Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation, to promote viral infection and symptom development in N. benthamiana

The phytohormone gibberellin (GA) is a vital plant signaling molecule that regulates plant growth and defense against abiotic and biotic stresses. To date, the molecular mechanism of the plant responses to viral infection mediated by GA is still undetermined. DELLA is a repressor of GA signaling and is recognized by the F-box protein, a component of the SCFSLY1/GID2 complex. The recognized DELLA is degraded by the ubiquitin-26S proteasome, leading to the activation of GA signaling. Here, we report that ageratum leaf curl Sichuan virus (ALCScV)-infected N. benthamiana plants showed dwarfing symptoms and abnormal flower development. The infection by ALCScV significantly altered the expression of GA pathway-related genes and decreased the content of endogenous GA in N. benthamiana. Furthermore, ALCScV-encoded C4 protein interacts with the DELLA protein NbGAI and interferes with the interaction between NbGAI and NbGID2 to prevent the degradation of NbGAI, leading to inhibition of the GA signaling pathway. Silencing of NbGAI or exogenous GA3 treatment significantly reduces viral accumulation and disease symptoms in N. benthamiana plants. The same results were obtained from experiments with the C4 protein encoded by tobacco curly shoot virus (TbCSV). Therefore, we propose a novel mechanism by which geminivirus C4 proteins control viral infection and disease symptom development by interfering with the GA signaling pathway.


Introduction
Geminiviruses are a class of DNA viruses with circular single-stranded genomes that encode limited numbers of proteins [1][2][3][4]. Geminiviruses recruit host factors to ensure their genome replication, virion assembly, movement, insect transmission, and other processes [2]. Plants infected with geminiviruses often develop disease symptoms such as yellow veins, leaf curling, enation, and curly shoots [4][5][6][7]. During virus-plant arms races, host plants have evolved specific proteins targeting various viral infection steps. In response, viruses, including geminiviruses, have evolved specific strategies to hijack specific host factors to interfere with host defense responses [8].
C4 proteins (also known as AC4 proteins of bipartite geminiviruses), encoded by monopartite geminiviruses in the genus Curtovirus, Topocuvirus, Turncurtovirus, and Begomovirus, are multifunctional proteins [9,10]. To date, multiple C4 proteins have been reported as disease symptom determinants that can disrupt host cell development to cause symptom formation [11][12][13][14]. Mutational analyses of C4/AC4 proteins have shown that mutations can alleviate disease symptoms and suppress virus accumulation in infected plants [15]. Overexpression of C4/ AC4 in plants induces hyperplasia, callus-like tissues, and curly leaves due to changes in cell cycle-related gene expression [13,16]. It was reported that the C4 protein encoded by tomato leaf curl Yunnan virus (TLCYnV) could interact with NbSKη to reduce its nuclear accumulation. Cytoplasmic NbSKη is less phosphorylated and has a reduced ability to degrade the cell cycle regulator NbCycD1;1 [17]. Sweet potato leaf curl virus (SPLCV) was reported to interact with brassinosteroid-insensitive 2 (AtBIN2) to relocate AtBIN2-interacting proteins, AtBES1/ AtBZR1, into the nucleus to induce abnormal plant development via activation of the BR signaling pathway [18]. Recently, C4 has been proven to suppress RNA silencing through its interaction with receptor-like kinase BARELY ANY MERISTEM 1 (BAM1) and BAM2. These interactions inhibit the functions of BAM1 and BAM2 in spreading RNAi signals to adjacent cells [19]. Cotton leaf curl Multan virus (CLCuMuV) C4 could interact with S-adenosyl methionine synthetase (SAMS) to inhibit its ability to suppress transcriptional and post-transcriptional gene silencing, resulting in more substantial CLCuMuV infection [20]. Geminivirus C4 can also regulate systemic viral infection in plants. For example, the introduction of mutations into beet severe curly top virus (BSCTV) C4 resulted in a systemic movement defective virus [21].
Gibberellins (GAs) are plant hormones that play crucial roles in plant growth and development and defense responses against abiotic and biotic stresses [22][23][24]. GA-defective mutant plants are stunted, and this stunted phenotype can be restored by spraying the plants with exogenous GA. Although GA-insensitive mutant plants also exhibit a dwarfing phenotype, similar to the GA-defective mutant plants, this phenotype is not reversible through the application of exogenous GA [25]. In contrast, mutant plants that produce constitutively activated GA have elongated stems relative to wild-type plants. The phenotype of these plants directly correlated with the GA content. In the past two decades, the main elements involved in the GA signaling pathway, including GA receptor protein GID1 (GIBBERELLIN INSENSITIVE DWARF1), DELLA (aspartic acid-glutamic acid-leucine-leucine-alanine), F-box protein SLY1 (SLEEPY1), and SNZ (SNEEZY), have been identified through genetic screening of rice and Arabidopsis mutant lines [26].
DELLA is a member of the GRAS family and is an inhibitor of plant growth and development, which functions at the nexus in the GA signaling pathway to negatively regulate GA biosynthesis [27,28]. Similar to other GRAS family proteins, DELLA has a conserved C-terminal GRAS domain, which is necessary for protein-protein interactions and transcriptional regulation, and an N-terminal DELLA domain. Deletion of the DELLA domain affects its binding to the GA receptor GID1 and results in a semidominant GA-insensitive dwarfing phenotype [29]. DELLA proteins of different plant species, including Arabidopsis, wheat, corn, rice and barley, are highly conserved. Arabidopsis is known to encode five DELLA proteins (e.g., GAI, RGA, RGL1, RGL2 and RGL3) playing slightly different, but redundant, functions in the inhibition of GA-dependent responses [30]. However, tomato has only one DELLA gene, called PROCERA (PRC) [31].
Based on the transmission mode, GA can be divided into two major groups: the group utilizing the F-box protein-dependent transmission mode and the other group utilizing the Fbox protein-independent transmission mode. After receiving the signal, the receptor protein GID1 binds the active GA and interacts with the negative regulatory DELLA to form a GA-GID1-DELLA complex. DELLA is then recognized by the F-box protein in the SCF SLY1/ GID2 complex and degraded by the ubiquitin-26S proteasome, leading to the removal of the repressive effect of DELLA [32][33][34][35][36]. For those plants that lack functional F-box proteins, a large amount of DELLA will accumulate in cells, leading to abnormal plant growth and development [37].
Ageratum leaf curl Sichuan virus (ALCScV) is a member of the genus Begomovirus, family Geminiviridae. ALCScV was first reported in Ageratum conyzoides in Sichuan Province, China, in 2018 and can cause severe symptoms on multiple plants [7,38]. Although ALCScV C4 has been discovered as a disease symptom determinant, the molecular mechanism underpinning this action is unclear. In this study, we found determined that upon ALCScV infection, the expression of GA pathway-related genes and the content of endogenous GA were significantly affected. Further analyses revealed that ALCScV C4 could interact with NbGAI in vitro and in vivo, which thwarts the interaction of NbGAI and NbGID2, thereby preventing degradation of NbGAI. In addition, silencing of NbGAI or spraying plants with exogenous GA 3 greatly enhanced N. benthamiana resistance to ALCScV infection. Meanwhile, tobacco curly shoot virus (TbCSV)-encoded C4 can also interact with NbGAI, and silencing of NbGAI expression can inhibit TbCSV infection. In summary, we propose that one of the specific functions of geminivirus C4 proteins is to interfere with the GA signaling pathway to promote virus infection and disease symptom development, a mechanism that has not been reported previously. expression of GA signaling pathway-associated genes were investigated through qRT-PCR. The results showed that the relative expression levels of NbGA20, NbGA-3β, NbPIF3 and NbPIF4 were significantly down-regulated in the ALCScV-inoculated N. benthamiana plants (Fig 1B). Similarly, at 14 days post-agroinfiltration (dpi), the content of endogenous GA was also significantly decreased in the ALCScV-inoculated N. benthamiana plant (2.19 μg g -1 FW) leaves compared to that in the mock-(3.04 μg g -1 FW) or the ALCScV-mC4-inoculated N. benthamiana plant (2.77 μg g -1 FW) leaves (Fig 1C). In a separate study, the plants inoculated with PVX-C4 also displayed significant dwarfing and delayed flowering (S1 Fig). As expected, the content of endogenous GA in the PVX-C4-inoculated plants (0.63 μg g -1 FW) was significantly decreased compared to that in the PVX-inoculated plants (1.05 μg g -1 FW) ( Fig 1D). Consequently, we speculated that the C4 protein is a regulator of the GA pathway-associated genes as well as the biosynthesis of endogenous GA.

ALCScV C4 directly interacts with NbGAI
To understand how ALCScV C4 regulates the GA pathway, Y2H assays were performed to screen host protein(s) that could interact with ALCScV C4. The results showed that a GA signaling pathway repressor, NbGAI, interacted with C4 in the yeast cells (Fig 2A). The GAI proteins belong to the family of GRAS transcription factors, of which only one member has been identified in Solanaceae plants [31]. To verify this interaction in planta, BiFC assays were performed, and co-expressed of cYFP-C4 and nYFP-NbGAI resulted in a YFP fluorescence signal in both the nucleus and cytoplasm at 48 hours post-incubation (hpi). No YFP fluorescence was observed in the leaves co-infiltrated with cYFP-C4 and nYFP or cYFP and nYFP-NbGAI ( Fig  2B). Then the interaction between C4 and NbGAI was further confirmed by a Co-IP assays

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation after transient co-expression of C4-His and NbGAI-GFP in N. benthamiana leaves ( Fig 2C). Taken together, these results demonstrate that the ALCScV C4 protein directly interacts with the NbGAI protein both in vitro and in vivo.

The central region of NbGAI is responsible for its interaction with C4
The coding sequence of NbGAI contains 1701 nucleotides and is predicted to encode a hydrophilic protein with 567 amino acids and a molecular mass of approximately 62 kDa (https://web. expasy.org/cgi-bin/protparam/protparam). Amino acid sequence alignment using GAI sequences from S. lycopersicum (NP_001234365), C. baccatum (PHT30960), C. maxima (XP_022998067), I. triloba (XP_031108300), N. benthamiana (AMO02501), and N. sylvestris (XP_009796771) showed that like other GAIs, NbGAI also contains two conserved domains (e.g., the N-terminal DELLA domain and the C-terminal GRAS domain) (S2A Fig). The phylogenetic tree also revealed that NbGAI is more closely related to the GAIs from other Solanaceae plants (S2B Fig).
To explore which region in NbGAI is responsible for the interaction with C4, five mutant plasmids were constructed based on the conserved domain predictions (Fig 3A). The Y2H assays showed that NbGAI-M2 (aa residues 98-566), but not the other four fragments, Results of the BiFC assay show the interaction between ALCScV C4 and NbGAI in vivo. Leaves co-expressing cYFP-C4 and nYFP, cYFP and nYFP-NbGAI, or cYFP-C4 and nYFP-NbGAI were examined and imaged under a confocal microscope equipped with a FITC filter. Scale bar = 100 μm. (C) Results of the Co-IP assay show the interaction between ALCScV C4 and NbGAI in vivo. C4-His and NbGAI-GFP or C4-His and GFP (control) were co-expressed in N. benthamiana leaves. Total protein was extracted from the infiltrated leaf samples at 2 dpi. The input and the coimmunoprecipitated proteins were analyzed through Western blot analyses using an anti-GFP or an anti-His antibody. https://doi.org/10.1371/journal.ppat.1010217.g002

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation interacted with C4 ( Fig 3B). This interaction was also confirmed in planta through BiFC assays ( Fig 3C). Meanwhile, the protein expression of NbGAI-M2 was confirmed by Western blot (S3 Fig). These results suggest that the 98-566 amino acid positions of NbGAI are responsible for the interaction with ALCScV C4.

The interaction between NbGAI and NbGID2 causes the degradation of NbGAI via the ubiquitin-26S proteasome
Previous studies have shown that the GAI and GID2 interaction results in the degradation of GAI and activation of the GA signaling pathway [22]. In this study, the interaction between NbGAI and NbGID2 was analyzed via Y2H and BiFC assays. The results demonstrated that NbGAI interacted with NbGID2 in yeast cells ( Fig 4A) and in N. benthamiana leaf cells (Fig 4B).
Because treatment of plants with exogenous GA 3 can promote the degradation of GAI via the ubiquitin-26S proteasome [33,40], we investigated whether NbGAI degradation was also ubiquitin-26S proteasome dependent. cYFP-NbGID2 and nYFP-NbGAI were co-expressed transiently in N. benthamiana leaves; at 48 hpi, the green fluorescence signal was observed in the nuclei of the N. benthamiana leaf cells ( Fig 4C). As expected, after the infiltrated leaves were treated with 50 μM MG132 solution (MG132, a specific inhibitor of the 26S proteasome), the YFP fluorescence intensity in the MG132-treated leaves was significantly stronger than that in the 0.8% ethanol solution (mock)-treated leaves (Fig 4C and 4D). In contrast, the YFP fluorescence intensity in the GA 3 -treated leaves was significantly decreased compared to the mock-treated leaves (Fig 4C and 4D). Together, these results indicated that the degradation pathway of NbGAI protein in N. benthamiana depends on the ubiquitin-26S proteasome.
Further analyses using various NbGAI deletion constructs indicated that the middle region of NbGAI, encompassing amino acids 98-566, is responsible for the interaction between NbGAI and NbGID2 (Fig 4E and 4F).

ALCScV C4 interferes with the interaction between NbGAI and NbGID2
Because both ALCScV C4 and NbGID2 interacted with NbGAI-M2, but C4 and NbGID2 did not interact with each other (S4 Fig), we speculated that ALCScV C4 and NbGID2 may be

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation competitively combining with NbGAI. To test this hypothesis, a competitive BiFC assay was performed. cYFP-NbGID2, nYFP-NbGAI and C4-His or cYFP-NbGID2, nYFP-NbGAI and pCV empty vector were co-expressed in N. benthamiana leaves through agroinfiltration. At 48 hpi, the result showed that the YFP fluorescence intensity was significantly decreased in the leaves co-expressing cYFP-NbGID2, nYFP-NbGAI and C4-His compared to that in the leaves co-expressing cYFP-NbGID2, nYFP-NbGAI and pCV empty vector (Fig 5A and 5B). Meanwhile, the Western blot assay confirmed that C4-His was in deed expressed in the infiltrated leaves ( Fig 5C). To further verify that ALCScV C4 can interfere with the interaction between NbGAI and NbGID2, a competitive pull-down assay was conducted, and the result indicated that, in the presence of C4-His, the amount of NbGAI-GFP pulled down by NbGID2-Flag was significantly reduced (Fig 5D). In addition, as the amount of C4-His was gradually reduced, the amount of NbGAI-GFP pulled down by NbGID2-Flag was gradually increased, indicating the negative effect of C4 on the interaction between NbGAI and NbGID2.

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation

ALCScV C4 can inhibit NbGAI degradation
The interaction between GAI and GID2 can enhance GAI degradation via the ubiquitin-26S proteasome [41]. In this study, to test whether ALCScV C4 could also affect the stability of NbGAI, NbGAI-GFP was transiently expressed in N. benthamiana leaves through agro-infiltration, and the green fluorescence from NbGAI-GFP was found predominantly in the nuclei, with a small amount of fluorescence in the cytoplasm at 48 hpi. Meanwhile, the NbGAI-GFP fusion protein was confirmed by the Western blot experiments (S5 Fig). Then, NbGAI-GFP was co-expressed with C4-His or the empty vector in N. benthamiana leaf cells, and stronger green fluorescence was observed in the leaves co-expressing NbGAI-GFP and C4-His than in the leaves expressing NbGAI-GFP alone (e.g., NbGAI-GFP and empty vector) (Fig 6A, left two images). However, whether plants were treated with 50 μM GA 3 or MG132, the green fluorescence was enhanced when co-expressed with pCV-C4-His ( Fig 6A). Then, the relative accumulation levels of NbGAI-GFP protein were analyzed by Western blot assay. The NbGAI-GFP protein accumulated to significantly higher levels in N. benthamiana leaves co-infiltrated with NbGAI-GFP and pCV-C4-His compared to co-infiltration with NbGAI-GFP and pCV empty vector. These results proved that the C4 protein inhibits the degradation of NbGAI (Fig 6B). Furthermore, qRT-PCR and semi-qRT-PCR results indicated that transient expression of C4 did not affect the mRNA expression level of NbGAI (Fig 6C and 6D). Meanwhile, N. benthamiana expressing GFP alone was used as a control, and the results showed that the C4 protein does not affect the stability of the GFP protein (Fig 6E-6H). In addition, Western blot assays confirmed that the C4 protein did not affect the stability of NbGID2 (S6 Fig). Taken together, these results suggest that the ALCScV C4 protein inhibits the degradation of NbGAI.

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation To further confirm the ALCScV C4-mediated inhibition of NbGAI degradation, agrobacterium culture carrying pNbGAI-GFP was co-infiltrated with pC4-His, at various concentrations, into N. benthamiana leaves. At 2 dpi, Western blot analyses showed that the expression level of NbGAI-GFP increased when the concentration of Agrobacterium culture expressing C4-His was increased, suggesting that the C4-mediated stabilization of NbGAI is C4 dosedependent ( Fig 7A). As expected, C4-His had no clear effect on the expression of GFP (Fig 7B).

Silencing of NbGAI expression in N. benthamiana inhibits ALCScV infection
To determine the role of NbGAI in ALCScV infection, NbGAI expression was silenced in N. benthamiana using a TRV-based VIGS vector and then inoculated with TRV-PDS or TRV-GUS as controls. By 7 dpi, the TRV-PDS-inoculated plants showed photobleaching phenotypes, whereas no obvious symptoms were observed in TRV-GUS-inoculated plants ( Fig  8A). As expected, the TRV-NbGAI-inoculated plants resulted in excessive growth compared to TRV-GUS-inoculated plants, which is consistent with a GA-sensitive growth phenotype (Fig 8A). qRT-PCR analysis revealed that the mean transcription level of NbGAI in the

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation TRV-NbGAI-inoculated plants was down-regulated by approximately 80% compared to that in the TRV-GUS-inoculated control plants (S7A Fig). In the TRV-NbGAI-inoculated plants, the GA content increased significantly (3.01 μg g -1 FW) compared to that in the TRV-GUSinoculated plants (2.14 μg g -1 FW) (S7B Fig). At 14 dpi, the mean height of the TRV-NbGAIinoculated plants was significantly greater than that of the TRV-GUS-inoculated plants (S7C and S8 Figs). Furthermore, the NbGAI-silenced plants showed a significantly earlier flowering than that of the control plants (S7D and S8 Figs). Then, the effect of NbGAI on ALCScV accumulation and symptom induction was investigated. By 14 dpi, the TRV-GUS-and ALCScVinoculated N. benthamiana plants exhibited severe leaf curling and plant dwarfing symptoms, whereas only mild leaf curling symptoms were observed on the TRV-NbGAI-and ALCScVinoculated N. benthamiana plants (Fig 8B). The qRT-PCR results showed that, compared to the control plants, the accumulation level of ALCScV DNA was significantly reduced in the NbGAI-silenced plants (Fig 8C). Similarly, silencing NbGAI can alleviate the ALCScV-induced dwarfing symptoms and promote early flowering (S9 Fig). Taken together, these results indicate that NbGAI plays important roles in viral accumulation and disease symptom development in infected plants.

Application of exogenous GA 3 enhances N. benthamiana resistance to ALCScV infection
To investigate how GA 3 affects ALCScV infection in plants, N. benthamiana plants were inoculated with ALCScV at 12 hours post 50 μM GA 3 or 0.8% ethanol treatment (control treatment). By 14 dpi, the control plants showed severe leaf curling and dwarfing symptoms, while the GA 3 -treated and ALCScV-inoculated plants exhibited only mild leaf curling symptoms (Fig 9A). To evaluate ALCScV accumulation in these plants, the systemic leaf tissues were

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation harvested at 7, 14, and 21 dpi and analyzed individually through qPCR. The results showed that the accumulation level of ALCScV DNA in the GA 3 -treated and ALCScV-inoculated plants was significantly reduced compared with that in the control plants (Fig 9B). In addition, ALCScV-induced dwarfing symptoms and delayed flowering were significantly reduced by the application of exogenous GA 3 (S10 Fig).

NbGAI can also inhibit the infection of tobacco curly shoot virus (TbCSV) infection in N. benthamiana
To further examine whether other begomovirus-encoded C4s could also interact with NbGAI, TbCSV, also a member of the genus Begomovirus, was selected as the test virus. Y2H and BiFC assays demonstrated that TbCSV-encoded C4 also interacted with NbGAI and NbGAI-M2 (Figs 10A, 10B and S11). To investigate the effect of NbGAI on TbCSV infection, the NbGAIsilenced (TRV-NbGAI) or non-silenced (TRV-GUS) N. benthamiana plants were inoculated with TbCSV. The results showed that the disease symptoms as well as the TbCSV DNA levels in the NbGAI-silenced N. benthamiana plants were clearly suppressed compared to those in the non-silenced control plants (Fig 10C and 10D). Similarly, TbCSV C4 could also inhibit the degradation of NbGAI (S12 Fig). Consequently, we propose that begomovirus-encoded C4 proteins are inhibitors of NbGAI degradation and that NbGAI is an important regulator of begomovirus infection and disease symptom development in plants.

Discussion
As the smallest protein of ALCScV, C4 plays an important role in disease symptom development [38]. To elucidate the molecular mechanism underlying C4-dependent pathogenesis, we

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation analyzed the function of ALCScV C4 in virus infection and symptom development in N. benthamiana and found that ALCScV C4 is a regulator of the GA signaling pathway. Further analyses demonstrated that C4 could directly interact with the negative regulator NbGAI of the GA signaling pathway in vitro and in vivo, and this interaction prevents the degradation of NbGAI, resulting in a block of the GA signaling pathway, causing infected plants to exhibit typical dwarfing and delayed flowering (Fig 11). Meanwhile, the similar role of the C4 protein encoded by tobacco curly shoot virus (TbCSV), another species of Begomovirus [42], was also revealed in this study. It is speculated that the C4-mediated induction of delayed flowering and plant dwarfing via the suppression of the GA signaling pathway are probably common mechanisms among all begomoviruses.
Virus infection in plants causes significant changes in physiological processes, leading to the onset of various disease symptoms [43]. Numerous reports have indicated that GA plays important roles in plant growth and flower development, and plants defective in GA production often show severe plant dwarfing and the development of abnormal flowering [22]. For example, the infection of barley yellow dwarf virus (BYDV) in barley causes a significant reduction in endogenous GA content, leading to plant dwarfing symptoms [44]. Similarly, rice plants infected with rice tungro spherical virus (RTSV) produce significantly less GA, which is considered the main reason for plant dwarfing [45]. Rice dwarf virus (RDV) P2 protein interacted with rice kauri oxidase to suppress GA biosynthesis, which caused rice dwarfing symptoms. In contrast, treatment of rice plants with exogenous GA alleviated rice dwarfing symptoms [39]. In this study, we found that ALCScV infection also causes dwarfing symptoms and delayed flowering, and the infected plants produced significantly less GA. Meanwhile, the ALCScV-induced dwarfing symptoms and delayed flowering were significantly reduced by the application of exogenous GA 3 , suggesting a direct correlation between dwarfing symptoms and delayed flowering with a reduced endogenous GA content.
DELLA is a key negative regulator of the GA signaling pathway [28]. GA-mediated plant responses are usually regulated by the SCF SLY1/GID2 complex, which senses the GA signal and recruits DELLA followed by ubiquitination and then degradation of DELLA through the ubiquitin-26S proteasome, to activate the GA pathway [22,46]. Sequences of DELLA proteins from different plant species are relatively conserved, suggesting that their functions are similar [47,48]. DELLA proteins contain two conserved domains. The C-terminal GRAS domain is important for DELLA stability [49]. In this study, we demonstrated that ALCScV C4 can

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation directly interact with NbGAI, which interferes with the interaction between NbGAI and NbGID2, leading to the prevention of NbGAI degradation and suppression of the GA signaling pathway. Previous studies have demonstrated that to achieve infection in plants, viruses have evolved multiple mechanisms to defeat the plant defense machinery. For example, the RDV P2 protein hijacks OsIAA10 to block the interaction between OsIAA10 and OsTIR1, thereby preventing ubiquitin-26S proteasome-mediated OsIAA10 degradation [50]. Cotton leaf curl Multan betasatellite-encoded βC1 has been proven to interact with NbSKP1, which interferes with the interaction between SKP1 and CUL1 and the function of the SCF complex, resulting in the prevention of GAI degradation and repression of the plant response to GA [51]. Rice black-streaked dwarf virus (RBSDV)-encoded P5-1 can regulate the ubiquitination activity of SCF E3 ligases and inhibit the jasmonate signaling pathway to benefit its infection in rice [52]. Turnip mosaic virus (TuMV) P1 protein interacts with cpSRP54 and mediates its degradation via the 26S proteosome and autophagy pathways, thereby inhibiting the cpSRP54-facilitated delivery of AOCs to the thylakoid membrane and manipulation of JAmediated defense [53]. Tobacco mosaic virus (TMV) replication-associated protein has been demonstrated to affect the localization and stability of auxin/indole-3-acetic acid (IAA) proteins in Arabidopsis to promote its infection [54,55]. The interaction of cucumber mosaic virus (CMV) 2b protein with JAZ can prevent the recruitment of JAZ by COI1 and the degradation of JAZ, resulting in inhibition of the JA pathway [56]. DELLA not only plays a role directly in the GA signaling pathways but also in other hormone signal transduction pathways in plants. Whether the C4 protein impacts other hormone pathways by inhibiting the degradation of the DELLA protein remains to be further studied.
Previous studies have reported that TbCSV infection can cause a certain degree of dwarf symptoms in plants [6]. In this study, we also found that the TbCSV C4 protein interacts with the NbGAI protein and inhibits its protein degradation, which probably explains why the presence of TbCSV causes a dwarf plant phenotype.
Phytohormones are not only significant signaling molecules that regulate plant growth and development but they also regulate plant abiotic and biotic stress responses [57,58]. It was reported that auxin signaling plays a positive role in rice defense against RBSDV infection [59]. Application of methyl jasmonate (MeJA) or brassinazole (BRZ) to rice leaves causes significant reductions in RBSDV incidence and virus accumulation [60]. In this study, we found that silencing NbGAI expression or applying of exogenous GA 3 greatly enhanced N. benthamiana resistance to ALCScV infection. Crosstalk between plant hormones is the core of the plant stress response [61]. GA does not work independently but acts in a complex signaling network combined with other plant hormone signaling pathways. For example, application of exogenous GA to Arabidopsis plants can increase SA biosynthesis as well as the expression of the NPR1 gene to enhance plant resistance to abiotic stresses [62]. Navarro et al., found that pretreatment with exogenous GA promotes the resistance of Arabidopsis to Pto DC3000 by degrading DELLAs and thus changing the balance of SA/JA signaling [63]. It has been shown that DELLAs can interact with BZR1, leading to inhibition of the DNA-binding ability of BZR1, and GA-induced DELLA degradation enhances BR signaling [64,65]. BR has been shown to play a key role in coping with viral infections. For example, BL (the most active BR) treatment can increase systemic resistance to TMV through the production of reactive oxygen species (ROS) in N. benthamiana [66]. Exogenous application of BR has been shown to alleviate tomato yellow leaf curl virus (TYLCV)-induced symptoms in tomato and BCTV C4-induced developmental abnormalities in Arabidopsis [67,68]. In addition, GAs and DEL-LAs contribute to the fine-tuning of ROS production; among them, GA can induce oxidative stress in plants, lead to programmed cell death, and contribute to enhanced disease resistance

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation [69]. Therefore, we speculate that GAs influence viral DNA accumulation through crosstalk with other plant endogenous hormones (such as SA, JA, and BR) or regulate the ROS balance.
In summary, the results presented here reveal that geminivirus-encoded C4 proteins can directly interact with NbGAI, which interrupts the interaction between NbGAI and NbGID2, leading to inhibition of NbGAI degradation and blockage of the GA signaling pathway; therefore, the infected plants display symptoms of typical dwarfing and delayed flowering. This is a novel mechanism by which geminivirus C4 proteins influence viral pathogenicity by interfering with the GA signaling pathway.

Plant growth and virus inoculation
N. benthamiana plants were grown inside a growth chamber set at 26˚C and 16 h/8 h (light/ dark) photoperiod. ageratum leaf curl Sichuan virus (ALCScV), ALCScV-mC4 (e.g., ALCScV lacks the C4 gene), potato virus X (PVX) and PVX-C4 (PVX with an inserted ALCScV C4 gene) were from a previously published source and maintained inside the laboratory [37]. These viruses were inoculated to the leaves of 4-6-leaf-old N. benthamiana plants though agroinfiltration method.
To silence NbGAI expression in N. benthamiana, a fragment of NbGAI sequence 437 bp was amplified and cloned into the pTRV2 vector (kindly provided by Professor Fei Yan, Ningbo University, Zhejiang, China) to generate pTRV2-NbGAI. Plasmid pTRV1, pTRV2-GUS, and pTRV2-PDS were also from the same source.

Yeast two-hybrid assay
The recombinant plasmids were co-transformed into Saccharomyces cerevisiae strain AH109 cells as indicated in the legends of Figs 2, 3 and 4 using the lithium acetate method. The transformed cells were grown on the SD/-Trp-Leu medium. After 2-3 days, positive colonies were selected and cultured on the SD/-Leu-His-Trp-Ade medium plate after being diluted to the concentration of 10 −1 , 10 −2 , 10 −3 , and 10 −4 , respectively.

Co-IP assay
For Co-IP assays, mixed agrobacterium cultures carrying plasmid pC4-His and pNbGAI-GFP, or pC4-His and pGFP were infiltrated into N. benthamiana leaves. At 2 dpi, the infiltrated leaves were harvested for total protein extraction in an extraction buffer (50 mM Tris-HCl, pH 8.0, 0.5 M sucrose, 1 mM MgCl 2 , 10 mM EDTA, 5 mM dithionthreitol). The resulting protein samples were individually incubated with anti-GFP agarose beads (Sigma) for 4 h at 4˚C followed by five rinses in an ice-cold PBS buffer. The input and co-immunoprecipitated protein samples were analyzed through Western blot assays using an anti-His or an anti-GFP antibody.

In vitro competitive pull-down assay
For competitive pull-down assay in vitro, C4-His, NbGAI-GFP, and NbGID2-Flag fusion proteins were purified by in vitro translation system (The Wheat Germ Extract Systems), respectively. The purified NbGAI-GFP (4 μg) was mixed with 0, 2, 4, or 8 μg purified C4-His, and then incubated with 4 μg immobilized NbGID2-Flag for 2 h at 4˚C. After centrifugation and several rinses, the bead-bound proteins and the proteins in the supernatants from different treatments were analyzed through Western blot assays using an anti-Flag, anti-His, or an anti-GFP antibody.

VIGS and transient gene expression assays
For VIGS, plasmid pTRV1, pTRV2, pTRV2-NbGAI, pTRV2-GUS, and pTRV2-PDS were individually transformed into A. tumefaciens strain EHA105 cells. After overnight growth in the liquid LB medium, the cultures were pelleted individually, resuspended in an induction buffer followed by 3 h incubation at room temperature. The agrobacterium culture harboring pTRV1 was diluted to OD 600 = 1.0 and mixed with an equal volume of diluted agrobacterium culture harboring pTRV2 (refers to as TRV), pTRV2-NbGAI (TRV-NbGAI), pTRV2-GUS (TRV-GUS) or pTRV2-PDS (TRV-PDS). The mixed agrobacterium cultures were individually infiltrated into the leaves of N. benthamiana plants at the 3-5-leaf-stage using needless syringes. The plants inoculated with TRV-GUS or TRV-PDS were used as the controls. At 7 dpi, systemic leaves of the assayed plants were harvested for total RNA isolation followed by qRT-PCR analyses. The expression level of NbActin in individually tissue samples was used as the internal control. This experiment was repeated three times.
For transient gene expression assays, agrobacterium cultures harboring specific expression vectors were individually grown, pelleted and induced as described above. Each agrobacterium culture was diluted to OD 600 = 1.0 before infiltration into leaves of N. benthamiana plants at 4-6-leaf-stage. This experiment was repeated three times with 15 plants per treatment.

Hormone level analysis
Leaves of the TRV-GUS-and TRV-NbGAI-inoculated N. benthamiana plants were collected at 7 dpi, and the leaves of the mock-, ALCScV-, ALCScV-mC4-, PVX-, and PVX-C4-inoculated N. benthamiana plants were collected at 14 dpi. The harvested leaf tissues were weighed, stored at -80˚C, and analyzed for endogenous hormone contents by Suzhou Grace Biotechnolgy Co., Ltd through high performance liquid chromatography (HPLC).

GA 3 treatment and ALCScV inoculation
To test the effect of GA 3 on ALCScV infection, N. benthamiana plants at the 4-6-leaf stage were sprayed with a solution containing 50 μM GA 3 or 0.8% ethanol (mock treatment). After 12 h, the sprayed plants were inoculated with ALCScV through agroinfiltration method. The inoculated plants were grown inside the growth chamber and sprayed with the 50 μM GA 3 solution or the 0.8% ethanol solution once every other day till 7 dpi. Leaves of these plants were harvested and analyzed for ALCScV accumulation through qPCR assays.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the harvested leaf tissues using the RNAiso Plus reagent (TaKaRa, Dalian, China). Complementary DNA was synthesized using the RT reagent Kit supplemented with a gDNA Eraser (TaKaRa, Dalian, China) and random primers. Quantitative PCR was conducted using the NovoStart SYBR qPCR SuperMix Plus kit (Novoprotein, Shanghai, China). Relative gene expression levels were calculated using the 2 -ΔΔCt method [70]. The expression level of NbActin gene was used as an internal control during the assays. For each treatment, three technical replicates were used and each treatment was repeated three times. The final result was presented as the mean of three experiments.

DNA extraction and viral DNA accumulation analysis
Total DNA was extracted using the CTAB method. The relative accumulation levels of viral DNA were measured using the qPCR assays as described previously [37].

Western blot analysis
Total protein was extracted from leaf samples in a lysis buffer (Biotime, Shanghai, China), separated in 12% sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gels through electrophoresis, and transferred to PVDF membranes. The membranes were probed individually with a specific rabbit antibody followed by a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody. The detection signal was visualized through incubation of the membranes in an ECL solution.

PLOS PATHOGENS
Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation S1