Upland cotton (Gossypium hirsutum), an allotetraploid, contains At- and Dt- subgenome and most genes have multiple homologous copies, which pose a huge challenge to investigate genes’ function due to the functional redundancy. Therefore, it is of great significance to establish effective techniques for the functional genomics in cotton. In this study, we tested two novel genome editing vectors and compared them with the CRISPR/Cas9 system (pRGEB32-GhU6.7) developed in our laboratory previously. In the first new vector, the sgRNA transcription unite was constructed into the replicon (LIR-Donor-SIR-Rep-LIR) of the bean yellow dwarf virus (BeYDV) and named as pBeYDV-Cas9-KO and in the second vector, the ubiquitin promoter that drives Cas9 protein was replaced with a constitutive CaMV 35S promoter and defined as pRGEB32-35S. The results from transgenic cotton calli/plants revealed that pBeYDV-Cas9-KO vector showed the highest editing efficiency of GhCLA1 in At and Dt subgenomes edited simultaneously up to 73.3% compared to the 44.6% of pRGEB32-GhU6.7 and 51.2% of pRGEB32-35S. The editing efficiency of GhCLA1 in At and Dt subgenome by pBeYDV-Cas9-KO was 85.7% and 97.2%, respectively, whereas the efficiency by pRGEB32-GhU6.7 and pRGEB32-35S vectors was 67.7%, 86.5%, 84%, and 87.2%, respectively. The editing profile of pBeYDV-Cas9-KO was mainly composed of fragment deletion, accounting for 84.0% and ranging 1–10 bp in length. The main editing sites are located at positions 11–17 upstream of PAM site. The off-target effects were not detected in all potential off-target sites. Taken together, the pBeYDV-Cas9-KO system has high editing efficiency and specificity with wide editing range than the traditional CRISPR/Cas9 system, which provides a powerful tool for cotton functional genomics research and molecular breeding.

Thesium chinense Turcz. (T. chinense), a perennial herb in the Santalaceae family, exhibits potent antibacterial and anti-inflammatory properties. Transcriptome sequencing was performed on one- and two-year-old T. chinense plants across seedling, flowering, and fruiting stages (all sampled from the same location) using the illumina NovaSeq 6000 platform. A total of 58,706 unigenes were identified, including 1656 transcription factors (TFs). Further analysis classified these TFs into seven functional categories, enabling the reconstruction of a representative TF regulatory network. Differential expression analysis revealed that the number of differentially expressed genes (DEGs) ranged from 2000 to 5000 during different developmental stages in first-year plants, while varying between 1000 and 2000 in second-year plants. Comparative analysis of DEGs between one- and two-year-old plants showed that they were primarily associated with sesquiterpene, triterpene, and terpene skeleton biosynthesis, as well as other metabolic pathways. Additionally, analysis of key genes involved in flavonoid biosynthesis—the major bioactive compounds in T. chinense—revealed their predominant accumulation during the first year of growth. This study provides valuable insights into the developmental biology of T. chinense and establishes a foundation for future research on flavonoid biosynthesis pathway genes and their therapeutic applications.

Calcium‐dependent protein kinases (CDPKs) in plants play crucial roles in mediating responses to both biotic and abiotic stresses. However, the molecular mechanism through which CDPKs regulate antiviral immunity in plants remains largely elusive. In this study, we identified ZmCDPK7 as a key player in response to various stimuli. Lines overexpressing ZmCDPK7 exhibited enhanced resistance to maize chlorotic mottle virus (MCMV) infection, while ZmCDPK7 knock‐out lines demonstrated increased sensitivity, indicating that ZmCDPK7 positively regulates maize immunity against MCMV. Furthermore, ZmCDPK7 interacts with ZmAPX1 and enhances its enzymatic activity by phosphorylating ZmAPX1 at Thr164, thereby reducing reactive oxygen species (ROS) production. The scaffold protein ZmRACK1 interacts with both ZmCDPK7 and ZmAPX1, facilitating the formation of a ternary complex that enhances ZmCDPK7‐mediated ZmAPX1 enzymatic activity. Lines overexpressing ZmAPX1 also showed increased resistance to MCMV infection, while ZmAPX1 mutant lines ( Zmapx1 MU1 and Zmapx1 MU2 ) exhibited increased sensitivity. Additionally, CRISPR/Cas9‐mediated genome editing of ZmRACK1 in maize resulted in increased susceptibility to MCMV. To counteract plant defense, the MCMV protein P31 interacts with ZmCDPK7 and ZmRACK1, disrupting the interaction between ZmRACK1–ZmCDPK7 and ZmCDPK7–ZmAPX1, thereby blocking the ternary complex formation. Moreover, ZmCDPK7 is also implicated in defense against drought stress and corn stalk rot. Overall, our findings provide valuable insights into the molecular mechanisms by which the ZmRACK1–ZmCDPK7–ZmAPX1 module regulates plant immunity, identifying the potential new targets for the genetic control of maize viral diseases.