During plant-pathogen interactions, plants use intracellular proteins with nucleotide-binding site and leucine-rich repeat (NBS-LRR) domains to detect pathogens. NBS-LRR proteins represent a major class of plant disease resistance genes (R-genes). Whereas R-genes have been well characterized in angiosperms, little is known about their origin and early diversification. Here we perform comprehensive evolutionary analyses of R-genes in plants and report the identification of R-genes in basal-branching streptophytes, including charophytes, liverworts, and mosses. Phylogenetic analyses suggest that plant R-genes originated in charophytes and R-proteins diversified into TIR-NBS-LRR proteins (TNLs) and non-TIR-NBS-LRR proteins (nTNLs) in charophytes. Moreover, we show that plant R-proteins evolved in a modular fashion through frequent gain or loss of protein domains. Most of the R-genes in basal-branching streptophytes underwent adaptive evolution, indicating an ancient involvement of R-genes in plant-pathogen interactions. Our findings provide novel insights into the origin and evolution of R-genes and the mechanisms underlying colonization of terrestrial environments by plants.

Citrus HLB caused by Candidatus Liberibacter asiaticus is a pathogen-triggered immune disease. Here, we identified putative genetic determinants of HLB pathogenicity by integrating citrus genomic resources to characterize the pan-genome of accessions that differ in their response to HLB. Genome-wide association mapping and analysis of allele-specific expression between susceptible, tolerant, and resistant accessions further refined candidates underlying the response to HLB. We first developed a phased diploid assembly of Citrus sinensis 'Newhall' genome and produced resequencing data for 91 citrus accessions that differ in their response to HLB. These data were combined with previous resequencing data from 356 accessions for genome-wide association mapping of the HLB response. Genes determinants for HLB pathogenicity were associated with host immune response, ROS production, and antioxidants. Overall, this study has provided a significant resource of citrus genomic data and identified candidate genes to be further explored to understand the genetic determinants of HLB pathogenicity.

Navel orange cv. Gannanzao is a variant of the navel orange cv. Newhall (Citrus sinensis Osbeck cv. Newhall) that exhibits an earlier maturation, making it commercially valuable. However, the mechanisms underlying its early maturation remain obscure. To address this question, we conducted genome sequencing and de novo assembly of navel orange cv. Gannanzao. The assembled genome sequence is 334.57 Mb in length with a GC content of 31.48%. It comprises 318 contigs (N50 = 3.23 Mb) and 187 scaffolds (N50 = 31.86 Mb). The Benchmarking Universal Single-Copy Orthologs test demonstrates 94.6% completeness. The annotation revealed 23,037 gene models, 164.95 Mb of repetitive sequences, and 2,554 noncoding RNAs. A comparative analysis identified 323 fruit ripening–related genes in navel orange cv. Gannanzao genome, while navel orange cv. Newhall genome contained 345 such genes. These genes were organized into 320 orthologous gene families, with 30.3% of them exhibiting differences in gene copy numbers between the 2 genomes. Additionally, we identified 15 fruit ripening–related genes that have undergone adaptive evolution, suggesting their potential role in advancing fruit maturation in navel orange cv. Gannanzao. Whole-genome sequencing and annotation of navel orange cv. Gannanzao provides a valuable resource to unravel the early maturation mechanism of citrus and enriches the genomic resources for citrus research.

NLR genes are crucial components of the effector-triggered immunity (ETI) system, responsible for recognizing pathogens and initiating immune responses. Although NLR genes in many plant species have been extensively studied, the diversity of NLR genes in citrus remains largely unknown. Our analysis revealed significant variations in the copy numbers of NLR genes among these species. Gene duplication and recombination were identified as the major driving forces behind this diversity. Additionally, horizontal gene transfer (HGT) emerged as the principal mechanism responsible for the increase in NLR gene copy number in A. buxifolia. The citrus NLR genes were classified into four categories: TIR-NBS-LRR (TNL), CC-NBS-LRR (CNL), RPW8-NBS-LRR (RNL), and NL. Our findings indicate that TNL, RNL, and CNL genes originated from NL genes through the acquisition of TIR and RPW8 domains, along with CC motifs, followed by the random loss of corresponding domains. Phylogenetic analysis suggested that citrus NLR genes originated alongside the species and underwent adaptive evolution, potentially playing crucial roles in the global colonization of citrus. This study provides important insights into the diversity of citrus NLR genes and serves as a foundational dataset for future research aimed at breeding disease-resistant citrus varieties.

HLB is a severe and devastating disease affecting citrus plants, for which no cure is currently available. The pathogen CLas is an unculturable, phloem-limited α-proteobacterium associated with HLB. Phages, known for their ability to directly lyse pathogenic bacteria, have been widely recognized as effective control agents in both medical and agricultural fields. In this study, we identified 191 prophages across 48 Liberibacter genomes using comparative genomics and phylogenetic methods. These prophages were classified into 17 distinct types, with 13 novel types, named NF1 to NF13. Diversity analysis revealed that the number of prophages per genome ranged from one to eight, with an average of four. Annotation showed that the genome sizes of NF1–NF13 ranged from 10,160 bp to 72,736 bp, with an average size of 27,616 bp, containing between 7 and 66 genes (average: 27 genes). Further functional enrichment of the sequences within 5 kb upstream and downstream of the integration site identified six main aspects, involving four core biological processes: genome structure modification, gene expression regulation, stress response activation, and metabolic pathway modulation. It is speculated that the insertion of the prophage significantly impacts the host. Six virulence factors, including carB, clpC, flhA, algW, rffG, and IlpA, were identified in the prophages. Interestingly, prophages containing carB or clpC are predominantly found in CLas, which may contribute to the global colonization of CLas. Notably, among the 35 identified Type 4 prophages, 19 were found to be active. Type 4 is the only active prophage type, making it a promising candidate for developing phage therapy against CLas. This study is significant for a deeper understanding of the diversity of Liberibacter and the interactions between prophages and their hosts.

Introduction: The MADS-box gene family comprises key transcription factors that regulate plant growth and development, particularly flower and fruit morphogenesis. However, the evolutionary characteristics of this gene family in sweet orange ( Citrus sinensis ) and their specific regulatory roles in secondary fruit development remain unclear. This study aimed to systematically investigate these aspects by comparing cultivars with distinct fruit phenotypes. Methods: Newhall navel orange ( C. sinensis ‘Newhall’), which exhibits a typical secondary fruit phenotype, and Hamlin sweet orange ( C. sinensis ‘Hamlin’), which lacks this trait, were used as experimental materials. A high-quality, chromosome-level de novo genome assembly was generated for Hamlin sweet orange. Comparative genomic analysis was performed to identify drivers of divergence between the two cultivars. Additionally, transcriptomic sequencing was conducted across bud, full bloom, and post-bloom stages, followed by weighted gene co-expression network analysis (WGCNA) to identify gene modules associated with secondary fruit development. Results: The Hamlin sweet orange genome assembly achieved an N50 of 32.89 Mb. Comparative genomics revealed that tandem duplication is a primary driver of divergence between the two cultivars. Gene family identification showed that Newhall possesses more MADS-box genes (139) than Hamlin (119). Transcriptomic and WGCNA analyses identified gene modules significantly correlated with secondary fruit development, pinpointing CSN SEP 3 and CSN AG 6 as key candidate regulatory genes. Discussion: This study provides a high-quality reference genome for Hamlin sweet orange and offers valuable transcriptomic insights into the role of MADS-box genes in citrus differentiation and fruit morphogenesis. The identification of CSN SEP 3 and CSN AG 6 as critical regulators of secondary fruit development lays a theoretical foundation for molecular breeding programs targeting fruit morphology in citrus.