Soybean Mosaic Virus 6K1 Interactors Screening and GmPR4 and GmBI1 Function Characterization.

Host proteins are essential during virus infection, and viral factors must target numerous host factors to complete their infectious cycle. The mature 6K1 protein of potyviruses is required for viral replication in plants. However, the interaction between 6K1 and host factors is poorly understood. The present study aims to identify the host interacting proteins of 6K1. Here, the 6K1 of Soybean mosaic virus (SMV) was used as the bait to screen a soybean cDNA library to gain insights about the interaction between 6K1 and host proteins. One hundred and twenty-seven 6K1 interactors were preliminarily identified, and they were classified into six groups, including defense-related, transport-related, metabolism-related, DNA binding, unknown, and membrane-related proteins. Then, thirty-nine proteins were cloned and merged into a prey vector to verify the interaction with 6K1, and thirty-three of these proteins were confirmed to interact with 6K1 by yeast two-hybrid (Y2H) assay. Of the thirty-three proteins, soybean pathogenesis-related protein 4 (GmPR4) and Bax inhibitor 1 (GmBI1) were chosen for further study. Their interactions with 6K1 were also confirmed by bimolecular fluorescence complementation (BiFC) assay. Subcellular localization showed that GmPR4 was localized to the cytoplasm and endoplasmic reticulum (ER), and GmBI1 was located in the ER. Moreover, both GmPR4 and GmBI1 were induced by SMV infection, ethylene and ER stress. The transient overexpression of GmPR4 and GmBI1 reduced SMV accumulation in tobacco, suggesting their involvement in the resistance to SMV. These results would contribute to exploring the mode of action of 6K1 in viral replication and improve our knowledge of the role of PR4 and BI1 in SMV response.

GmGSTU13 Is Related to the Development of Mosaic Symptoms in Soybean Plants Infected with Soybean Mosaic Virus.

The leaves of soybean cultivar ZheA8901 show various symptoms (necrosis, mosaic, and symptomless) when infected with different strains of soybean mosaic virus (SMV). Based on a proteomic analysis performed with tandem mass tags (TMT), 736 proteins were differentially expressed from soybean samples that showed asymptomatic, mosaic, and necrosis symptoms induced by SMV strains SC3, SC7, and SC15, respectively. Among these, GmGSTU13 and ascorbate peroxidase (APX) were only upregulated in mosaic and symptomless leaves, respectively. The protein level of GmGSTU13 determined by western blot analysis was consistent with TMT analysis, and quantitative reverse transcriptase PCR analysis showed that GmGSTU13 mRNA levels in mosaic plants were 5.26- and 3.75-fold higher than those in necrotic and symptomless plants, respectively. Additionally, the expression of the viral coat protein (CP) gene was increased, and serious mosaic symptoms were observed in GmGSTU13-overexpressing plants inoculated with all three SMV strains. These results showed that GmGSTU13 is associated with the development of SMV-induced mosaic symptoms in soybean and that APX is upregulated in symptomless leaves at both the transcriptional and protein levels. In APX gene-silenced soybean plants, the relative expression of the viral CP gene was 1.50, 7.59, and 1.30 times higher than in positive control plants inoculated with the three SMV strains, suggesting that the upregulation of APX may be associated with lack of symptoms in soybean infected with SMV. This work provides a useful dataset for identifying key proteins responsible for symptom development in soybean infected with different SMV strains.

The 40S Ribosomal Protein S6 Response to Blue Light by Interaction with SjAUREO in Saccharina japonica.

Blue light (BL) plays an important role in regulation of the growth and development of aquatic plants and land plants. Aureochrome (AUREO), the recent BL photoreceptor identified in photosynthetic stramenopile algae, is involved in the photomorphogenesis and early development of Saccharina japonica porophytes (kelp). However the factors that interact with the SjAUREO under BL conditions specifically are not clear. Here in our study, three high quality cDNA libraries with CFU over 5 × 106 and a recombination rate of 100% were constructed respectively through white light (WL), BL and darkness (DK) treatments to the juvenile sporophytes. Based on the constructed cDNA libraries, the interactors of SjAUREO were screened and analyzed. There are eighty-four genes encoding the sixteen predicted proteins from the BL cDNA library, sixty-eight genes encoding eighteen predicted proteins from the DK cDNA library, and seventy-four genes encoding nineteen proteins from the WL cDNA library. All the predicted proteins are presumed to interact with SjAUREO when co-expressed with SjAUREO seperately. The 40S ribosomal protein S6 (RPS6), which only exists in the BL treated cDNA library except for two other libraries, and which is essential for cell proliferation and is involved in cell cycle progression, was selected for detailed analysis. We showed that its transcription was up-regulated by BL, and was highly transcribed in the basal blade (meristem region) of juvenile sporophytes but less in the distal part. Taken together, our results indicated that RPS6 was highly involved in BL-mediated kelp cellular division and photomorphogenesis by interacting with SjAUREO.

Comprehensive Analysis of Soybean Mosaic Virus P3 Protein Interactors and Hypersensitive Response-Like Lesion-Inducing Protein Function.

Soybean mosaic virus (SMV) is one of the most prevalent and important pathogens of soybean, which produces 11 proteins, and the third protein, P3, was suggested to be involved in virus movement and replication, as well as host infection. During the virus infection, host proteins are essential in the virus cycle. However, there is no comprehensive report on the network of host proteins that interact with P3. Fifty-one interactors were identified by using the P3 protein as the bait against the SMV SC15 strain-challenged soybean cDNA library. These proteins were classified into five groups, including transport and protein transport-related proteins, defense and disease-related proteins, photosynthesis proteins, cellular metabolic proteins, and unknown proteins. Among these proteins, the protein defined as hypersensitive response-like lesion-inducing (HRLI) appeared multiple times and showed strong affinity with P3, which indicated its important role in SMV infection. Thus, it was chosen for further investigation. Phylogenetic classification showed that paralog proteins GmHRLI-1 and GmHRLI-2 clustered together and shared 90% homologous identity. Bimolecular fluorescence complementation (BiFC) assay was carried out to confirm the interaction, and fluorescence was detected at the cell periplasmic as well as at the nucleus. Subcellular localization showed that GmHRLI was localized to the cell periplasmic, while the co-localization of GmHRLI and P3 signals was also observed in the nucleus, suggesting that GmHRLI could interact with P3 and promoted the translation of P3 to the nucleus. Moreover, the gene expression of GmHRLI was abundant in the roots, leaves, and flowers, and could be induced by SMV infection, suggesting its involvement in SMV infection. Our results together lay the foundation to explore the mechanisms of P3 in the HR process and the HRLI protein function in SMV response.

The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis.

The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other's nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection.

Soybean actin-depolymerizing factor 2 interacts with Soybean mosaic virus-encoded P3 protein.

Soybean mosaic virus (SMV), a member of the Potyvirus genus, is one of the most prevalent and devastating viral pathogens in soybean-growing regions worldwide. It is generally accepted that symptom development of a viral plant disease results from molecular interactions between the virus and its host plant. P3 protein is the most variable polyprotein in potyviruses, which potentially plays an important role in the process of the evolution of virus type specialization. However, P3 not only plays a major role in virus replication and movement, but it is also responsible for symptom development in SMV-infected plants. This study provides evidence that actin-depolymerizing factor 2 (designated as ADF2) of soybean interacts with SMV P3 via a two-hybrid yeast system by screening a soybean cDNA library. Bimolecular fluorescence complementation assay further confirmed the interaction, which occurred in both the cytomembrane and cytoskeleton of Nicotiana benthamiana cells. The results support the hypothesis that SMV P3 might have a role in virus movement within cells.

GmCYB5-4 inhibit SMV proliferation by targeting P3 protein.

Soybean mosaic virus (SMV) is a potyvirus found worldwide in soybean (Glycine max). GmCYB5-4 is a strong candidate interactor of P3. In this study, we comprehensively analyzed the GmCYB5 family in soybeans, including its distribution on chromosomes, promoter analysis, conserved motifs, phylogenetic analysis, and expression patterns. We cloned the full-length GmCYB5-4 and examined its interaction with P3 in yeast, which was later confirmed using bimolecular fluorescence complementation (BiFc). We silenced GmCYB5-4 using a bean pottle mosaic viris (BPMV) based system to generate SilCYB5-4 tissues, which surprisingly knocked down four isoforms of GmCYB5s for functional characterization. SilCYB5-4 plants were challenged with the SC3 strain to determine its involvement in SMV infection. Silencing GmCYB5-4 increased SMV accumulation, indicating that GmCYB5-4 inhibited SMV proliferation. However, further experiments are needed to elucidate the mechanism underlying the involvement of GmCYB5-4 in SMV infection.

Soybean 40S Ribosomal Protein S8 (GmRPS8) Interacts with 6K1 Protein and Contributes to Soybean Susceptibility to Soybean Mosaic Virus.

Soybean mosaic virus (SMV), a member of Potyvirus, is the most destructive and widespread viral disease in soybean production. Our earlier studies identified a soybean 40S ribosomal protein S8 (GmRPS8) using the 6K1 protein of SMV as the bait to screen a soybean cDNA library. The present study aims to identify the interactions between GmRPS8 and SMV and characterize the role of GmRPS8 in SMV infection in soybean. Expression analysis showed higher SMV-induced GmRPS8 expression levels in a susceptible soybean cultivar when compared with a resistant cultivar, suggesting that GmRPS8 was involved in the response to SMV in soybean. Subcellular localization showed that GmRPS8 was localized in the nucleus. Moreover, the yeast two-hybrid (Y2H) experiments showed that GmRPS8 only interacted with 6K1 among the eleven proteins encoded by SMV. The interaction between GmRPS8 and 6K1 was further verified by a bimolecular fluorescence complementation (BiFC) assay, and the interaction was localized in the nucleus. Furthermore, knockdown of GmRPS8 by a virus-induced gene silencing (VIGS) system retarded the growth and development of soybeans and inhibited the accumulation of SMV in soybeans. Together, these results showed that GmRPS8 interacts with 6K1 and contributes to soybean susceptibility to SMV. Our findings provide new insights for understanding the role of GmRPS8 in the SMV infection cycle, which could help reveal potyviral replication mechanisms.

Transgenic plant generated by RNAi-mediated knocking down of soybean Vma12 and soybean mosaic virus resistance evaluation.

Soybean mosaic virus (SMV) is one of the most destructive viral diseases in soybean and causes severe reduction of soybean yield and destroys the seed quality. However, the production of SMV resistant plants by transgenic is the most effective and economical means. Based on our previous yeast two-hybrid assay, the GmVma12 was selected as a strong candidate gene for further function characterization. Here we transformed soybean plants with a construct containing inverted repeat of-GmVma12 sequence to analyze the role of GmVma12 during SMV invasion. Totals of 33 T0 and 160 T1 plants were confirmed as positive transgenic plants through herbicide application, PCR detection and LibertyLink® strip screening. Based on the segregation ratio and Southern Blot data, T1 lines No. 3 and No. 7 were selected to generate T2 plants. After SMV-SC15 inoculation, 41 T1 and 38 T2 plants were identified as highly resistant, and their quantification disease levels were much lower than non-transformed plants. The transcript level of GmVma12 in T2 plants decreased to 70% of non-transformed plants. The expression level of SMV-CP transcript in T2 transgenic plants was lower than that in non-transformed plants and SMV CP protein in T2 plants could not be detected by Enzyme-linked Immunosorbent assay, which indicated that SMV production would be inhibited in transgenic plants. Moreover, coat mottles of T2 seeds were obliterated significantly. In conclusion, inverted repeat of the hairpin structure of GmVma12 interfered with the transcription of GmVma12, which can induce resistance to SMV in soybean. This research lays the foundation for the mechanism of SMV pathogenesis, and provides new ideas for SMV prevention and control.

Soybean Cytochrome b5 Is a Restriction Factor for Soybean Mosaic Virus.

Soybean mosaic virus (SMV) is one of the most destructive viral diseases in soybeans (Glycine max). In this study, an interaction between the SMV P3 protein and cytochrome b5 was detected by yeast two-hybrid assay, and bimolecular fluorescence complementation assay showed that the interaction took place at the cell periphery. Further, the interaction was confirmed by co-immunoprecipitation analysis. Quantitative real-time polymerase chain reaction analysis revealed that GmCYB5 gene was differentially expressed in resistant and susceptible soybean plants after inoculation with SMV-SC15 strain. To test the involvement of this gene in SMV resistance, the GmCYB5 was silenced using a bean pod mottle virus (BPMV)-based vector construct. Results showed that GmCYB5-1 was 83% and 99% downregulated in susceptible (NN1138-2) and resistant (RN-9) cultivars, respectively, compared to the empty vector-treated plants. Silencing of GmCYB5 gene promotes SMV replication in soybean plants. Our results suggest that during SMV infection, the host CYB5 protein targets P3 protein to inhibit its proliferation. Taken together, these results suggest that CYB5 is an important factor in SMV infection and replication in soybeans, which could help soybean breeders develop SMV resistant soybean cultivars.