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/sgRNA technology to modify the canker susceptibility gene CsLOB1 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.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.12677

Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker.

"Candidatus Liberibacter" species are associated with economically devastating diseases of citrus, potato, and many other crops. The importance of these diseases as well as the proliferation of new diseases on a wider host range is likely to increase as the insects vectoring the "Ca. Liberibacter" species expand their territories worldwide. Here, we review the progress on understanding pathogenesis mechanisms of "Ca. Liberibacter" species and the control approaches for diseases they cause. We discuss the Liberibacter virulence traits, including secretion systems, putative effectors, and lipopolysaccharides (LPSs), as well as other important traits likely to contribute to disease development, e.g., flagella, prophages, and salicylic acid hydroxylase. The pathogenesis mechanisms of Liberibacters are discussed. Liberibacters secrete Sec-dependent effectors (SDEs) or other virulence factors into the phloem elements or companion cells to interfere with host targets (e.g., proteins or genes), which cause cell death, necrosis, or other phenotypes of phloem elements or companion cells, leading to localized cell responses and systemic malfunction of phloem. Receptors on the remaining organelles in the phloem, such as plastid, vacuole, mitochondrion, or endoplasmic reticulum, interact with secreted SDEs and/or other virulence factors secreted or located on the Liberibacter outer membrane to trigger cell responses. Some of the host genes or proteins targeted by SDEs or other virulence factors of Liberibacters serve as susceptibility genes that facilitate compatibility (e.g., promoting pathogen growth or suppressing immune responses) or disease development. In addition, Liberibacters trigger plant immunity response via pathogen-associated molecular patterns (PAMPs, such as lipopolysaccharides), which leads to premature cell death, callose deposition, or phloem protein accumulation, causing a localized response and/or systemic effect on phloem transportation. Physical presence of Liberibacters and their metabolic activities may disturb the function of phloem, via disrupting osmotic gradients, or the integrity of phloem conductivity. We also review disease management strategies, including promising new technologies. Citrus production in the presence of Huanglongbing is possible if the most promising management approaches are integrated. HLB management is discussed in the context of local, area-wide, and regional Huanglongbing/Asian Citrus Psyllid epidemiological zones. For zebra chip disease control, aggressive psyllid management enables potato production, although insecticide resistance is becoming an issue. Meanwhile, new technologies such as clustered regularly interspaced short palindromic repeat (CRISPR)-derived genome editing provide an unprecedented opportunity to provide long-term solutions.

The Candidatus Liberibacter-Host Interface: Insights into Pathogenesis Mechanisms and Disease Control.

The NON EXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) and related NPR1-like proteins are a functionally similar, yet surprisingly diverse family of transcription co-factors. Initially, NPR1 in Arabidopsis was identified as a positive regulator of systemic acquired resistance (SAR), paralogs NPR3 and NPR4 were later shown to be negative SAR regulators. The mechanisms involved have been the subject of extensive research and debate over the years, during which time a lot has been uncovered. The known roles of this protein family have extended to include influences over a broad range of systems including circadian rhythm, endoplasmic reticulum (ER) resident proteins and the development of lateral organs. Recently, important advances have been made in understanding the regulatory relationship between members of the NPR1-like protein family, providing new insight regarding their interactions, both with each other and other defense-related proteins. Most importantly the influence of salicylic acid (SA) on these interactions has become clearer with NPR1, NPR3 and NPR4 being considered bone fide SA receptors. Additionally, post-translational modification of NPR1 has garnered attention during the past years, adding to the growing regulatory complexity of this protein. Furthermore, growing interest in NPR1 overexpressing crops has provided new insights regarding the role of NPR1 in both biotic and abiotic stresses in several plant species. Given the wealth of information, this review aims to highlight and consolidate the most relevant and influential research in the field to date. In so doing, we attempt to provide insight into the mechanisms and interactions which underly the roles of the NPR1-like proteins in plant disease responses.

/pdf/the-nonexpressor-of-pathogenesis-related-genes-1-npr1-and-62yy1ecmez.pdf

The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) and related family: mechanistic insights in plant disease resistance.

NPR1, nonexpressor of pathogenesis-related gene 1, is a master regulator involved in plant defense response to pathogens, and its regulatory mechanism in the defense pathway has been relatively clear. However, information about the function of NPR1 in plant response to abiotic stress is still limited. Tomato is the fourth most economically crop worldwide and also one of the best-characterized model plants employed in genetic studies. Because of the lack of a stable tomato NPR1 (SlNPR1) mutant, little is known about the function of SlNPR1 in tomato response to biotic and abiotic stresses. Here we isolated SlNPR1 from tomato ‘Ailsa Craig’ and generated slnpr1 mutants using the CRISPR/Cas9 system. Analysis of the cis-acting elements indicated that SlNPR1 might be involved in tomato plant response to drought stress. Expression pattern analysis showed that SlNPR1 was expressed in all plant tissues, and it was strongly induced by drought stress. Thus, we investigated the function of SlNPR1 in tomato-plant drought tolerance. Results showed that slnpr1 mutants exhibited reduced drought tolerance with increased stomatal aperture, higher electrolytic leakage, malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels, and lower activity levels of antioxidant enzymes, compared to wild type (WT) plants. The reduced drought tolerance of slnpr1 mutants was further reflected by the down-regulated expression of drought related key genes, including SlGST, SlDHN, and SlDREB. Collectively, the data suggest that SlNPR1 is involved in regulating tomato plant drought response. These results aid in further understanding the molecular basis underlying SlNPR1 mediation of tomato drought sensitivity.

/pdf/crispr-cas9-mediated-slnpr1-mutagenesis-reduces-tomato-plant-5fec7w6w2z.pdf

CRISPR/Cas9-Mediated SlNPR1 mutagenesis reduces tomato plant drought tolerance.

Genetic engineering technique offers myriads of applications in improvement of horticultural crops for biotic and abiotic stress tolerance, and produce quality enhancement. During last two decades, a large number of transgenic horticultural crops has been developed and more are underway. A number of genes including natural and synthetic Cry genes, protease inhibitors, trypsin inhibitors and cystatin genes have been used to incorporate insect and nematode resistance. For providing protection against fungal and bacterial diseases, various genes like chitinase, glucanase, osmotin, defensin and pathogenesis-related genes are being transferred to many horticultural crops world over. RNAi technique has been found quite successful in inducing virus resistance in horticultural crops in addition to coat protein genes. Abiotic stresses such as drought, heat and salinity adversely affect production and productivity of horticultural crops and a number of genes encoding for biosynthesis of stress protecting compounds including mannitol, glycine betaine and heat shock proteins have been employed for abiotic stress tolerance besides various transcription factors like DREB1, MAPK, WRKY, etc. Antisense gene and RNAi technologies have revolutionized the pace of improvement of horticultural crops, particularly ornamentals for color modification, increasing shelf-life and reducing post-harvest losses. Precise genome editing tools, particularly CRISPR/Cas9, have been efficiently applied in tomato, petunia, citrus, grape, potato and apple for gene mutation, repression, activation and epigenome editing. This review provides comprehensive overview to draw the attention of researchers for better understanding of genetic engineering advancements in imparting biotic and abiotic stress tolerance as well as on improving various traits related to quality, texture, plant architecture modification, increasing shelf-life, etc. in different horticultural crops.

Genetic engineering strategies for biotic and abiotic stress tolerance and quality enhancement in horticultural crops: a comprehensive review

Commercial sweet orange cultivars lack resistance to Huanglongbing (HLB), a serious phloem limited bacterial disease that is usually fatal. In order to develop sustained disease resistance to HLB, transgenic sweet orange cultivars ‘Hamlin’ and ‘Valencia’ expressing an Arabidopsis thaliana NPR1 gene under the control of a constitutive CaMV 35S promoter or a phloem specific Arabidopsis SUC2 (AtSUC2) promoter were produced. Overexpression of AtNPR1 resulted in trees with normal phenotypes that exhibited enhanced resistance to HLB. Phloem specific expression of NPR1 was equally effective for enhancing disease resistance. Transgenic trees exhibited reduced diseased severity and a few lines remained disease-free even after 36 months of planting in a high-disease pressure field site. Expression of the NPR1 gene induced expression of several native genes involved in the plant defense signaling pathways. The AtNPR1 gene being plant derived can serve as a component for the development of an all plant T-DNA derived consumer friendly GM tree.

/pdf/transgenic-citrus-expressing-an-arabidopsis-npr1-gene-467fhooo0w.pdf

Transgenic Citrus Expressing an Arabidopsis NPR1 Gene Exhibit Enhanced Resistance against Huanglongbing (HLB; Citrus Greening).

Evaluation of transgenic citrus for disease resistance to hlb and canker

Author(s): Dutt, M.; Omar, A.; Barthe, G. A.; Orbovic, V.; Irey, M.; Grosser, J. W. | Abstract: Transgenic citrus scion (mostly grapefruit and sweet orange) and rootstock cultivars (Carrizo and experimental complex tetraploids) were transformed with gene(s) encoding antimicrobial peptides or systemic acquired resistance (SAR) proteins. Each transgene was under control of an enhanced CaMV 35S promoter. Several genes were also under control of a phloem specific Arabidopsis SUC2 (AtSUC2) promoter. A number of clones of each transgenic line (at least 3 replicate plants per clone) were evaluated for resistance to Huanglongbing (HLB, caused by Candidatus Liberibacter asiaticus). 650 clones, from over 180 individual transgenic lines planted in spring 2009 in a heavily HLB infected Martin County grove were tested using qPCR for infection to HLB after 30 months in field. 396 trees tested negative for the HLB bacterium. Approximately 200 clones were observed to be healthy and flushing after 40 months in the field and were again evaluated using qPCR during June 2012. We did not detect the Ca. Liberibacter asiaticus bacterium in a majority of these trees. In a separate trial in St. Lucie County, 300 clones, from over 80 individual transgenic lines planted during 2010 were evaluated in October 2012. Similar trends were observed to that seen in our Martin County site. 345 transgenic clones and controls containing the same transgene(s) were also placed in a greenhouse containing free flying HLB-infected Asian citrus psyllids (ACP) during April 2011. All trees were evaluated for infection after 12 months by qPCR, and 80% of the transgenic trees tested negative for the bacterium. These results suggest that some of the antimicrobial peptides and SAR-inducing proteins can provide long-term resistance against HLB.

/pdf/screening-of-transgenic-citrus-for-hlb-resistance-2fi656ek4q.pdf

Michael Irey

Papers

Transgenic Citrus Expressing an Arabidopsis NPR1 Gene Exhibit Enhanced Resistance against Huanglongbing (HLB; Citrus Greening).

Evaluation of transgenic citrus for disease resistance to hlb and canker

Screening of Transgenic Citrus for HLB Resistance