Tobacco curly shoot virus Down-Regulated the Expression of nbe-miR167b-3p to Facilitate Its Infection in Nicotiana benthamiana

Tobacco curly shoot virus (TbCSV) belongs to the genus Begomovirus of the family Geminiviridae, and causes leaf curling and curly shoot symptoms in tobacco and tomato crops. MicroRNAs (miRNAs) are pivotal modulators of plant development and host-virus interactions. However, the relationship between TbCSV infection and miRNAs accumulation has not been well investigated. The present study was conducted to analyze different expressions of miRNAs in Nicotiana benthamiana in response to the infection of TbCSV via small RNAs sequencing. The results showed that 15 up-regulated miRNAs and 12 down-regulated miRNAs were differentially expressed in TbCSV infected N. benthamiana, and nbe-miR167b-3p was down-regulated. To decipher the relationship between nbe-miR167b-3p expression and the accumulations of TbCSV DNA, pCVA mediation of miRNA overexpression and PVX based short tandem target mimic (STTM) were used in this study. It was found that overexpression of nbe-miR167b-3p attenuated leaf curling symptom of TbCSV and decreased viral DNA accumulation, but suppression of nbe-miR167b-3p expression enhanced the symptoms and accumulation of TbCSV. PRCP, the target gene of nbe-miR167b-3p, was silenced in plants using VIGS and this weakened the viral symptoms and DNA accumulation of TbCSV in the plants. Overall, this study clarified the effect of nbe-miR167b-3p on plant defense during TbCSV infection, and provided a framework to reveal the molecular mechanisms of miRNAs between plants and viruses.

Characterization of Pathogenicity-Associated V2 Protein of Tobacco Curly Shoot Virus

V2 proteins encoded by some whitefly-transmitted geminiviruses were reported to be functionally important proteins. However, the functions of the V2 protein of tobacco curly shoot virus (TbCSV), a monopartite begomovirus that causes leaf curl disease on tomato and tobacco in China, remains to be characterized. In our report, an Agrobacterium infiltration-mediated transient expression assay indicated that TbCSV V2 can suppress local and systemic RNA silencing and the deletion analyses demonstrated that the amino acid region 1–92 of V2, including the five predicted α-helices, are required for local RNA silencing suppression. Site-directed substitutions showed that the conserved basic and ring-structured amino acids in TbCSV V2 are critical for its suppressor activity. Potato virus X-mediated heteroexpression of TbCSV V2 in Nicotiana benthamiana induced hypersensitive response-like (HR-like) cell death and systemic necrosis in a manner independent of V2′s suppressor activity. Furthermore, TbCSV infectious clone mutant with untranslated V2 protein (TbCSV∆V2) could not induce visual symptoms, and coinfection with betasatellite (TbCSB) could obviously elevate the viral accumulation and symptom development. Interestingly, symptom recovery occurred at 15 days postinoculation (dpi) and onward in TbCSV∆V2/TbCSB-inoculated plants. The presented work contributes to understanding the RNA silencing suppression activity of TbCSV V2 and extends our knowledge of the multifunctional role of begomovirus-encoded V2 proteins during viral infections.

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

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.

Suppression of nbe-miR1919c-5p Expression in Nicotiana benthamiana Enhances Tobacco Curly Shoot Virus and Its Betasatellite Co-Infection

MicroRNAs (miRNAs) are non-coding but functional RNA molecules of 21–25 nucleotides in length. MiRNAs play significant regulatory roles in diverse plant biological processes. In order to decipher the relationship between nbe-miR1919c-5p and the accumulations of tobacco curly shoot virus (TbCSV) and its betasatellite (TbCSB) DNAs, as well as viral symptom development, we investigated the function of nbe-miR1919c-5p during TbCSV and TbCSB co-infection in plants using a PVX-and a TRV-based short tandem target mimic (STTM) technology. Suppression of nbe-miR1919c-5p expression using these two technologies enhanced TbCSV and TbCSB co-infection-induced leaf curling symptoms in Nicotiana benthamiana plants. Furthermore, suppression of nbe-miR1919c-5p expression enhanced TbCSV and TbCSB DNA accumulations in the infected plants. Our results can advance our knowledge on the nbe-miR1919c-5p function during TbCSV and TbCSB co-infection.

Identification of microRNAs regulated by tobacco curly shoot virus co-infection with its betasatellite in Nicotiana benthamiana

Background MicroRNAs (miRNAs) are a class of 21–24 nucleotide endogenous non-coding small RNAs that play important roles in plant development and defense responses to biotic and abiotic stresses. Tobacco curly shoot virus (TbCSV) is a monopartite begomovirus, cause leaf curling and plant stunting symptoms in many Solanaceae plants. The betasatellite of TbCSV (TbCSB) induces more severe symptoms and enhances virus accumulation when co-infect the plants with TbCSV. Methods In this study, miRNAs regulated by TbCSV and TbCSB co-infection in Nicotiana benthamiana were characterized using high-throughput sequencing technology. Results Small RNA sequencing analysis revealed that a total of 13 known miRNAs and 42 novel miRNAs were differentially expressed in TbCSV and TbCSB co-infected N. benthamiana plants. Several potential miRNA-targeted genes were identified through data mining and were involved in both catalytic and metabolic processes, in addition to plant defense mechanisms against virus infections according to Gene Ontology (GO) analyses. In addition, the expressions of several differentially expressed miRNAs and their miRNA-targeted gene were validated through quantitative real time polymerase chain reaction (qRT-PCR) approach. Conclusions A large number of miRNAs are identified, and their target genes, functional annotations also have been explored. Our results provide the information on N. benthamiana miRNAs and would be useful to further understand miRNA regulatory mechanisms after TbCSV and TbCSB co-infection.

Cryo-EM Structure of a Begomovirus Geminate Particle

Tobacco curly shoot virus, a monopartite begomovirus associated with betasatellite, causes serious leaf curl diseases on tomato and tobacco in China. Using single-particle cryo-electron microscopy, we determined the structure of tobacco curly shoot virus (TbCSV) particle at 3.57 Å resolution and confirmed the characteristic geminate architecture with single-strand DNA bound to each coat protein (CP). The CP–CP and DNA–CP interactions, arranged in a CP–DNA–CP pattern at the interface, were partially observed. This suggests the genomic DNA plays an important role in forming a stable interface during assembly of the geminate particle.

Iterons Homologous to Helper Geminiviruses Are Essential for Efficient Replication of Betasatellites

ABSTRACTBetasatellites associated with geminiviruses can be replicated promiscuously by distinct geminiviruses but exhibit a preference for cognate helper viruses. However, theciselements responsible for betasatellite origin recognition have not been characterized. In this study, we identified an iteron-like repeated sequence motif, 5′-GAGGACC-3′, in a tobacco curly shoot betasatellite (TbCSB) associated with tobacco curly shoot virus (TbCSV). Competitive DNA binding assays revealed that two core repeats (5′-GGACC-3′) are required for specific binding to TbCSV Rep; TbCSB iteron mutants accumulated to greatly reduced levels and lost the cognate helper-mediated replication preference. Interestingly, TbCSV also contains identical repeated sequences that are essential for specific Rep binding andin vivoreplication. In order to gain insight into the mechanism by which TbCSB has acquired the cognate iterons, we performed a SELEX (systematic evolution of ligands by exponential enrichment) assay to identify the high-affinity Rep binding ligands from a large pool of randomized sequences. Analysis of SELEX winners showed that all of the sequences contained at least one core iteron-like motif, suggesting that TbCSB has evolved to contain cognate iterons for high-affinity Rep binding. Further analyses of various betasatellite sequences revealed a region upstream of the satellite conserved region replete with iterative sequence motifs, including species-specific repeats and a general repeat (5′-GGTAAAT-3′). Remarkably, the species-specific repeats in many betasatellites are homologous to those in their respective cognate helper begomoviruses, whereas the general repeat is widespread in most of the betasatellite molecules analyzed. These data, taken together, suggest that many betasatellites have evolved to acquire homologous iteron-like sequences for efficient replication mediated by cognate helper viruses.IMPORTANCEThe geminivirus-encoded replication initiator protein (Rep) binds to repeated sequence elements (also known as iterons) in the origin of replication that serve as essentialciselements for specific viral replication. Betasatellites associated with begomoviruses can be replicated by cognate or noncognate helper viruses, but theciselements responsible for betasatellite origin recognition have not been characterized. Using a betasatellite (TbCSB) associated with tobacco curly shoot virus (TbCSV) as a model, we identify two tandem repeats (iterons) in the Rep-binding motif (RBM) that are required for specific Rep binding and efficient replication, and we show that identical iteron sequences present in TbCSV are also necessary for Rep binding and the replication of helper viruses. Extensive analysis of begomovirus/betasatellite sequences shows that many betasatellites contain iteron-like elements homologous to those of their respective cognate helper begomoviruses. Our data suggest that many betasatellites have evolved to acquire homologous iteron-like sequences for efficient replication mediated by cognate helper viruses.