Screening for Soybean Host Factors that Interact with Soybean Mosaic Virus Nuclear Inclusion Proteins Using the Yeast Two-Hybrid System

Soybean mosaic virus (SMV) is one of the most damaging viral diseases of soybean, which seriously affects soybean yield and quality. Identification of host proteins interacting with SMV nuclear inclusion proteins (NIa-Pro and NIb) using yeast two-hybrid library screening, aiming to establish a theoretical foundation and propose novel perspectives insights into the molecular mechanisms of SMV infection and soybean resistance.

Firstly, the coding sequences of NIa-Pro and NIb were cloned from the SMV strain SMV-HN and recombined into the pGBKT7 vector to construct the bait plasmids, and then soybean proteins interacting with the two viral functional proteins were identified by yeast library screening. Secondly, the host gene GmOEP16 encoding Outer Envelope Pore Protein 16 (OEP16) was cloned, and the interactions of GmOEP16 with NIa-Pro and NIb were clarified by yeast two-hybrid (Y2H) and luciferase complementation assay (LCA). Quantitative real-time PCR (qRT-PCR) was used to analyse the expression pattern of GmOEP16 under SMV treatment and exogenous hormone induction. Finally, virus-induced gene silencing (VIGS) was used to validate the function of GmOEP16 gene in SMV disease response.

pGBKT7-NIa-Pro and pGBKT7-NIb recombinant plasmids were successfully constructed, and 12 soybean host proteins were screened for interactions with NIa-Pro and NIb, respectively. The Y2H assay was further used to verify that NIa-Pro interacted with GmOEP16 and GmDEG5, and NIb interacted with GmOEP16, GmZC3H18 and GmAHP1. The LCA assay was further used to clarify that GmOEP16 interacted with both NIa-Pro and NIb. Expression analysis revealed that GmOEP16 was induced by SMV infection and responded rapidly to salicylic acid (SA) and abscisic acid (ABA) stimuli during early response. The VIGS assay showed that effectively silencing of GmOEP16 resulted in no obvious susceptibility phenotype in leaf tissues relative to the wild-type controls. Meanwhile, the expression of SMV-CP was significantly reduced in the GmOEP16-silenced plants, suggesting that the soybean resistance to SMV was enhanced. Collectively, these findings demonstrated that GmOEP16 could function as a negative regulator of SMV resistance in soybean.

The pGBKT7-NIa-Pro and pGBKT7-NIb bait vectors were successfully constructed, and each 12 soybean host proteins that respectively interacted with pGBKT7-NIa-Pro and pGBKT7-NIb were identified. Among them, GmOEP16 interacted with both NIa-Pro and NIb. GmOEP16 responded to SMV induction and negatively regulated SMV resistance, which promoted SMV infection on soybeans.