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Background: Cold is a major abiotic stress and Huanglongbing and citrus canker disease are two devastating bacterial diseases for citrus. The Ca2+-CBL-CIPK network is known to regulate different types of stress signalling in plants. How do CBL–CIPK signalling networks function in response to cold and infection by CLas or Xcc in citrus? Results: Eight calcineurin B-like proteins (CBLs) and seventeen CBL-interacting protein kinases (CIPKs) were identified from the cold-tolerant satsuma mandarin 'Guijing2501' (Citrus. unshiu) and CLas/Xcc-sensitive sweet orange (C. sinensis). Phylogenetic analysis revealed that both CBL and CIPK family members in citrus were classified into an ancient and a recent clade according to their conserved domain characteristics and/or intron/exon structures. Genome duplication analysis suggested that both tandem and segmental duplications contributed to the amplification of the CBL and CIPK gene families in citrus under intense purifying selection, and the duplication events only existed in the recent clades. Expression comparison of the duplicated gene pairs indicated that the duplicated CBL and CIPK genes underwent functional differentiation. Further expression analysis identified that CBL1, 5, 6, and 8 and CIPK2, 8, 12, 15, 16, and 17 were significantly regulated by multiple stresses, including cold, Xcc infection and/or CLas infection, in citrus, whereas CBL2/7 and CIPK1/4/5/11/13/14 were independently highly regulated by cold and CIPK3 was uniquely responsive to Xcc infection. The combination analyses of targeted Y2H assay and expression analysis revealed that CBL6-CIPK8 was the common signalling network in response to cold and Xcc infection, while CBL6/CBL8-CIPK14 was uniquely responsive to cold in citrus. Further stable transformation and cold tolerance assay indicated that overexpression of CuCIPK16 enhanced the cold tolerance of transgenic Arabidopsis with higher POD activity and lower MDA content. Conclusions: In this study, evolution, gene expression and protein‒protein interaction analyses of citrus CBLs and CIPKs were comprehensively conducted over a genome-wide range. The results will facilitate future functional characterization of individual citrus CBLs and CIPKs under specific stresses and provide clues for the clarification of cold tolerance and disease susceptibility mechanisms in corresponding citrus cultivars. [ABSTRACT FROM AUTHOR]

• Various genetic transformation methods are important for citrus genetic breeding. • A simple, rapid and efficient root transformation technique is established in a variety of citrus varieties. • Protein interactions and FT gene signal transduction are investigated using root transformation method. • Overexpression of CjCAD promoted lignin accumulation in transgenic citrus roots. Although several methods of citrus genetic transformation have been studied and published, it is still necessary to further improve the efficiency of genetic transformation and increase the varieties used for citrus genetic transformation. Here, we report a simple, rapid and efficient root transformation technique for citrus to facilitate gene function studies. We found that infestation of Agrobacterium rhizogenes into citrus seedlings of five different species could produce stable transgenic roots in a short period after comparing and optimizing the selection of citrus species, bacterium concentration, cutting time and infection method. PCR and fluorescence observations showed a positive root transformation efficiency of approximately 13%-36% in the five tested citrus species. To demonstrate the utility of this method in the functional verification of citrus genes, a bimolecular fluorescence complementation (BiFC) assay was used to prove the interaction between CrCIPK6/9 and CrCBL5/6 in Citrus reticulata. Additionally, the PCR results showed that CrFT-RFP was transferred from the transgenic positive root to the stem in Citrus reticulata. Finally, overexpression of CjCAD from Citrus junos promoted lignin accumulation in transgenic citrus roots. This method could not only be used to study genes in roots but also various genes involved in plant growth and development. Taken together, we demonstrated that this method allows fast study of gene function in a wide range of citrus species, which would facilitate the understanding of novel genes in citrus. [ABSTRACT FROM AUTHOR]