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Journal of Virology logoLink to Journal of Virology
. 2020 Sep 29;94(20):e01307-20. doi: 10.1128/JVI.01307-20

Divergent Symptoms Caused by Geminivirus-Encoded C4 Proteins Correlate with Their Ability To Bind NbSKη

Yuzhen Mei a,#, Fanfan Zhang a,#, Mingyu Wang a, Fangfang Li b, Yaqin Wang a,✉, Xueping Zhou a,b,✉
Editor: Rozanne M Sandri-Goldinc
PMCID: PMC7527059  PMID: 32759325

Geminiviruses constitute the largest group of known plant viruses and cause devastating diseases in many economically important crops worldwide. Geminivirus-encoded C4 protein is a multifunctional protein. In this study, we found that the C4 proteins from different geminiviruses showed differential abilities to interact with NbSKη, which correlated with their symptom determinant capabilities. Moreover, a minidomain of tomato leaf curl Yunnan virus (TLCYnV) C4 that is indispensable for interacting with NbSKη and tethering it to the plasma membrane, thus leading to symptom induction, was determined. Supporting these findings, a recombinant geminivirus carrying the minidomain of TLCYnV C4 induced more-severe symptoms than the wild type. Therefore, these findings expand the scope of the interaction of NbSKη and C4-mediated symptom induction and thus contribute to further understanding of the multiple roles of C4.

KEYWORDS: C4, geminivirus, NbSKη, binding, symptom, tomato leaf curl Yunnan virus

ABSTRACT

Geminiviruses induce severe developmental abnormalities in plants. The C4/AC4 protein encoded by geminiviruses, especially those not associated with betasatellites, functions as a symptom determinant by hijacking a shaggy-related protein kinase (SKη) and interfering with its functions. Here, we report that the symptom determinant capabilities of C4 proteins encoded by different geminiviruses are divergent and tightly correlated with their abilities to interact with SKη from Nicotiana benthamiana (NbSKη). Swap of the minidomain of tomato leaf curl Yunnan virus (TLCYnV) C4 critical for the interaction with NbSKη increases the capacities of the C4 proteins encoded by tomato yellow leaf curl China virus (TYLCCNV) or tobacco curly shoot virus (TbCSV) to induce symptoms. The severity of symptoms induced by recombinant TYLCCNV C4 or TbCSV C4 correlates with the amount of NbSKη tethered to the plasma membrane by the viral protein. Moreover, a recombinant TYLCCNV harboring the minidomain of TLCYnV C4 induces more-severe symptoms than wild-type TYLCCNV. Thus, this study provides new insights into the mechanism by which different geminivirus-encoded C4 proteins possess divergent symptom determinant capabilities.

IMPORTANCE Geminiviruses constitute the largest group of known plant viruses and cause devastating diseases in many economically important crops worldwide. Geminivirus-encoded C4 protein is a multifunctional protein. In this study, we found that the C4 proteins from different geminiviruses showed differential abilities to interact with NbSKη, which correlated with their symptom determinant capabilities. Moreover, a minidomain of tomato leaf curl Yunnan virus (TLCYnV) C4 that is indispensable for interacting with NbSKη and tethering it to the plasma membrane, thus leading to symptom induction, was determined. Supporting these findings, a recombinant geminivirus carrying the minidomain of TLCYnV C4 induced more-severe symptoms than the wild type. Therefore, these findings expand the scope of the interaction of NbSKη and C4-mediated symptom induction and thus contribute to further understanding of the multiple roles of C4.

INTRODUCTION

Geminiviruses cause devastating diseases in the field and economic losses worldwide. They have single-stranded DNA (ssDNA) genomes encapsidated in twinned particles with quasi-icosahedral morphology (1). The family Geminiviridae is divided into nine genera, Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus, and Turncurtovirus, based on genome organization, host range, and insect vector (2). Begomovirus is the largest genus within the Geminiviridae family. Begomoviruses are divided into two groups based on their genome structures: monopartite begomoviruses, comprising one circular ssDNA molecule, and bipartite begomoviruses, including two ssDNA molecules (3–5). Some monopartite begomoviruses are associated with betasatellites, which have circular ssDNA genomes that are half the size (∼1.3 kb) of those of their helper viruses (6, 7).

Tomato leaf curl Yunnan virus (TLCYnV) is a naturally occurring recombinant monopartite begomovirus originating from Yunnan, China. TLCYnV evolved from two geminivirus species: tomato yellow leaf curl China virus (TYLCCNV) as the major parent and pepper yellow leaf curl China virus (PepYLCCNV) as the donor of the C4 gene and the partial intergenic region. TLCYnV C4 is a symptom determinant causing downward leaf curling and callus-like tissue formation (8). TYLCCNV is a typical monopartite begomovirus associated with a betasatellite, which is required for full symptom induction and encodes the βC1 protein as the symptom determinant. TYLCCNV alone can systemically infect host plants, such as Nicotiana benthamiana, but infected plants are symptomless (9), which suggests that C4 encoded by TYLCCNV does not act as a symptom determinant. Tobacco curly shoot virus (TbCSV) is another monopartite begomovirus associated with a betasatellite. TbCSV alone is able to induce typical geminiviral symptoms, but coinfection with a betasatellite intensifies symptom severity (10). Although the C4 proteins encoded by these geminiviruses seem to play different roles in viral symptom development, the molecular mechanisms underpinning these divergent symptom determinant abilities is poorly understood.

Shaggy-related protein kinase (SKη) is a homologue of mammalian glycogen synthase kinase 3 (GSK3), which plays critical roles in glycogen metabolism (11, 12). The plant SKη, also known as BIN2, is a negative regulator in the brassinosteroid (BR) signal transduction pathway (13). SKη phosphorylates downstream transcription factors, such as brassinazole-resistant 1 (BZR1) (14, 15) and BRI1-EMS suppressor 1 (BES1, also named BZR2) (16, 17), to regulate BR signaling. SKη is a target of geminivirus C4 (18, 19). Our previous study reported that the relocalization of SKη from Nicotiana benthamiana (NbSKη) caused by the interaction between C4 and NbSKη is critical for viral symptom development (19). Here, we found that C4 proteins from different geminiviruses have divergent symptom determinant abilities. We further found that the minidomain in C4 protein essential for the interaction with NbSKη determines its symptom determinant ability and that swap of the minidomain between C4 proteins could alter the symptom determinant abilities of those proteins. Furthermore, we confirmed that the divergent symptom determinant abilities of geminivirus C4 proteins are tightly coupled to their abilities to tether NbSKη to the plasma membrane.

RESULTS

The divergent symptom determinant abilities of different geminivirus C4 proteins correlate with their capacities to interact with NbSKη.

Since the highly virulent TLCYnV is a recombinant virus derived from TYLCCNV as the major donor and PepYLCCNV as the donor of the C4 gene (8), we wondered whether the abilities of the C4 proteins encoded by the different geminiviruses to act as virulence factors are divergent. We selected three geminiviruses isolated from China, including TLCYnV, TYLCCNV, and TbCSV. To evaluate the symptom determinant abilities of these C4 proteins, we expressed them by using a potato virus X (PVX)-based vector in N. benthamiana plants. Our results show that TLCYnV C4 induces severe symptoms, such as foliar distortion and chlorotic spots, while milder symptoms or almost none could be observed in N. benthamiana plants expressing TbCSV C4 or TYLCCNV C4 (Fig. 1A). These results suggest that C4 proteins encoded by different geminiviruses differ in their symptom determinant abilities.

FIG 1.

FIG 1

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Divergent symptom determinant abilities of the C4 proteins encoded by different geminiviruses result from their differential capacities to interact with NbSKη. (A) Phenotypes of N. benthamiana plants infected with PVX-based vectors harboring C4 from TbCSV, TYLCCNV, or TLCYnV. Photographs were taken at 12 days postinoculation (dpi). (B) Detection of the interaction between NbSKη and the C4 protein encoded by TbCSV, TYLCCNV, or TLCYnV. Yeast strain Gold cotransformed with the indicated plasmids was subjected to 10-fold serial dilutions and grown on an SD/–Leu/–Trp/–His/–Ade medium. (C) Coimmunoprecipitation analysis of the interaction between NbSKη and the C4 protein encoded by TbCSV, TYLCCNV, or TLCYnV in vivo. (D) Distribution pattern of GFP-NbSKη in the presence of CFP, TbCSV C4-, TYLCCNV C4-, or TLCYnV C4-CFP transiently expressed in epidermal cells of H2B-RFP transgenic N. benthamiana plants. Bars, 50 μm. (E) Nuclear-cytoplasmic fractionation analysis of the accumulation of nucleus-localized NbSKη in plant tissues under different treatments. Western blotting was conducted with antibodies specific to the indicated proteins. N and C indicate nuclear and cytoplasmic extracts, respectively. Cytoplasmic extracts of wild-type N. benthamiana plants were used as the positive control for the cytoplasmic fraction.

We next conducted yeast two-hybrid (Y2H) assays to detect the interactions between NbSKη and the C4 proteins encoded by the above geminiviruses. Interestingly, the abilities of these C4 proteins to interact with NbSKη are significantly different. TLCYnV interacted with NbSKη, and TYLCCNV C4 did not, while a weak interaction was detected for TbCSV C4 (Fig. 1B). A similar interaction ability between C4 and NbSKη was confirmed by coimmunoprecipitation (co-IP) analysis (Fig. 1C).

To explore the influence of C4 on the subcellular localization of NbSKη, we coexpressed GFP-NbSKη (intact green fluorescent protein [GFP] fused to the N terminus of NbSKη) with cyan fluorescent protein (CFP) or TYLCCNV C4-, TbCSV C4-, or TLCYnV C4-CFP (CFP fused to the C terminus of C4) in H2B-RFP (red fluorescent protein) transgenic N. benthamiana plants. Confocal micrographs show that the native nuclear accumulation of GFP-NbSKη could rarely be observed in the presence of TLCYnV C4-CFP (Fig. 1D). In contrast, coexpression of GFP-NbSKη with TYLCCNV C4-CFP or TbCSV C4-CFP had no obvious impact, or only a mild impact, on its localization (Fig. 1D).

To confirm the above results, we expressed CFP, TbCSV C4-, TLCYnV C4-, and TYLCCNV C4-CFP in leaves of N. benthamiana plants and then performed nuclear-cytoplasmic fractionation assays to detect the accumulation level of NbSKη in the nucleus. The accumulation level of NbSKη in the nucleus in leaves expressing TLCYnV C4-CFP is lower than that in leaves expressing CFP, TbCSV C4-, or TYLCCNV C4-CFP. TYLCCNV C4- or TbCSV C4-CFP had no obvious impact, or only a mild impact, on NbSKη nuclear accumulation (Fig. 1E). These results indicate that the divergent symptom determinant capacities of geminivirus-encoded C4 proteins correlate with, and hence might be caused by, their different abilities to interact with NbSKη.

To further explore whether the abilities of C4 proteins encoded by different geminiviruses to directly bind NbSKη are divergent, we prokaryotically expressed and purified His-NbSKη and glutathione S-transferase (GST)-TYLCCNV C4, -TbCSV C4, and -TLCYnV C4 from Escherichia coli (strain BL21). We then performed indirect enzyme-linked immunosorbent assays (ELISAs), showing that more His-NbSKη could be bound by GST-TLCYnV C4 than by GST-TbCSV C4 or GST-TYLCCNV C4 (Fig. 2A). Additionally, we performed protein binding assays, in which GST-TYLCCNV C4, GST-TbCSV C4, and GST-TLCYnV C4 were blotted onto a nitrocellulose filter membrane and then incubated with His-NbSKη. In agreement with the previous results, significantly more His-NbSKη could be bound by GST-TLCYnV C4 than by GST-TbCSV C4, while barely any His-NbSKη was bound by GST-TYLCCNV C4 in vitro (Fig. 2B). Similar results could be obtained in renatured blot overlay assays (Fig. 2C). These experiments demonstrate that the physical binding ability of geminivirus C4 to NbSKη correlates with the symptom determinant ability of C4.

FIG 2.

FIG 2

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Abilities of the C4 proteins encoded by different geminiviruses to directly bind NbSKη. (A) Indirect ELISA analysis of the abilities of different geminiviral C4 proteins to bind NbSKη. The relative amount of GST-C4-bound His-NbSKη is normalized to the amount of loaded GST-C4. (B) Abilities of C4 proteins encoded by different geminiviruses to bind NbSKη. The upper panel shows the His-NbSKη bound by different geminiviral C4 proteins; the lower panel shows the input. GST-C4 proteins encoded by different geminiviruses were spotted onto nitrocellulose membranes with repetition. (C) Renatured blot overlay analysis of the divergent binding capabilities of C4 proteins encoded by different geminiviruses with NbSKη. The upper panel shows the His-NbSKη binding to geminiviral C4; the lower panel indicates the loaded geminivirus C4 proteins that serve as the input.

A minidomain in C4 essential for the interaction with NbSKη determines its symptom determinant capacity.

The divergent binding abilities of geminivirus C4 proteins to NbSKη raise the possibilities that the specific ability of C4 to trigger symptoms might result from these differences in binding. In order to test this hypothesis, the sequences of the C4 proteins previously used were aligned and compared. The sequence from amino acid (aa) 28 to aa 37 of TLCYnV C4 is different from those of TYLCCNV C4 and TbCSV C4 (Fig. 3A). Interestingly, we previously identified some key sites of TLCYnV C4 essential for its interaction with NbSKη (Pro32, Asn34, and Thr35) (19), all of which are localized in this aa 28-to-37 stretch. We next exchanged the aa 28-to-37 sequence of TLCYnV C4 for that of TYLCCNV C4 or TbCSV C4 to create recombinant C4 proteins (Fig. 3B). Expression of the C4 or recombinant C4 proteins (Flag tag fused to C terminus of C4 protein) by using PVX-based expression vectors in N. benthamiana showed that the recombinant C4 proteins harboring the sequence from aa 28 to aa 37 of TLCYnV C4 have stronger symptom determinant abilities. However, TLCYnV C4 carrying the aa 28–to-37 sequence of TLCCNV C4 could not induce obvious symptoms (Fig. 3C). Western blot analysis indicated that all the C4 or recombinant C4 proteins were expressed well in the PVX vector (Fig. 3D).

FIG 3.

FIG 3

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Identification of the minidomain critical for geminivirus C4 symptom determinant ability. (A) Alignment of different C4 protein sequences by using the MegAlign software. The red rectangle indicates a divergent amino acid stretch between TLCYnV C4 and TYLCCNV C4 or TbCSV C4. (B) Schematic representation of the recombinant geminivirus C4 proteins through exchanging the minidomain critical for geminivirus C4 symptom determinant ability. (C) Phenotype of systemic leaves in N. benthamiana plants infected with a PVX-based vector harboring C4 or recombinant C4 proteins. Photographs were taken at 12 days postinoculation. Over 150 N. benthamiana plants in three independent experiments were used to observe the symptom development. (D) Western blot analysis of the accumulation of C4 or recombinant C4 proteins in N. benthamiana plants infected with a PVX-based vector harboring C4 or recombinant C4 proteins with antibodies specific to the indicated proteins. Black and red arrowheads indicate nonphosphorylated and phosphorylated C4 proteins, respectively. (E) Y2H analysis of the interaction between NbSKη and geminivirus C4 or the recombinant C4 proteins. Yeast strain Gold cotransformed with the indicated plasmids was subjected to 10-fold serial dilutions and grown on an SD/–Leu/–Trp/–His medium.

To test if recombinant C4 proteins containing the aa 28-to-37 sequence of TLCYnV C4 can interact with NbSKη strongly, we performed Y2H assays. Recombinant TLCYnV C4 harboring the TYLCCNV C4 aa 28-to-37 sequence loses its ability to interact with NbSKη (Fig. 3E). Also, a point mutation (T35A) in the recombinant TYLCCNV or TbCSV C4 proteins could compromise their interactions with NbSKη. The above results indicated that the aa 28-to-37 sequence of TLCYnV C4 is essential for both the interaction with NbSKη and symptom development.

The interaction of C4 with NbSKη is critical for the tethering of NbSKη to the plasma membrane.

In further investigation of the molecular mechanism by which geminivirus-encoded C4 proteins induce symptoms with different severity, we performed coexpression assays to measure the influence of TLCYnV C4 or recombinant C4 proteins on the subcellular localization of NbSKη. GFP-NbSKη was coexpressed with TLCYnV C4-CFP or recombinant C4-CFP proteins in leaves of H2B-RFP transgenic N. benthamiana plants, and CFP, GFP, and RFP fluorescence was observed by confocal microscope at 48 h postinfiltration (hpi). Confocal micrographs show that the changes in the localization of NbSKη caused by the different recombinant C4 proteins closely correlate with their abilities to interact with NbSKη (Fig. 4A).

FIG 4.

FIG 4

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The changed subcellular localization of NbSKη caused by C4 proteins is coupled to their abilities to interact with NbSKη. (A) Distribution pattern of GFP-NbSKη in the presence of CFP, TbCSV C4-, TYLCCNV C4-, or TLCYnV C4-CFP, or recombinant C4 proteins tagged with CFP, transiently expressed in epidermal cells of H2B-RFP transgenic N. benthamiana plants. Bars, 50 μm. (B) Nuclear-cytoplasmic fractionation analysis of the accumulation of nucleus-localized NbSKη in plant tissues expressing C4 or recombinant C4 proteins. Western blotting was conducted with antibodies specific to the indicated proteins. N and C indicate nuclear and cytoplasmic extracts, respectively. Cytoplasmic extracts of wild-type N. benthamiana plants were used as the positive control for the cytoplasmic fraction.

To validate the above confocal results, we expressed CFP, TLCYnV C4-CFP, or recombinant C4-CFP in leaves of N. benthamiana plants. Nuclear-cytoplasmic fractionation assays were conducted by using plant tissues expressing CFP, TLCYnV C4-CFP, or recombinant C4-CFP proteins at 48 hpi. Immunoblot analysis showed that the reduced nuclear accumulation level of NbSKη caused by the expression of different C4 or recombinant C4 proteins is tightly coupled to their abilities to bind NbSKη (Fig. 4B).

We then explored the subcellular distribution of different C4 proteins and their influence on the subcellular localization of NbSKη. Subcellular fractionation shows that TLCYnV C4 is mostly associated with the membrane; in contrast, a significant proportion of TbCSV C4 and most of TYLCCNV C4 can be detected in the S30 fraction (Fig. 5A). GFP, which is soluble, appeared only in the S30 fraction, while PIP2A acts as a membrane-localized marker that could be detected only in the P30 fraction (Fig. 5A). Subcellular fractionation assays were performed to detect the influence of C4 or recombinant C4 proteins on the subcellular distribution of NbSKη. We coexpressed GFP-NbSKη with TLCYnV C4-GFP, TYLCCNV C4-GFP, or TbCSV C4-GFP in leaves of N. benthamiana plants; plant tissues were harvested at 48 hpi; and protein extracts were prepared from plant tissues and separated by high-speed centrifugation into pellet (P30) and supernatant (S30) fractions. Western blot results show that unlike TLCYnV C4, which led to the presence of NbSKη in the membrane fraction (P30), TbCSV C4 could tether NbSKη to membranes only partially, and TYLCCNV C4 could only residually tether NbSKη to membranes (Fig. 5B). Although most GFP-NbSKη was detected in the P30 fraction in the presence of TLCYnV C4-CFP, when GFP-NbSKη was coexpressed with recombinant TLCYnVTYLCCNV 28–37 aa C4, containing aa 28 to 37 of TYLCCNV C4, most GFP-NbSKη was present in the S30 fraction (Fig. 5C). In contrast, GFP-NbSKη could hardly be detected in the P30 fraction when coexpressed with TYLCCNV C4. However, a significant proportion of GFP-NbSKη could be detected in the P30 fraction when coexpressed with recombinant TYLCCNVTLCYnV 28–37 aa C4 protein containing aa 28 to 37 of TLCYnV C4, which resulted from the increased interaction between recombinant TYLCCNVTLCYnV 28–37 aa C4 and NbSKη (Fig. 5D). Remarkably, an increased amount of GFP-NbSKη in the P30 fraction could also be detected when this protein was coexpressed with recombinant TbCSVTLCYnV 28–37 aa C4, containing aa 28 to 37 of TLCYnV C4 (Fig. 5E). As expected, coexpression of GFP-NbSKη with TYLCCNVTLCYnV 28–37 aa T35A C4 or TbCSVTLCYnV 28–37 aa T35A C4, both of which are deficient in NbSKη binding, could not tether NbSKη to the membrane (Fig. 5D and E). These results indicate that the abilities of C4 proteins to tether NbSKη to the plasma membrane are significantly different, and replacement of amino acids 28 to 37 of TYLCCNV C4 or TbCSV C4 by the corresponding amino acid stretch of TLCYnV C4, which increases the ability of these proteins to interact with NbSKη, results in alterations in NbSKη subcellular distribution.

FIG 5.

FIG 5

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The divergent symptom determinant capabilities of C4 proteins are attributed to their differential abilities to interact with NbSKη. (A) Subcellular fractionation analysis of the cellular distribution patterns of C4 proteins encoded by TLCYnV, TYLCCNV, and TbCSV. (B to E) Subcellular fractionation analysis of the cellular distribution pattern of NbSKη in the presence of TLCYnV C4, TYLCCNV C4, TbCSV C4, or the recombinant C4 proteins. Plant tissues expressing the proteins indicated above were fractionated into soluble (S) and membrane-enriched (P) fractions. GFP and PIP2A-DsRed acted as markers of the soluble and membrane-enriched fractions, respectively. 3K, extracts following centrifugation at 3,000 × g; 30K, extracts following centrifugation at 30,000 × g. GFP-NbSKη, TLCYnV C4, TYLCCNV C4-CFP, TbCSV C4-CFP, and recombinant geminiviral C4-CFP were detected using a rabbit monoclonal antibody raised against GFP. Double asterisks indicate GFP-NbSKη; single asterisks represent C4- or recombinant C4-CFP.

Recombinant geminivirus variants producing NbSKη-interacting C4 proteins develop more-severe symptoms.

To investigate whether the ability of C4 to interact with NbSKη determines geminivirus pathogenicity, we constructed recombinant virus infectious clones producing recombinant versions of C4 containing the swapped minidomains. Wild-type TLCYnV induced severe symptoms at 15 days postinoculation (dpi) in N. benthamiana, such as leaf curling and vein thickening. However, recombinant TLCYnV (TLCYnVTYLCCNV 28–37 aa) producing a mutated C4 protein carrying aa 28 to 37 of TYLCCNV C4 could not infect N. benthamiana plants systemically (Fig. 6A). These results are consistent with our previous data showing that a TLCYnV infectious clone carrying the mutated C4 deficient in NbSKη binding could not infect N. benthamiana plants (19). Although wild-type TYLCCNV alone induced very mild symptoms, we found that the recombinant TYLCCNV (TYLCCNVTLCYnV 28–37 aa) producing C4 carrying aa 28 to 37 of TLCYnV C4, which is able to interact with NbSKη, could induce more-severe symptoms (Fig. 6A). Southern blot analysis showed that N. benthamiana plants infected with recombinant TYLCCNV (TYLCCNVTLCYnV 28–37 aa) accumulated more DNA than those infected with TYLCCNV, and N. benthamiana plants infected with TLCYnV accumulated large amounts of viral DNA, while no viral DNA was detected in plants infected with recombinant TLCYnV (TLCYnVTYLCCNV 28–37 aa) (Fig. 6B). These results suggest that the divergent pathogenicities of these geminiviruses are due to the differential abilities of their corresponding C4 proteins to interact with NbSKη.

FIG 6.

FIG 6

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The minidomain of C4 that is essential for its interaction with NbSKη is critical for geminivirus-induced symptom development. (A) Phenotypes of N. benthamiana plants infected with TLCYnV, TYLCCNV, or recombinants. Photographs were taken at 15 days postinoculation (dpi). (B) Southern blot analysis of viral accumulation in systemic leaves of N. benthamiana plants infected with TLCYnV, TYLCCNV, or recombinants. Plant tissues (1 g) harvested from at least 5 geminivirus-infected N. benthamiana plants were prepared for DNA extraction Total nucleic acids (25 μg) were extracted at 15 dpi. Blots were hybridized with TLCYnV C4 and TYLCCNV C4 probes, respectively. Total genomic DNA visualized by ethidium bromide staining is shown below as the loading control.

DISCUSSION

The geminivirus C4/AC4 protein acts as a multifunctional effector to induce symptom development and to counter plant defense responses during viral infection (20–25). Many different geminivirus-encoded C4/AC4 proteins could serve as symptom determinants (18, 19, 26–29). For example, transgenic tobacco and tomato plants expressing tomato yellow leaf curl virus C4 under the control of the cauliflower mosaic virus 35S promoter develop virus-disease-like phenotypes (30). C4 encoded by beet severe curly top virus (BSCTV) induces abnormal cell division by upregulating the expression of KRP, which interacts with and might degrade the cyclin kinase inhibitor (ICK2) to promote the mitotic cycle (26). Moreover, the S-acylated form of BSCTV C4 interacts with CLAVATA 1 (CLV1) to regulate the CLAVATA pathway for cell division interference (28). TLCYnV C4, a pathogenicity determinant, is able to interact with SKη kinase to inhibit the degradation of cyclin CycD1;1 to enable the transition of the cell from G1 to S phase, which facilitates viral genome replication (19). These findings showed that geminivirus C4/AC4 functions as a viral “oncogene,” similar to those encoded by animal viruses (31), regulating cell cycle progression and causing development defects.

Our previous work demonstrated that TLCYnV C4 induced viral symptoms by interacting with NbSKη and tethering it to the plasma membrane to impede the phosphorylation-mediated degradation of NbCycD1;1 in the nucleus (19). Furthermore, the nucleocytoplasmic shuttling of TLCYnV C4 is critical for viral pathogenicity: TLCYnV C4 can be phosphorylated by NbSKη in the nucleus, which promotes N-myristoylation of this viral protein. Myristoylation of phosphorylated C4 favors its interaction with exportin-α (XPO I), which in turn promotes the nuclear export of the C4/NbSKη complex (29). In this study, we analyzed the C4 functions of three typical monopartite begomoviruses, TLCYnV, TYLCCNV, and TbCSV, in symptom induction and their abilities to interact with NbSKη. Our results showed that the symptom induction capabilities of these C4 proteins positively correlate with their binding to NbSKη. Also, we identified a minidomain in TLCYnV C4 required for NbSKη binding; this minidomain includes some key sites of TLCYnV C4 previously proven essential for its interaction with NbSKη (Pro32, Asn34, and Thr35). Swap of the TLCYnV C4 minidomain increased the ability of the C4 proteins encoded by TYLCCNV or TbCSV to induce symptoms. These results are consistent with previous results showing that the interaction between C4 protein and NbSKη is important for abnormal cell division induction, which is essential for geminivirus replication and infection (19, 26).

Although recombinant TYLCCNV C4 and TbCSV C4 carrying the minidomain of TLCYnV C4 could induce more-severe symptoms than wild-type TYLCCNV C4 and TbCSV C4 (Fig. 3C), replacement of the minidomain of TYLCCNV C4 with that of TLCYnV C4 does not fully recover the intensity of the symptoms induced by TLCYnV C4 (Fig. 6A). We think the milder symptoms induced by TYLCCNVTLCYnV 28–37 aa C4 relative to those induced by TLCYnV C4 are attributable to their divergent abilities for tethering NbSKη to the plasma membrane (Fig. 4). However, we could not exclude the possibility that other regions of the C4 protein might also contribute to NbSKη binding.

The recombinant TLCYnV producing a mutated C4 protein carrying aa 28 to 37 of TYLCCNV C4 could not infect N. benthamiana plants systemically (Fig. 6). The C4 open reading frame (ORF) is located within the ORF for Rep protein, so domain replacement of the C4 protein might affect Rep protein functions, which are important in virus replication. Further efforts are necessary to explore the influence of recombinant C4 on Rep functions.

In summary, we provide evidence that the C4 proteins encoded by different geminiviruses have divergent symptom determinant abilities and that these are dependent mainly on their differential capabilities to interact with NbSKη via a minidomain to tether the interaction complex to the plasma membrane, removing NbSKη from the nucleus (Fig. 7). This study reveals the molecular mechanism by which different geminivirus-encoded C4 proteins possess divergent symptom determinant capabilities, and it provides new insights into the mechanisms underpinning the diversity of geminivirus pathogenicity.

FIG 7.

FIG 7

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Proposed model for the divergent symptom determinant capabilities of C4 proteins encoded by different geminiviruses. C4 proteins encoded by different geminiviruses have differential capacities to interact with NbSKη and divergent membrane-binding abilities. After replacement of the corresponding amino acids of TYLCCNV C4 or TbCSV C4 with the TLCYnV C4 minidomain, which mediates the interaction with NbSKη, more NbSKη is restricted to the membrane, which results in more-severe pathogenicity. The right panel shows the phenotype of systemic leaves in N. benthamiana plants infected with PVX-based vectors harboring C4 encoded by TYLCCNV, TbCSV, or TLCYnV.

MATERIALS AND METHODS

Plasmid construction.

TYLCCNV C4, TbCSV C4, or TLCYnV C4 was cloned into the pGR106, pGBKT7, pCHF3-CFP, pGD-GFP, and pGEX4T-3 vectors. NbSKη was cloned into pGADT7, pGD-GFP, and pET-32a. The recombinant C4 mutants were cloned individually into the pGR106, pGBKT7, pCHF3-CFP, and pGD-GFP vectors. The recombinant infectious clones were constructed in pBinplus to generate region-directed infectious clones. All cDNAs were PCR amplified using the KOD-Plus-Neo High-Fidelity DNA polymerase (Toyobo). The resulting PCR fragments were first cloned individually into the pMD18-T vector (TaKaRa) and then ligated into expression vectors. To generate recombinant geminivirus infectious clones, overlapping PCR methods were used as described previously (32). The primer sequences will be available upon request.

Plant materials and growth condition.

Transgenic N. benthamiana plants expressing the nuclear marker H2B-RFP (full-length red fluorescent protein fused to the C terminus of histone 2B) (33) were kindly provided by Michael M. Goodin (University of Kentucky, Lexington, KY, USA). N. benthamiana plants were grown in an insect-free chamber at 26°C under a 16-h-light/8-h-dark photoperiod.

Protein binding assay.

GST-C4 proteins encoded by different geminiviruses and His-NbSKη were prokaryotically expressed and purified from Escherichia coli (strain BL21). Equal amounts of geminivirus C4 proteins were spotted onto nitrocellulose membranes and allowed to dry. The membrane harboring proteins was first incubated with 3% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween 20 (TBST) buffer for 1 h at room temperature and then incubated with TBST containing His-NbSKη (10 μg/ml) at 4°C overnight. The membrane was then washed with TBST three times and incubated with 5% BSA containing an anti-His monoclonal antibody (1:5,000) for 2 h at room temperature. Following three washes with TBST, the membrane was incubated with 5% BSA containing the secondary antibody for 2 h at room temperature. After four washes with TBST, His-NbSKη bound on the membrane was detected with the ECL Western blotting kit (4A Biotech).

Indirect ELISAs.

Indirect ELISAs were conducted as described previously (34). The 96-well microtiter plate was incubated with GST-TYLCCNV C4, -TbCSV C4, or TLCYnV C4 at 4°C overnight. The plate coated with GST-C4 proteins was washed with phosphate-buffered saline with Tween 20 (PBST) buffer three times; then His-NbSKη was added to the wells coated with GST-C4 proteins and was incubated at 37°C for 2 h. After the removal of GST-C4 proteins, antigen-coated ELISA (ACP-ELISA) was performed to detect the His-NbSKη bound by GST-C4 proteins using an anti-His alkaline phosphatase (AP)-conjugated monoclonal antibody. Another 96-well microtiter plate coated with the same volume of GST-TYLCCNV C4, -TbCSV C4, or TLCYnV C4 was not incubated with His-NbSKη; then AP-ELISA was performed by using an AP-conjugated anti-GST monoclonal antibody as a loading control.

Renatured blot overlay assay.

The renatured blot overlay assay was conducted as described previously (35). Purified GST, GST-TYLCCNV C4, GST-TbCSV C4, or GST-TLCYnV C4 was resolved on a 12.5% SDS-PAGE gel in running buffer (26 mM Tris base, 191 mM glycine, 0.01% SDS) and electroblotted (100 V for 2 h at room temperature) onto a 5- by 8.5-cm polyvinylidene difluoride (PVDF) Immobilon P membrane in transfer buffer (26 mM Tris base, 191 mM glycine, 20% methanol). After electroblotting, the membrane was washed for 15 min with gentle shaking in buffer (30 mM Tris-HCl [pH 7.4], 0.05% Tween 20) and then denatured for 2 h at room temperature in 50 ml of denaturation buffer (7 M guanidine hydrochloride, 2 mM EDTA, 50 mM dithiothreitol [DTT], 50 mM Tris-HCl [pH 8.3]). Following a 5-min wash in TBS (140 mM NaCl, 30 mM Tris-HCl [pH 7.4]), the blotted proteins were renatured by an overnight incubation at 4°C in 250 ml of renaturing buffer (140 mM NaCl, 10 mM Tris-HCl [pH 7.4], 2 mM DTT) with gentle shaking (10 rpm). After renaturation, the membrane containing the blotted proteins was blocked in the presence of 1% BSA to block potential nonspecific protein adsorption sites on the membrane. Then the renatured proteins on the membrane were incubated with purified His-NbSKη for 90 min at room temperature with 10 ml renaturing buffer containing His-NbSKη (10 μg/ml). After incubation with His-NbSKη, the membrane was washed five times (5 min at room temperature) with TBST, and then Western blot analysis was performed with a mouse monoclonal antibody raised against His.

Agroinfection assays in N. benthamiana.

Agroinfection assays were conducted as described previously (19). The constructs in the pgR106 backbone were transformed into Agrobacterium tumefaciens (strain GV3101) by electroporation; others were transformed into A. tumefaciens (strain C58C1) by electroporation. The plasmids carrying virus infectious clones in pBinplus were transformed into A. tumefaciens (strain EHA105) by electroporation. The transformed bacterial cultures were grown individually until the optical density at 600 nm (OD600) reached approximately 0.5 to 0.8. The cultures were collected, resuspended in induction buffer (10 mM MgCl2, 100 mM morpholineethanesulfonic acid [MES] [pH 5.7], 2 mM acetosyringone), and incubated for 3 h at room temperature. The suspensions were adjusted to an OD600 of 0.5. For colocalization experiments, the individual cultures were adjusted to an OD600 of 1.0, and equal volumes were mixed before leaf infiltration. The suspensions were infiltrated into leaves of 4- to 6-week-old N. benthamiana plants using 1-ml needleless syringes.

Y2H assay.

Yeast two-hybrid (Y2H) assays were conducted as described previously (19, 29). AD-NbSKη and BD-TbCSV C4, BD-TYLCCNV C4, or BD-TLCYnV C4 were transformed into Saccharomyces cerevisiae strain Gold to detect the interaction. All transformants were grown at 30°C for 72 h on synthetic defined (SD) medium lacking Leu and Trp and were then transferred to the medium lacking Leu, Trp, His, and adenine (Ade). AD-T and BD-p53 were transformed into S. cerevisiae strain Gold to serve as positive controls; AD-T and BD-Lam were transformed into S. cerevisiae strain Gold to serve as negative controls.

Co-IP assays.

Co-IP assays were conducted essentially as described previously (19).

Subcellular fractionation assay.

Subcellular fractionation assays were conducted as described previously (36–38). Leaves (0.5 g) expressing the corresponding proteins by agroinfiltration were harvested at 48 hpi and gently ground in 1 ml homogenization buffer (50 mM Tris-HCl [pH 8.0], 10 mM KCl, 3 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.1% BSA, 0.3% dextran, and 13% [wt/vol] sucrose). The homogenate was centrifuged at 3,000 × g for 20 min at 4°C to remove nuclei and large cellular debris, and the resulting supernatant solution was ultracentrifuged at 30,000 × g for 1 h at 4°C to generate the soluble (S30) and microsomal (P30) fractions. The P30 fraction was resuspended in the homogenization buffer. All fractions were analyzed by SDS-PAGE and Western blotting.

Southern blot assays.

Southern blot assays were conducted as described previously (39).

Nuclear-cytoplasmic fractionation assay.

Nuclear-cytoplasmic fractionation assays were conducted as described previously (40).

ACKNOWLEDGMENTS

The work was supported by grants from the National Natural Science Foundation of China (31720103914 and 31390422) to X.Z.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

REFERENCES

  • 1.Fauquet CM, Briddon RW, Brown JK, Moriones E, Stanley J, Zerbini M, Zhou X. 2008. Geminivirus stain demarcation and nomenclature. Arch Virol 153:783–821. doi: 10.1007/s00705-008-0037-6. [DOI] [PubMed] [Google Scholar]
  • 2.Varsani A, Roumagnac P, Fuchs M, Navas-Castillo J, Moriones E, Idris A, Briddon RW, Rivera-Bustamante R, Murilo Zerbini F, Martin DP. 2017. Capulavirus and Grablovirus: two new genera in the family Geminiviridae. Arch Virol 162:1819–1831. doi: 10.1007/s00705-017-3268-6. [DOI] [PubMed] [Google Scholar]
  • 3.Harrison B, Robinson D. 1999. Natural genomic and antigenic variation in whitefly-transmitted geminiviruses (begomoviruses). Annu Rev Phytopathol 37:369–398. doi: 10.1146/annurev.phyto.37.1.369. [DOI] [PubMed] [Google Scholar]
  • 4.Boulton MI. 2003. Geminiviruses: major threats to world agriculture. Ann Appl Biol 142:143–143. doi: 10.1111/j.1744-7348.2003.tb00239.x. [DOI] [Google Scholar]
  • 5.Brown JK, Fauquet CM, Briddon RW, Zerbini M, Moriones E, Navas-Castillo J. 2011. Geminiviridae, p 351–373. In King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (ed), Virus taxonomy. Classification and nomenclature of viruses. Ninth report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San Diego, CA. [Google Scholar]
  • 6.Zhou X. 2013. Advances in understanding begomovirus satellites. Annu Rev Phytopathol 51:357–381. doi: 10.1146/annurev-phyto-082712-102234. [DOI] [PubMed] [Google Scholar]
  • 7.Hu T, Song Y, Wang YQ, Zhou XP. 2020. Functional analysis of a novel βV1 gene identified by a geminivirus betasatellite. Sci China Life Sci 63:688–696. doi: 10.1007/s11427-020-1654-x. [DOI] [PubMed] [Google Scholar]
  • 8.Xie Y, Zhao L, Jiao X, Jiang T, Gong H, Wang B, Briddon RW, Zhou X. 2013. A recombinant begomovirus resulting from exchange of the C4 gene. J Gen Virol 94:1896–1907. doi: 10.1099/vir.0.053181-0. [DOI] [PubMed] [Google Scholar]
  • 9.Cui X, Tao X, Xie Y, Fauquet CM, Zhou X. 2004. A DNAβ associated with tomato yellow leaf curl China virus is required for symptom induction. J Virol 78:13966–13974. doi: 10.1128/JVI.78.24.13966-13974.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Li Z, Xie Y, Zhou X. 2005. Tobacco curly shoot virus DNAβ is not necessary for infection but intensifies symptoms in a host-dependent manner. Phytopathology 95:902–908. doi: 10.1094/PHYTO-95-0902. [DOI] [PubMed] [Google Scholar]
  • 11.Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785–789. doi: 10.1038/378785a0. [DOI] [PubMed] [Google Scholar]
  • 12.Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, Woodgett JR. 2000. Requirement for glycogen synthase kinase-3β in cell survival and NF-κB activation. Nature 406:86–90. doi: 10.1038/35017574. [DOI] [PubMed] [Google Scholar]
  • 13.Li J, Nam KH. 2002. Regulation of brassinosteroid signaling by a GSK3/SHAGGY-like kinase. Science 295:1299–1301. doi: 10.1126/science.1065769. [DOI] [PubMed] [Google Scholar]
  • 14.Wang ZY, Nakano T, Gendron J, He J, Chen S, Vafeados D, Yang Y, Fujioka S, Yoshida S, Asami T, Chory J. 2002. Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell 2:505–513. doi: 10.1016/S1534-5807(02)00153-3. [DOI] [PubMed] [Google Scholar]
  • 15.He JX, Gendron JM, Sun Y, Gampala SS, Gendron N, Sun CQ, Wang ZY. 2005. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science 307:1634–1638. doi: 10.1126/science.1107580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Yin Y, Wang ZY, Mora-Garcia S, Li J, Yoshida S, Asami T, Chory J. 2002. BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell 109:181–191. doi: 10.1016/s0092-8674(02)00721-3. [DOI] [PubMed] [Google Scholar]
  • 17.Yin Y, Vafeados D, Tao Y, Yoshida S, Asami T, Chory J. 2005. A new class of transcription factors mediates brassinosteroid regulated gene expression in Arabidopsis. Cell 120:249–259. doi: 10.1016/j.cell.2004.11.044. [DOI] [PubMed] [Google Scholar]
  • 18.Piroux N, Saunders K, Page A, Stanley J. 2007. Geminivirus pathogenicity protein C4 interacts with Arabidopsis thaliana shaggy-related protein kinase AtSKη, a component of the brassinosteroid signalling pathway. Virology 362:428–440. doi: 10.1016/j.virol.2006.12.034. [DOI] [PubMed] [Google Scholar]
  • 19.Mei Y, Yang X, Huang C, Zhang X, Zhou X. 2018. Tomato leaf curl Yunnan virus-encoded C4 induces cell division through enhancing stability of Cyclin D 1.1 via impairing NbSKη-mediated phosphorylation in Nicotiana benthamiana. PLoS Pathog 14:e1006789. doi: 10.1371/journal.ppat.1006789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Park J, Hwang HS, Buckley KJ, Park JB, Auh CK, Kim DG, Lee S, Davis KR. 2010. C4 protein of Beet severe curly top virus is a pathomorphogenetic factor in Arabidopsis. Plant Cell Rep 29:1377–1389. doi: 10.1007/s00299-010-0923-8. [DOI] [PubMed] [Google Scholar]
  • 21.Yang X, Xie Y, Raja P, Li S, Wolf JN, Shen Q, Bisaro DM, Zhou X. 2011. Suppression of methylation-mediated transcriptional gene silencing by βC1-SAHH protein interaction during geminivirus-betasatellite infection. PLoS Pathog 7:e1002329. doi: 10.1371/journal.ppat.1002329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Li F, Huang C, Li Z, Zhou X. 2014. Suppression of RNA silencing by a plant DNA virus satellite requires a host calmodulin-like protein to repress RDR6 expression. PLoS Pathog 10:e1003921. doi: 10.1371/journal.ppat.1003921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Ismayil A, Haxim Y, Wang Y, Li H, Qian L, Han T, Chen T, Jia Q, Yihao Liu A, Zhu S, Deng H, Gorovits R, Hong Y, Hanley-Bowdoin L, Liu Y. 2018. Cotton leaf curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase. PLoS Pathog 14:e1007282. doi: 10.1371/journal.ppat.1007282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Rosas-Diaz T, Zhang D, Fan P, Wang L, Ding X, Jiang Y, Jimenez-Gongora T, Medina-Puche L, Zhao X, Feng Z, Zhang G, Liu X, Bejarano ER, Tan L, Zhang H, Zhu JK, Xing W, Faulkner C, Nagawa S, Lozano-Duran R. 2018. A virus-targeted plant receptor-like kinase promotes cell-to-cell spread of RNAi. Proc Natl Acad Sci U S A 115:1388–1393. doi: 10.1073/pnas.1715556115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Mei Y, Ma Z, Wang Y, Zhou X. 2020. Geminivirus C4 antagonizes the HIR1-mediated hypersensitive response by inhibiting the HIR1 self-interaction and promoting degradation of the protein. New Phytol 225:1311–1326. doi: 10.1111/nph.16208. [DOI] [PubMed] [Google Scholar]
  • 26.Lai J, Chen H, Teng K, Zhao Q, Zhang Z, Li Y, Liang L, Xia R, Wu Y, Guo H, Xie Q. 2009. RKP, a RING finger E3 ligase induced by BSCTV C4 protein, affects geminivirus infection by regulation of the plant cell cycle. Plant J 57:905–917. doi: 10.1111/j.1365-313X.2008.03737.x. [DOI] [PubMed] [Google Scholar]
  • 27.Mills-Lujan K, Andrews DL, Chou CW, Deom CM. 2015. The roles of phosphorylation and SHAGGY-like protein kinases in geminivirus C4 protein induced hyperplasia. PLoS One 10:e0122356. doi: 10.1371/journal.pone.0122356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Li H, Zeng R, Chen Z, Liu X, Cao Z, Xie Q, Yang C, Lai J. 2018. S-acylation of a geminivirus C4 protein is essential for regulating the CLAVATA pathway in symptom determination. J Exp Bot 69:4459–4468. doi: 10.1093/jxb/ery228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Mei Y, Wang Y, Hu T, Yang X, Lozano-Duran R, Sunter G, Zhou X. 2018. Nucleocytoplasmic shuttling of geminivirus C4 protein mediated by phosphorylation and myristoylation is critical for viral pathogenicity. Mol Plant 11:1466–1481. doi: 10.1016/j.molp.2018.10.004. [DOI] [PubMed] [Google Scholar]
  • 30.Krake LR, Rezaian MA, Dry IB. 1998. Expression of the tomato leaf curl geminivirus C4 gene produces virus like symptoms in transgenic plants. Mol Plant Microbe Interact 11:413–417. doi: 10.1094/MPMI.1998.11.5.413. [DOI] [Google Scholar]
  • 31.Fondong VN. 2019. The ever-expanding role of C4/AC4 in geminivirus infection: punching above its weight? Mol Plant 12:145–147. doi: 10.1016/j.molp.2018.12.006. [DOI] [PubMed] [Google Scholar]
  • 32.Tao X, Zhou X, Li G, Yu J. 2002. The pathogenicity on legumes of cucumber mosaic virus was determined by 243 nucleotides on 2a polymerase gene of viral RNA2. Chinese Sci Bull 47:748–750. doi: 10.1360/02tb9169. [DOI] [Google Scholar]
  • 33.Martin K, Kopperud K, Chakrabarty R, Banerjee R, Brooks R, Goodin MM. 2009. Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta. Plant J 59:150–162. doi: 10.1111/j.1365-313X.2009.03850.x. [DOI] [PubMed] [Google Scholar]
  • 34.Zhang M, Chen R, Zhou X, Wu J. 2018. Monoclonal antibody-based serological detection methods for wheat dwarf virus. Virol Sin 33:173–180. doi: 10.1007/s12250-018-0024-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Chen MH, Sheng J, Hind G, Handa AK, Citovsky V. 2000. Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement. EMBO J 19:913–920. doi: 10.1093/emboj/19.5.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Peremyslov VV, Pan YW, Dolja VV. 2004. Movement protein of a closterovirus is a type III integral transmembrane protein localized to the endoplasmic reticulum. J Virol 78:3704–3709. doi: 10.1128/jvi.78.7.3704-3709.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Genoves A, Pallas V, Navarro JA. 2011. Contribution of topology determinants of a viral movement protein to its membrane association, intracellular traffic, and viral cell-to-cell movement. J Virol 85:7797–7809. doi: 10.1128/JVI.02465-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Sun Z, Yang D, Xie L, Sun L, Zhang S, Zhu Q, Li J, Wang X, Chen J. 2013. Rice black-streaked dwarf virus P10 induces membranous structures at the ER and elicits the unfolded protein response in Nicotiana benthamiana. Virology 447:131–139. doi: 10.1016/j.virol.2013.09.001. [DOI] [PubMed] [Google Scholar]
  • 39.Xu X, Qian Y, Wang Y, Li Z, Zhou X. 2018. Iterons homologous to helper geminiviruses are essential for efficient replication of betasatellites. J Virol 93:e01532-18. doi: 10.1128/JVI.01532-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Wang W, Ye R, Xin Y, Fang X, Li C, Shi H, Zhou X, Qi Y. 2011. An importin β protein negatively regulates microRNA activity in Arabidopsis. Plant Cell 23:3565–3576. doi: 10.1105/tpc.111.091058. [DOI] [PMC free article] [PubMed] [Google Scholar]

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