mpg, the quantification of chlorophyll a fluorescence parameter (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) revealed an increased Performance Index (PI) ABS at 35 and 42 DPI in PMeV-complex-infected \u003cem\u003eC. papaya\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PI ABS reflects the energy absorption by PSII antenna pigments, derived from terms expressing energy bifurcations from absorption to the reduction of the electron transport chain (Tsimill-Michael, 2020). This energy cascade involves light absorption (ABS), trapping (TR) (primary photochemistry), reduction of pheophytin (Phe) and quinone A (QA), electron transport (ET) after Qa- to intersystem electron acceptors, and energy dissipation (DI) (Tsimill-Michael, 2020). In the PMeV-complex-infected leaf cross-section (CS), the plants exhibited a reduction in ABS (ABS/CS), DI (DI/CS), and TR (TR/CS) at 28 and 35 DPI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). ET (ET/CS) was reduced only at 28 DPI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, compared to the control, there was a significant increase in oxygen-evolving complex (OEC) activity in infected \u003cem\u003eC. papaya\u003c/em\u003e after 28 and 35 DPI. OEC activity returned to normal at 42 and 49 DPI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). OEC in photosystem II catalyzes the oxidation of water into dioxygen, protons, and electrons (Ferreira et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePI total extends beyond PI ABS by incorporating the γ reaction center (γRC) parameter, reflecting the energy bifurcation until the reduction of PSI end electron acceptors (Tsimill-Michael, 2020). In infected plants, both PItotal and γRC/(1 \u0026ndash; γRC) increased at 35, 42, and 49 DPI compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, the indicator of reduction of end acceptors at the PSI electron acceptor side, RE/CS, significantly increased at 35, 42, and 49 DPI (Supplemental Figure \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e). These findings collectively suggest a heightened efficiency in PSII and PSI energy flux in PMeV-infected juvenile \u003cem\u003eC. papaya\u003c/em\u003e.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBright-field optical microscopy (BFM) and scanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eBright-field microscopy (BFM) and scanning electron microscopy (SEM) were employed to examine potential morphological and ultrastructural changes in PMeV-infected cells. BFM of semi-thin sections was additionally conducted to facilitate the selection of sections for transmission electron microscopy analysis. Initial differences between asymptomatic and symptomatic samples were noted via BFM (Supplemental Figure \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e). In healthy plant leaves, numerous laticifer cells filled with intracellular content were observed (Supplemental Figure \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003eC, arrows), some displaying anastomosis. Conversely, diseased samples exhibited empty laticifer cells (Supplemental Figure \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003eD, arrow). This observation was supported by SEM imaging (Supplemental Figure \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e), wherein laticifers in asymptomatic plants appeared replete with intracellular content, while those in symptomatic plants appeared empty. Possible cellular alterations were also discerned (Supplemental Figure \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eTEM analysis revealed that laticifer cells in asymptomatic leaves exhibited numerous granules with varying electron densities (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Conversely, examination of laticifers in symptomatic plants indicated a notable difference in intracellular contents compared to healthy plants. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, diseased laticifer cells displayed a reduced number of granules, signifying a substantial alteration in cytoplasmic content relative to healthy laticifers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). At higher magnification, the ultrastructure of laticifer cell walls appeared well-defined and preserved in asymptomatic plants, encompassing regions of the primary wall, secondary wall, and median lamella akin to those observed in healthy plants. In the inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, the parallel organization of cellulose microfibrils in the laticifer wall of asymptomatic plants is discernible. Conversely, symptomatic plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) exhibited several differences, including disorganization of the laticifer cell wall (thin arrow) and vacuolization in the region of the secondary wall (arrowheads). In the inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, notable vacuolization of the cell wall is evident, indicating signs of cellulose microfibril degradation and loss of ultrastructural organization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMorphometric analysis was conducted to quantify changes in the cell wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Cell wall thickness was measured across different cells and regions, with a total sample size (N) of 25 measurements. The analysis revealed an average wall thickness of approximately 662 nm in symptomatic laticifers, whereas asymptomatic laticifers exhibited an average thickness of around 300 nm. This twofold increase underscores the degree of organization of cellulose microfibrils, which were observed as juxtaposed and parallel in asymptomatic cells. Conversely, microfibrils in symptomatic cells appeared more loosened, occasionally degraded, and disorganized (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTransmission electron microscopy provided insight into the presence of numerous viral particles within the laticifer cells of symptomatic plants (Supplemental Figure \u003cspan refid=\"MOESM11\" class=\"InternalRef\"\u003eS11\u003c/span\u003e, indicated by arrows), often found in proximity to membrane structures, potentially the endoplasmic reticulum. Further magnified images enabled the determination of viral particle size, estimated to be around 50 nm, thereby confirming the observed structures as PMeV.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn a preliminary investigation (Soares et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the proteome of PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e leaf samples during pre-flowering was examined. Expanding upon this research, the current study extends the proteomic analysis to samples collected at four distinct phenological phases (i.e., 3-, 4-, 7-, and 9-mpg) to elucidate the underlying mechanisms contributing to PSD symptom development. Notably, symptoms of PMeV infection manifest post-flowering initiation, specifically at the 7 and 9 mpg time points. Therefore, the 3 and 4 mpg time points represent the plant pre-flowering, despite being infected, and are characterized as asymptomatic.\u003c/p\u003e \u003cp\u003eTo our knowledge, the protein dataset presented in this study represents the most extensive proteomic coverage of \u003cem\u003eC. papaya\u003c/em\u003e to date. Combined with complementary physiological and microscopy experiments, this dataset enhances the understanding of the interaction between PMeV-complex and \u003cem\u003eC. papaya\u003c/em\u003e. In the following sections, particular attention is given to proteomic changes associated with photosynthesis and cell wall remodeling. Changes in other areas will be explored in subsequent publications.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChanges in photosynthesis-related proteins\u003c/h2\u003e \u003cp\u003eThe reduction of photosynthetic activity is a characteristic feature of symptom development in virus-infected plants (Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In our study, a significant accumulation of photosynthesis-related proteins in PMeV-complex infected \u003cem\u003eC. papaya\u003c/em\u003e at 3 and 4 mpg, corresponding to the pre-flowering phase, compared to controls was observed. Notably, PsbQ-like2, a crucial component of the oxygen evolving complex (OEC), exhibited up-accumulation in asymptomatic plants at 4 mpg but down-accumulation in symptomatic plants at 7 mpg. Alongside PsbP (up-accumulated at 4 and 7 mpg), PsbO, and PsbR, PsbQ-like2 positively influences photosystem II (PSII) efficiency (Sasi et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, physiological experiments measuring chlorophyll a fluorescence revealed an increased OEC activity in PMeV-infected juvenile \u003cem\u003eC. papaya\u003c/em\u003e leaves. PsbQ is known to play a critical role in maintaining and enhancing PSII function and stability under stress conditions (Ifuku et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Yi et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). These findings suggest that the enhanced accumulation and activity of PSII components may contribute to the inhibition of PSD symptom development by juvenile, pre-flowering, and infected plants.\u003c/p\u003e \u003cp\u003eSome chloroplast proteins have been reported to directly interact with viruses, influencing both virus accumulation and symptom development. For instance, PsbP from \u003cem\u003eArabidopsis\u003c/em\u003e was found to interact with the coat protein of Alfalfa mosaic virus (AMV), leading to the inhibition of reactive oxygen species (ROS) production in the chloroplast and facilitating virus accumulation (Balasubramaniam et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Similarly, the disease-specific protein (SP) encoded by rice stripe virus (RSV) interacts with and reduces the accumulation of PsbP in the chloroplast of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e protoplasts, thereby enhancing virus accumulation and RSV-induced symptoms. Conversely, PsbP overexpression has been shown to suppress AMV replication (Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), while overexpression of PsbP reduced chlorosis in leaves caused by RSV (Kong et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, the accumulation of PsbQ-like 1, PsbQ-like 2, and PsbP proteins in asymptomatic PMeV complex-infected plants was observed. PsbQ protein stabilizes PsbP binding to PSII (Kakiuchi et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and their increased levels may potentially interfere with PMeV-complex replication. Consistent with this hypothesis, a reduction in PsbQ-like accumulation was associated with the onset of PSD symptoms.\u003c/p\u003e \u003cp\u003eSeveral chloroplast proteins, including those crucial for photosynthesis, have been implicated in plant immunity processes (J\u0026auml;rvi et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). For instance, in the compatible interaction between mungbean yellow mosaic India virus (MYMIV) and \u003cem\u003eVigna mungo\u003c/em\u003e, there is a decrease in the photosynthetic rate and chlorophyll content of plants (Kundu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Conversely, incompatible interactions lead to an increase in photosynthetic proteins such as rubisco activase, PSII OEC protein, and ribulose-1,5-bisphosphate carboxylase small subunit (Kundu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This suggests that successful plant resistance against virus infection involves an upregulation of photosynthetic proteins.\u003c/p\u003e \u003cp\u003eIn line with this concept, our study revealed the accumulation of rubisco activase, light-harvesting complex photosystem II, and photosystem II subunit proteins in PMeV complex-infected, but symptomless, plants. This accumulation mirrors findings from previous studies (Yoshioka et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; S\u0026aacute;nchez-Vicente et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Caplan et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), supporting the notion that chloroplasts play a pivotal role in PSD symptom development. This role could be attributed to the accumulation of reactive oxygen species (ROS), which contribute to programmed cell death (PCD) (Madro\u0026ntilde;ero et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and/or the production of salicylic acid (SA), a key regulator of systemic acquired resistance (SAR) (Spoel et al., 2012).\u003c/p\u003e \u003cp\u003eThe light-driven reactions of photosynthesis, particularly at the level of PSI acceptors, play a crucial role in regulating reactive oxygen species (ROS) production and are highly sensitive to stress conditions. Among physiological parameters, the photosynthesis performance indexes (PI\u003csub\u003eabs\u003c/sub\u003e and PIt\u003csub\u003eotal\u003c/sub\u003e) have emerged as efficient tools for quantifying stress levels (Strasser et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Huther et al., 2016). PI\u003csub\u003etotal\u003c/sub\u003e, which reflects the performance of PSI end electron acceptors through the δRo / (1\u0026thinsp;\u0026minus;\u0026thinsp;δRo) parameter, provides insights into the efficiency of electron transfer processes (Tsimilli-Michael \u0026amp; Strasser, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, juvenile PMeV-complex infected \u003cem\u003eC. papaya\u003c/em\u003e leaves exhibited significant increases in PSI activity and end acceptors reduction, as evidenced by elevated PIt\u003csub\u003eotal\u003c/sub\u003e, δRo / (1\u0026thinsp;\u0026minus;\u0026thinsp;δRo), and REo/CSo values. These findings suggest that the accumulation of proteins related to PSI, PSII, and plastocyanin (which were up-accumulated in infected pre-flowering \u003cem\u003eC. papaya\u003c/em\u003e) enhances maximum energy flow and electron transfer efficiency (Strasser et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Interestingly, infected but asymptomatic plants appeared to modulate light absorption, energy dissipation, and trapping, as indicated by reduced ABS/CS0, DI0/CS0, and TR0/CS0 parameters.\u003c/p\u003e \u003cp\u003eThe accumulation of two antioxidant enzymes, thioredoxin M-type 4 and NADPH-dependent thioredoxin reductase C, at 4 mpg in PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e compared to controls suggests a potential role for redox balance in the chloroplast of juvenile infected plants, contributing to symptom development tolerance. Interestingly, previous studies have shown that the abundance of maize thioredoxin (ZmTrxh) transcripts strongly correlates with the latency of sugarcane mosaic virus (SCMV) symptoms (Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, ZmTrxh overexpression has been demonstrated to suppress SCMV RNA replication and accumulation, indicating its involvement in plant resistance against SCMV at early infection stages (Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eChanges in cell wall-related proteins\u003c/h2\u003e \u003cp\u003eMicroscopic examination of leaf sections post-flowering initiation revealed distinct disparities between asymptomatic and symptomatic plants. In bright-field microscopy, laticifers in asymptomatic leaves were visibly filled with latex, while those in symptomatic leaves appeared mostly empty, consistent with 'meleira' symptoms. This observation was further confirmed through scanning electron microscopy. Additionally, transmission electron microscopy revealed noticeable variations in the structure of laticifer cell walls. Healthy laticifer cell walls exhibited well-defined primary, secondary, and median lamella regions, with cellulose microfibrils organized in parallel. In contrast, sections from symptomatic leaves displayed significant disorganization of the laticifer wall, including degradation of cellulose microfibrils and loss of ultrastructural organization, leading to vacuolization within the secondary cell wall. This structural deterioration, coupled with the high osmotic pressure within infected laticifers (Rodrigues et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), likely contributes to latex leakage.\u003c/p\u003e \u003cp\u003eTerrestrial plants exhibit a primary cell wall composition that shares similarities across species, primarily comprised of cellulose microfibrils, hemicellulose, and pectic substances (H\u0026ouml;fte \u0026amp; Voxeur \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Cellulose microfibrils form the backbone of the wall and are intertwined with hemicellulose xyloglucans, which act as non-covalent connectors. Xyloglucans feature a (1,4)-β-linked glucan backbone regularly substituted with (1\u0026ndash;6)-α-xylosyl residues. Together, cellulose and xyloglucans constitute two-thirds of the dry mass of the cell wall, forming a network crucial for withstanding tensile forces. However, the cleavage of xyloglucan chains, without simultaneous synthesis, can weaken the cell wall, potentially leading to rupture. This process can be facilitated by endo β-1,4 glucanases, which modify the viscosity and porosity of the wall, enabling the action of other enzymes such as expansins (Scheller \u0026amp; Ulvskov \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, PMeV-complex infected pre-flowering \u003cem\u003eC. papaya\u003c/em\u003e exhibited reduced accumulation of several cell-wall degrading enzymes involved in cellulose and hemicellulose degradation, including β-1,4 glucanases, β-D-xylosidase, β-glucosidase, and β-galactosidase, compared to uninfected plants. This reduction in enzyme levels is noteworthy considering their role in host resistance to various pathogens in other plant species. For instance, in tomato plants, endo-β-1,4-glucanase has been shown to influence host resistance by modulating the expression of PR1 and callose deposition. Decreased levels of endo-β-1,4-glucanase are associated with enhanced resistance to \u003cem\u003eBotrytis cinerea\u003c/em\u003e but increased susceptibility to \u003cem\u003ePseudomonas syringae\u003c/em\u003e (Flors et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Similarly, infected \u003cem\u003eC. papaya\u003c/em\u003e exhibited elevated levels of PR1 mRNA and protein, suggesting potential activation of defense responses. Additionally, upregulation of callose synthases mRNA and downregulation of the callose-degrading enzyme glucan endo-1,3-beta-glucosidase indicate possible callose accumulation, which could restrict PMeV-complex movement within the host. Furthermore, the decreased accumulation of β-1,3 glucanase, a member of the PR2 family involved in callose degradation and symplastic trafficking regulation, further supports the notion of host defense mechanisms limiting virus spread.\u003c/p\u003e \u003cp\u003eIn addition to the changes in cell-wall degrading enzymes, infected juvenile plants exhibited increased accumulation of pectin lyase-like protein compared to controls. This protein is involved in the production of 4,5-unsaturated oligogalacturonides (OGs), which serve as important elicitors of plant defense responses against various pathogens (C\u0026ocirc;t\u0026eacute; et al. 1994). Studies in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e have shown that OGs accumulate prior to symptom development caused by fungal infection. However, the onset of symptoms coincides with increased OG oxidation, reducing their elicitor activity. This oxidation is mediated by OG oxidases, primarily belonging to the Berberine Bridge Enzyme-like (BBE-like) protein family. Interestingly, our study identified a BBE-like enzyme, methyl esterase 10, which accumulated in PMeV/PMeV2-infected \u003cem\u003eC. papaya\u003c/em\u003e plants at 4 and 7 mpg. This finding suggests a potential role for OGs and their oxidation products in modulating the plant's response to PMeV infection. The balance between the accumulation and processing of cell-wall degrading enzymes' products, such as OGs, may contribute to the tolerance of pre-flowering \u003cem\u003eC. papaya\u003c/em\u003e plants to PSD symptoms by preserving the integrity of the cell wall structure.\u003c/p\u003e \u003cp\u003eThe onset of PSD symptom development during the plant post-flowering coincides with disruptions in the laticifer cell wall, as observed in our study. Interestingly, despite these changes, there was no significant alteration in the abundance of wall degradation enzymes between infected and control plants at 7 mpg, suggesting a progressive accumulation of these enzymes over the course of symptom development. Concurrently, infected plants exhibited an induction of several enzymes involved in the synthesis of cell wall precursors, such as Glucose-1-phosphate adenylyl transferase (GPA), UDP-glucose 6-dehydrogenase (UGD), NAD(P)-binding Rossmann-fold superfamily protein, nucleotide-rhamnose synthase, and phosphomannomutase. GPA catalyzes the initial step in starch synthesis, while UGD plays a crucial role in the biosynthesis of nucleotide sugars, including UDP-glucuronic acid, a precursor for various cell wall components (Reboul et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Notably, mutants lacking UGD activity display swollen cell walls, indicating the importance of this enzyme in maintaining cell wall integrity. One plausible hypothesis is that infected plants respond to the increasing osmotic pressure within laticifers by upregulating wall synthesis enzymes and remodeling enzymes. However, this compensatory response becomes inadequate by the flowering phase, leading to cell wall disruption and leakage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eInteraction with jasmonic and salicylic acid-mediated pathways\u003c/h2\u003e \u003cp\u003eSalicylic acid (SA) is known to be a key regulator in systemic acquired resistance (SAR), a defense mechanism against pathogens in plants (Spoel et al., 2012). Previous studies (Madro\u0026ntilde;ero et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) have demonstrated the induction of several SA-activated genes, including pathogenesis-related proteins (PRs), WRKY transcription factors, reactive oxygen species (ROS), and callose genes in PMeV-infected pre-flowering \u003cem\u003eC. papaya\u003c/em\u003e plants. This suggests the involvement of SA signaling in the delayed onset of symptoms observed in these plants. Moreover, treatment of pre-flowering \u003cem\u003eC. papaya\u003c/em\u003e with exogenous SA led to a reduction in PMeV and PMeV2 loads compared to control infected plants. SA has been shown to induce the expression of PR proteins, which are known to reduce virus loads in plants (Spoel et al., 2012). This effect is likely mediated through the oxidation of NPR1 (nonexpressor of pathogenesis-related genes 1), a master immune coactivator (Kinkema et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Tada et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Fu \u0026amp; Dong, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Consistent with this, activated forms of NPR1 were observed in PMeV-infected \u003cem\u003eC. papaya\u003c/em\u003e plants at 4 mpg, coinciding with the upregulation of SA/NPR1-dependent transcripts during this developmental phase (Madro\u0026ntilde;ero et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, an accumulation of photosynthesis-related proteins in infected plants at 3 and 4 mpg, accompanied by an increase in chlorophyll a fluorescence parameter was observed. This finding is consistent with previous research (Yoshioka et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; S\u0026aacute;nchez-Vicente et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Caplan et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), suggesting a crucial role of chloroplasts in conferring viral tolerance. It is likely that this tolerance mechanism involves the accumulation of reactive oxygen species (ROS), which have been shown to contribute to programmed cell death (PSD) in infected plants (Madro\u0026ntilde;ero et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), as well as the production of salicylic acid (SA). Supporting this hypothesis, an accumulation of antioxidant proteins at 4 mpg, indicating a potential defense response against viral infection was observed.\u003c/p\u003e \u003cp\u003eThe dynamics of the jasmonic acid (JA) pathway in PMeV-infected \u003cem\u003eC. papaya\u003c/em\u003e before flowering are intricate. Previous research has demonstrated the accumulation of transcripts encoding a candidate NPR1-inhibitor (NPR1-I/NIM1-I), UDP-glycosyltransferases (UGTs), and genes involved in the ethylene pathway in infected plants (Madro\u0026ntilde;ero et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), all of which are known negative regulators of salicylic acid (SA) signaling. Additionally, at 4 mpg, infected plants accumulate a pectin-hydrolytic enzyme that can enhance the release of cell-wall defense response triggers, potentially leading to increased biosynthesis of JA (Gouveia et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, our findings reveal lower levels of UDP-glucosyltransferase proteins than controls before flowering, but higher levels post-flowering (7 mpg). Furthermore, it is noteworthy that one of the most down-regulated proteins in infected plants at 3 mpg is urate oxidase, which produces allantoin, an activator of the JA pathway (Kaur et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdding to the complexity is the role of oligogalacturonides (OGs). An accumulation of a protein associated with OG production, pectin lyase-like protein, in juvenile infected plants compared to controls was observed. Conversely, an OG oxidation protein, methyl esterase 10, accumulated in infected plants at 4 to 7 mpg. This mirrors findings from \u003cem\u003eA. thaliana\u003c/em\u003e, where OGs were found to accumulate before symptom development but were oxidized just before symptoms appeared during fungal infection (Benedetti et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Notably, OGs have been shown to induce both the salicylic acid (SA) and jasmonic acid (JA) pathways in \u003cem\u003eA. thaliana\u003c/em\u003e (Howlader et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn post-flowering, characterized by symptom development, we observed a reduction in the overproduction of photosynthesis-related proteins compared to controls. This is reminiscent of findings in symptomatic pecan plants infected with the Badnavirus Pecan Virus, where inhibited SA biosynthesis and reduced photosynthesis were reported (Zhang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Conversely, contrasting responses have been observed in soybean plants infected with different forms of the Soybean Mosaic Virus. The virulent form induced the jasmonic acid pathway, while the avirulent form stimulated callose production and upregulated photosynthesis genes, resulting in rapid elimination (Alazem et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaken together, these responses suggest that SA signaling likely dominates at the pre-flowering phase of PMeV complex-infected \u003cem\u003eC. papaya\u003c/em\u003e, inhibiting the development of PSD symptoms. However, the induction of negative regulators of SA and concurrent activation of the JA pathway prevent full-scale and long-lasting tolerance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eP.M.B. Fernandes and J.A. Ventura acknowledge the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico, CNPq for their research productivity award (# 303432/2018-7 and # 307905/2020-9). This work was supported by FAPES grants # 76437906/16 and # 80598609/17 awarded to P.M.B.F., and E. Soares and thank the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Esp\u0026iacute;rito Santo, FAPES (# 76437906/16), and T. F. S\u0026aacute;- Antunes, and M. Maurastoni thank the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior, CAPES for their scholarships (# 88882.315885/2019-01 and, 88887.467501/2019-00).\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eS.P.R., J.A.V., and P.M.B.F. conceived and designed the study. E.A.S., T.S.A., M.M., S.G.B., L.E.C.N., and B.R.F.V conducted the experiments. All authors analyzed and discussed the results. S.P.R., E.A.S., T.S.A., M.M., D.B., and P.M.B.F. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThe authors extend their gratitude to Emily G. Werth and Leslie M. Hicks from the Department of Chemistry, University of North Carolina at Chapel Hill, for their invaluable technical and scientific assistance with proteomic analysis. Additionally, the authors acknowledge the contributions of Leidy J. Madro\u0026ntilde;ero and Karla V. A. Hern\u0026aacute;ndez, former graduate students at Universidade Federal do Esp\u0026iacute;rito Santo, for their valuable support in proteomics data mining and photosynthesis data acquisition, respectively.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlazem M, Tseng KC, Chang WC, Seo JK, Kim KH (2018) Elements involved in the Rsv3-mediated extreme resistance against an avirulent strain of soybean mosaic virus. Viruses 10:581\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalasubramaniam M, Kim BS, Hutchens-Williams HM, Loesch-Fries LS (2014) The photosystem II oxygen-evolving complex protein PsbP interacts with the coat protein of \u003cem\u003eAlfalfa mosaic virus\u003c/em\u003e and inhibits virus replication, Mol. 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Int J Mol Sci 23(21):13576\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Label-free quantitative proteomics, mass spectrometry, papaya meleira virus, photosynthesis, laticifer","lastPublishedDoi":"10.21203/rs.3.rs-4523827/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4523827/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of Papaya Sticky Disease (PSD), caused by the papaya meleira virus (PMeV) complex, only occurs after flowering, suggesting the presence of tolerance mechanisms during the transition from juvenile to adult papaya plants (\u003cem\u003eC. papaya\u003c/em\u003e). In this study, 1,609 leaf proteins of \u003cem\u003eC. papaya\u003c/em\u003e were quantified using a label-free strategy. Differentially accumulated proteins\u0026mdash;38, 130, 160, and 17 at 3, 4, 7, and 9 months post-germination, respectively\u0026mdash;indicated modulation of biological processes at each development phase, mainly involving photosynthesis and cell wall remodeling. Juvenile \u003cem\u003eC. papaya\u003c/em\u003e plants infected with the PMeV complex showed an accumulation of photosynthetic proteins. Correspondingly, chlorophyll fluorescence results suggested enhanced efficiency in photosystem (PS) II and PSI energy flux in these plants. In parallel, pre-flowering plants exhibited a reduction in cell wall-degrading enzymes, followed by an accumulation of proteins involved in the synthesis of wall precursors post-flowering. These findings, combined with ultrastructural data on laticifers, suggest that \u003cem\u003eC. papaya\u003c/em\u003e struggles to maintain the integrity of laticifer walls, ultimately failing to do so after the juvenile-adult transition and resulting in latex exudation, thereby supporting initiatives for the genetic improvement of \u003cem\u003eC. papaya\u003c/em\u003e to enhance resistance against the PMeV complex.\u003c/p\u003e","manuscriptTitle":"Insights on Carica papaya L. proteomic, ultrastructural and physiological changes associated with pre-flowering-related tolerance to papaya sticky disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-12 15:05:18","doi":"10.21203/rs.3.rs-4523827/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90287d14-2791-4ef8-9290-3a4faf10cdfa","owner":[],"postedDate":"July 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-21T00:00:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-12 15:05:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4523827","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4523827","identity":"rs-4523827","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}