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Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide.  It is caused by unculturable phloem-limited bacteria that belong to the Candidatus Liberibacter genus including Ca. L. asiaticus (CLas), Ca. L. africanus, and Ca. L. americanus.  Currently, there is no effective control strategy for HLB and no known cure for the disease.  We have previously generated transgenic ‘Duncan’ grapefruit and ‘Hamlin’ sweet orange expressing the Arabidopsis NPR1 (AtNPR1) gene, which encodes a master regulator of systemic acquired resistance.  Characterization of the transgenic lines indicated that overexpression of AtNPR1 confers resistance to citrus canker, another serious bacterial disease in citrus.  In this study, we intensively screened these transgenic lines for resistance or tolerance to HLB under greenhouse conditions.  Three independent transgenic lines (one line in the ‘Duncan’ grapefruit background and two lines in the ‘Hamlin’ sweet orange background) only occasionally displayed mild HLB symptoms in the presence of C Las and have kept growing normally over a period of nine years.  Significantly, all vegetatively propagated progeny plants of these lines have retained the same levels of HLB tolerance.  Furthermore, immunoblot analysis revealed that the three transgenic lines accumulate high levels of AtNPR1 protein.  These results, together with the previous finding that the same three transgenic lines are resistant to citrus canker, demonstrate that overexpression of the AtNPR1 protein in citrus is able to provide robust tolerance to HLB.

Citrus canker is a devastating disease caused by Xanthomonas axonopodis pv. citri (Xac).  The NPR1 gene plays a pivotal role in systemic acquired resistance (SAR) in Arabidopsis.  We report the isolation and characterization of an NPR1 homolog from citrus, namely Citrus NPR1 homolog 1 (CtNH1).  When over-expressed in citrus, CtNH1 confers resistance to Xac and leads to constitutive expression of the pathogenesis-related (PR) gene chitinase 1 (Chi1), suggesting that CtNH1 is orthologous to NPR1.  In addition, we recently identified two closely-related citrus genes, named Xbct31 and Xbct32.  Database searching and sequence analyses reveals that other plant species, including rice, Arabidopsis, tomato, and Nicotiana benthamiana, contain homologs of these two citrus genes and they share high levels of sequence identity at the amino acid level.  When overexpressed in Nicotiana benthamiana via Agrobacterium infiltration-mediated transient expression, the citrus genes as well as their closely-related homologs from rice and Arabidopsis all trigger (HR)-like cell death hypersensitive response.  Since HR is often associated with resistance (R) gene-mediated immunity, our data suggests Xbct3s represent a family of evolutionarily conserved defense regulators and could be used to heighten defense against citrus diseases including greening and canker.

Summary: Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), causes dramatic losses to the citrus industry worldwide. Transcription activator‐like effectors (TALEs), which bind to effector binding elements (EBEs) in host promoters and activate transcription of downstream host genes, contribute significantly to Xcc virulence. The discovery of the biochemical context for the binding of TALEs to matching EBE motifs, an interaction commonly referred to as the TALE code, enabled the in silico prediction of EBEs for each TALE protein. Using the TALE code, we engineered a synthetic resistance (R) gene, called the Xcc‐TALE‐trap, in which 14 tandemly arranged EBEs, each capable of autonomously recognizing a particular Xcc TALE, drive the expression of Xanthomonas avrGf2, which encodes a bacterial effector that induces plant cell death. Analysis of a corresponding transgenic Duncan grapefruit showed that transcription of the cell death‐inducing executor gene, avrGf2, was strictly TALE‐dependent and could be activated by several different Xcc TALE proteins. Evaluation of Xcc strains from different continents showed that the Xcc‐TALE‐trap mediates resistance to this global panel of Xcc isolates. We also studied in planta‐evolved TALEs (eTALEs) with novel DNA‐binding domains and found that these eTALEs also activate the Xcc‐TALE‐trap, suggesting that the Xcc‐TALE‐trap is likely to confer durable resistance to Xcc. Finally, we show that the Xcc‐TALE‐trap confers resistance not only in laboratory infection assays but also in more agriculturally relevant field studies. In conclusion, transgenic plants containing the Xcc‐TALE‐trap offer a promising sustainable approach to control CBC. [ABSTRACT FROM AUTHOR]

Citrus is a highly valued tree crop worldwide, while, at the same time, citrus production faces many biotic challenges, including bacterial canker and Huanglongbing ( HLB). Breeding for disease-resistant varieties is the most efficient and sustainable approach to control plant diseases. Traditional breeding of citrus varieties is challenging due to multiple limitations, including polyploidy, polyembryony, extended juvenility and long crossing cycles. Targeted genome editing technology has the potential to shorten varietal development for some traits, including disease resistance. Here, we used CRISPR/Cas9/sg RNA technology to modify the canker susceptibility gene Cs LOB1 in Duncan grapefruit. Six independent lines, DLOB2, DLOB3, DLOB9, DLOB10, DLOB11 and DLOB12, were generated. Targeted next-generation sequencing of the six lines showed the mutation rate was 31.58%, 23.80%, 89.36%, 88.79%, 46.91% and 51.12% for DLOB2, DLOB3, DLOB9, DLOB10, DLOB11 and DLOB12, respectively, of the cells in each line. DLOB2 and DLOB3 showed canker symptoms similar to wild-type grapefruit, when inoculated with the pathogen Xanthomonas citri subsp. citri (Xcc). No canker symptoms were observed on DLOB9, DLOB10, DLOB11 and DLOB12 at 4 days postinoculation ( DPI) with Xcc. Pustules caused by Xcc were observed on DLOB9, DLOB10, DLOB11 and DLOB12 in later stages, which were much reduced compared to that on wild-type grapefruit. The pustules on DLOB9 and DLOB10 did not develop into typical canker symptoms. No side effects and off-target mutations were detected in the mutated plants. This study indicates that genome editing using CRISPR technology will provide a promising pathway to generate disease-resistant citrus varieties. [ABSTRACT FROM AUTHOR]

A Mature Citrus Facility (MCF) was established at the University of Florida's Citrus Research and Education Center (CREC) to produce transgenic citrus (Citrus spp.) tolerant to canker and greening bacterial diseases. Plants produced from mature tissue should flower and fruit early. Budsticks of 'Hamlin', 'Valencia', and 'Pineapple' sweet orange [C. sinensis (L.) Osbeck] and 'Ray Ruby' grapefruit (C. paradisi Macfad.) were obtained from the Florida Division of Plant Industry (DPI) and introduced into the facility through shoot-tip grafting (STG). These plants were tested for bacteria, phloem-limited Huanglongbing (HLB), and indexed for viruses to ensure freedom from disease. Mature buds were grafted onto immature rootstock and the first two flushes of mature scion used in transformation feasibility studies with Agrobacterium strain EHA105 harboring pCAMBIA2301 and strain EHA101 harboring pTLAB21, which carry the β-glucuronidase (GUS) and green fluorescent protein (GFP) reporters, respectively. β-glucuronidase assays or GFP fluorescence was used to identify transgenic shoots, which developed through organogenesis from stem explants. Transgenic shoots were micrografted onto immature rootstock, and secondarily micrografted onto 4- to 5-mo-old rootstock. NPTII enzyme expression in older trees was confirmed using the immunostrip and enzyme-linked immunosorbent assays (ELISA). Southern blots of a representative group of transgenics showed that most were low copy number with 1 to 3 copies of the T-DNA integrated into the genome. There were a large number of escapes on kanamycin, and many transgenic shoots were lost in micrografting. Several transgenic trees flowered 18 to 19 mo after cocultivation with Agrobacterium. Transformation of mature citrus with disease resistance genes would be a feasible way to produce citrus tolerant to canker and greening. [ABSTRACT FROM AUTHOR]