Huanglongbing (HLB) is one of the most severe citrus diseases in the world, causing huge economic losses. However, efficient methods of protecting citrus from HLB have not yet been developed. microRNA (miRNA)-mediated regulation of gene expression is a useful tool to control plant diseases, but the miRNAs involved in regulating resistance to HLB have not yet been identified. In this study, we found that miR171b positively regulated resistance to HLB in citrus. Upon infection with HLB bacteria, the bacteria were detected in the second month in the control plants. However, in the miR171b-overexpressing transgenic citrus plants, the bacteria could not be detected until the 24th month. RNA-seq data indicated that multiple pathways, such as photosynthesis, plant–pathogen interaction, the MAPK signaling pathway, etc., might be involved in improving the resistance to HLB in miR171b-overexpressing plants compared with the control. Finally, we determined that miR171b could target SCARECROW-like (SCL) genes to downregulate their expression, which then led to promoted resistance to HLB stress. Collectively, our results demonstrate that miR171b plays a positive regulatory role in resistance to citrus HLB, and provides a new insight into the role of miRNAs in the adaptation of citrus to HLB stress.

The study focuses on the Cyclic nucleotide-gated ion channels (CNGCs) proteins in citrus, aiming to investigate their potential roles. A total of 33 CcCNGC proteins were identified and characterized in Citrus clementina using a genome-wide method. The study revealed that these proteins share a conserved CNGC domain structurally but exhibit significant differences in their primary sequence and motif composition. Phylogenetic analysis classified the CcCNGC proteins into 13 subgroups. The cis-elements present in all CcCNGCs promoters were identified and classified, and the number of elements was determined. The results suggested that these genes play important roles in citrus growth and development, as well as in response to biotic and abiotic stresses. Gene expression analysis further supported these findings, demonstrating that CNGC genes were responsive to various plant hormones and Phytophthora nicotianae infection, which causes citrus foot rot. Overall, the study indicated that members of the CcCNGC gene family exhibit structural and functional diversity. Further research is needed to validate the specific functions of individual family members and their roles in citrus physiology and response to stress conditions.

Citrate is a major determinant of fruit flavor quality. Currently, citrus species and/or varieties with significant alterations in citrate level have greatly advanced the molecular basis of citrate accumulation in fruit. However, in-depth dissections of the molecular mechanism specific to citrate accumulation are still limited due to the lack of mutants, especially within one single variety. In this study, a fresh-sweet ‘Shatangju’ mutant (Citrus reticulata cv.) was obtained during a survey of citrus resources in Guangdong, China, and the phenotype, fruit morphology, and primary flavor profiles were comparatively analyzed. Unlike the wild-type ‘Shatangju’ (WT), the mutant (MT) material exhibited a dwarfed and multi-branched tree shape, delayed flowering and fruit ripening at maturity, a prolonged fruit tree-retention time, and a decreased single fruit weight at maturity. Dynamic measurement of the metabolite levels further suggested that the contents and fluctuation patterns of vitamin C, malate, quinate, and oxalate showed no obvious difference between MT and MT fruits, while the citrate level in MT fruits significantly decreased over various developmental stages, ranging from 0.356 to 1.91 mg g⁻¹ FW. In addition, the accumulation patterns of the major soluble sugars (sucrose, fructose, and glucose), as well as the sugar/acid ratio, were also altered in MT fruits during development. Taken together, this study provides a novel acid-free ‘Shatangju’ mutant, which can serve as a powerful tool for the research of fruit flavor quality, especially for the comprehensive understanding of the molecular mechanism of citrate accumulation in fruits.

The citrus industry has been threatened by Huanglongbing (HLB) for over a century. Here, an HLB-induced Arabidopsis RPM1-interacting protein 4 (RIN4) homologous gene was cloned from Citrus clementina, and its characteristics and function were analyzed to determine its role during citrus–Candidatus Liberibacter asiaticus (CLas) interactions. Quantitative real-time PCR showed that RIN4 was expressed in roots, stems, leaves and flowers, with the greatest expression level in leaves. Its expression was suppressed by gibberellic acid, indole-3-acetic acid, salicylic acid and jasmonic acid treatments, but was induced by abscisic acid and salt treatments, as well as wounding. The transient expression of a RIN4-GFP showed that RIN4 was localized in the cell membrane. RIN4-overexpressing transgenic C. maxima cv. ‘Shatianyou’ plants were obtained, and some transgenic plants showed greater sensitivity to CLas infection and earlier HLB symptoms appearance than non-transgenic controls. Results obtained in this study indicated that the upregulated expression of RIN4 in HLB diseased citrus may aid CLas infection.

The Dof transcription factor family plays crucial roles in plant growth and stress responses. In this study, we identified 24 Dof genes (CcDof1–CcDof24) from the genome of Citrus clementina (Hort. ex Tan.). Phylogenetic analysis classified these proteins into six distinct clades, revealing evolutionary conservation with Dof members from Arabidopsis and tomato. Analysis of gene structure and conserved motifs showed that most CcDof genes are intronless or contain only a few introns, and their motif compositions are largely consistent with their phylogenetic relationships. Promoter analysis revealed a variety of cis-regulatory elements associated with light responsiveness, hormone signaling, and abiotic/biotic stress responses. Expression profiling demonstrated that CcDof genes exhibit tissue-specific expression patterns and are differentially regulated by various phytohormones (including ABA, SA, GA, and MeJA), low temperature stress, and infection by Phytophthora parasitica. Notably, transient overexpression of CcDof4 and CcDof6 in citrus leaves significantly enhanced resistance to P. parasitica, accompanied by upregulation of SA pathway markers NPR1 and PR1. Our findings provide a systematic characterization of the CcDof family and highlight the important roles of CcDof4 and CcDof6 in mediating citrus disease resistance, likely through modulation of the SA signaling pathway.