Citrus bacterial canker disease, caused by Xanthomonas citri subsp. citri (Xcc), poses a significant global threat to the citrus industry. Lateral organ boundaries 1 (Lob1) is confirmed as a citrus susceptibility gene that induces pathogenesis by interaction with the PthA4 effector of Xcc. Citron C-05 (Citrus medica) is a Citrus genotype resistant to Xcc. However, there is little information available on the regulation of Lob1 in resistant genotypes, which is important for the breeding of citrus cultivars resistant to canker disease. This study aimed to identify upstream regulatory factors of Lob1 in Citron C-05 and to investigate its function in disease resistance. ‘Bingtang’ sweet orange (C. sinensis), a susceptible genotype, was utilized as the control. cDNA yeast libraries of Xcc-induced Citron C-05 and ‘Bingtang’ sweet orange were constructed. The capacities of ‘Bingtang’ and Citron C-05 were 1.896 × 107 and 2.154 × 107 CFU, respectively. The inserted fragments ranged from 500 to 2000 bp with a 100% recombination rate. The promoter of Lob1 was segmented into two pieces and the P1 fragment from both genotypes was used to construct a bait yeast (PAbAi-CsLob1-P1; PAbAi-CmLob1-P1). Through library screening with the bait yeast, upstream regulators interacting with the Lob1-P1 promoter were identified and then validated using Y1H and dual-luciferase tests. The expression analysis of the three transcript factors indicated that RMA3 was upregulated by inoculation with Xcc in the resistant Citron C-05, but not in the susceptible sweet orange. The overexpression of CsRMA3 in ‘Bingtang’ sweet orange led to reduced canker symptoms, with a significantly lower pathogen density in the leaves following Xcc inoculation. When CmRMA3 was silenced by virus-induced gene silencing (VIGS) in Citron C-05, typical canker symptoms appeared on the CmRMA3-silenced leaves at 15 days post-inoculation with Xcc. Further expression analyses revealed that the CmRMA3 transcription factor suppressed the expression of Lob1. These results suggest that RMA3 participates in the resistant reaction of Citron C-05 to Xcc infection, and such a response might be in relation to its suppression of the expression of the pathogenic gene Lob1.

Citrus canker is a quarantined disease caused by the bacterial plant pathogen Xanthomonas citri subsp. citri (Xcc), which causes persistent surface damage, leaf and fruit drop, and tree decline in citrus plants. The citrus cultivar Citron C-05 (Citrus medica L.) is a disease-resistant genotype identified after years of screening at the National Center for Citrus Improvement (Changsha), which displays allergic, necrotic, and disease-resistant responses to Xcc. In this study, the BAK1 gene was identified in this cultivar to be a disease resistance gene involved in plant-microbe interaction between citrus and Xcc. Functional investigations of this gene revealed that both CsBAK1 (C. sinensis BAK1) or CmBAK1(C. medica BAK1) could inhibit the growth of Xcc to some extent when transiently expressed in the susceptible ‘Bingtang’ genotype of sweet orange. Critical regions of the CmBAK1 promoter sequence were identified by creating downstream deletions and exposing mutants to Xcc to determine effects on the resistance phenotype; a 426 bp region (−2000~–1574) was identified as a key functional region responsible for eliciting the hypersensitive response in plants. Through screening arrayed Citron C-05 cDNA libraries by yeast one-hybrid assays, a basic APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor of CmRAP2-13 that binds directly to the 426 bp key sequence and activates expression of CmBAK1 was identified. Moreover, transcriptional analysis revealed an obvious increase in transcript levels of CsRAP2-13 in Citron C-05, American citron, and Finger citron. In this study, we present the identification of transcriptional activators that are found to interact with BAK1 proteins in response to Xcc. These results reveal a coordinated regulatory mechanism of RAP2-13, which may be involved in defence responses through the regulation of BAK1.

BACKGROUND Bactrocera minax, one of the most important citrus pests, oviposits exclusively on citrus fruit. In the insect olfactory system, odorant-binding proteins (OBPs) facilitate the initial recognition role of host odor molecules. The aim of this study was to characterize the functional OBPs ofB. minaxand identify specific volatile organic compounds in theCitrusgenus as OBP targets. RESULTS BminOBP9(BminGOBP99a), a closely related homolog ofBdorGOBP99a, which reduces the egg-laying behavior ofBactrocera dorsalisthrough silencing technology, was cloned, expressed, and purified. The binding ability of BminOBP9 to 11 citrus volatiles was then examined using fluorescence competition binding assays (FCBA). The results demonstrated that BminOBP9 could bind to all tested citrus volatiles, as could BdorGOBP99a, ZcucGOBP99a, and ZtauGOBP99a. Interestingly, the binding ability of BminOBP9 was the strongest among the four, suggesting that BminOBP9 may have a function in the specific recognition of citrus volatiles. Furthermore, we aligned the above four proteins and found nine distinctive amino acid sites in BminOBP9. To identify the unique binding sites of BminOBP9, we produced the nine mutants using site-directed mutagenesis. Further FCBA showed that the binding ability of the nine mutants to citrus volatiles significantly reduced, and six of them (substitutes S24P, L36F, E53K, N68D, D112A, and S118R) had the weakest binding ability. CONCLUSION The results demonstrated that BminOBP9 was the specific protein involved in the perception of citrus host volatiles byB. minax. Moreover, BminOBP9 could prove efficient in screening the candidate odors for pest management.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a serious bacterial disease that affects citrus production worldwide. Citron C-05 (Citrus medica) is the only germplasm in the Citrus genus that has been identified to exhibit strong resistance to Xcc. However, it has not been determined when, where, and how Xcc is restricted in the tissues of Citron C-05 during the infection process. In the present study, we investigated the spatiotemporal growth dynamics of an eGFP-labeled virulent Xcc (eGFP-Xcc) strain in Citron C-05 along with five susceptible biotypes (i.e., lemon, pummelo, sour orange, sweet orange, and ponkan mandarin) upon inoculation via the spraying or leaf infiltration of a bacterial suspension. The results from extensive confocal laser scanning microscopy analyses showed that while Xcc grew rapidly in plants of all five susceptible genotypes, Xcc was severely restricted in the epidermal and mesophyll cell layers of the leaves of Citron C-05 in the early stage of infection. Not surprisingly, resistance against Xcc in Citron C-05 was found to be associated with the production of reactive oxygen species and hypersensitive response-like cell death, as well as greater upregulation of several defense-related genes, including a pathogenesis-related gene (PR1) and a glutathione S-transferase gene (GST1), compared with sweet orange as a susceptible control. Taken together, our results not only provide further valuable details of the spatiotemporal dynamics of the host entry, propagation, and spread of Xcc in both resistant and susceptible citrus plants but also suggest that resistance to Xcc in Citron C-05 may be attributed to the activation of multiple defense mechanisms.