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 (PIabs and PItotal) have emerged as efficient tools for quantifying stress levels (Strasser et al., 2010; Huther et al., 2016). PItotal, 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 PItotal, 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.