The ability of various pests and diseases to adapt to a single plant resistance gene over time leads to loss of resistance in transgenic rice. Therefore, introduction of different pest and disease resistance genes is critical for successful cultivation of transgenic rice strains with broad-spectrum resistance to multiple pathogens. Here, we produced resistance rice lines with multiple, stacked resistance genes by stacking breeding and comprehensively evaluated their resistance to Chilo suppressalis (striped rice stemborer), Magnaporthe oryzae (rice blast), and Nilaparvata lugens (brown planthopper) in a pesticide-free environment. CRY1C and CRY2A are exogenous genes from Bacillus thuringiensis. Pib, Pikm, and Bph29 are natural genes in rice. CH121TJH was introduced into CRY 1C, Pib, Pikm, and Bph29. CH891TJH and R205XTJH were introduced into CRY 2A, Pib, Pikm, and Bph29. Compared with those observed in their recurrent parents, CH121TJH significantly increased the mortality of borers. The other two lines CH891TJH and R205XTJH are the same result. Three lines introduction of Pib and Pikm significantly reduced the area of rice blast lesions, and introduction of Bph29 significantly reduced seedling mortality from N. lugens. Introduction of the exogenous genes had relatively few effects on agronomic and yield traits of the original parents. These findings suggest that stacking of rice resistance genes through molecular marker-assisted backcross breeding can confer broad spectrum and multiple resistance in differently genetic backgrounds.

The extent of molecular diversity and differentially expressed proteins (DEPs) in transgenic lines provide valuable information to understand the phenotypic performance of transgenic crops compared with their parents. Here, we compared the differences in the phenotypic variation of twelve agronomic and end-use quality traits, the extent of microsatellite diversity, and DEPs of a recurrent parent line with three transgenic rice restorer lines carrying either CRY1C gene on chromosome 11 or CRY2A gene on chromosome 12 or both genes. The three transgenic lines had significantly smaller stem borer infestation than the recurrent parent without showing significant differences among most agronomic traits, yield components, and end-use quality traits. Using 512 microsatellite markers, the three transgenic lines inherited 2.9–4.3% of the Minghui 63 donor genome and 96.3–97.1% of the CH891 recurrent parent genome. As compared with the recurrent parent, the number of upregulated and down-regulated proteins in the three transgenic lines varied from 169 to 239 and from 131 to 199, respectively. Most DEPs were associated with the secondary metabolites biosynthesis transport and catabolism, carbohydrate transport and metabolism, post-translational modification, and signal transduction mechanisms. Although several differentially expressed proteins were observed between transgenic rice and its recurrent parent, the differences may not have been associated with grain yield and most other phenotypic traits in transgenic rice.

Fruit ripening in tomato (Solanum lycopersicum) has been extensively studied at the transcriptomic level. However, comprehensive profiling of the tomato fruit proteome and phosphoproteome remains limited. In this study, we performed large-scale proteome and phosphoproteome profiling of tomato (Ailsa Craig) fruits across five ripening stages using tandem mass tags (TMT)-based quantitative proteomics. Our analysis quantified over 8800 proteins and 20,000 high-confidence phosphorylation sites. Ripening-associated phosphorylation and dephosphorylation events were identified in diverse ripening regulators, including transcription factors, ethylene biosynthesis and signaling proteins, and epigenetic modifiers. Weighted gene co-expression network analysis (WGCNA) revealed a tetratricopeptide repeat protein, REDUCED CHLOROPLAST COVERAGE 1a (REC1a), as a key regulator of fruit ripening. Parallel reaction monitoring (PRM)-based targeted proteomic analysis validated the expression profiles of REC1a and its three phosphorylation sites. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout of REC1a resulted in reduced lycopene accumulation and slower chlorophyll degradation, highlighting its role in the chloroplast-to-chromoplast transition, which is critical for fruit pigmentation during ripening. Quantitative proteomic analyses of rec1a mutants demonstrated reduced levels of Clp proteases and chaperones, proteins known to regulate plastid transitions. Additionally, co-immunoprecipitation and split-luciferase complementation assays revealed that REC1a interacts with the eukaryotic translation initiation factor subunits eIF2α and eIF2Bβ, suggesting its role in regulating protein synthesis during ripening. This study provides the most comprehensive quantitative proteome and phosphoproteome atlas of tomato fruits to date and identifies REC1a as a regulator of fruit ripening, offering new insights into the underlying molecular mechanisms.