CsPrx25, a class III peroxidase in Citrus sinensis, confers resistance to citrus bacterial canker through the maintenance of ROS homeostasis and cell wall lignification.

Citrus bacterial canker (CBC) results from Xanthomonas citri subsp. citri (Xcc) infection and poses a grave threat to citrus production. Class III peroxidases (CIII Prxs) are key proteins to the environmental adaptation of citrus plants to a range of exogenous pathogens, but the role of CIII Prxs during plant resistance to CBC is poorly defined. Herein, we explored the role of CsPrx25 and its contribution to plant defenses in molecular detail. Based on the expression analysis, CsPrx25 was identified as an apoplast-localized protein that is differentially regulated by Xcc infection, salicylic acid, and methyl jasmone acid in the CBC-susceptible variety Wanjincheng (C. sinensis) and the CBC-resistant variety Calamondin (C. madurensis). Transgenic Wanjincheng plants overexpressing CsPrx25 were generated, and these transgenic plants exhibited significantly increased CBC resistance compared with the WT plants. In addition, the CsPrx25-overexpressing plants displayed altered reactive oxygen species (ROS) homeostasis accompanied by enhanced H2O2 levels, which led to stronger hypersensitivity responses during Xcc infection. Moreover, the overexpression of CsPrx25 enhanced lignification as an apoplastic barrier for Xcc infection. Taken together, the results highlight how CsPrx25-mediated ROS homeostasis reconstruction and cell wall lignification can enhance the resistance of sweet orange to CBC.

CsWAKL08, a pathogen-induced wall-associated receptor-like kinase in sweet orange, confers resistance to citrus bacterial canker via ROS control and JA signaling.

Citrus bacterial canker (CBC) is a disease resulting from Xanthomonas citri subsp. citri (Xcc) infection and poses a grave threat to citrus production worldwide. Wall-associated receptor-like kinases (WAKLs) are proteins with a central role in resisting a range of fungal and bacterial diseases. The roles of WAKLs in the context of CBC resistance, however, remain unclear. Here, we explored the role of CsWAKL08, which confers resistance to CBC, and we additionally analyzed the molecular mechanisms of CsWAKL08-mediated CBC resistance. Based on systematic annotation and induced expression analysis of the CsWAKL family in Citrus sinensis, CsWAKL08 was identified as a candidate that can be upregulated by Xcc infection in the CBC-resistant variety. CsWAKL08 can also be induced by the phytohormones salicylic acid (SA) and methyl jasmonic acid (MeJA) and spans the plasma membrane. Overexpression of CsWAKL08 resulted in strong CBC resistance in transgenic sweet oranges, whereas silencing of CsWAKL08 resulted in susceptibility to CBC. The peroxidase (POD) and superoxide dismutase (SOD) activities were significantly enhanced in the CsWAKL08-overexpressing plants compared to the control plants, thereby mediating reactive oxygen species (ROS) homeostasis in the transgenic plants. Moreover, the JA levels and the expression of JA biosynthesis and JA responsive genes were substantially elevated in the CsWAKL08 overexpression plants relative to the controls upon Xcc infection. Based on these findings, we conclude that the wall-associated receptor-like kinase CsWAKL08 positively regulates CBC resistance through a mechanism involving ROS control and JA signaling. These results further highlight the importance of this kinase family in plant pathogen resistance.

Genomewide analysis of the CIII peroxidase family in sweet orange (Citrus sinensis) and expression profiles induced by Xanthomonas citri subsp. citri and hormones.

Class III peroxidase (CIII prx) is a plant-specific multigene family that regulates the physiological and stress responses. This research aimed to exhaustively annotate and analyse the CIII prx family in sweet orange and to explore the regulated expression profiles by Xanthomonas citri subsp. citri (Xcc) and plant hormones. We further assessed the relationship between CIII prxs and citrus bacterial canker. The phylogeny, gene structure, conserved motifs, gene duplications and microsynteny of the CIII prx family were analysed. Expression profiles of specific CsPrxs induced by Xanthomonas citri subsp. citri and plant hormones were detected by quantitative reverse transcription-polymerase chain reaction. Subcellular localization was analysed through transient expression assessments. A total of 72 CIII prx members were identified from the genomes of sweet orange. In all chromosomes of sweet orange, the CsPrxs could be detected except in chromosome 8. In addition, three segmental duplications, four tandem duplications and 11 whole-genome duplications occurred among the CsPrxs, contributing to the family size expansion. From the Ka/Ks ratios, 15 of 18 duplicated CsPrxs pairs have experienced purifying selection process. A total of 15 conserved motifs were detected in CsPrxs, four of which were detected in all complete CsPrxs. A total of 12 expressed genes were identified from the EST database. The expression trends of 12 CsPrxs were differently expressed at different stages of infection by Xcc, five of which were potential candidate genes involved in Xcc resistance. These genes could be induced by salicylic acidand methyl jasmonate, and were extracellular proteins. These results further support our understanding of CIII prxs in citrus, particularly incitrus bacterial canker studies.

CitGVD: a comprehensive database of citrus genomic variations.

Citrus is one of the most important commercial fruit crops worldwide. With the vast genomic data currently available for citrus fruit, genetic relationships, and molecular markers can be assessed for the development of molecular breeding and genomic selection strategies. In this study, to permit the ease of access to these data, a web-based database, the citrus genomic variation database (CitGVD, http://citgvd.cric.cn/home) was developed as the first citrus-specific comprehensive database dedicated to genome-wide variations including single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs). The current version (V1.0.0) of CitGVD is an open-access resource centered on 1,493,258,964 high-quality genomic variations and 84 phenotypes of 346 organisms curated from in-house projects and public resources. CitGVD integrates closely related information on genomic variation annotations, related gene annotations, and details regarding the organisms, incorporating a variety of built-in tools for data accession and analysis. As an example, CitGWAS can be used for genome-wide association studies (GWASs) with SNPs and phenotypic data, while CitEVOL can be used for genetic structure analysis. These features make CitGVD a comprehensive web portal and bioinformatics platform for citrus-related studies. It also provides a model for analyzing genome-wide variations for a wide range of crop varieties.

Systematic Analysis and Functional Validation of Citrus XTH Genes Reveal the Role of Csxth04 in Citrus Bacterial Canker Resistance and Tolerance.

In this study, we performed a comprehensive survey of xyloglucan endotransglucosylase/hydrolase (XTH) and a functional validation of Citrus sinensis (Cs) XTH genes to provide new insights into the involvement of XTHs in Xanthomonas citri subsp. citri (Xcc) infection. From the genome of sweet orange, 34 CsXTH genes with XTH characteristic domains were identified and clustered into groups I/II, IIIA, and IIIB. Except for chromosome 9, the CsXTH genes were unevenly distributed and duplicated among all chromosomes, identifying a CsXTH duplication hot spot on chromosome 4. With Xcc induction, a group of citrus canker-related CsXTHs were detected. CsXTH04 was identified as a putative candidate gene, which is up-regulated in citrus bacterial canker (CBC)-resistant varieties and induced by exogenous treatment with salicylic acid (SA) and methyl jasmonate (MeJA). CsXTH04 overexpression conferred CBC susceptibility to transgenic citrus, while CsXTH04 silencing conferred CBC resistance. Taken together, the annotation of the CsXTH family provides an initial basis for the functional and evolutionary study of this family as potential CBC-susceptible genes. CsXTH04, validated in this study, can be used in citrus breeding to improve CBC resistance.

CsBZIP40, a BZIP transcription factor in sweet orange, plays a positive regulatory role in citrus bacterial canker response and tolerance.

Citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc) is a systemic bacterial disease that affects citrus plantations globally. Biotic stress in plants has been linked to a group of important transcription factors known as Basic Leucine Zippers (BZIPs). In this study, CsBZIP40 was functionally characterized by expression analysis, including induction by Xcc and hormones, subcellular localization, over-expression and RNAi silencing. CsBZIP40 belongs to group D of the CsBZIP family of transcription factors and localizes in the nucleus, potentially serving as a transcriptional regulator. In wild type (WT) plants CsBZIP40 can be induced by plant hormones in addition to infection by Xcc which has given insight into its involvement in CBC. In the present study, over-expression of CsBZIP40 conferred resistance to Xcc while its silencing led to Xcc susceptibility. Both over-expression and RNAi affected salicylic acid (SA) production and expression of the genes involved in the SA synthesis and signaling pathway, in addition to interaction of CsBZIP40 with CsNPR1, as detected by a GST pull-down assay. Taken together, the results of this study confirmed the important role of CsBZIP40 in improving resistance to citrus canker through the SA signaling pathway by the presence of NPR1 to activate PR genes. Our findings are of potential value in the breeding of tolerance to CBC in citrus fruits.