In plant signal transduction, protein phosphorylation is an essential posttranslational modification that plays a vital role in various aspects of plant life, including growth, development, and response to environmental stresses. During plant growth and development, from seed germination to mature organ formation, protein phosphorylation acts as a master regulator controlling the activity of key proteins involved in cell division, differentiation, and expansion. Additionally, plants encounter stresses such as drought, high salinity, extreme temperatures, and pathogen attack. Protein phosphorylation quickly triggers stress-responsive signaling cascades in response to these conditions, activating stress-related gene expression, regulating the synthesis of osmoprotectants to preserve cellular water balance, and modulating antioxidant enzyme activity to scavenge reactive oxygen species produced under heightened stress. Therefore, protein phosphorylation is vital for plant adaptation and survival in harsh environments.

In recent years, as interest in understanding the molecular basis of plant biology has grown, numerous methods have been used to detect dynamic changes in protein phosphorylation and explore its functional mechanisms. These methods range from traditional biochemical techniques to advanced high-throughput technologies, each offering unique advantages for studying protein phosphorylation. This review provides a comprehensive summary of the diverse methods employed in researching plant protein phosphorylation signaling networks, along with recent advancements. First, an overview of the process, types, and classification of protein phosphorylation is presented. Protein phosphorylation involves the transfer of a phosphate group from a donor molecule, typically ATP, to specific amino acid residues on target proteins via protein kinases. The main types include serine, threonine, and tyrosine phosphorylation, each with distinct features and biological roles. Additionally, different amino acids can result in diverse phosphorylation forms. A classification system is also introduced, categorizing protein phosphorylation based on factors like the functional groups of target proteins and signaling pathways involved. Moreover, the methods for detecting protein phosphorylation are summarized in detail. Traditional techniques such as radioactive labeling, which tracks phosphate incorporation using radioactive isotopes, have been widely used but pose safety concerns and limit large-scale analysis. Modern approaches, including immunoblotting with phosphorylation-specific antibodies, enable specific detection of phosphorylated proteins through antigen-antibody interactions. High-throughput techniques like mass spectrometry-based phosphoproteomics have revolutionized the field, allowing large-scale identification and quantification of phosphorylated proteins within a single experiment, providing a global view of phosphorylation changes under different conditions. Finally, the progress in studying protein phosphorylation in plants is outlined, highlighting its roles in hormone signaling and resistance to salt, drought, cold, and disease stresses.

By summarizing recent key findings, this review offers a valuable reference for further investigation into plant protein phosphorylation. In the future, it may inspire new research directions and enhance our understanding of plant biology.

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.

Soybean mosaic virus (SMV) disease causes by SMV seriously restricts soybean yields and qualities. In this study, we cloned GmRHF1, which encodes a Ring-H2 type zinc finger protein (RHF), and identified its function in SMV resistance through the sequence variation analysis and the virus induced gene silencing (VIGS) assay. Yeast two-hybrid and luciferase complementation assay (LCA) were used to screen and verify the interacting proteins of GmRHF1. The research lay a foundation for further exploring the mechanism of GmRHF1 in soybean resistance to SMV.

Firstly, GmRHF1 was cloned from soybean varieties of Xudou 14 (highly resistant to SMV) and Shishanheidou (highly susceptible to SMV), and the consistency of sequence variation was then analyzed in representative resistant and susceptible soybean resources, so as to excavate the excellent alleles with SMV resistance. Secondly, the expression abundance of GmRHF1 in different soybean tissues and under SMV treatment were analyzed by real-time quantitative PCR (RT-qPCR), respectively. At the same time, the function of GmRHF1 in SMV resistance was identified by the VIGS assay. The interacting proteins of GmRHF1 were screened and verified by yeast cDNA library screening, point to point verification and LCA experiments. Finally, the function of GmClpP6 on SMV resistance was verified, and the biological significance based on the interaction of GmRHF1 and GmClpP6 for soybean resistance to SMV was clarified.

GmRHF1 contained a conserved RING-H2 domain of the E3 ubiquitin protein ligase, and belongs to the typical RING-H2 finger family. The results of sequence variation analysis showed that a natural non-synonymous mutation existed in the CDS region of GmRHF1 among various soybean varieties, providing a probable relevance to the SMV resistance or sensibility. After SMV inoculations, the expression level of GmRHF1 in resistant soybean was significantly higher than that in susceptible material by RT-qPCR assay. Through the VIGS experiments, GmRHF1 was effectively silenced and the soybean leaves showed a weakened SMV resistance than the normal plants, indicating that GmRHF1 could enhance soybean resistance to SMV. Based on the yeast cDNA library, 30 potential interacting proteins of GmRHF1 were screened. The interaction between GmRHF1 and GmClpP6 was confirmed by the two-hybrid and LCA experiments. Furthermore, the soybean resistance to SMV was also reduced in soybean leaves with the silenced GmClpP6 by the VIGS experiments.

The non-synonymous mutation identified in the coding sequence of GmRHF1 may be a key SNP variation related to the SMV resistance and sensibility; and GmRHF1 can interact with GmClpP6. GmRHF1 plays an important role in SMV resistance.