Soybean Mosaic Virus Strains Research Articles

A comprehensive analysis of the WRKY family in soybean and functional analysis of GmWRKY164-GmGSL7c in resistance to soybean mosaic virus

BackgroundSoybean mosaic disease caused by soybean mosaic virus (SMV) is one of the most devastating and widespread diseases in soybean producing areas worldwide. The WRKY transcription factors (TFs) are widely involved in plant development and stress responses. However, the roles of the GmWRKY TFs in resistance to SMV are largely unclear.ResultsHere, 185 GmWRKYs were characterized in soybean (Glycine max), among which 60 GmWRKY genes were differentially expressed during SMV infection according to the transcriptome data. The transcriptome data and RT-qPCR results showed that the expression of GmWRKY164 decreased after imidazole treatment and had higher expression levels in the incompatible combination between soybean cultivar variety Jidou 7 and SMV strain N3. Remarkably, the silencing of GmWRKY164 reduced callose deposition and enhanced virus spread during SMV infection. In addition, the transcript levels of the GmGSL7c were dramatically lower upon the silencing of GmWRKY164. Furthermore, EMSA and ChIP-qPCR revealed that GmWRKY164 can directly bind to the promoter of GmGSL7c, which contains the W-box element.ConclusionOur findings suggest that GmWRKY164 plays a positive role in resistance to SMV infection by regulating the expression of GmGSL7c, resulting in the deposition of callose and the inhibition of viral movement, which provides guidance for future studies in understanding virus-resistance mechanisms in soybean.

Identification of soybean mosaic virus strain SC7 resistance loci and candidate genes in soybean [Glycine max (L.) Merr.

Soybean [Glycine max (L.) Merr.] is an important legume crop worldwide, which provides abundant plant protein and oil for human beings. Soybean mosaic virus (SMV) can cause serious damage to the yield and quality of soybean, but it is difficult to control SMV with chemicals, breeding SMV-resistant varieties has become the most effective way to control the disease. Therefore, it is important to identify SMV resistance genes from soybean resources and apply them to soybean breeding. In this study, the disease rates (DRs) of 219 soybean accessions to SMV strain SC7 in two environments were investigated. A high-density NJAU 355K SoySNP array was used for genome-wide association study (GWAS) of DR. A 274kb region on chromosome 15 (1,110,567bp to 1,384,173bp) was repeatedly detected in two environments. Six new significant single nucleotide polymorphisms (SNPs) on chromosome 15 were identified. Four of these six SNPs were located within two candidate genes, Glyma.15G015700 and Glyma.15G015800. The elite haplotype Glyma.15G015700Hap I with low DR exhibited strong resistance to SC7. The expression of Glyma.15G015700 in the SMV-resistant accession increased significantly after inoculation with SC7. Furthermore, most of the proteins predicted to interact with Glyma.15G015700 are heat shock proteins, which have been shown to be related to disease resistance. In summary, new SMV resistance loci and a new candidate gene, Glyma.15G015700, were identified and might be utilized in further soybean disease resistance breeding.

GmTOC1b negatively regulates resistance to Soybean mosaic virus

Soybean (Glycine max) is a major oil and feed crop worldwide. Soybean mosaic virus (SMV) is a globally occurring disease that severely reduces the yield and quality of soybean. Here, we characterized the role of the clock gene TIMING OF CAB EXPRESSION 1b (GmTOC1b) in the resistance of soybean to SMV. Homozygous Gmtoc1b mutants exhibited increased tolerance to SMV strain SC3 due to the activation of programmed cell death triggered by a hypersensitive response. Transcriptome deep sequencing and RT-qPCR analysis suggested that GmTOC1b likely regulates the expression of target genes involved in the salicylic acid (SA) signaling pathway. GmTOC1b binds to the promoter of GmWRKY40, which encodes a protein that activates the expression of SA-mediated defense-related genes. Moreover, we revealed that the GmTOC1bH1 haplotype, which confers increased tolerance to SMV, was artificially selected in improved cultivars from the Northern and Huang-Huai regions of China. Our results therefore identify a previously unknown SMV resistance component that could be deployed in the molecular breeding of soybean to enhance SMV resistance.

RSC3 K of soybean cv. Kefeng No.1 confers resistance to soybean mosaic virus by interacting with the viral protein P3.

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 RSC3 K) 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 RSC3 K, LOC100526921, and LOC100812666. The recombinant RSC3 K 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 RSC3 K 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.

Characterization and allelism of soybean mosaic virus strain SC3 resistance in ‘Qihuang‐1’ derived soybean cultivars

AbstractSoybean mosaic virus is a severe constraint of soybean production in China. A total of country‐wide 22 SMV strains (SC1‐SC22) were identified. Of these, SC3 is a major strain widely distributed in Huanghuai and Yangtze River Valley region of China. Soybean cultivar ‘Qihuang‐1’ contains RSC3Q locus conditioning the resistance to SC3 and is an important parental line extensively used to breed the soybean cultivars in China. The objective of this study was to elucidate the genetic pattern of SC3 resistance genes in cultivars developed from ‘Qihuang‐1’ or its derivative lines. Hence, we have evaluated the SC3 resistance in 91 cultivars developed from ‘Qihuang‐1’ or its derivative lines. The results showed that a total of 43 cultivars exhibited resistance to the SC3 strain. Among them, 37 cultivars were derived from ‘Qihuang‐1’. Then, we have detected the RSC3Q loci in these cultivars using four SSR markers (Satt334, Sct_033, BARCSOYSSR_13_1114 and BARCSOYSSR_13_1136). It revealed that, among the 37 resistant cultivars derived from ‘Qihuang‐1’, there are 20 cultivars containing RSC3Q loci. Moreover, the allelic relationship of resistance genes was analysed using the crosses from resistance × resistance between ‘Qihuang‐1’ and its resistant derived cultivars. The results showed that the resistance genes of ‘Qihuang‐1’ and its 20 cultivars were allelic. But it is not allelic with those of the other 17 cultivars, different from ‘Qihuang‐1’, and also, RSC3Q does not condition the resistance. These results will be beneficial to exploring the transmission of resistance genes of ‘Qihuang‐1’ and will be useful to the disease resistance breeding of soybean.

Fine Mapping the Soybean Mosaic Virus Resistance Gene in Soybean Cultivar Heinong 84 and Development of CAPS Markers for Rapid Identification.

Heinong 84 is one of the major soybean varieties growing in Northeast China, and is resistant to the infection of all strains of soybean mosaic virus (SMV) in the region including the most prevalent strain, N3. However, the resistance gene(s) in Heinong 84 and the resistant mechanism are still elusive. In this study, genetic and next-generation sequencing (NGS)-based bulk segregation analysis (BSA) were performed to map the resistance gene using a segregation population from the cross of Heinong 84 and a susceptible cultivar to strain N3, Zhonghuang 13. Results show that the resistance of Heinong 84 is controlled by a dominant gene on chromosome 13. Further analyses suggest that the resistance gene in Heinong 84 is probably an allele of Rsv1. Finally, two pairs of single-nucleotide-polymorphism (SNP)-based primers that are tightly cosegregated with the resistance gene were designed for rapidly identifying resistant progenies in breeding via the cleaved amplified polymorphic sequence (CAPS) assay.

Rapid and visual detection of soybean mosaic virus SC7 with a loop-mediated isothermal amplification strategy

Soybean mosaic virus (SMV) is the most prevalent viral pathogen of soybean worldwide that reduces seed quality and causes serious losses in yields. The accurate, rapid and early detection of SMV strains is crucial for screening SMV-resistant soybean varieties and controlling soybean mosaic disease timely. In this study, we report a loop-mediated isothermal amplification (LAMP) assay for visual and rapid detection of SMV-SC7, the major prevalent SMV strain in China, in soybean leaves. Specific primers were rationally designed and tested to detect the SMV-SC7 strain. The detection limit for SMV-SC7 can be as low as 10−4 ng/μL by the developed LAMP assay. These results were visualized by three indicators (SYBR green I, neutral red and hydroxy naphthol blue) with high fidelity. The high analytical performance of the LAMP assay was demonstrated with 21 soybean cultivars, among which 8 soybean cultivars were further inoculated with consistent results to those achieved by LAMP. This visual and rapid LAMP assay is highly promising for early detection of SMV-SC7-associated plant diseases.

Deep decoding of codon usage strategies and host adaption preferences of soybean mosaic virus

Soybean mosaic virus (SMV) has threatened the global yield of Leguminosae crops, but the mechanism of its infection, spread, and evolution remains unknown. A systemic analysis of 107 SMV strains was performed to explore the genome-wide codon usage profile and the various factors influencing the codon usage patterns of SMV, which provides insight into its molecular evolution and elucidates its unknown host adaptation pattern. The overall nucleotide composition and correlation analysis revealed that the preferred synonymous codons mostly end with A/U. Clustering by RSCU value of each strain and phylogenetic tree analysis showed that the SMV isolates studied were divided into four clades, with a low overall extent of codon usage bias (CUB) in SMV. According to the ENC, PR2, neutrality plot, and correspondence analysis, natural selection of geographical diversity may play a critical role in the CUB. Higher adaptability was shown in Glycine with SMV and more pressure was received by clade III. These findings could not only provide valuable information about the overall codon usage pattern of the SMV genome, but could also aid in the clarification of the involved mechanisms that dominate the codon usage patterns and genetic evolution of the SMV genome.

Comparative Transcriptome Analyses between Resistant and Susceptible Varieties in Response to Soybean Mosaic Virus Infection

Soybean mosaic virus (SMV) is a worldwide and hardly controlled virus disease in soybean. Kefeng-1 is an elite variety resistant to SMV in China. In order to discover resistance genes and regulation networks in Kefeng-1, we analyzed transcriptome data of resistant (Kefeng-1) and susceptible (NN1138-2) soybean varieties in response to infection of the SMV strain SC18 at 0, 6, and 48 hours post-inoculation (hpi) and 5 days post-inoculation (dpi). Many differentially expressed genes (DEGs) were identified with Kefeng-1 and NN 1138-2. Based on the enrichment analysis for gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, we found that 48 hpi was the best time point for the defense response of the two soybean varieties in response to the SMV infection. The expression of seven candidate genes was further verified by qRT-PCR and was relatively consistent with the results of RNA-Seq. The expression of genes for Glyma.11G239000 and Glyma.18G018400, members of the ethylene-insensitive 3/ethylene-insensitive3-like (EIN3/EIL) protein family involved in ETH, were downregulated in NN1138-2 but not in Kefeng-1 and the expression of Glyma.14G041500 was upregulated in Kefeng-1 at 5 dpi. The expression of jasmonic acid repressor genes (TIFY/JAZ) was downregulated in NN1138-2 but not in Kefeng-1. NPR1 involved in the salicylic acid signaling pathway was downregulated in NN1138-2 at 48 hpi but upregulated in Kefeng-1. It shows that ethylene, jasmonic acid, and salicylic acid signaling pathways may be involved in the disease resistance process to the SMV strain SC18. Our findings would help to understand the molecular mechanism of soybean resistance to SMV.

Decades of Genetic Research on Soybean mosaic virus Resistance in Soybean.

This review summarizes the history and current state of the known genetic basis for soybean resistance to Soybean mosaic virus (SMV), and examines how the integration of molecular markers has been utilized in breeding for crop improvement. SVM causes yield loss and seed quality reduction in soybean based on the SMV strain and the host genotype. Understanding the molecular underpinnings of SMV–soybean interactions and the genes conferring resistance to SMV has been a focus of intense research interest for decades. Soybean reactions are classified into three main responses: resistant, necrotic, or susceptible. Significant progress has been achieved that has greatly increased the understanding of soybean germplasm diversity, differential reactions to SMV strains, genotype–strain interactions, genes/alleles conferring specific reactions, and interactions among resistance genes and alleles. Many studies that aimed to uncover the physical position of resistance genes have been published in recent decades, collectively proposing different candidate genes. The studies on SMV resistance loci revealed that the resistance genes are mainly distributed on three chromosomes. Resistance has been pyramided in various combinations for durable resistance to SMV strains. The causative genes are still elusive despite early successes in identifying resistance alleles in soybean; however, a gene at the Rsv4 locus has been well validated.

Mapping Locus RSC11K and predicting candidate gene resistant to Soybean mosaic virus strain SC11 through linkage analysis combined with genome resequencing of the parents in soybean

Soybean mosaic virus (SMV) strain SC11 was prevalent in middle China. Its resistance was controlled by a Mendelian single dominant gene RSC11K in soybean Kefeng-1. This study aimed at mapping RSC11K and identifying its candidate gene. RSC11K locus was mapped ~217 kb interval between two SNP-linkage-disequilibrium-blocks (Gm02_BLOCK_11273955_11464884 and Gm02_BLOCK_11486875_11491354) in W82.a1.v1 genome using recombinant inbred lines population derived from Kefeng-1 (Resistant) × NN1138-2 (Susceptible), but inserted with a ~245 kb segment in W82.a2.v1 genome. In the entire 462 kb RSC11K region, 429 SNPs, 142 InDels and 34 putative genes were identified with more SNPs/InDels distributed in non-functional regions. Thereinto, ten genes contained SNP/InDel variants with high and moderate functional impacts on proteins, among which Glyma.02G119700 encoded a typical innate immune receptor-like kinase involving in virus disease process and responded to SMV inoculation, therefore was recognized as RSC11K's candidate gene. The novel RSC11K locus and candidate genes may help developing SMV resistance germplasm.

Identifying Quantitative Trait Loci and Candidate Genes Conferring Resistance to Soybean Mosaic Virus SC7 by Quantitative Trait Loci-Sequencing in Soybean.

Soybean mosaic virus (SMV) is detrimental to soybean (Glycine max) breeding, seed quality, and yield worldwide. Improving the basic resistance of host plants is the most effective and economical method to reduce damage from SMV. Therefore, it is necessary to identify and clone novel SMV resistance genes. Here, we report the characterization of two soybean cultivars, DN50 and XQD, with different levels of resistance to SMV. Compared with XQD, DN50 exhibits enhanced resistance to the SMV strain SC7. By combining bulked-segregant analysis (BSA)-seq and fine-mapping, we identified a novel resistance locus, RSMV-11, spanning an approximately 207-kb region on chromosome 11 and containing 25 annotated genes in the reference Williams 82 genome. Of these genes, we identified eleven with non-synonymous single-nucleotide polymorphisms (SNPs) or insertion-deletion mutations (InDels) in their coding regions between two parents. One gene, GmMATE68 (Glyma.11G028900), harbored a frameshift mutation. GmMATE68 encodes a multidrug and toxic compound extrusion (MATE) transporter that is expressed in all soybean tissues and is induced by SC7. Given that MATE transporter families have been reported to be linked with plant disease resistance, we suggest that GmMATE68 is responsible for SC7 resistance in DN50. Our results reveal a novel SMV-resistance locus, improving understanding of the genetics of soybean disease resistance and providing a potential new tool for marker-assisted selection breeding in soybean.

Fine mapping and genetic analysis of resistance genes, Rsc18, against soybean mosaic virus

Soybean mosaic virus (SMV) affects seed quality and production of soybean (Glycine max (L.) Merr.) worldwide. SC18 is one of the dominant SMV strains in South China, and accession Zhonghuang 24 displayed resistance to SC18. The F1, F2 and 168 F11 recombinant inbred lines (RILs) population derived from a hybridization between Zhonghuang 24 (resistant, R) and Huaxia 3 (susceptible, S) were used in this study. According to the segregation ratios of the F2 generation (3R:1S) and the recombinant inbred lines (RILs) population (1R:1S), one dominant locus may regulate the resistance to SC18 in Zhonghuang 24. By using composite interval mapping (CIM), Rsc18 was mapped to a 415.357-kb region on chromosome 13. Three candidate genes, including one NBS-LRR type gene and two serine/threonine protein type genes, were identified according to the genetic annotations, which may be related to the resistance to SC18. The qRT-PCR demonstrated that these genes were up-regulated in the R genotype compared to the control. In conclusion, the findings of this research enhanced the understanding about the R genes at the Rsc18 locus. Moreover, our results will provide insights for designing molecular markers to improve marker-assisted selection and developing new varieties with resistance to SC18.

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.

Pathogenicity and genome-wide sequence analysis reveals relationships between soybean mosaic virus strains.

Soybean mosaic virus (SMV) is the most prevalent viral pathogen in soybean. In China, the SMV strains SC and N are used simultaneously in SMV resistance assessments of soybean cultivars, but the pathogenic relationship between them is unclear. In this study, SMV strains N1 and N3 were found to be the most closely related to SC18. Moreover, N3 was found to be more virulent than N1. A global pathotype classification revealed the highest level of genetic diversity in China. The N3 type was the most frequent and widespread worldwide, implying that SMV possibly originated in China and spread across continents through the dissemination of infected soybean. It also suggests that the enhanced virulence of N3 facilitated its spread and adaptability in diverse geographical and ecological regions worldwide. Phylogenetic analysis revealed prominent geographical associations among SMV strains/isolates, and genomic nucleotide diversity analysis and neutrality tests demonstrated that the whole SMV genome is under negative selection, with the P1 gene being under the greatest selection pressure. The results of this study will facilitate the nationwide use of SMV-resistant soybean germplasm and could provide useful insights into the molecular variability, geographical distribution, phylogenetic relationships, and evolutionary history of SMV around the world.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-021-05271-z.

Photosynthesis-related genes induce resistance against soybean mosaic virus: Evidence for involvement of the RNA silencing pathway.

Increasing lines of evidence indicate that chloroplast‐related genes are involved in plant–virus interactions. However, the involvement of photosynthesis‐related genes in plant immunity is largely unexplored. Analysis of RNA‐Seq data from the soybean cultivar L29, which carries the Rsv3 resistance gene, showed that several chloroplast‐related genes were strongly induced in response to infection with an avirulent strain of soybean mosaic virus (SMV), G5H, but were weakly induced in response to a virulent strain, G7H. For further analysis, we selected the PSaC gene from the photosystem I and the ATP‐synthase α‐subunit (ATPsyn‐α) gene whose encoded protein is part of the ATP‐synthase complex. Overexpression of either gene within the G7H genome reduced virus levels in the susceptible cultivar Lee74 (rsv3‐null). This result was confirmed by transiently expressing both genes in Nicotiana benthamiana followed by G7H infection. Both proteins localized in the chloroplast envelope as well as in the nucleus and cytoplasm. Because the chloroplast is the initial biosynthesis site of defence‐related hormones, we determined whether hormone‐related genes are involved in the ATPsyn‐α‐ and PSaC‐mediated defence. Interestingly, genes involved in the biosynthesis of several hormones were up‐regulated in plants infected with SMV‐G7H expressing ATPsyn‐α. However, only jasmonic and salicylic acid biosynthesis genes were up‐regulated following infection with the SMV‐G7H expressing PSaC. Both chimeras induced the expression of several antiviral RNA silencing genes, which indicate that such resistance may be partially achieved through the RNA silencing pathway. These findings highlight the role of photosynthesis‐related genes in regulating resistance to viruses.

A MADS-box gene is involved in soybean resistance to multiple Soybean mosaic virus strains

Soybean mosaic virus (SMV) is a member of the genus Potyvirus that extensively impairs global soybean production. The full-length coding sequence of the MADS-box transcription factor GmCAL was cloned from the SMV-resistant soybean cultivar Kefeng 1. SMV-induced expression analysis indicated that GmCAL responded quickly to SMV-SC8 infection in Kefeng 1 but not in NN1138-2. GmCAL was expressed at high levels in flowers and pods but at lower levels in leaves. The gene was localized to the nucleus by subcellular localization assay. Virus-induced gene silencing did not increase the accumulation of SMV in GmCAL-silenced Kefeng 1 plants (with silencing efficiency ∼ 80%) after SC8 inoculation. GmCAL-silencing plants still conferred resistance to SC8 that might be owing to incomplete silencing of genes with lower expression. SMV content decreased significantly in GmCAL-overexpressing NN1138-2 plants after SMV-SC3, SMV-SC7, and SMV-SC8 inoculation in comparison with a vector control, showing that overexpression of GmCAL conferred broad-spectrum resistance to multiple SMV strains. These results confirm that GmCAL, a key regulator but not a specific SC8 resistance gene (Rsc8), is a positive regulatory transcription factor involved in soybean resistance to SMV.

Optimizing RNAi-Target by Nicotiana benthamiana-Soybean Mosaic Virus System Drives Broad Resistance to Soybean Mosaic Virus in Soybean.

Soybean mosaic virus (SMV) is a prevalent pathogen of soybean (Glycine max). Pyramiding multiple SMV-resistance genes into one individual is tedious and difficult, and even if successful, the obtained multiple resistance might be broken by pathogen mutation, while targeting viral genome via host-induced gene silencing (HIGS) has potential to explore broad-spectrum resistance (BSR) to SMV. We identified five conserved target fragments (CTFs) from S1 to S5 using multiple sequence alignment of 30 SMV genome sequences and assembled the corresponding target-inverted-repeat constructs (TIRs) from S1-TIR to S5-TIR. Since the inefficiency of soybean genetic transformation hinders the function verification of batch TIRs in SMV-resistance, the Nicotiana benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS pathosystems combined with Agrobacterium-mediated transient expression assays were invented and used to test the efficacy of these TIRs. From that, S1-TIR assembled from 462 bp CTF-S1 with 92% conservation rate performed its best on inhibiting SMV multiplication. Accordingly, S1-TIR was transformed into SMV-susceptible soybean NN1138-2, the resistant-healthy transgenic T1-plants were then picked out via detached-leaf inoculation assay with the stock-plants continued for progeny reproduction (T1 dual-utilization). All the four T3 transgenic progenies showed immunity to all the inoculated 11 SMV strains under individual or mixed inoculation, achieving a strong BSR. Thus, optimizing target for HIGS via transient N. benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS assays is crucial to drive robust resistance to SMV in soybean and the transgenic S1-TIR-lines will be a potential breeding source for SMV control in field.

Overexpression of purple acid phosphatase GmPAP2.1 confers resistance to Soybean mosaic virus in a susceptible soybean cultivar.

A purple acid phosphatase, GmPAP2.1, from the soybean (Glycine max) cultivar L29 may function as a resistance factor acting against specific strains of Soybean mosaic virus (SMV). In this study, we found that overexpression of GmPAP2.1 from L29 conferred SMV resistance to a susceptible cultivar, Lee 74. We determined that GmPAP2.1 interacted with the SMV protein P1 in the chloroplasts, resulting in the up-regulation of the ICS1 gene, which in turn promoted the pathogen-induced salicylic acid (SA) pathway. SA accumulation was elevated in response to the co-expression of GmPAP2.1 and SMV, while transient knockdown of endogenous SA-related genes resulted in systemic infection by SMV strain G5H, suggesting that GmPAP2.1-derived resistance depended on the SA-pathway for the activation of a defense response. Our findings thus suggest that GmPAP2.1 purple acid phosphatase of soybean cultivar L29 functions as an SA-pathway-dependent resistance factor acting against SMV.

A cell wall-localized NLR confers resistance to Soybean mosaic virus by recognizing viral-encoded cylindrical inclusion protein

Soybean mosaic virus (SMV) causes severe yield losses and seed quality reduction in soybean (Glycine max) production worldwide. Rsc4 from cultivar Dabaima is a dominant genetic locus for SMV resistance, and its mapping interval contains three nucleotide-binding domain leucine-rich repeat-containing (NLR) candidates (Rsc4-1, Rsc4-2, and Rsc4-3). The NLR-type resistant proteins were considered as important intracellular pathogen sensors in the previous studies. In this study, based on transient expression assay in Nicotiana benthamiana leaves, we found that the longest transcript of Rsc4-3 is sufficient to confer resistance to SMV, and CRISPR/Cas9-mediated editing of Rsc4-3 in resistant cultivar Dabaima compromised the resistance. Interestingly, Rsc4-3 encodes a cell-wall-localized NLR-type resistant protein. We found that the internal polypeptide region responsible for apoplastic targeting of Rsc4-3 and the putative palmitoylation sites on the N terminus are essential for the resistance. Furthermore, we showed that viral-encoded cylindrical inclusion (CI) protein partially localizes to the cell wall and can interact with Rsc4-3. Virus-driven or transient expression of CI protein of avirulent SMV strains is enough to induce resistance response in the presence of Rsc4-3, suggesting that CI is the avirulent gene for Rsc4-3-mediated resistance. Taken together, our work identified a unique NLR that recognizes plant virus in the apoplast, and provided a simple and effective method for identifying resistant genes against SMV infection.