New Results

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

Pengbai Li, Liuming Guo, Xinyuan Lang, Mingjun Li, Gentu Wu, Rui Wu, Lvxin Wang, Meisheng Zhao, Ling Qing
doi: https://doi.org/10.1101/2021.12.20.473426

Abstract

Phytohormone gibberellin (GA) is an important 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 the GA signaling. Here, we report that ageratum leaf curl Sichuan virus (ALCScV)-infected N. benthamiana plants showed dwarfing symptom and abnormal flower development. The infection of ALCScV alters the expressions of GA pathway-related genes and decreases the content of endogenous GA significantly in N. benthamiana. Further, 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 the 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 proved by the experiments with C4 protein encoded by tobacco curly shoot virus (TbCSV). Therefore, we propose a novel mechanism of geminivirus C4 proteins controling virus infection and disease symptom development through interfering GA signaling pathway.

Author Summary Gibberellins (GAs) are plant hormones that are essential for many developmental processes in plants. It has indicated that plant virus infection can induce abnormal flower development and influence GA pathway resulting the plant dwarfing symptom, but the underlying mechanisms is still not well described. Here, we demonstrate that geminivirus - encoded C4 protein regulates the GA signaling pathway to promote viral accumulation and disease symptom development. Through directly interacting with NbGAI, the C4 protein interferes with the interaction between NbGAI and NbGID2, which inhibits the degradation of NbGAI. As a result, the GA signaling pathway is blocked, and the infected plants display symptoms of typical dwarfing and delayed flowering. Our results reveal a novel mechanism by which geminivirus C4 proteins influence viral pathogenicity via interfering the GA signaling pathway, and provide new insights into the interaction between virus and host.

Introduction

Geminiviruses are a class of DNA viruses with circular single-stranded genomes and encode limited numbers proteins [1-4]. Geminiviruses are known to be capable of recruiting host factors to ensure their genome replications, virion assembly, movement, insect transmission, and other processes [5]. Plants infected with geminiviruses often develop disease symptoms such as yellow vein, leaf curling, enation, and curly shoots [4,6-8]. During virus-plant arms races, host plants have evolved specific proteins that can target various viral infection steps. On the other hand, viruses, including geminiviruses, have evolved specific strategies to hijack specific host factors to interfere host defense responses [9].

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 [10,11]. To date, multiple C4 proteins have been reported as disease symptom determinates that can disrupt host cell development to cause symptom formation [12-14]. Mutational analyses of C4/AC4 proteins have shown that the mutations can alleviate disease symptoms and suppress virus accumulation in infected plants [15]. It has shown that overexpression of C4/AC4 in plants induces hyperplasia, callus-like tissues, and curly leaves, due to the changes of cell cycle-related gene expressions [13,16]. It was reported that C4 protein encoded by tomato leaf curl Yunnan virus (TLCYnV) can interact with NbSKη to reduce its nuclear accumulation. The cytoplasmic NbSKη is less phosphorylated and has a reduced ability to degrade cell cycle regulator NbCycD1;1 [17]. sweet potato leaf curl virus (SPLCV) was reported to interact with brassinosteroid-insensitive 2 (AtBIN2) to re-locate AtBIN2-interacting proteins, AtBES1/AtBZR1, into nucleus to induce abnormal plant development, via the activation of the BR signaling pathway [18]. Recently, C4 has been proved 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 to spread RNAi signal to adjacent cells [19]. cotton leaf curl Multan virus (CLCuMuV) C4 can interact with S-adenosyl methionine synthetase (SAMS) to inhibit its ability to suppress transcriptional and post-transcriptional gene silencing, resulting in a stronger CLCuMuV infection [20]. Geminivirus C4 can also regulate virus systemic infection in plants. For example, 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 plays crucial roles in plant growth and development, and defense responses against abiotic and biotic stresses [22-24]. For example, 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 application of exogenous GA [25]. In contrast, mutant plants that produce constitutively activate GA showed elongated stems than the wild-type plants. The phenotype of this plant is, however, correlated with the content of GA. 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 an inhibitor of plant growth and development, which is considered to function at the nexus in the GA signaling pathway to negatively regulate GA biosynthesis [27,28]. Like other GRAS family proteins, DELLA has a conserved C-terminal GRAS domain, necessary for the protein-protein interaction 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 semi-dominant 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 different, but redundant, functions in the inhibition of GA-dependent responses [30]. Whereas, 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 F-box 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. The DELLA is then recognized by the F-box protein in the SCFSLY1/GID2 complex and then degraded by the ubiquitin-26S proteasome, leading to the removal of the repressive effect of DELLA [32-36]. For those plants that lack of functional F-box proteins, large amount of DELLA will accumulate in cells, leading to an abnormal plant growth and development [37].

Ageratum leaf curl Sichuan virus (ALCScV) is a member in the genus Begomovirus, family Geminiviridae. ALCScV was first reported in Ageratum conyzoides in the Sichuan Province, China in 2018, and can cause severe symptoms on multiple plants [8,38]. Although ALCScV C4 has been discovered as a disease symptom determinant, the molecular mechanism underpinning this action is unclear. In this study, we have now determined that upon ALCScV infection, the expressions of GA pathway-related genes as well as the content of endogenous GA were significantly affected. Further analyses have revealed that ALCScV C4 can 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 GA3 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 function of geminivirus C4 proteins is to interfere GA signaling pathway to promote virus infection and disease symptom development, a mechanism that has not been reported previously.

Results

ALCScV infection affects the expressions of GA pathway-associated genes and biosynthesis of endogenous GA

The ALCScV-infected N. benthamiana plants showed strong dwarfing and abnormal flower development, which were not observed in the mock-inoculated plants or the plants inoculated with ALCScV-mC4 (Fig 1A). The result of statistical analysis supported the symptom observations (S1 Fig). Because previous studies have demonstrated that GA can regulate plant height and flower development [39], therefore, the effect of ALCScV infection on GA biosynthesis and the expressions 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 display 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.

Fig 1.
Fig 1. ALCScV infection influences gibberellin (GA) pathway in N. benthamiana plants.

(A) Photographs of the mock-, ALCScV- and ALCScV-mC4-inoculated N. benthamiana plants, taken at 28 dpi. (B) qRT-PCR analyses of relative expressions of the GA pathway-associated genes in the mock- or ALCScV-inoculated N. benthamiana plants. ** indicates a significant difference between the two treatments at the P < 0.01 level, determined by the Student’s t-test; * indicates a significant difference between the two treatments at the P < 0.05 level. This experiment had at least three plants per treatment. (C) and (D) Results of HPLC analyses showing the contents of endogenous GA in the PVX-, PVX-C4-, mock-, ALCScV-, and ALCScV-mC4-inoculated N. benthamiana plants at 14 dpi, respectively. Different letters above the bars indicate the significant differences (p < 0.05).

ALCScV C4 directly interact with NbGAI

To understand how ALCScV C4 regulates the GA pathway, Y2H assays was performed to screen host protein(s) that can interact with ALCScV C4. The result showed that a GA signaling pathway repressor, NbGAI, interacted with the C4 in 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 assay were performed, co-expressed of cYFP-C4 and nYFP-NbGAI resulted in a YFP fluorescence signal in both 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 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 NbGAI protein both in vitro and in vivo.

Fig 2.
Fig 2. ALCScV C4 interacts with NbGAI in vitro and in vivo.

(A) Result of Y2H assay showing the interaction between ALCScV C4 and NbGAI in yeast cells. Recombinant plasmids were co-transformed, in various combinations, into S. cerevisiae strain AH109 cells using the lithium acetate method. The transformants were 10-fold serially diluted and then grown on the SD/-Trp/-Leu or the SD/-Trp/-Leu/-His/-Ade selective medium. Images were taken at 72 hpi. (B) Results of BiFC assay showing 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 an FITC filter. Bars = 50 μm. (C) Result of Co-IP assay showing 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 co-immunoprecipitated proteins were analyzed through Western blot analyses using an anti-GFP or an anti-His antibody.

The central region of NbGAI is responsible for the interaction with C4

The coding sequence of NbGAI contains 1701 nucleotides, and is predicted to encode a hydrophilic protein with 567 amino acids and of a molecular mass of about 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 prediction (Fig 3A). The Y2H assays showed that NbGAI-M2 (aa residue 98–566), but not the other four fragments, interacted with C4 (Fig 3B). This interaction was also confirmed in planta through BiFC assays (Fig 3C). These results suggest that the 98-566 amino acid positions of NbGAI is responsible for the interaction with ALCScV C4.

Fig 3.
Fig 3. The middle region of NbGAI that is responsible for the interaction with ALCScV C4.

(A) Schematic representations of NbGAI constructs. The deletion mutants were constructed based on the predictions of the conserved domains in NbGAI. (B) Result of Y2H assay showing the positive interaction between the C4 and NbGAI or C4 and NbGAI-M2. (C) Result of BiFC assay showing the interaction between the NbGAI-M2 and C4 in planta. Procedures of the Y2H and BiFC assays are similar to those described in Figure 2.

The interaction between NbGAI and NbGID2 causes the degradation of NbGAI via the ubiquitin-26S proteasome

Previous studies have shown that GAI and GID2 interaction results in the degradation of GAI and activation of GA signaling pathway [22]. In this study, the interaction between NbGAI and NbGID2 were analyzed via Y2H and BiFC assays. The result demonstrated that NbGAI did interact with NbGID2 in yeast cells (Fig 4A) and in N. benthamiana leaf cells (Fig 4B).

Fig 4.
Fig 4. The interaction between NbGAI and NbGID2 enhances the ubiquitin-26S proteasome-dependent degradation of NbGAI.

(A) Result of Y2H assay showing the interaction between NbGAI and NbGID2. (B) Result of BiFC assay showing the interaction between NbGAI and NbGID2 in N. benthamiana leaf cells. Leaves co-expressing cYFP-NbGAI and nYFP, nYFP-NbGID2 and cYFP or cYFP-NbGAI and nYFP-NbGID2 were examined and imaged under a confocal microscope at 48 hpi. (C) Degradation of NbGAI is ubiquitin-26S proteasome-dependent. Fusion cYFP-NbGAI and nYFP-NbGID2 were transiently co-expressed in N. benthamiana leaves. The infiltrated leaves were sprayed with a solution containing 50 μM MG132 at 12 h before Confocal Microscopy or with a solution containing 50 μM GA3 or 0.8% ethanol (control) at 2 h before Confocal Microscopy. (D) Fluorescence intensities from the BiFC assays were quantified based on the numbers of nuclei with GFP fluorescence. Different letters above the bars indicate the significant differences among the treatments at the P < 0.05 level. This experiment had at least three plants per treatment. (E) Result of Y2H assay showing the positive interaction between NbGAI-M2 and NbGID2. (F) Result of BiFC assay showing the positive interaction between NbGAI-M2 and NbGID2 in planta. Procedures of the Y2H and BiFC assays are similar to those described in Figure 2.

Because treatment of plants with exogenous GA3 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 was 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 result showed that 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 Fig 4D). In contrast, the YFP fluorescence intensity in the GA3-treated leaves was significantly decreased compared to the mock-treated leaves (Fig 4C and Fig 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 acid 98-566, is responsible for the interaction between NbGAI and NbGID2 (Fig 4E and Fig 4F).

ALCScV C4 interferes 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 (S3 Fig), we speculated that ALCScV C4 and NbGID2 may be competitively combined 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 Fig 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 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.

Fig 5.
Fig 5. ALCScV C4 can inhibit the interaction between NbGAI and NbGID2.

(A) Result of competitive BiFC assay showing the inhibitory effect of ALCScV C4 on the interaction between NbGAI and NbGID2. Fusion cYFP-NbGID2, nYFP-NbGAI and C4-His or cYFP-NbGID2, nYFP-NbGAI and empty vector were co-expressed in N. benthamiana leaves through agroinfiltration. The infiltrated leaves were harvested at 48 hpi and examined under the confocal microscope. (B) Result of statistical analyses showing the number of nuclei with YFP fluorescence per examined area. ** indicates the significant difference between the two treatments at the P < 0.01 level, determined using the Student’s t-test. This experiment had at least three plants per treatment. (C) Result of Western blot analysis showing the expression level of ALCScV C4. After electrophoresis, the blot was probed with an anti-His antibody. The coomassie brilliant blue (CBB)-stained RuBisCo large subunit gel was used to showing sample loadings. (D) Result of competitive pull-down assay showing the interaction between NbGAI-GFP and NbGID2-Flag in vitro. In this assay, the purified NbGAI-GFP (4 μg) was mixed with 0, 2, 4, or 8 μg purified C4-His, respectively, and then incubated with 4 μg immobilized NbGID2-Flag.

ALCScV C4 can inhibit NbGAI degradation

It is known that 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, the green fluorescence from NbGAI-GFP was found predominantly in nuclei, with small amount of fluorescence in the cytoplasm at 48 hpi. Meanwhile, NbGAI-GFP fusion protein was confirmed by the Western blot experiment (Fig S4). Then, NbGAI-GFP was co-expressed with C4-His or the empty vector in N. benthamiana leaf cells, the stronger green fluorescence was observed in the leaves co-expressing NbGAI-GFP and C4-His compared to 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 GA3 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-infiltration with NbGAI-GFP and pCV-C4-His compared to co-infiltration with NbGAI-GFP and pCV empty vector. These results proved that 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 C4 protein does not affect the stability of the GFP protein (Fig 6E - 6H). Taken together, these results suggest that that the ALCScV C4 protein inhibited the degradation of NbGAI.

Fig 6.
Fig 6. ALCScV C4 can inhibit NbGAI degradation in vivo.

(A) NbGAI-GFP was transiently co-expressed with empty vector or C4-His in N. benthamiana leaves. GA3 (50 μM) and MG132 (50 μM) were applied into the infiltrated leaves 2 h and 12 h before observation, respectively. At 48 hpi, the assayed leaves were harvested and examined under the confocal microscope. (B) Result of Western blot analysis showing the relative accumulation level of NbGAI-GFP. The blots were probed with an anti-GFP or an anti-His antibody. The CBB-stained RuBisCo large subunit gel was used to showing sample loadings. (C) and (D) Results of qRT-PCR and semi-qRT-PCR analyses showing the relative expression level of NbGAI. The expression level of NbActin was used as an internal control. The result of qRT-PCR was analyzed using the 2-ΔΔCt method. (E) Result showing that ALCScV C4 does not affect stability of GFP in vivo. (F) Result of Western blot analysis showing the relative accumulation level of GFP in the mock-, GA3- or MG132-treated plant leaves. (G) and (H) Results of qRT-PCR and semi-qRT-PCR showing the relative expression level of GFP gene. ns indicates not significant according to Student’s t-test.

To further confirm the ALCScV C4-mediated inhibition of NbGAI degradation, agrobacterium culture carrying pNbGAI-GFP was co-infiltrated with the 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 dosage dependent (Fig 7A). As expected, C4-His had no clear effect on the expression of GFP (Fig 7B).

Fig 7.
Fig 7. Inhibition of NbGAI degradation is C4 concentration-dependent.

(A) Agrobacterium culture carrying pGAI-GFP and agrobacterium culture expressing C4-His (diluted to OD600 = 0, 0.25, 0.5 and 1.0, respectively) were co-infiltrated into N. benthamiana leaves. Two days later, total protein was extracted from different tissue samples and analyzed through Western blot analyses using an anti-GFP or an anti-His antibody. The CBB-stained RuBisCo large subunit gel was used to show sample loadings. (B) Result of Western blot analysis showing that ALCScV C4 does not affect the stability of GFP in vivo.

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, this is consistent with a GA-sensitive growth phenotype. qRT-PCR analysis revealed that the mean transcription level of NbGAI in the TRV-NbGAI-inoculated plants was down-regulated by about 80% compared to that in the TRV-GUS-inoculated control plants (Fig 8B). In the TRV-NbGAI-inoculated plants, the GA content increased significantly (3.01 μg g-1 FW) compared to that in the TRV-GUS-inoculated plants (2.14 μg g-1 FW) (Fig 8C). At 14 dpi, the mean height of the TRV-NbGAI-inoculated plants was significantly greater than the TRV-GUS-inoculated plants (Fig 8D). Furthermore, the NbGAI-silenced plants showed a significantly earlier flowering than that of the control plants (Fig 8E and S5). Then, the effect of NbGAI on ALCScV accumulation and symptom induction were investigated. By 14 dpi, the TRV-GUS and ALCScV inoculated N. benthamiana plants exhibited severe leaf curling and plant dwarfing symptoms, whereas only mild leaf curling symptoms were observed on the TRV-NbGAI and ALCScV inoculated N. benthamiana plants (Fig 8F). The result of qRT-PCR showed that, compared to that in the control plants, the accumulation level of ALCScV DNA was significantly reduced in the NbGAI-silenced plants (Fig 8G). Similarly, silencing NbGAI can alleviate the ALCScV-induced dwarfing symptom and promote early flowering (Fig S6). Taken together, these results indicate that NbGAI plays important roles in the viral accumulation and disease symptoms development in the infected plants.

Fig 8.
Fig 8. Silencing of NbGAI expression in N. benthamiana plants through VIGS inhibits ALCScV infection.

(A) Symptoms shown by the N. benthamiana plants inoculated with TRV-PDS, TRV-GUS or TRV-NbGAI at 7 dpi. (B) Result of qRT-PCR analysis showing the relative expression level of NbGAI in the TRV-GUS- or TRV-NbGAI-inoculated N. benthamiana plants at 7 dpi. The relative expression level of NbActin was used as the internal control. Each treatment had three biological replicates with three technical replicates each. (C) Result of HPLC analysis showing the GA content in the TRV-GUS- and TRV-NbGAI-inoculated N. benthamiana plants, respectively, at 7 dpi. (D) Measurement of the height of the TRV-GUS- or TRV-NbGAI-inoculated N. benthamiana plants at 14 dpi. ** indicates a significant difference between the two treatments at the P < 0.01 level, determined by the Student’s t-test. (E) Statistical analysis of flowering time of the TRV-GUS- or TRV-NbGAI-inoculated N. benthamiana plants. (F) The TRV-GUS- and ALCScV-inoculated N. benthamiana plants showing stronger symptoms of leaf curling and plant dwarfing than the TRV-NbGAI- and ALCScV-inoculated N. benthamiana plants at 14 dpi. (G) Result of qPCR analysis showing that the accumulation of ALCScV DNA in the TRV-NbGAI- and ALCScV-inoculated plants was significantly reduced compared to the TRV-GUS- and ALCScV-inoculated plants. * indicates a significant difference between the two treatments at the P < 0.05 level. This experiment had at least three plants.

Application of exogenous GA3 enhances N. benthamiana resistance to ALCScV infection

To investigate how GA3 affects ALCScV infection in plant, N. benthamiana plants was inoculated with ALCScV at 12 hours post 50 μM GA3 or 0.8 % ethanol treatment (control treatment). By 14 dpi, the control plants showed severe leaf curling and dwarfing symptoms, while the GA3-treated and ALCScV-inoculated plants exhibited only mild leaf curling symptoms (Fig 9A). To determine ALCScV accumulation in these plants, the systemic leaf tissues was harvested at 7, 14, and 21 dpi, and analyzed individually through qPCR. The result showed that the accumulation level of ALCScV DNA in the GA3-treated and ALCScV-inoculated plants was significantly reduced than that in the control plants (Fig 9B). In addition, the ALCScV-induced dwarfing symptom and delayed flowering were significantly reduced by the application of exogenous GA3 (Fig S7).

Fig 9.
Fig 9. Application of exogenous GA3 enhances N. benthamiana plant resistance to ALCScV infection.

(A) N. benthamiana plants were treated with exogenous GA3 or 0.8% ethanol (control), and then inoculated with ALCScV. After ALCScV inoculation, the plants were treated again with GA3 or 0.8% ethanol (once every other day). The plants were photographed at 14 dpi. (B) Result of qPCR showing the relative accumulation level of ALCScV DNA in the GA3 or 0.8% ethanol treated plants. ** indicates a significant difference between the two treatment at the P < 0.01 level, determined by the Student’s t-test. This experiment had at least three biological replicates (plants) per treatment.

NbGAI can also inhibits the infection of tobacco curly shoot virus (TbCSV) in N. benthamiana

To further examine whether other begomovirus-encoded C4s can also interact with NbGAI, TbCSV, also a member in the genus Begomovirus, was selected as the test virus. Y2H and BiFC assays demonstrated that the TbCSV-encoded C4 also interacted with NbGAI and NbGAI-M2 (Fig 10A, 10B and S8). To investigate the effect of NbGAI on TbCSV infection, the NbGAI-silenced (TRV-NbGAI) or non-silenced (TRV-GUS) N. benthamiana plants was inoculated with TbCSV. The results showed that the disease symptoms as well as TbCSV DNA level 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 (S9 Fig). Consequently, we propose that the begomovirus-encoded C4 proteins are inhibitors of NbGAI degradation, and NbGAI is an important regulator of begomovirus infection and disease symptom development in plants.

Fig 10.
Fig 10. TbCSV C4 interacts with NbGAI in vitro and in vivo.

(A) Result of Y2H assay showing the interaction between TbCSV C4 and NbGAI in yeast cells. (B) Result of BiFC assay showing the interaction between TbCSV C4 and NbGAI in N. benthamiana leaf cells. The agroinfiltrated leaves were harvested at 48 hpi and examined under the confocal microscope equipped with an FITC filter. Bars = 50 μm. (C) N. benthamiana plants were inoculated with TRV-GUS and TbCSV or TRV-NbGAI and TbCSV. The TRV-GUS and TbCSV-inoculated plants showed severe leaf curling symptom, while the TRV-NbGAI and TbCSV-inoculated N. benthamiana plants showed mild leaf curling symptom at 7 dpi. (D) Result of qPCR analysis showing the accumulation level of TbCSV DNA. ** indicates a significant difference between the two treatments at the P < 0.01 level, determined by the Student’s t-test. This experiment had at least three biological replicates (plants) per treatment.

Discussion

As the smallest protein of ALCScV, C4 plays an important role in disease symptom development [38]. To elucidate the molecular mechanism underlying the C4-dependent pathogenesis, we analyzed the function of ALCScV C4 in virus infection and symptom development in N. benthamiana, and found that ALCScV C4 is a regulator of GA signaling pathway. Further analyses demonstrated that C4 can directly interact with the negative regulator NbGAI of GA signaling pathway in vitro and in vivo, and this interaction prevents the degradation of NbGAI, resulting in the block of GA signaling pathway, and causing infected plants typical dwarfing and delayed flowering (Fig 11). Meanwhile, the similar role of C4 protein encoded by Tobacco curly shoot virus (TbCSV), another species of Begomovirus [42], was revealed in this study. It is speculated that the C4-mediated induction of delayed flowering and plant dwarfing via the suppression of GA signaling pathway are probably common for begomoviruses.

Fig 11.
Fig 11. Proposed model.

In mock N. benthamiana plants, the interaction between NbGAI and NbGID2 leads to the ubiquitination and the degradation of NbGAI by 26S proteasome, releasing the specific transcription factors. Then genes of gibberellin (GA) signaling pathway were adequately regulated by corresponding transcription factors, promoting normal growth of N. benthamiana plants. Under ALCScV infection condition, C4 directly interacts with NbGAI, which interferes the interaction between NbGAI and NbGID2 to prevent the degradation of NbGAI and block the GA signaling pathway, ultimately leading to the symptoms of plant dwarfing and abnormal flower development.

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, plants defective in GA production often show severe plant dwarfing and development of abnormal flowering [22]. For example, the infection of barley yellow dwarf virus (BYDV) in barley causes significant reduction of endogenous GA content, leading to plant dwarfing symptom [44]. Similarly, rice plants infected with rice tungro spherical virus (RTSV) produces significantly less GA, which was considered as the main reason of plant dwarfing [45]. rice dwarf virus (RDV) P2 protein interacted with rice kauri oxidase to suppress GA biosynthesis, which caused rice dwarfing symptom. In contrast, treatment of rice plants with exogenous GA alleviated rice dwarfing symptom [39]. In this study, we found that ALCScV infection also causes dwarfing symptom and delayed flowering, and the infected plants produced significantly less GA. Meanwhile, the ALCScV-induced dwarfing symptom and delayed flowering were significantly reduced by the application of exogenous GA3, suggesting a direct correlation between the dwarfing symptom and delayed flowering with the reduced endogenous GA content.

DELLA is a key negative regulator of GA signaling pathway [28]. The GA-mediated plant responses are usually regulated by the SCFSLY1/GID2 complex, which is known to sense the GA signal and to recruit 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 have determined that the ALCScV C4 can directly interact with NbGAI, which interferes the interaction between NbGAI and NbGID2, leading to the prevention of NbGAI degradation and suppression of GA signaling pathway. Previous studies have demonstrated that to achieve infection in plants, viruses have evolved multiple mechanisms to defeat plant defense machinery. For example, RDV P2 protein hijacks OsIAA10 to block the interaction between OsIAA10 and OsTIR1, thereby prevents the ubiquitin-26S proteasome-mediated OsIAA10 degradation [50]. cotton leaf curl Multan betasatellite-encoded βC1 has been proved to interact with NbSKP1, which interferes the interaction between SKP1 and CUL1 and the function of SCF complex, resulting in the prevention of GAI degradation and repression of 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 interacted with cpSRP54 and mediated its degradation via the 26S proteosome and autophagy pathways, thereby inhibiting the cpSRP54-facilitated delivery of AOCs to the thylakoid membrane and manipulation of JA-mediated 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 an inhibition of the JA pathway [56]. DELLA not only plays role directly in the GA signaling pathways, but also in other hormone signal transduction pathways in plants. Whether C4 protein, through inhibiting the degradation of DELLA protein, impacts other hormone pathways remains to be further studied.

Previous studies have reported that TbCSV infection can also cause plants a certain degree of dwarf symptom [7]. In this study, we also found that the TbCSV C4 protein interacts with the NbGAI protein and inhibits its protein degradation, which probably explained why the presence of TbCSV causes plant dwarf phenotype.

Phytohormones are not only significant signaling molecules that regulate plant growth and development, but also regulate plant abiotic and biotic stress responses [57,58]. For example, application of exogenous GA to Arabidopsis plants can increase SA biosynthesis as well as the expression of NPR1 gene to enhance plant resistance to abiotic stresses [59]. It was reported that auxin signaling plays a positive role in rice defense against RBSDV infection [60]. In addition, application of methyl jasmonate (MeJA) or brassinazole (BRZ) to rice leaves causes significant reductions in RBSDV incidence and virus accumulation [61]. In this study, we found that silencing of NbGAI expression or application of exogenous GA3 greatly enhanced N. benthamiana resistance to ALCScV infection, which further confirms the role of plant hormone in plant anti-virus resistance. However, the molecular mechanism of how GA defense against ALCScV infection is still unknown, and which needs to be investigated next.

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 the inhibition of NbGAI degradation and the block 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 through interfering GA signaling pathway.

Materials and methods

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 [38]. These viruses were inoculated to the leaves of 4-6-leaf-old N. benthamiana plants though agroinfiltration method.

Plasmid construction

For yeast two-hybrid (Y2H) assays, the full-length ALCScV C4 gene sequence was PCR-amplified using gene specific primers (S1 Table), and then cloned into the pGBKT7 vector to produce pBK-C4. The full-length NbGID2, NbGAI, NbGAI deletion derivatives (refer to as NbGAI-M1, NbGAI-M2, NbGAI-M3, NbGAI-M4, and NbGAI-M5, these mutants encode NbGAI amino acid 1-97, 98–566, 191–566, 1–190, and 98–190, respectively) were PCR-amplified and cloned into the pGAD-T7 vector to produce pAD-NbGID2, pAD-NbGAI, pAD-NbGAI-M1, pAD-NbGAI-M2, pAD-NbGAI-M3, pAD-NbGAI-M4, and pAD-NbGAI-M5, respectively.

For bimolecular fluorescence complementation (BiFC) assays, the full length sequence of C4 was amplified using the primer pair cYFP-C4-F/cYFP-C4-R, and then inserted into the pCV-nYFP vector to generate pcYFP-C4. The full-length NbGAI, NbGAI-M2, and NbGID2 sequences were also amplified and inserted individually into the pCV-nYFP vector to generate pnYFP-NbGAI, pnYFP-NbGAI-M2, and pcYFP-NbGID2, respectively.

For Co-immunoprecipitation (Co-IP) assays, DNA fragments of C4-His, GFP, and NbGAI-GFP were amplified, individually, double digested with restriction enzymes, and cloned into the pCV vector to generate pC4-His, pGFP, and pNbGAI-GFP, respectively.

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 Figure 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.

BiFC assay

Plasmid pcYFP-C4 and pnYFP-NbGAI were individually transformed into Agrobacterium tumefaciens strain EHA105 cells, and cultured overnight in a LB medium supplemented with kanamycin (50 μg/mL) and rifampicin (20 μg/mL). The cultures were pelleted, resuspended in an induction buffer (10 mM MgCl2, 100 mM MES, pH 5.7, and 2 mM acetosyringone, in distilled water) till OD600 = 0.8-1.0 followed a 3 h incubation at room temperature. The two agrobacterium cultures were mixed at a 1:1 (v/v) ratio, and then infiltrated into N. benthamiana leaves with needleless syringes. Mixed agrobacterium cultures carrying pcYFP-C4 and pnYFP (empty vector) or pcYFP (empty vector) and pnYFP-NbGAI were served as controls. At 36-48 hpi, the leaves were collected and examined under a confocal microscope (LEICA, Germany) equipped qith an FITC filter.

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 MgCl2, 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 TNT® 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 OD600 =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 OD600 =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).

GA3 treatment and ALCScV inoculation

To test the effect of GA3 on ALCScV infection, N. benthamiana plants at the 4-6-leaf stage were sprayed with a solution containing 50 μM GA3 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 GA3 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.

Protein sequence alignment

Sequences of GAI proteins from Solanum lycopersicum (GenBank No. NP_001234883.1), N. tabacum (GenBank No. BAG68655.1), Ipomoea batatas (GenBank No. XP_031118124.1), Capsicum annuum (GenBank No. XP_016540318.1), Camellia sinensis (GenBank No. ANB66339.1) and A. thaliana (GenBank No. NP_001319041.1) were downloaded from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) in the US. Alignment of these sequences was performed using the Clustal V software in the MEGA7.0 and the phylogenetic tree was constructed using the MEGA7.0 softwear. Conserved domains in these GAI proteins are then identified.

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 PrimeScript™ 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 [62]. 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 [38].

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.

Supporting information

S1 Fig. ALCScV C4 influences plant height and flower development. (A) Photographs of the mock-, ALCScV-, and ALCScV-mC4-inoculated N. benthamiana plants at 28 dpi. (B) Result of statistical analysis showing the height of the mock-, ALCScV-, and ALCScV-mC4-inoculated N. benthamiana plants at 28 dpi. Different letters above the bars indicate significant differences at the P < 0.05 level. (C) Result of statistical analysis showing the flowering time of the mock-, ALCScV-, and ALCScV-mC4-inoculated N. benthamiana plants. (D) Photographs of the PVX- and PVX-C4-infected N. benthamiana plants at 14 dpi. (E) Result of statistical analysis showing the height of the PVX- and PVX-C4-inoculated N. benthamiana plants at 14 dpi. (F) Result of statistical analysis showing the flowering time of the PVX- and PVX-C4-inoculated N. benthamiana plants. This experiment had at least three plants per treatment.

S2 Fig. Sequence alignment and phylogenetic analysis of GAI proteins from different plant species. (A) Analysis of conserved domains in the GAI proteins from different plant species. The selected plant species and gene accession numbers are Solanum lycopersicum (NP_001234365), Capsicum baccatum (PHT30960), Cucurbita maxima (XP_022998067), Ipomoea triloba (XP_031108300), Nicotiana benthamiana (AMO02501), and N. sylvestris (XP_009796771). (B) Phylogenetic tree was constructed using the GAI protein sequences from N. benthamiana (AMO02501), N. attenuata (XP_019232709), N. tomentosiformis (XP_009590329), N. sylvestris (XP_009796771), S. tuberosum (NP_001305514), S. lycopersicum (NP_001234365), C. baccatum (PHT30960), I. triloba (XP_031108300), Sesamum indicum (XP_011099949), Actinidia deliciosa (AHB17746), A. thaliana (CAA75492), Glycine soja (XP_028212758), C. maxima (XP_022998067), Zea mays (NP_001306661), and Oryza sativa (AAR31213). The red triangle indicates the GAI protein of N. benthamiana obtained in this study.

S3 Fig. ALCScV C4 does not interact with NbGID2. The recombinant plasmids were co-transformed, in various combinations, into S. cerevisiae strain AH109 cells using the lithium acetate method. The transformants were 10-fold serially diluted and then grown on the SD/-Trp/-Leu or the SD/-Trp/-Leu/-His/-Ade medium plates for 3 days. The result showing that the C4 does not interact with NbGID2.

S4 Fig. Analysis of NbGAI subcellular localization. (A) Leaf tissues expressing GFP (top image) or NbGAI-GFP (bottom image) area shown in the left column. Images in the left column were captured under the UV light. The images in the middle column were captured under the bright field. The images in the right column are merged images. Scale bar = 50 μm. (B) Result of Western blot analysis shows the expression level of GFP and NbGAI-GFP fusion protein. The blot was probed with an anti-GFP antibody. The CBB-stained RuBisCo large subunit gel was used to show sample loadings.

S5 Fig. Photographs of the TRV-GUS- and TRV-NbGAI-inoculated N. benthamiana plants at 28 dpi. The result showing that silencing NbGAI expression in N. benthamiana plants through VIGS significantly increases plant height, but early flowering.

S6 Fig. Silencing NbGAI can alleviate the ALCScV-induced dwarfing symptom and delayed flowering. (A) Statistical analysis of plant height of mock-inoculated and ALCScV-inoculated N. benthamiana plants at 14 dpi. (B) Statistical analysis of flowering time of mock-inoculated and ALCScV-inoculated N. benthamiana plants.

S7 Fig. Application of exogenous GA3 increases plant height, but early flowering. (A) The mock-or ALCScV-inoculated N. benthamiana plants were treated with GA3 or 0.8% ethanol (control), respectively. The plants were photographed at 14 dpi. (B) Result of statistical analysis showing the height of the mock- and ALCScV-inoculated N. benthamiana plants, respectively, at 14 dpi. (C) Result of statistical analysis showing the flowering time of the mock- and ALCScV-inoculated N. benthamiana plants, respectively.

S8 Fig. NbGAI-M2 is responsible for the interaction with TbCSV C4. (A) Result of Y2H assay showing that NbGAI-M2 is responsible for the interaction with TbCSV C4. (B) Result of BiFC assay agrees with the Y2H assay result.

S9 Fig. TbCSV C4 inhibits the degradation of NbGAI.

S1 Table. Sequence of primers used in this study.

Acknowledgments

We gratitude Professor Fei Yan (Ningbo University, Zhejiang, China) for providing the expression vector TRV and pCV, and Professor Xiaofeng Cui (Shanghai Institutes for Biological Sciences, Shanghai, China) for providing suggestions for research idea, and Professor Xinshun Ding (The Samuel Roberts Noble Foundation, USA) and Mengji Cao (Southwest University, Chongqing, China) for revising this manuscript.

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Posted December 22, 2021.
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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation, to promote viral infection and symptom development in N. benthamiana
Pengbai Li, Liuming Guo, Xinyuan Lang, Mingjun Li, Gentu Wu, Rui Wu, Lvxin Wang, Meisheng Zhao, Ling Qing
bioRxiv 2021.12.20.473426; doi: https://doi.org/10.1101/2021.12.20.473426
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