Huanglongbing (HLB), a bacterial disease caused by Candidatus Liberibacter asiaticus (CLas), is a major threat to the citrus industry. In a previous study conducted by our laboratory, several citrus transgenic trees expressing the Arabidopsis thaliana NPR1 (AtNPR1) gene remained HLB-free when grown in a field site under high HLB disease pressure. To determine the molecular mechanisms behind AtNPR1-mediated tolerance to HLB, a transcriptome analysis was performed using AtNPR1 overexpressing transgenic trees and non-transgenic trees as control, from which we identified 57 differentially expressed genes (DEGs). Data mining revealed the enhanced transcription of genes encoding pathogen-associated molecular patterns (PAMPs), transcription factors, leucine-rich repeat receptor kinases (LRR-RKs), and putative ankyrin repeat-containing proteins. These proteins were highly upregulated in the AtNPR1 transgenic line compared to the control plant. Furthermore, analysis of protein–protein interactions indicated that AtNPR1 interacts with CsNPR3 and CsTGA5 in the nucleus. Our results suggest that AtNPR1 positively regulates the innate defense mechanisms in citrus thereby boosting resistance and effectively protecting the plant against HLB.

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.

Pear black spot (PBS) disease, which is caused by Alternaria alternata (Aa), is one of the most serious diseases affecting sand pear (Pyrus pyrifolia Nakai) cultivation worldwide. To investigate the defense mechanisms of sand pear in response to Aa, the transcriptome of a sand pear germplasm with differential resistance to Aa was analyzed using Illumina paired-end sequencing. Four libraries derived from PBS-resistant and PBS-susceptible sand pear leaves were characterized through inoculation or mock-inoculation. In total, 20.5 Gbp of sequence data and 101,632,565 reads were generated, representing 44717 genes. Approximately 66% of the genes or sequenced reads could be aligned to the pear reference genome. A large number (5213) of differentially expressed genes related to PBS resistance were obtained; 34 microsatellites were detected in these genes, and 28 genes were found to be closely related to PBS resistance. Using a transcriptome analysis in response to PBS inoculation and comparison analysis to the PHI database, 4 genes (Pbr039001, Pbr001627, Pbr025080 and Pbr023112) were considered to be promising candidates for sand pear resistance to PBS. This study provides insight into changes in the transcriptome of sand pear in response to PBS infection, and the findings have improved our understanding of the resistance mechanism of sand pear to PBS and will facilitate future gene discovery and functional genome studies of sand pear.

C-repeat/dehydration-responsive element binding factor (CBF) plays important roles in cold response network in plants. Here, one member of CBF coding gene family in trifoliate orange (Poncirus trifoliata), designated as PtCBF, was isolated. Semi-quantitative reverse transcription-polymerase chain reactions showed up-regulation of PtCBF not only under low temperature but also induced by abscisic acid. Additionally, the CBF genomic fragments in four citrus species including trifoliate orange, sweet orange (Citrus sinensis), pummel (Citrus grandis) and rough lemon (Citrus jambhiri) were isolated with complete open reading frames. According to the results of alignment analysis between full length cDNA and genomic DNA sequences in trifoliate orange, there were no introns in PtCBF. Moreover, the results of multiple sequence alignment analysis and phylogenetic analysis on putative protein sequences suggested that the AP2 DNA binding domains and CBF signature sequences were highly conserved in four citrus CBF proteins. Finally, the CBF promoters in above citrus species were isolated, which provides some information concerning promoter function.

Chestnut (Castanea mollissima Blume) is an important economic tree owing to its tasty fruit and adaptability to environmental stresses, especially drought. Currently, there is limited information about non-specific lipid transfer protein (nsLTP) genes that respond to abiotic stress in chestnuts. Here, a chestnut nsLTP, named CmnsLTP6.9, was identified and analyzed. The results showed that the CmnsLTP6.9 protein localized in the extracellular matrix had two splicing variants (CmnsLTP6.9L and CmnsLTP6.9S). Compared with CmnsLTP6.9L, CmnsLTP6.9S had an 87 bp deletion in the 5′-terminal. Overexpression of CmnsLTP6.9L in Arabidopsis enhanced tolerance to osmotic and drought stress. Upon exposure to osmotic and drought treatment, CmnsLTP6.9L could increase reactive oxygen species (ROS)-scavenging enzyme activity, alleviating ROS damage. However, CmnsLTP6.9S-overexpressing lines showed no significant differences in phenotype, ROS content, and related enzyme activities compared with the wild type (WT) under osmotic and drought treatment. Moreover, lipid metabolism analysis confirmed that, unlike CmnsLTP6.9S, CmnsLTP6.9L mainly altered and upregulated many fatty acyls and glycerophospholipids, which implied that CmnsLTP6.9L and CmnsLTP6.9S played different roles in lipid transference in the chestnut. Taken together, we analyzed the functions of CmnsLTP6.9L and CmnsLTP6.9S, and demonstrated that CmnsLTP6.9L enhanced drought and osmotic stress tolerance through ROS scavenging and lipid metabolism.