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

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.

RNA interference‐based exogenous double‐stranded RNAs confer resistance to Rhizoctonia solaniAG‐3 on Nicotiana tabacum

“…37 HIGS has been reported to control a variety of plant diseases caused by viruses, fungi, insects, nematodes and parasitic plants. [38][39][40][41][42][43] Therefore, HIGS offers a new, pesticide-free and potentially sustainable option for plant protection, provided that transcript abundance can be downregulated sufficiently and efficiently. 44 Nevertheless, HIGS is limited to plants with established transformation methods, thus restricting the plant species that can apply the strategy.…”

“…Host‐induced gene silencing (HIGS) is an RNAi‐based approach in which siRNAs are produced in the host plant and subsequently enter the pathogens to silence their genes 37 . HIGS has been reported to control a variety of plant diseases caused by viruses, fungi, insects, nematodes and parasitic plants 38–43 . Therefore, HIGS offers a new, pesticide‐free and potentially sustainable option for plant protection, provided that transcript abundance can be downregulated sufficiently and efficiently 44 .…”

Hairpin-RNA Spray Confers Resistance to Mungbean Yellow Mosaic India Virus in Mungbean

“…In the present study, a non-transgenic approach was employed to induce resistance to YMD in mungbean. Initially, three hpRNAi constructs were transiently expressed in the MYMIV-susceptible mungbean cultivar K851, akin to previous studies (41,58), to evaluate their efficacy in providing protection against MYMIV infection. The most efficient hpRNAi clone was then selected, and from this single construct, highly efficient in vivo -produced hpRNA molecules were derived.…”