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.

H_(2)O_(2)mediates transcriptome reprogramming during Soybean mosaic virus-induced callose deposition in soybean

The main defense response to Soybean mosaic virus(SMV)infection in soybean[Glycine max(L.)Merr.]is thought to be blockage of intercellular virus transport by callose deposition on plasmodesmata.But the specific regulatory mechanism remains largely unknown.In this study,we found that hydrogen peroxide(H_(2)O_(2))signal downstream of NO was associated with the regulation of callose accumulation.Abundant H_(2)O_(2)was produced on the cell membrane and cell wall in the incompatible combination of soybean cultivar Jidou 7 and SMV strain N3,whereas no obvious H_(2)O_(2)was observed in the compatible combination of Jidou 7 and strain SC-8.When H_(2)O_(2)production was inhibited,callose accumulation induced by SMV infection decreased to a level insufficient to restrict virus transport in the incompatible combination.The H_(2)O_(2)-associated transcriptome dynamics of soybean during SMV infection was investigated.Transcriptome and functional analysis using virus-induced gene silencing showed that Gm SEOB and Gm PAP27,two genes regulated by H_(2)O_(2),functioned in resistance by positively regulating the accumulation of callose in response to SMV infection.These results lay a foundation for further research on the signal transduction and molecular regulation of callose deposition during soybean resistance to SMV infection.

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 Gm CAL was cloned from the SMV-resistant soybean cultivar Kefeng 1.SMV-induced expression analysis indicated that Gm CAL responded quickly to SMV-SC8 infection in Kefeng 1 but not in NN1138-2.Gm CAL 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 Gm CAL-silenced Kefeng 1 plants(with silencing efficiency~80%)after SC8 inoculation.Gm CAL-silencing plants still conferred resistance to SC8 that might be owing to incomplete silencing of genes with lower expression.SMV content decreased significantly in Gm CAL-overexpressing NN1138-2 plants after SMVSC3,SMV-SC7,and SMV-SC8 inoculation in comparison with a vector control,showing that overexpression of Gm CAL conferred broad-spectrum resistance to multiple SMV strains.These results confirm that Gm CAL,a key regulator but not a specific SC8 resistance gene(Rsc8),is a positive regulatory transcription factor involved in soybean resistance to SMV.

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 F_(1),F_(2) and 168 F_(11) 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 F_(2) generation(3 R:1 S) and the recombinant inbred lines(RILs) population(1 R:1 S),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 q RT-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.

R_(SC3)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 SMVresistance 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 F_(2)-derived F_(3)(F_(2:3)) 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 R_(SC3)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 R_(SC3)K, LOC100526921, and LOC100812666. The recombinant R_(SC3)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 R_(SC3)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.

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 research, based on transient expression assay in Nicotiana benthamiana leaves, we found that the longest transcript of Rsc4-3 is sufficient to induce resistance response to SMV;and CRISPR/Cas9-mediated Rsc4-3 knockout in resistant cultivar Dabaima compromised the resistance. These indicate that Rsc4-3 confers resistance to SMV. Interestingly, Rsc4-3 encodes a cell wall localized NLR-type resistant protein (Rsc4-3). 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 response. 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. Our work exhibited a case of NLR recognizing virus in the apoplast and provided a simple and effective method for identifying resistant genes against SMV infection.

Grading method of soybean mosaic disease based on hyperspectral imaging technology

Soybean is a crop with a long cultivation history that occupies an important position in agricultural production.Soybean mosaic virus disease(SMV)has caused a rapid decline in soybean yields,causing huge losses to the soybean industry,wherefrom its early detec-tion is particularly important.This study proposes a new classification method for the early SMV,dividing its severity into grades 0,1 and 2.In the case of a small number of experi-mental samples of soybeans,this study proposes a combined convolutional neural network and support vector machine(CNN-SVM)method for the early detection of SMV.Experimen-tal results showed that the accuracy of the training set of the CNN-SVM model reached 96.67%,and the accuracy rate of the test set reached 94.17%.The experiment proved the feasibility of using the proposed CNN-SVM model to classify early SMV under the new clas-sification method,and provided a new direction for early SMV detection based on hyper-spectral images.