The P2 nucleic acid binding protein of Sugarcane bacilliform virus is a viral pathogenic factor.

Background: Saccharum spp. is the primary source of sugar and plays a significant role in global renewable bioenergy. Sugarcane bacilliform virus (SCBV) is one of the most important viruses infecting sugarcane, causing severe yield losses and quality degradation. It is of great significance to reveal the pathogenesis of SCBV and resistance breeding. However, little is known about the viral virulence factors or RNA silencing suppressors and the molecular mechanism of pathogenesis. Methods: To systematically investigate the functions of the unknown protein P2 encoded by SCBV ORF2. Phylogenetic analysis was implemented to infer the evolutionary relationship between the P2 of SCBV and other badnaviruses. The precise subcellular localization of P2 was verified in the transient infiltrated Nicotiana benthamiana epidermal mesophyll cells and protoplasts using the Laser scanning confocal microscope (LSCM). The post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) RNA silencing suppressor activity of P2 was analyzed, respectively. Furthermore, restriction digestion and RT-qPCR assays were conducted to verify the probable mechanism of P2 on repressing DNA methylation. To explore the pathogenicity of P2, a potato virus X-based viral vector was used to heterologously express SCBV P2 and the consequent H2O2 accumulation was detected by the 3,3'-diaminobenzidine (DAB) staining method. Results: Phylogenetic analysis shows that SCBV has no obvious sequence similarity and low genetic relatedness to Badnavirus and Tungrovirus representatives. LSCM studies show that P2 is localized in both the cytoplasm and nucleus. Moreover, P2 is shown to be a suppressor of PTGS and TGS, which can not only repress ssRNA-induced gene silencing but also disrupt the host RNA-directed DNA methylation (RdDM) pathway. In addition, P2 can trigger an oxidative burst and cause typical hypersensitive-like response (HLR) necrosis in systemic leaves of N. benthamiana when expressed by PVX. Overall, our results laid a foundation for deciphering the molecular mechanism of SCBV pathogenesis and made progress for resistance breeding.

Screening and Identification of Host Factors Interacting with the Virulence Factor P0 Encoded by Sugarcane Yellow Leaf Virus by Yeast Two-Hybrid Assay.

Sugarcane yellow leaf virus (SCYLV), a member of the genus Polerovirus in the family Luteoviridae, causes severe damage and represents a great threat to sugarcane cultivation and sugar industry development. In this study, inoculation of Nicotiana benthamiana plants with a potato virus X (PVX)-based vector carrying the SCYLV P0 gene induced typical mosaic, leaf rolling symptoms and was associated with a hypersensitive-like response (HLR) necrosis symptom, which is accompanied with a systemic burst of H2O2 and also leads to higher PVX viral genome accumulation levels. Our results demonstrate that SCYLV P0 is a pathogenicity determinant and plays important roles in disease development. To further explore its function in pathogenic processes, a yeast two-hybrid assay was performed to screen the putative P0-interacting host factors. The recombinant plasmid pGBKT7-P0 was constructed as a bait and transformed into the yeast strain Y2HGold. The ROC22 cultivar (an important parental resource of the main cultivar in China) cDNA prey library was constructed and screened by co-transformation with the P0 bait. We identified 28 potential interacting partners including those involved in the optical signal path, plant growth and development, transcriptional regulation, host defense response, and viral replication. To our knowledge, this is the first time we have reported the host proteins interacting with the P0 virulence factor encoded by sugarcane yellow leaf virus. This study not only provides valuable insights into elucidating the molecular mechanism of the pathogenicity of SCYLV, but also sheds light on revealing the probable new pathogenesis of Polerovirus in the future.

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 Šresolution 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.

Betasatellites associated with geminiviruses can be replicated promiscuously by distinct geminiviruses but exhibit a preference for cognate helper viruses. However, the cis elements 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 and in vivo replication. 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.IMPORTANCE The geminivirus-encoded replication initiator protein (Rep) binds to repeated sequence elements (also known as iterons) in the origin of replication that serve as essential cis elements for specific viral replication. Betasatellites associated with begomoviruses can be replicated by cognate or noncognate helper viruses, but the cis elements 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.

Gene Expression Profiling Shows That NbFDN1 Is Involved in Modulating the Hypersensitive Response-Like Cell Death Induced by the Oat dwarf virus RepA Protein.

In this study, we used high-throughput deep nucleotide sequencing to characterize the global transcriptional response of Nicotiana benthamiana plants to transient expression of the RepA protein from Oat dwarf virus (ODV). We identified 7,878 significantly differentially expressed genes (DEG) that mapped to 125 pathways, suggesting that comprehensive networks are involved in regulation of RepA-induced cell death. Of the 202 DEG associated with photosynthesis, expression of 195 was found to be downregulated, indicating a significant inhibition of photosynthesis in response to RepA expression, which is associated with chloroplast disruption and physiological changes. We focused our analysis on NbFDN1, a member of the ferredoxin protein family that participates in the chloroplast electron transport chain performing oxygenic photosynthesis, which was identified to directly interact with NbTsip1. We separately knocked down the expression of NbFDN1 and NbTsip1 using virus-induced gene silencing, and found that NbFDN1 silencing speeded up the development of RepA-induced cell death, unlike NbTsip1 silencing, which showed an opposite effect on RepA-induced response. Further study showed increased H2O2 accumulation and a negative correlation between the transcripts of NbFDN1 and NbTsip1 in NbFDN1-silenced plants. Hence, we speculate that NbFDN1 has an effect on RepA-induced hypersensitive response-like response by modulating NbTsip1 transcription as well as H2O2 production.

A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana.

A recently characterized calmodulin-like protein is an endogenous RNA silencing suppressor that suppresses sense-RNA induced post-transcriptional gene silencing (S-PTGS) and enhances virus infection, but the mechanism underlying calmodulin-like protein-mediated S-PTGS suppression is obscure. Here, we show that a calmodulin-like protein from Nicotiana benthamiana (NbCaM) interacts with Suppressor of Gene Silencing 3 (NbSGS3). Deletion analyses showed that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and NbCaM suppressor activity. Overexpression of NbCaM reduced the number of NbSGS3-associated granules by degrading NbSGS3 protein accumulation in the cytoplasm. This NbCaM-mediated NbSGS3 degradation was sensitive to the autophagy inhibitors 3-methyladenine and E64d, and was compromised when key autophagy genes of the phosphatidylinositol 3-kinase (PI3K) complex were knocked down. Meanwhile, silencing of key autophagy genes within the PI3K complex inhibited geminivirus infection. Taken together these data suggest that NbCaM acts as a suppressor of RNA silencing by degrading NbSGS3 through the autophagy pathway.

A Novel DNA Motif Contributes to Selective Replication of a Geminivirus-Associated Betasatellite by a Helper Virus-Encoded Replication-Related Protein.

UNLABELLED: Rolling-circle replication of single-stranded genomes of plant geminiviruses is initiated by sequence-specific DNA binding of the viral replication-related protein (Rep) to its cognate genome at the replication origin. Monopartite begomovirus-associated betasatellites can be trans replicated by both cognate and some noncognate helper viruses, but the molecular basis of replication promiscuity of betasatellites remains uncharacterized. Earlier studies showed that when tomato yellow leaf curl China virus (TYLCCNV) or tobacco curly shoot virus (TbCSV) is coinoculated with both cognate and noncognate betasatellites, the cognate betasatellite dominates over the noncognate one at the late stages of infection. In this study, we constructed reciprocal chimeric betasatellites between tomato yellow leaf curl China betasatellite and tobacco curly shoot betasatellite and assayed their competitiveness against wild-type betasatellite when coinoculated with TYLCCNV or TbCSV onto plants. We mapped a region immediately upstream of the conserved rolling-circle cruciform structure of betasatellite origin that confers the cognate Rep-mediated replication advantage over the noncognate satellite. DNase I protection and in vitro binding assays further identified a novel sequence element termed Rep-binding motif (RBM), which specifically binds to the cognate Rep protein and to the noncognate Rep, albeit at lower affinity. Furthermore, we showed that RBM-Rep binding affinity is correlated with betasatellite replication efficiency in protoplasts. Our data suggest that although strict specificity of Rep-mediated replication does not exist, betasatellites have adapted to their cognate Reps for efficient replication during coevolution. IMPORTANCE: Begomoviruses are numerous circular DNA viruses that cause devastating diseases of crops worldwide. Monopartite begomoviruses are frequently associated with betasatellites which are essential for induction of typical disease symptoms. Coexistence of two distinct betasatellites with one helper virus is rare in nature. Our previous research showed that begomoviruses can trans replicate cognate betasatellites to higher levels than noncognate ones. However, the molecular mechanisms of betasatellites selective replication remain largely unknown. We investigated the interaction between the begomovirus replication-associated protein and betasatellite DNA. We found that the replication-associated protein specifically binds to a motif in betasatellites, with higher affinity for the cognate motif than the noncognate motif. This preference for cognate motif binding determines the selective replication of betasatellites. We also demonstrated that this motif is essential for betasatellite replication. These findings shed new light on the promiscuous yet selective replication of betasatellites by helper geminiviruses.

The AC5 protein encoded by Mungbean yellow mosaic India virus is a pathogenicity determinant that suppresses RNA silencing-based antiviral defenses.

It is generally accepted that begomoviruses in the family Geminiviridae encode four proteins (from AC1/C1 to AC4/C4) using the complementary-sense DNA as template. Although AC5/C5 coding sequences are increasingly annotated in databases for many begomoviruses, the evolutionary relationships and functions of this putative protein in viral infection are obscure. Here, we demonstrate several important functions of the AC5 protein of a bipartite begomovirus, Mungbean yellow mosaic India virus (MYMIV). Mutational analyses and transgenic expression showed that AC5 plays a critical role in MYMIV infection. Ectopic expression of AC5 from a Potato virus X (PVX) vector resulted in severe mosaic symptoms followed by a hypersensitive-like response in Nicotiana benthamiana. Furthermore, MYMIV AC5 effectively suppressed post-transcriptional gene silencing induced by single-stranded but not double-stranded RNA. AC5 was also able to reverse transcriptional gene silencing of a green fluorescent protein transgene by reducing methylation of promoter sequences, probably through repressing expression of a CHH cytosine methyltransferase (DOMAINS REARRANGED METHYLTRANSFERASE2) in N. benthamiana. Our results demonstrate that MYMIV AC5 is a pathogenicity determinant and a potent RNA silencing suppressor that employs novel mechanisms to suppress antiviral defenses, and suggest that the AC5 function may be conserved among many begomoviruses.