Highly efficient genetic transformation technology is greatly beneficial for crop gene function analysis and precision breeding. However, the most commonly used genetic transformation technology for woody plants, mediated by Agrobacterium tumefaciens, is time-consuming and inefficient, which limits its utility for gene function analysis. In this study, a simple, universal, and highly efficient genetic transformation technology mediated by A. rhizogenes K599 is described. This technology can be applied to multiple citrus genotypes, and only 2–8 weeks were required for the entire workflow. Genome-editing experiments were simultaneously conducted using 11 plasmids targeting different genomic positions and all corresponding transformants with the target knocked out were obtained, indicating that A. rhizogenes-mediated genome editing was highly efficient. In addition, the technology is advantageous for investigation of specific genes (such as ACD2) for obtaining “hard-to-get” transgenic root tissue. Furthermore, A. rhizogenes can be used for direct viral vector inoculation on citrus bypassing the requirement for virion enrichment in tobacco, which facilitates virus-induced gene silencing and virus-mediated gene expression. In summary, we established a highly efficient genetic transformation technology bypassing tissue culture in citrus that can be used for genome editing, gene overexpression, and virus-mediated gene function analysis. We anticipate that by reducing the cost, required workload, experimental period, and other technical obstacles, this genetic transformation technology will be a valuable tool for routine investigation of endogenous and exogenous genes in citrus.

The overuse of pesticides has led to resistance in phytopathogens, posing significant threats to global food security and environmental health. Antimicrobial peptides (AMPs), small molecules produced by various organisms as part of their innate immune defense, exhibit broad-spectrum antimicrobial activity with a lower risk of resistance development. These properties make AMPs promising candidates for sustainable agricultural practices. However, challenges such as high production costs, instability, and potential toxicity to plant cells have hindered their widespread application. This review provides a comprehensive overview of the discovery, classification, and antimicrobial mechanisms of AMPs, focusing on their roles in plant protection. It also explores strategies for identifying and optimizing AMPs, including structural modifications, targeted delivery systems, and production methods using plant- and microbe-based expression systems. Additionally, the review highlights the potential of transgenic approaches to enhance crop resistance by expressing AMP genes in plants. Despite the challenges, AMPs offer a transformative opportunity for modern agriculture, providing innovative solutions to combat plant diseases while reducing reliance on conventional pesticides. Continued research and technological advancements are essential to fully realize the potential of AMPs in sustainable plant protection.

The genetic transformation of plants has provided fundamental insights into plant biology. However, the genetic transformation systems for most horticultural plants remain incomplete. Genome editing has significantly contributed to the improvement of crop traits, but it heavily relies on effective genetic transformation. Currently, reducing costs and improving the efficiency of genetic transformation are crucial for promoting the widespread application of genome editing in plants. Here, we review the advances in plant genetic transformation research, performing analysis of three methods for plant gene function analysis that bypass tissue culture: Agrobacterium rhizogenes-mediated root genetic transformation, developmental regulators (DRs)-mediated genetic transformation, and virus-mediated genome editing. We analyzed transformation efficiency in strawberry and citrus using the A. rhizogenes infiltration method, employing GFP to label different subcellular locations to investigate the morphology of microfilaments, nuclei, and peroxisomes in strawberry cells. Sequence analysis revealed that a series of developmental regulators critical for enhancing genetic transformation efficiency in specific species are highly conserved across different plant species. Additionally, we successfully edited the endogenous Pds gene in Cas9-overexpressing transgenic tobacco using TRV and CLBV containing the gRNA module. These three methods offer the benefits of being cost-effective and time-efficient, providing valuable technical insights for the application of plant genome editing.

Alternaria brown spot, caused by the tangerine pathotype of Alternaria alternata, is a destructive fungal disease affecting citrus production worldwide. Nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes constitute a major class of plant immune receptors; however, their genome-wide characteristics and potential association with Alternaria brown spot resistance loci in citrus remain poorly understood. In this study, we performed a comprehensive genome-wide identification and comparative analysis of NBS-LRR genes across representative citrus species. A total of 417 and 326 NBS-LRR genes were identified in Citrus reticulata and Citrus clementina, respectively, and were classified into NL, CNL, TNL, and RNL subfamilies based on domain architecture. Phylogenetic reconstruction, gene structure analysis, conserved motif composition, chromosomal distribution, synteny relationships, and promoter cis-element profiling collectively revealed considerable structural variation and lineage-specific expansion of the NBS-LRR gene family in citrus genomes. By integrating previously reported quantitative trait locus (QTL) data for Alternaria brown spot, we identified several NBS-LRR genes located within a resistance-associated genomic interval on chromosome 3. Among these, a candidate gene, designated LRR2, exhibited differential transcriptional responses upon pathogen inoculation and displayed distinct sequence variations between citrus genotypes. Structural modeling and molecular docking analyses suggested potential binding interfaces between LRR2 and multiple host-selective toxins, although the biological relevance of these interactions requires further experimental validation. Subcellular localization assays in Nicotiana benthamiana showed that LRR2 is distributed in both the nucleus and cytoplasm. Notably, transient overexpression of LRR2 triggered hypersensitive response-like cell death and H₂O₂ accumulation. Collectively, this study provides a comprehensive overview of the citrus NBS-LRR gene family and presents a multifaceted characterization of a QTL-anchored candidate gene. These findings establish a genomic and molecular framework for further functional investigations of citrus–Alternaria interactions.