Effective infection of tobacco and cowpea leaves by CPSMVCE was detected based on the presence of the corresponding cDNA fragments of the CPSMV-coat protein (Fig. 1A). Somehow, tobacco accumulated less viral particles than the susceptible cowpea genotype. Severe mosaic and chlorosis, which are typical symptoms observed in the virus-susceptible cowpea genotype CE-31 inoculated with CPSMVCE, were not noticed in the tobacco leaves (Fig. 1B), even at 30 days after inoculation (figure not shown). CPSMVCE accumulation was previously reported in CE-31 (Souza et al., 2017; Varela et al., 2017), which characterize a compatible interaction. This suggests that the studied tobacco variety (Xanthi) is tolerant to CPSMVCE, as it limits the virus disease severity and established a style of asymptomatic co-existence (Sanfaçon, 2020) probably through induction of specific mechanisms, most of them still not well understood because tolerance, in comparison with resistance to plant pathogens, has received much less attention. Consequently, experimental results and data on the mechanism of tolerance that helps plants to prevent the severe symptoms observed in susceptible hosts are scarce in the pertinent literature (Pagán and Garcia-Arenal, 2018). Nevertheless, in tolerant interactions, considerable accumulation of viruses has been observed in asymptomatic plants (Sanfaçon, 2020). Then, what biochemical responses are induced by CPSMVCE in tobacco (var. Xanthi) plants to attain tolerance is an open question. To address this demand, we challenged tobacco plants with CPSMVCE and compared with mock-inoculated controls.
Lower accumulation of soluble proteins and RNA in CPSMVCE-inoculated tobacco leaves (Fig. 1B, C) might be a plant strategy to reduce the available cellular machinery for virus replication. Lower protein and RNA contents is a broadly-known plant response against virus infection in consequence of gene silencing and protein target to degradation (Garcia-Ruiz, 2018; Souza et al., 2017). Protein degradation and the resulting constituent amino acids are linked to the supply of energy and metabolic intermediates necessary for an effective defense strategy (Rojas et al., 2014). Recently, a proteomic study carried out by our research group showed that proteins involved in RNA metabolism, in addition to specific transcription factors, were less accumulated in resistant cowpea plants inoculated with CPSMVCE (Varela et al., 2017).
Part of the defense mechanism of tobacco plants against CPSMVCE infection consisted of increased H2O2 content at 1 and 6 DAI (Fig. 2). H2O2 accumulation in tobacco leaves is similar to that in cowpea, either in susceptible or resistant genotypes (Paiva et al., 2016; Silva et al., 2016; Souza et al., 2017), possibly as an attempt to limit the CPSMVCE spread in the host plants at the earlier stages of infection. At this infection phase, H2O2 accumulation may act as a microbicidal agent at the site of pathogen invasion and also as a signaling molecule to trigger the plant defense responses (Deng et al., 2016; Lei et al., 2016; Zhang et al., 2020).
One of the sources of H2O2 production in plants is dismutation of superoxide anions (O2• −) catalysed by SOD (Smirnoff and Arnaud, 2019). The SOD activity of the CPSMVCE-inoculated tobacco plants (Fig. 3A) increased remarkably (180%) at 6 DAI in comparison to the respective mock-inoculated control. Changes in SOD activity and H2O2 content in CPSMVCE-inoculated tobacco plants apparently did not match, apart from 6 DAI when both SOD and H2O2 increased concomitantly. However, the net amount of H2O2 is also dependent on CAT, APX, and POX activities as they use H2O2 as substrate and thus are scavenge enzymes. As shown in Fig. 3B, 3C, and 4C, whereas CAT activity is overall lower, APX and POX are much higher, particularly at 6 DAI, in comparison to the respective tobacco plant controls. Together, these data would suggest that H2O2 should be lower at 6 DAI. However, SOD is regarded as the main intracellular antioxidant defense against superoxide anions, which are converted into H2O2 and O2 (Stephenie et al., 2020), and may have overwhelmed APX and POX activities to explain the net high amount of H2O2 at 6 DAI. Moreover, there are other many biochemical ways, non- and enzymatic, by which H2O2 is produced in plants such as mitochondrial generation through the electron transport chain (ETC), oxidases involved in photorespiration, photosynthesis, fatty acid oxidation, amine/polyamine oxidation, and purine catabolism (Smirnoff and Arnaud, 2019). As previously mentioned, H2O2 accumulation may act as a direct toxic agent against CPSMVCE at the site of pathogen invasion. For instance, H2O2 was considered an effective agent for in vitro inactivation of adenovirus types 3 and 6, adenoassociated virus type 4, rhinoviruses 1A, 1B, and type 7, myxoviruses, influenza A and B, respiratory syncytial virus, and coronavirus strain 229E (Mentel et al., 1977). In addition of being antimicrobial, H2O2 acts as a signalling molecule in the induction of systemic acquired resistance (SAR) and defence-associated genes in plants (Smirnoff and Arnaud, 2019). Lei and collaborators (2016) showed that the M strain of Cucumber mosaic virus (M-CMV) induced H2O2 accumulation in inoculated leaves of Nicotiana tabacum (cv. white burley) during systemic infection, compared with mock-inoculated leaves. Previous works suggested that the higher activities of SOD, APX, and POX, and the lower activity of CAT, compared to mock-inoculated controls, were associated to plant defense mechanisms against virus infection (Gonçalves et al., 2013; Souza et al., 2017; Varela et al., 2017).
In our study, the phenolic compound content increased at 1 DAI and 6 DAI (Fig. 4A) and the PAL activity was considerably higher at all studied time points (Fig. 4B) in CPSMVCE-inoculated tobacco leaves, compared with the respective mock-inoculated controls. Therefore, both the increased PAL activity and the biosynthesis of secondary compounds (phenols) appeared to be important in conferring tolerance of tobacco plants to CPSMVCE. In most plants, the key step of the biosynthesis of phenolics is the non-oxidative deamination of phenylalanine by PAL to trans-cinnamate, which constitutes the initial reaction of the phenylpropanoid pathway. In wheat plants infected with Wheat streak mosaic virus the phenylpropanoid pathway was stimulated upon infection, but lignification was not (Kofalvi and Nassuth, 1995). When the cotyledons of 3–true leaf potted seedlings of a common Japanese tomato cultivar (cv. Fukuju No. 2) were inoculated with potato Virus X (PVX) and with an attenuated strain (L11A) of tobacco mosaic virus (TMV-L11A), the time-course analysis of the methanol-extractable free, and saponifiable ester-bound phenol contents accumulated significantly in the primary leaves during the first 3 days, continued to increase steadily, and peaked between 6 and 10 days postinoculation, compared to uninoculated controls. These results indicated that PVX and TMV-L11A infection not only affected the quantity, but also altered the type of phenol components of the infected tomato plants (Balogun and Teraoka, 2004). Other studies indicated that increased PAL activity and elevated phenolic contents play active roles in virus defense as verified in resistant cowpea plants infected with CPSMVCE (Varela et al., 2017), Capsicum baccatum infected with Pepper yellow mosaic virus (PEPYMV) (Gonçalves et al., 2013), and Gossypium arboreum and Gossypium herbaceum both infected with Cotton leaf curl burewala virus (CLCuBuV) (Siddique et al., 2014)
Our study showed that the GLU specific activity in CPSMVCE-inoculated tobacco leaves was higher than in mock-inoculated controls, at 6 DAI (Fig. 5A). This increase of GLU activity in CPSMVCE-inoculated tobacco, may have been induced by the virus as an attempt to degrade deposited callose, a β-1,3-glucan polysaccharide, at the neck region of plasmodesmata (Zavalievet al., 2013). GLU is an enzyme that degrades callose. Callose accumulation regulates plasmodesmata permeability and limits the cell-to-cell movement of plant viruses, whereas its degradation allows virus spread (Harries and Ding, 2011; Zavaliev et al., 2013; Walsh and and Mohr, 2011). It is well known that viruses subvert the host protein synthesis machinery to their own purpose. The increased GLU activity observed in our study may be an example of such interference on the tobacco protein metabolism.
The cysteine proteinase inhibitor (PIN) of tobacco leaves infected with CPSMVCE was strongly induced at the early stage of infection (Fig. 5B). Plausibly, increased PIN activity constitutes an attempt of tobacco to inhibit the CPSMVCE cysteine proteinase that is required for the proteolytic cleavage of the CPSMVCE RNA 1-encoded polyprotein precursor (208 kDa molecular mass), which is processed to yield five mature, functional protein sub-units (a 32 kDa proteinase cofactor, a 58 kDa presumed helicase, a viral genome 5′-linked protein [VPg] of the CPSMV genomic RNAs [RNA-1 and RNA-2], a 24 kDa proteinase, and a 87 kDa presumed RNA-dependent RNA polymerase) that are required for replication, a crucial event for successful viral infection (Chen and Bruening, 1992; Ponz et al., 1988; Rodamilans et al., 2018). For instance, increased proteinase inhibitor activity was reported in the incompatible interactions of both G. arboreum and G. herbaceum with CLCuBuV (Siddique et al., 2014) and transgenic tobacco plants infected with Tobacco etch virus (TEV) and Potato virus Y (PTY) (Gutierrez-Campos et al., 1999). Therefore, inhibitors of cysteine proteinases could be further studied as potential antiviral agents against plant viruses that use cysteine proteinases to process genomic precursor proteins for replication.
Viruses depend on the host cellular translation initiation factors to synthesize their proteins to regulate replication, the systemic movement into the plant cells, and complete their cycle (Keima et al., 2017; Sanfaçon, 2015; Shopan et al., 2020; Walsh and Mohr, 2011). The transcript content of the elongation factor EF1α (also referred as eEF1A, in the literature) was down-regulated in CPSMVCE-inoculated tobacco at 6 DAI, whereas eIF4E was up-regulated and its eIF(iso)4E isoform did not change. eIF4E and eIF(iso)4E have similar activities, but may have distinct physiological functions and the lack of eIF4E or eIFiso4E does not influence the viability of plants. However, deficiency of eIF4E or eIFiso4E decreases the infectivity of plant viruses (Keima et al., 2017). For instance, natural recessive resistance to potyvirus and several other viruses has been often linked to mutations in eIF4E or eIF(iso)4E that impede their interactions with the VPg protein (Sanfaçon, 2015; Shopan et al., 2020). Presumably, CPSMVCE induced the increase of the eIF4E gene products (Fig. 6) in tobacco as an attempt to support infection. Therefore, increase of the EF1α level was expected, together with eIF4E, to enhance the synthesis of the CPSMVCE polyprotein. However, in response to the augmented eIF4E expression, the CPSMVCE-inoculated tobacco plants down-regulated EF1α (Fig. 6). Plausibly, it represents an attempt to control CPSMVCE replication. For instance, CPSMVCE is also a positive-strand RNA virus. EF1α is frequently found in association with eIF4E/eIF(iso)4E and PABP (poly(A)-binding protein) to comprise the virus translation/replication complexes of positive-strand RNA viruses (Sanfaçon, 2015). EF1α is required to deliver selected aminoacylated tRNA to the 80S ribosome A site (Walsh and Mohr, 2011). The positive-strand RNA virus type constitutes the majority of the characterized infectious plant viruses (Sanfaçon, 2020). Proteomic analyses of a resistant cowpea genotype infected with CPSMVCE also showed lower EF1α accumulation (Varela et al., 2017). Moreover, recent findings by our research group revealed that a susceptible cowpea genotype up-regulated eIF(iso)4E when inoculated with CPSMVCE, while the resistant cowpea genotype exhibited no alteration in both eIF4E and eIF(iso)4E (data not published). eIF4E is a major target factor hijacked by Potyviruses, to which Comoviruses are similar, to enhance protein synthesis, cell-to-cell and systemic movements, and regulate replication (Sanfaçon, 2015).
BRI1 gene, which codes for a receptor kinase, was down-regulated in tobacco plants infected with CPSMVCE as compared to uninoculated controls (Fig. 6). BRI1 and BAK1 are receptors for brassinosteroid (BR) signalling and play important functions in infections caused by RNA virus, like those promoted by Turnip crinkle virus (TCV), Oilseed rape mosaic virus (ORMV), and TMV (Zhao and Li, 2021). Silencing of the BR biosynthetic and signaling genes NbBRI1 and NbBAK1, along with NbDWARF, NbBSK1, and NbBIK1, enhanced the susceptibility of N. benthamiana plants to TMV infection indicating that these genes also support the antiviral immune response (Deng et al., 2016a). Moreover, silencing of NbBRI1 compromised the BR-induced H2O2 and NO production associated with systemic virus resistance (Deng et al., 2016). Therefore, lower BRI1 expression induced in tobacco by CPSMVCE may represent an additional virus strategy by which the BR-induced resistance of tobacco to CPSMVCE is blocked.
The NR gene, which code for nitrite-dependent nitrate reductase, was down-regulated upon tobacco inoculation with CPSMVCE (Fig. 6). Deng et al. (2016) showed that silencing of NbNR or systemic pharmacological inhibition of NR compromised BR-triggered systemic nitric oxide (NO) accumulation that participates in the BR-induced general virus defence signalling in Nicotiniana benthamiana. Probably, down-regulation of NR in tobacco, induced by CPSMVCE, limits the BR-induced defence mechanism of tobacco as there is evidence that H2O2 and NO are involved in the BR-mediated systemic defense signaling pathway against plant viruses (Deng et al., 2016; Štolfa Čamagajevac et al., 2019). This suggests that CPSMVCE successfully inhibited the BR-dependent defense pathway in tobacco plants. For instance, BR levels and signalling were positively correlated with the tolerance of Arabidopsis thaliana to Cucumber mosaic virus (CMV) (Zhang et al., 2015b). Zou et al. (2018) showed that there is indeed a BR-dependent pathway that induces NO accumulation through NR activity to suppress viral infection in A. thaliana challenged with CMV.
We conclude that CPSMVCE modulated the activity of some enzymes and gene expression in tobacco to its own benefit, as proposed in the supplementary Figure S1. Indeed, the genes related to the plant defense pathways (NR, BRI1, and BAK1) had their expression down-regulated, whereas eIF4E, which is related to CPSMVCE protein synthesis, replication, and spread, was up-regulated. However, even with alterations induced by CPSMVCE to facilitate the viral action, N. tabacum overcomes and actively responds against the viral infection (Fig. S1). For example, the EF1α gene, which is also related to viral protein synthesis, was down-regulated. Moreover, the H2O2 and polyphenol contents (PC), as well as the activity of the enzymes SOD and PAL increased. Nevertheless, the severe mosaic disease symptoms caused by CPSMVCE in susceptible cowpea genotypes were not developed in N. tabacum (var. Xanthi), although CPSMVCE viral particles were present in the plant leaves at 6 DAI (Fig. 1). Taken together, these findings suggest that N. tabacum (var. Xanthi), a non-leguminous plant, exhibited the patterns of a typical tolerant host to CPSMVCE since this virus did not cause any harmful disease symptoms although it modulated some transcripts and enzyme activities crucial for successful viral replication. Nevertheless, other many biochemical and molecular aspects of this pathosystem need to be investigated to obtain a complete picture of the mechanisms driving the tolerance of N. tabacum (var. Xanthi) to CPSMVCE, which is of utmost importance to develop new cowpea genotypes tolerant or resistant to this viral disease.