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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. [ABSTRACT FROM AUTHOR]

Soybean mosaic virus (SMV) is one of the most devastating viral pathogens of soybean (Glycine max (L.) Merr). In total, 22 Chinese SMV strains (SC1–SC22) have been classified based on the responses of 10 soybean cultivars to these pathogens. However, although several SMV‐resistance loci in soybean have been identified, no gene conferring SMV resistance in the resistant soybean cultivar (cv.) Kefeng No.1 has been cloned and verified. Here, using F2‐derived F3 (F2:3) and recombinant inbred line (RIL) populations from a cross between Kefeng No.1 and susceptible soybean cv. Nannong 1138‐2, we localized the gene in Kefeng No.1 that mediated resistance to SMV‐SC3 strain to a 90‐kb interval on chromosome 2. To study the functions of candidate genes in this interval, we performed Bean pod mottle virus (BPMV)‐induced gene silencing (VIGS). We identified a recombinant gene (which we named RSC3K) harboring an internal deletion of a genomic DNA fragment partially flanking the LOC100526921 and LOC100812666 reference genes as the SMV‐SC3 resistance gene. By shuffling genes between infectious SMV DNA clones based on the avirulent isolate SC3 and virulent isolate 1129, we determined that the viral protein P3 is the avirulence determinant mediating SMV‐SC3 resistance on Kefeng No.1. P3 interacts with RNase proteins encoded by RSC3K, LOC100526921, and LOC100812666. The recombinant RSC3K conveys much higher anti‐SMV activity than LOC100526921 and LOC100812666, although those two genes also encode proteins that inhibit SMV accumulation, as revealed by gene silencing in a susceptible cultivar and by overexpression in Nicotiana benthamiana. These findings demonstrate that RSC3K mediates the resistance of Kefeng No.1 to SMV‐SC3 and that SMV resistance of soybean is determined by the antiviral activity of RNase proteins. [ABSTRACT FROM AUTHOR]

Key message: A putative candidate gene conferring resistance to SMV strain SC1 was identified on chromosome 2, and the linked marker was validated in soybean cultivars Soybean mosaic, caused by the soybean mosaic virus, is the most common disease in soybean and a significant impediment to soybean production in the Huanghuai and Yangtze River regions of China. Kefeng No.1, a soybean cultivar, showed high resistance to soybean mosaic virus strain (SC1) collected from Huanghuai and Yangtze River regions. Genetic analysis based on the Mendelian genic population derived from the cross Kefeng No.1 × Nannong 1138-2 revealed that Kefeng No.1 possesses a single dominant gene. Furthermore, genetic fine-mapping using an F2 population containing 281 individuals delimited resistant gene to a genomic region of 186 kb flanked by SSR markers BS020610 and BS020620 on chromosome 2. Within this region, there were 14 genes based on the Williams 82 reference genome. According to sequence analysis, six of the 14 genes have amino acid differences, and one of these genes is the Rsv4 allele designated as Rsc1-DR. The functional analysis of candidate genes using the bean pod mottle virus (BPMV)-induced gene silencing (VIGS) system revealed that Rsc1-DR was accountable for Kefeng No.1's resistance to SMV-SC1. Based on the genome sequence of Rsc1-DR, an Insertion/Deletion (InDel) molecular marker, JT0212, was developed and genotyped using 100 soybean cultivars, and the coincidence rate was 89%. The study enriched our understanding of the SMV resistance mechanism. The marker developed in this study could be directly used by the soybean breeders to select the genotypes with favorable alleles for making crosses, and also it will facilitate marker-assisted selection of SMV resistance in soybean breeding. [ABSTRACT FROM AUTHOR]

Gene silencing induced by hairpin RNA or virus infection expression is one of the major tools in genetics studies in plants. However, when dealing with essential genes, virus-induced gene silencing (VIGS) and transgenic expression of hairpin RNA could lead to plant death, while transient expression of hairpin RNA in leaves is often less competent in downregulating target gene mRNA levels. Here, we developed a transient double-stranded RNA (dsRNA) expression system assisted by a modified viral RNA-dependent RNA polymerase (RdRp) in plant leaves. We show that this system is more effective in inducing gene silencing than the intron-spliced hairpin RNA expression. Furthermore, by using this system, we tested the role of the early secretory pathway during infection of Soybean mosaic potyvirus (SMV). We found that key components of the coat protein complex II vesicles are required for the multiplication of SMV. Overall, this dsRNA-based gene silencing system is effective in downregulating plant gene expression and can be used to identify host genes involved in plant-virus interactions. [ABSTRACT FROM AUTHOR]

The Ferredoxin-NADP reductase (FNR) gene plays a significant role in NADPH production, carbon assimilation, antioxidation, and cross-talking between chloroplasts and mitochondria in plants. This study aims to know the functional response of the soybean FNR gene (GmFNR) during a soybean mosaic virus (SMV) infection. For this purpose, we developed the bean pod mottle virus (BPMV)-based gene construct (BPMV-GmFNR) and used it to silence the GmFNR gene in resistant and susceptible lines. The results showed that GmFNR expression decreased to 50% in the susceptible line, compared to 40% in the resistant line. The silencing of GmFNR reduces the photosynthetic capacity and CAT activity of both lines compared to their respective controls. In addition, the H2O2 content increased significantly in the susceptible line, whereas the resistant line did not exhibit any change. Further, an SMV infection in the silencing plants of the susceptible line resulted in serious morphological changes and increased the SMV NIa-protease transcript accumulation compared to its control plants. However, the same impact was not observed in the resistant line. The yeast two-hybrid system, BIFC assay, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that the GmFNR was interacting with EF1A and coincided with the increased SMV accumulation. The results obtained in this study improve the understanding of the soybean FNR gene response during SMV infection and provide a novel insight into the SMV resistance mechanism. [ABSTRACT FROM AUTHOR]

• Screening of soybean cDNA library by the Quantitative Interactor Screening with the next-generation Sequencing method. • A DnaJ protein (GmCPIP) from soybean interacts with soybean mosaic virus coat protein. • GmCPIP was effectively silenced via virus-induced gene silencing (VIGS) system. • GmCPIP acts as a susceptible factor to facilitate the accumulation of soybean mosaic virus in soybean. Soybean mosaic virus (SMV)-disease is one of the most serious and widespread diseases in soybean (Glycine max). In the present study, a DnaJ protein in soybean designated GmCPIP (SMV coat protein-interacting protein) was screened by the QIS-Seq (quantitative interactor screening with next-generation sequencing) method, and the interaction between SMV CP and GmCPIP was confirmed by the yeast two-hybrid (Y2H) system and bimolecular fluorescence complementation (BiFC) assay. Subcellular localization analysis indicated that both proteins are localized in the cytoplasm, cytomembrane and nucleus. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that infection with SMV-SC4 temporarily increased the transcription of GmCPIP. Virus-induced gene silencing (VIGS) down-regulated the GmCPIP gene by 82%, and the accumulation of SMV was decreased by 88.6% in GmCPIP-silenced plants inoculated with SMV-SC4. The interaction of GmCPIP with SMV CP seems to contribute to SMV infection in soybean. [ABSTRACT FROM AUTHOR]