1. Introduction
Tomato chlorosis virus (ToCV, genus
Crinivirus, family
Closteroviridae) is an emergent plant virus that causes a yellow leaf disorder in tomato, including interveinal yellowing chlorotic areas, thickening of leaves and bronzing and necrosis of the older leaves which may cause serious economic losses in crop yield [
1,
2]. ToCV has a bipartite genome of positive single-stranded RNA and like many other crinivirus, is restricted to phloem associated cells and transmitted in a semi-persistent manner by several whiteflies of the genera
Bemisia and
Trialeurodes, although its emergence has been associated mostly with the global spread of the whitefly
B. tabaci in tropical and warm regions worldwide [
1]. Furthermore, recent studies have demonstrated that ToCV, when present in a mixed infection with Tomato yellow leaf curl virus (TYLCV, genus
Begomovirus, family
Geminiviridae) also transmitted by
B. tabaci, might result into a synergistic interaction disease that could have a significant detrimental impact on tomato production [
3,
4,
5,
6].
Since plant viruses are obligate intracellular parasites that exploit host cellular machinery to establish systemic infections [
7], they generally induce a wide variety of alterations in host gene expression and cell physiology in order to facilitate infections. These alterations include not only defense-related pathways but also some others involved in photosynthesis, secondary metabolism, or regulation of plant hormone levels [
8,
9,
10,
11,
12]. Transcriptome sequencing using RNA-seq technology allows to explore gene expression changes that are either directly or indirectly associated with viral infection [
13,
14,
15]. Previous studies have followed this approach to characterize genome-wide gene expression profiles in tomato in response to ToCV infection [
16,
17]. These two papers focused on a single time-point after inoculation for their sampling and analysis. In the first work, Seo et al. (2018) [
16] performed transcriptome analysis of grafted plants from ToCV-infected tomato plants at 56 days post inoculation (dpi), pointing to genes potentially involved in the response affecting the development. Instead, Yue et al. (2021) [
17] analyzed plants infected by agroinoculation with an infectious clone and at 40 dpi, identifying in this case genes associated with the MAPK signaling pathway, the glyoxylate cycle, and photosynthesis processes. This variability in the observed responses might reflect experimental differences in terms of virus isolates, plant materials, and specially inoculation modes. Furthermore, it is important to consider the dynamic nature of the pathogenic interaction. In particular, the changes of gene expression in response to viral infection are highly dynamic, and plant viruses are known to induce changes in host gene expression at the early stages of infection, which leads to the activation of antiviral responses [
8,
18,
19]. Therefore, our understanding of how ToCV infection processes regulate gene expression during the early stages of natural vector-mediated infection remains limited.
RNA silencing is a well-established antiviral defense system in plants [
20,
21]. This antiviral defense involves production of virus-derived small interfering RNAs (vsRNAs) by RNase III Dicer-like (DCL) proteins processing viral RNA precursors into 21- to 24-nucleotide (nt) [
22,
23]. In Arabidopsis thaliana, virus resistance against positive-strand RNA viruses is initiated by either DCL4 or DCL2, which are involved in the biogenesis of 21-nt and 22-nt vsRNAs, respectively [
24,
25,
26]. These vsRNAs are loaded onto AGO proteins, leading to the formation of RNA-induced silencing complexes (RISCs) that repress complementary target RNA [
27]. Moreover, the 22-nt vsRNAs are likely to promote the amplification of RNA silencing, which includes the production of secondary vsRNAs by DCL proteins from the products of RNA-dependent RNA polymerases (RDRs) [
28,
29]. Characterization of viRNAs through deep sequencing techniques in response to various plant viruses has been previously conducted in multiple agronomically important crop species [
30,
31,
32,
33,
34,
35]. Nevertheless, the vsRNAs profile originating from ToCV infection in tomato plants has only been documented in a single study, which analyzed the tomato virome through sequencing sRNAs from field crop samples collected in China [
35]. Consequently, our understanding of the vsRNAs profile associated with ToCV infection in tomato plants remains limited.
In this study, we performed time-course transcriptome analysis using RNA-seq to analyze the dynamic changes of differentially expressed genes (DEGs) in tomato plants after ToCV infection when transmitted by its natural insect vector B. tabaci. Key genes were comprehensively identified and classified into essential pathways, providing new insight into ToCV pathogenesis and host immune response. Further analyses of the distribution of vsRNAs along the viral genome determined using sRNA sequencing indicated that RNA1 and RNA 2 were differentially targeted by vsRNAs. We also observed that genes involved in plant immunity, such as Hsp90 (heat shock protein 90) and its co-chaperone Sgt1 (suppressor of the G2 allele of Skp1) may contribute to the basal resistance to viral infection. These findings provide new insights into the molecular responses that occur in ToCV-infected tomato plants and may represent a step toward identifying potential genes for designing future disease control strategies against ToCV.
4. Discussion
In our study, our objective was to provide a comprehensive insight into the alterations within the tomato transcriptome throughout the course of ToCV infection. We accomplished this by analyzing the dynamic transcriptional responses of tomato plants at different time points following their infection by
B. tabaci. Our findings revealed that gene expression in plants undergoes significant changes over time. We observed a large number of DEGs at 2 dpi (2,009), which a substantial decrease at 7 dpi (561). However, at 14 dpi we observed a remarkable reprogramming of the plant transcript profile, with 5,937 genes exhibiting differential expression (
Figure 1B). These findings highlight the dynamic nature of the plant transcriptome during the progression of ToCV infection, and offer a snapshot of a particular stage in the plant's life cycle and the course of infection. On the other hand, the PCA analysis indicated that mock and ToCV samples formed a distinct cluster from naïve samples at 2 dpi (
Figure 1A), suggesting that the host can sense the whitefly infestation regardless of whether the whitefly was viruliferous or non-viruliferous. However, the significant deregulation of 2,009 genes observed at 2 dpi in the ToCV samples, as compared to the mock samples, strongly suggests that ToCV may have significant effects on whitefly-plant interaction. Interestingly, the pathways related to flavonoids [
54,
55] and the steroid biosynthesis involved in resistance to insect herbivories were repressed during ToCV infection (
Figure 3A). Yao et al, 2019 [
55] demonstrated that tomatoes with high flavonoid levels exhibited resistance to
B. tabaci, resulting in a decrease in both the primary and secondary spread of TYLCV. Furthermore, steroid plant hormones such as Brassinosteroids (BRs), which are involved in plant growth and development [
56], also play a role in plant-herbivore interactions likely by regulating glucosinolate biosynthesis [
57,
58,
59,
60]. Additionally, several studies have suggested that BRs also function in plant immunity by inducing plant defenses against viruses [
61,
62,
63]. Further studies are required to gain a deeper understanding of the specific mechanisms underlying the repression of flavonoids and BRs during ToCV infection, and to investigate how this repression may influence insect resistance and the spread of the virus.
The GO analysis revealed there was an up-regulation of genes associated with the microtubule-based process at 2 dpi (
Figure 2A), a phenomenon frequently observed in response to viral infections [
64,
65,
66]. Microtubules are known to play crucial roles in various biological processes, such as virus movement, the assembly of viral replication complexes, and the formation of transmission bodies that facilitate virus transmission between plants via insect vectors [
67,
68]. However, the rationale behind their up-regulation in ToCV-infected plants remains unknown, and further investigation will be necessary to uncover any underlying causes. GO annotation also revealed an enrichment of genes related to photosynthesis activity and chloroplast organization and structure at 2 and 7 dpi. However, an intriguing contrast emerged, as these same GO terms exhibited down-regulation at 14 dpi (
Figure 2 and
Figure 3). This underscores once again the need to conduct transcriptomic analyses at different time points during viral infection. Considering that chloroplasts function as factories for the synthesis of key signaling molecules such as salicylic acid (SA) and jasmonic acid (JA) for host defense responses against viruses [
69], it is reasonable to speculate that the activation of these pathways constitutes a direct reaction to viral infection. On the contrary, at 14 dpi we observed a downregulation of genes linked to chloroplasts and photosynthesis, consistent with previous findings reported by (Çevik et al., 2021). This downregulation was accompanied by an upregulation of genes involved in SA signaling pathway. Such patterns are frequently documented in virus-infected tissues and are believed to underlie the development of chlorosis symptoms commonly associated with viral infections [
9,
70]. Indeed, several genes related with leaf senescence and autophagy activity [
71,
72] were up-regulated during ToCV infection at 14 dpi (
Figure 4A). Similar findings were reported by [
17] in ToCV-infected plants, despite differences in experimental conditions, tomato cultivars, and virus isolates used in their study. These findings suggest that diverse genes related to defense pathways of tomato plants are expressed during late stages of ToCV infection [
73,
74].
We found that RNA2 spawns much more abundant vsRNAs than RNA1, which reflects the higher replication rate of the RNA2 (data not shown) potentially leads to the production of more abundant dsRNA replication intermediates for dicing. It has been well established that DCL4 function as the primary sensor of viral dsRNAs producing vsRNAs 21-nt in length [
23]. However, consistent with a previous report [
35], we found that vsRNAs originating from ToCV were predominantly 22-nt in length, likely orchestrated by tomato orthologs of DCL2. Considering that viral suppressors of RNA silencing (VSRs) have the potential to interfere with host factors involved in antiviral silencing [
75], the prevalence of 22-nt vsRNAs may be a consequence of the ability of ToCV-encoded VSRs [
76] to disrupt the functioning of DCL4. Similar mechanisms have been observed in other viruses, such as Turnip crinkle virus, where the VSR CP inhibits DCL4 activity, and consequently DCL2 becomes the major contributor for vsRNAs biogenesis [
24]. Interestingly, our transcriptome analysis revealed that single infections with ToCV led to a significant upregulation in the expression levels of DCL2b, DCL2d, and DCL4 when compared to mock-infected plants. Furthermore, Wang et al. (2018) [
77] documented that tomato DCL2b ranks among the most abundantly expressed members of the DCL2 family and offers enhanced protection against tobacco mosaic virus. Approximately equal amounts of vsRNAs were mapped respectively to the positive and negative strands of RNA1 and RNA2 viral genomic of ToCV (
Figure 4B), indicating vsRNAs were likely produced from double-stranded replicative intermediates.
The
Hsp90-
Sgt1 complex plays a critical role in regulating the plant immune system against pathogens, including plant viruses [
78]. In this study, we noted a substantial upregulation of
Hsp90 and
Sgt1 expression at 14 dpi following ToCV infection. Notably, the silencing of these genes resulted in a higher level of ToCV accumulation in tomato plants, suggesting their potential involvement in antiviral responses specific to the tomato-ToCV pathosystem. Additionally, silencing of
Sgt1 through VIGS led to cell death, whereas silencing of
Hsp90 did not cause noticeable differences when compared to control TRV-infected plants. Our results are in line with those of [
79], who demonstrated that silencing the tomato
Hsp90 and
Sgt1 genes resulted in increased TYLCV accumulation. However, while they reported that silencing of both genes led to cell death, we observed this phenotype only in
Sgt1 tomato plants (
Figure S1A). Interestingly, it has been reported that the infection of various RNA viruses, such as TSWV and potato virus X, is impaired in
Sgt1-silenced N. benthamiana, despite the fact that these viral infections strongly upregulate
Sgt1 expression in the plant (Ye et al., 2012; Qian et al., 2018). Therefore, The
Hsp90-
Sgt1 complex appears to serve dual functions, playing a crucial role in basal resistance against certain viruses while also potentially acting as a proviral factor for others.
We believe that our study enhances our comprehension of the molecular responses occurring in ToCV-infected tomato plants. This knowledge may aid in the identification of potential genes implicated in defensive responses, making them good candidates for future breeding efforts. These candidates could serve as targets for the development of new strategies aimed at controlling the disease through the modulation of endogenous pathways.
Author Contributions
Conceptualization, I.O., J.J.L-M. and J.A.D-P.; methodology, I.O., N.F-P., A.E-C., J.J.L-M. and J.A.D-P. ; software, I.O., N.F-P. and A.E-C. .; validation, I.O. and N.F-P.; formal analysis, J.J.L-M. and J.A.D-P.; investigation, I.O.; resources, J.J.L-M. and J.A.D-P.; data curation, I.O., and N.F-P.; writing—original draft preparation, I.O.; writing—review and editing, I.O., J.J.L-M. and J.A.D-P.; supervision, J.J.L-M. and J.A.D-P. ; project administration, J.J.L-M. and J.A.D-P.; funding acquisition, J.J.L-M. and J.A.D-P. All authors have read and agreed to the published version of the manuscript.