Abstract
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.
Highlights
Soybean mosaic virus (SMV) is a prevalent viral pathogen of soybean [Glycine max (L.) Merr.], especially in China (Hill and Whitham, 2014; Li and Zhi, 2016); it usually leads to soybean yield reductions that varied with cultivar, virus strain, location, and infection time (Hajimorad et al, 2018)
Focusing on the assumption of selecting relatively conserved sequences of SMV genome as the target of host-induced gene silencing (HIGS) may contribute to the success of broad-spectrum resistance (BSR) to SMV, the potential target sequences in the SMV genome were analyzed through multiple sequence alignment of the complete genome sequences of 30 SMV isolates (Supplementary Table 1)
S1-targetinverted-repeat constructs (TIRs) to S5-TIR assembled from conserved target fragments (CTFs) S1 to S5 was separately transiently expressed in N. benthamiana leaves, and the siRNAs produced from these constructs were detected by Northern blotting with respective DIG-labeled RNA probes
Summary
Soybean mosaic virus (SMV) is a prevalent viral pathogen of soybean [Glycine max (L.) Merr.], especially in China (Hill and Whitham, 2014; Li and Zhi, 2016); it usually leads to soybean yield reductions that varied with cultivar, virus strain, location, and infection time (Hajimorad et al, 2018). Soybean mosaic virus belongs to Potyvirus; its genome is a singlestranded, positive-sense (+) RNA, with a length of ∼ 9.6 Kb. It is prone to high mutation rates, generating genetic diversity and different SMV strains (Seo et al, 2009b). Based on the symptoms produced on a set of different soybean genotypes, SMV isolates can be classified into different strain groups. Seven SMV strains were reported in the United States based on the responses of a set of eight differential soybean genotypes to the virus (Cho and Goodman, 1979). In China, more than 4,500 country-wide SMV isolates were collected and grouped into twenty-two strains based on the responses of ten differential hosts to the virus at the National Center for Soybean Improvement (Li et al, 2010; Wang et al, 2014)
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